General Microbiology

COORDINATOR:

SIMONA IVANA

REVISED EDITION Plasticine Collection

INDIGO ROYAL

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

COORDINATOR: SIMONA IVANA AUTHORS: SIMONA IVANA NICOLAE STARCIUC NICOLETA ANDREESCU DANA MAGDALENA CAPLAN CRISTEA COSTACHE DELIA COSTACHE

GENERAL MICROBIOLOGY

REVISED EDITION Plasticine Collection

INDIGO ROYAL Publishing House, 2018

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

BOOK TEAM

COORDINATOR: SIMONA IVANA

AUTHORS: SIMONA IVANA NICOLAE STARCIUC NICOLETA ANDREESCU DANA MAGDALENA CAPLAN CRISTEA COSTACHE DELIA COSTACHE

EDITOR: Simona IVANA

COVER DESIGN AND BOOK ILLUSTRATION Original Product: Plasticine Collection by SIMONA IVANA (Animate Microorganisms): Clostridium perfringens (Lemon Face); Staphylococcus aureus (Grape Man); Bacteriophage T4 (Buddha Man); Escherichia coli (Vamp); Yersinia pestis (Black Plague); Bacillus athracis (Teutonic Knight); Stachybotrys chartarum (Gavroche), Vibrio parahaemolyticus (Seagull), Yellow Fever Virus (Yellow Jack) - http://academiamicrobilorbysimonaivana.com/

BOOK DESIGN IOANA IRINEL POPESCU

No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the INDIGO ROYAL PUBLISHING HOUSE.

Descrierea CIP a Bibliotecii Naţionale a României General microbiology / Simona Ivana (coord.), Nicolae Starciuc, Nicoleta Andreescu, .... - Revised ed.. - Bucureşti : Indigo Royal, 2018 Conţine bibliografie ISBN 978-606-94157-3-3

I. Ivana, Simona II. Starciuc, Nicolae III. Andreescu, Nicoleta

579

ISBN: 978-606-94157-3-3 2

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

In memoriam to Professor Dr. Constantin Ciufecu (1929-2015), for showing me the way! In memoriam to PhD., Scientific Researcher Iudith Ipate (1968-2017), my best friend!

To my students: I don’t love you for what you are, but for what I am when I am with you! You are those from whom I want and expect to receive comments, suggestions, and objections concerning possible difficulties you might have encountered while trying to understand certain mechanisms, processes, etc. in this book! By doing so, I will be able to adjust and correct for future editions. Simona Ivana

To IOANA IRINEL POPESCU: For her attention to detail during the development of this book!

Dr. Simona IVANA D.V.M., Ph.D. Associate Professor and Senior Researcher at Romanian Academy is the coordinator an first author of this book. The most representative paper is ”Treaty of Veterinary Medical Bacteriology and Introduction in Mycology”, published in Medical Sciences Publishing House, Bucharest, 768 pages. This paper was awarded by the Romanian Academy on December 18, 2008, with the ”Traian Săvulescu” prise. During 2011-2013 dr. Simona IVANA made six patents applicationsof which 3 have already been patented. The most representative for her didactic and scientific activity was the patent titled ”Interactive System for Teaching and Testing Students in Microbiology”, which she already included as teaching material in her syllabus.

e-mail: [email protected]

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

reface to Revised Edition

What is Microbiology and what are Microorganisms?

”Messieurs, c'est les microbes qui auront le dernier mot” (Louis Pasteur)

A century ago, Louis Pasteur said ”Life would not long remain possible in the absence of microbes”. Indeed, microorganisms have an impact upon the earth's ecosystems that rivals that of the sun, the oceans, and the activities of humans and other living things. Microbiology is emerging as the key biological science. Microorganisms provide the models used in molecular biology for research. There is growing recognition of the potential of microorganisms in many applied areas. The ability of microorganisms to decompose materials, the potential of microorganisms as food supplements, the exploitation of microbial activity to produce energy such as methane gas, and the potential of new therapeutic substances produced by microorganisms – these and other uses are becoming increasingly attractive. Recombinant DNA technology makes it feasible to consider genetically manipulated microorganisms for commercial production of new andvaluable products for a variety of purposes (medicinal, fuel and food). ”We cannot fathom the marvelous complexity of an organic being; but on the hypothesis here advanced this complexity is much increased. Each living creature must be looked at as a microcosm-a little universe, formed of a host of self-propagating organisms, inconceivably minute as the stars in heaven.” (Charles Darwin). Starting with this concept we became engaged in a race for leadership in science and technology. We are now experiencing a rapid shift of national and international priorities in research and development. In the field of science, and among the biological sciences, microbiology has gained a new stature. Microorganisms and their activities are increasingly central to many of the concerns of society, both nationally and internationally. ”The more formidable the contradiction between inexhaustible life-joy and inevitable fate, the greater the longing which reveals itself in the kingdom of poetry and in the self-created world of dreams hopes to banish the dark power of reality. The gods enjoy eternal youth and the search for the means of securing it was one of the occupations of the heroes of mythology and the sages, as it was of real adventurers in the middle ages and more recent times, but the fountain of youth has not been found, and cannot be found if it is sought in any particular spot on the earth. Yet it is no fable, no dream picture; it requires no adept to find it: it streams forth inexhaustible in all living nature.” (Ferdinand Cohn). In the light of this concept appears a unique perspective aiming to increase our awareness of microorganisms and their roles in illness, industry, and ecology, and that has the power to change people's lives. Although microbiology is primarily the study of microorganisms, it leads irrevocably to the study of animals, humans and their conditions.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Microbiology is rapidly becoming an important field of science, in which the geneticist, the cell physiologist, and the biochemist find, in the ground plowed by the pathologist, a fertile soil for new approaches to fundamental problems of the cell’s functioning and organization.

Content

When we first had the idea for this book we wanted to introduce microbiology in a way that left an exciting and indelible impression on young students and that made difficult concepts comprehensible. At the same time we aimed for a style and organization that produced a readable, current, accessible, and attractive book. Our primary emphasis is on a survey of general topics needed by students entering careers in allied health. My teaching of microbiology, and this book as a result of it, have been built around a central concept, that of the dual nature (”bad and good”) of microorganisms on the one hand, and of operating constituents of functional cells on the other hand. This manual attempts to be thorough without being exhaustive and sufficiently broad in scope to appeal to any interested student desiring a background on the topic.

Organization

Each course begins witha learning objective and key points, and a brief characterization of a microorganism to arouse curiosity in the field of Special Microbiology. This encourages students to develop an ever-expanding background of knowledge and improves their ability to understand increasingly sophisticated subjects. The text is organized into 15 courses grouped into informal units of information. The first group of courses is designed to develop the student's basic background and vocabulary. In Chapter 1 we explain the roles of microorganisms in ecosystem and biotechnology. Microbes serve many functions in almost any ecosystem on the earth. Some microbes (~10%) are pathogens. Chapter 2 contain the morphology of bacteria; shape of bacteria; bacterial classification and identification. It also surveys the basis morphological differences between bacteria; the forms of bacteria and their associations. Chapter 3 deals with bacterial cytology: the study of microscopic detail of bacteria; structures external to the cell wall; ultrastructure of a typical bacterial cell. Chapter 4 cover aspects of cell envelope structure and chemical composition; the three basic layers that can be identified in electron micrographs are the capsule, the cell wall and the cell membrane. Chapter 5 relies on the structure of cytoplasmic membrane and nuclear material; cytoplasmic inclusions, vacuoles, and nuclear material. Chapter 6 discusses about the certain species of bacteria that produce spores, either within the cell (endospores) or external to the cell (exospores); the spore is a metabolically dormant form which under appropriate conditions can undergo germination and outgrowth to form a vegetative cell. Chapter 7 discusses about reproduction and growth to bacteria, distinguish among the types of reproduction in prokaryotes, binary fission a type of reproduction in which the chromosome is replicated and resultant prokaryotic is an exact copy of the parental prokaryote, thus leaving no opportunity for genetic diversity. Chapter 8 is devoted to the essential nutrients: macronutrients and micronutrients; sources of essential nutrients; nutritional types of bacteria; autotrophs and heterotrophs. Chapter 9, 10 and 11 cover aspects of microbial metabolism and enzymes (Chapter 9); the influence of environmental factors on microbes (Chapter 10 and 11).

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Chapter 12 and 13 discuss about viruses; animal and plant viruses greatly vary in size and shape; virions range in size from 20 to 350 nm and represent the smallest and simplest infectious agents (course 12); bacterial viruses or bacteriophages that infect bacteria (Chapter 13). Chapter 14 is about the fungi (molds and yeasts); the fungi are a group of eukaryotic organisms that are of great practical and scientific interests to microbiologists; the science of study of fungi is called Micology. The optional Chapter 15 describes the microbiome; the gut microbiome is very important in maintaining both gastrointestinal and immune function. In this course students will be stepping into a new world with its own language. Educators have shown that providing students with the origins of terminology greatly enhances their recollection, usage, and proficiency with the subject matter. Because visual and mental figures can be extremely helpful in understanding a process, structure, or abstraction, we have tried to develop an unique approach using plasticine technique. Each course opens with a picture (animated plasticine microorganism) that focuses interest on one of the thought-provoking subjects about bacteria, viruses or molds. Numerous feature boxes (color: blue-aqua, 3D, shape: schoolbox) cover topics that are integral to the subject of the course but are separated from the text to conveniently summarize or call attention to the topic. Other boxes discuss interesting historical vignettes, diseases, unusual microorganisms, current developments in science, practical applications, laboratory correlates, and analogies. As an additional study help, we have included a question section (Chapter Quiz). The true-false, multiple choice, and incomplete sentences questions are meant to be a quick but somewhat selective self-test. The concept questions, bacterial jokes and quotes are designed as brain-teasers that challenge students to apply some of the principles in other contexts, to test their depth of understanding, to propose alternate theories, and to develop exercises and models. The concept questions include essay, type questions that challenge comprehension of the principles in that course as well as matching or short-answer questions. The three factors that most contribute to an effective learning experience revolve around the dynamic relationship between student, instructor, and textbook:  The students – Learn most effectively when they have an inherent interest in the topic, and approach it with an inquisitive and curious mind. The goals of study are threefold: 1. To improve the ability to memorize and recall factual information; 2. To increase your ability to interpret and analyze information; 3. To improve understanding. While reading, stop and test yourself on important concepts by closing the book. Because you will usually be evaluated by means of exams, one helpful learning tactic is to become actively involved in self-testing. Study in small groups that allow the free exchange of ideas and cross-quizzing. Do not make the mistake of thinking that, just because concepts are described as basic, they are also easy. The spacing of study time is also important, since devoting a couple of cours a day to study yields greater gains.  The instructor – Is another player who plays a key part in an effective learning environment. Your instructor will serve as the ultimate authority on which sections and topics will be covered and what will be omitted.  The textbook – Is the third learning support. We have purposely written the manual to be comprehensive and up-to-date so that not only will it be a valuable source of information now, but will continue to be so long after the course is over. The discipline of “General Microbiology” commits itself to provide the students within

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

the shortest possible time, additional, really useful study materials. Considering that General Microbiology is the basis of special microbiology, infectious diseases and food control, within the short time allocated to this subject (one semester) I thought that this approach into this new, miraculous and amazing world should help the student stepto go on with courage and curiosity. Microbiology is not only a new subject matter but also a unique and peculiar world that has its own language.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Expanded contents

Preface to Revised Edition ...... 4 Chapter 1. Introduction in Microbiology 1.1. What is Microbiology? What is General Microbiology?...... 11 1.2. Branches of microbiology...... 11 1.3. A brief history of microbiology...... 12 1.4. Importance of microorganisms...... 14 1.5. Eukaryotic and prokaryotic microorganisms ...... 17 1.6. Acellular and unicellular organisms ...... 18 1.7. Units of measurement for microbes...... 18 1.8. Spectrum of microorganisms ...... 19 Chapter 2. The morphology of bacteria 2.1. Shape of bacteria. Group patterns (arrangement of bacterial cell) ...... 27 2.2. Bacterial classification and identification systems...... 32 2.3. Identification of unknown bacteria ...... 33 Chapter 3. Prokaryotic cell structure 3.1. Extracellular (external) structures...... 40 3.1.1. Bacterial flagellum ...... 40 3.1.2. Bacterial fimbria……………………………………………………………………………………...... 45 3.1.3. Pilus ...... 46 3.2. Bacterial conjugation ...... 47 Chapter 4. The cell envelope: The outer wrapping of bacteria 4.1. Bacterial capsule ...... 52 4.2. The cell wall...... 53 4.2.1. The Gram positive cell wall ...... 55 4.2.2. The Gram negative cell wall ...... 56 4.2.3. Exceptions in the cell wall ...... 58 Chapter 5. Structures internal to the cell wall 5.1. The cytoplasmic membrane (plasma membrane)...... 64 5.2. Intracellular (internal) structures ...... 66 5.2.1. The prokaryotic cytoplasm ...... 66 5.2.2. Nuclear material (the bacterial DNA and plasmids) ...... 70 Chapter 6. Bacterial endospores 6.1.General characterization ...... 75 6.2.Endospore structure ...... 75 6.3.The position of the endospore in the bacterial cell...... 77 6.4. Endosporulation ...... 77 6.5. Germination ...... 78 6.6. Endospore resistance ...... 78 6.7. The importance of the bacterial endospore ...... 79 Chapter 7. Reproduction and growth 7.1.Reproduction (modes of cell division) binary fission ...... 84 7.2.Some unusual forms of reproduction in bacteria ...... 85 7.3. Sexual reproduction of bacteria ...... 88 7.3.1. Bacterial transformation ...... 88 7.3.2. Bacterial transduction ...... 89 7.3.3. Bacterial conjugation ...... 90 7.4. Normal growth cycle (growth curve of bacteria)………………………………………………………...... 91 Chapter 8. Bacterial nutrition 8.1. Mode of nutrition in bacteria ...... 98 8.2. Nutritional types of bacteria ...... 102 8.3. Symbiotic bacteria...... 106 8.4. Saprobes and parasitic bacteria ...... 106 8.5. Absorption of nutrients: transport mechanisms ...... 107 8.5.1. Passive transport ...... 107

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

8.5.2. Active transport ...... 109 8.5.3. Bulk transport ………………………………………………………………………………………....110 Chapter 9. Microbial metabolism and enzymes 9.1. Heterotrophic microbial metabolism...... 115 9.2. Fermentation ...... 116 9.3. Special metabolic properties ……………………………………………………………………………….…116 9.3.1. Methylotrophy ……………………………………………………………………………………..….116 9.3.2. Syntrophy …………………………………………………………………………………………..…116 9.4. Anaerobic respiration …………………………………………………………………………………………117 9.5. Enzymes ………………………………………………………………………………………………………119 Chapter 10. The influence of environmental factors on microbes (Part I) 10.1. Temperature ...... 130 10.2. Gas requirements ...... 133 10.3. Osmotic pressure ...... 135 10.4. Radiation ………………………………………………………………………………………...…………..135 Chapter 11. The influence of environmental factors on microbes (Part II) 11.1. Microbial interactions ...... 141 11.1.1. Neutralism ...... 142 11.1.2. Amensalism ...... 142 11.1.3. Competition ...... 142 11.1.4. Cooperation ...... 142 11.1.5. Mutualism ...... 143 11.1.6. Quorum sensing (QS) ...... 144 11.1.7. Parasitism or predation ...... 145 11.1.8. Bacterial intelligence ...... 145 11.1.8.1. Social IQ score of bacteria...... 146 11.1.9. Commensalism ...... 147 11.1.10. Satelitism ...... 148 11.1.11. Selfishness ………………………………………………………………………………...………..148 11.1.12. Altruism ……………………………………………………………………………………...……..149 11.1.13. Antagonism ...... 149 Chapter 12. An introduction to the viruses 12.1. History ...... 156 12.2. Structure and composition ...... 159 12.3. Virus replication ...... 167 12.4. Effect of virus infection on cells ...... 169 12.5. Medical importance of viruses ...... 170 12.6. Epidemiology ...... 170 Chapter 13. Bacterial viruses and prions 13.1. Bacterial viruses (bacteriophages) ...... 181 13.2. Prions ...... 187 Chapter 14. Fungi-Molds and Yeasts 14.1. The Kingdom of the Fungi ...... 199 14.2. Organization of microscopic fungi …………………………………………………………...……………..199 14.3. Asexual and sexual reproduction ...... 202 14.4. Physiology ...... 206 14.5. Molds ...... 207 14.6. Yeasts ...... 211 Chapter 15. The microbiome (optional course) 15.1. General characteristics ……………………………………………………………………………………....220 15.2. Beneficial microbes …………………………………………………………………………………………224 15.3. Definition of a healthy microbiome …………………………………………………………………………226 15.4. Enterotypes of human gut microbiome ……………………………………………………………………...228

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 1 Introduction in Microbiology

1.1. What is microbiology? What is general microbiology? 1.2. Branches of microbiology 1.3. A brief history of microbiology 1.4. Importance of microorganisms 1.5. Eukaryotic and prokaryotic microorganisms 1.6. Acellular and unicellular organisms 1.7. Units of measurement for microbes 1.8. Spectrum of microorganisms

S. aureus (from Greek ”grape-cluster berry”, Latin aureus, ”golden”, is a Gram positive coccal bacterium also known as ”golden staph” and ”oro saphira”. It appears as grape-like clusters when viewed through a microscope, and has large, round, golden- yellow colonies, often with hemolysis, when grown on blood agar plates. S. aureus is catalase-positive, this means, it can produce the enzyme catalase. S. aureus produces various enzymes and exotoxins. Staphylococcus aureus nickname ”Grape Man”

Learning objective

Explain the roles of microorganisms in the ecosystem and biotechnology

Key points

 Microbes serve many functions in almost any ecosystem on the earth  Some microbes (~10%) are pathogens

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

1.1. WHAT IS GENERAL MICROBIOLOGY?

Microbiology (From Greek mikros = small, bios = life, and logia = science) is the study of microscopic organisms, which can be unicellular (single cell), multicellular (cell colony), or acellular (lacking cells). Microbiology encompasses numerous subdisciplines including bacteriology, virology, mycology and parasitology [8].

1.2. BRANCHES OF MICROBIOLOGY

The branches of microbiology can be classified into pure and applied sciences.

A. PURE MICROBIOLOGY. a. Taxonomic arrangement:  Bacteriology: The study of bacteria  Mycology: The study of fungi  Virology: The study of viruses  Immunology: The study of the immune system b. Integrative arrangement:  Microbial cytology: The study of microscopic and submicroscopic details of microorganisms;  Microbial physiology: The study of how the microbial cell functions biochemically. Includes the study of microbial growth, microbial metabolism and microbial cell structure.  Microbial genetics: The study of how genes are organized and regulated in microbes in relation to their cellular functions. Closely related to the field of molecular biology.  Microbial ecology: The relationship between microorganisms and their environment.  Cellular microbiology: Is a discipline bridging microbiology and cell biology.  Evolutionary microbiology: Is the study of the evolution of microbes. This field can be subdivided into: - Microbial taxonomy: the naming and classification of microorganisms; - Microbial systematic: the study of the diversity and genetic relationship of microorganisms; - Generation microbiology: the study of those microorganisms that have the same characters as their parents; - Systems microbiology: a discipline bridging systems in biology and microbiology; - Molecular microbiology: the study of the molecular principles of the physiological processes in microorganisms. c. Other:  Nanomicrobiology: The study of those organisms at nanolevel.  Exomicrobiology (or Astromicrobiology).  Biological agents: The study of those microorganisms which are being used in weapon industries. B. APPLIED MICROBIOLOGY.  Medical microbiology: The study of the pathogenic microbes and the role of microbes in human illness: - microbial pathogenesis; - epidemiology.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 Pharmaceutical microbiology: The study of microorganisms that are related to the production of antibiotics, enzymes, vitamins, vaccines.  Industrial microbiology: - industrial fermentation; - wastewater treatment; - brewing.  Microbial biotechnology: The manipulation of microorganisms at a genetic and molecular level to generate useful products.  Food microbiology: The study of microorganisms causing food spoilage and foodborne illness using microorganisms to produce food (fermentation).  Agricultural microbiology: The study of agriculturally relevant microorganisms. - plant microbiology and plant pathology; - soil microbiology: the study of those microorganisms that are found in the soil.  Veterinary microbiology: The study of the role of microbes in veterinary medicine or animal taxonomy.  Environmental microbiology: The study of the function and diversity of microbes in their natural environments.  Branches: microbial ecology;microbial mediated nutrient cycling; geomicrobiology; microbial diversity; bioremediation.  Water microbiology (or Aquatic Microbiology): The study of those microorganisms that are found in water.  Aeromicrobiology (or Air Microbiology): The study of airborne microorganisms.

1.3. A BRIEF HISTORY OF MICROBIOLOGY

 The existence of unseen microbiological life was postulated by Jainism, which is based on Mahavira’s teachings written as early as 6th century BCE. Paul Dundas noted that Mahavira asserted the existence of unseen microbiological creatures living in the earth, water, air and fire.  Jain scriptures describe the Nigodas which are submicroscopic creatures living in large dusters and having a very short life and are said to pervade each part of the universe, even in tissues of plants and flesh of animals [6].

Feature 1.1. Nigodas-the Jain path of purification

Jain Hindi ancient scripture describe nigodas (microorganisms) as submicroscopic creatures living in large clusters and having a very short life and are said to pervade each and every part of universe, even in tissues of plants and flesh of animals. The Nigoda exist s in contrast to the Supreme Abode, also located at the top of the universe where liberated souls exist in omniscience and eternal bliss. According to Jain tradition, it is said that when a human being rises to this state after death it achieves liberation (Moksha). Every nigoda a Jain sucks in inhabits his or her body, and, because nigoda are young souls facing many earthly lives, their presence in the body increases the host's earthly attachments. This makes attaining mocksa, or Nirvana, much more difficult, forcing the Jain to return to Earth for yet another life [11].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 The Roman Marcus Terentius Varro: There are bred certain minute creatures which cannot be seen by the eyes, which float in the air and enter the body through the mouth and nose thereby causing serious diseases.  In the Medieval Islamic World: - Avicenna ”Ibn Sina” (also known as Avenzoar) in his book ”The Canon of Medicine” described scrabies genus. - Al-Razi in his book ”The Virtuous Life” (Al-Hawi) spoke of parasites.  In modern world: - Girolamo Fracastoro (1546) proposed that epidemic diseases were caused by transferable seedlike entities that could transmit infections by direct or indirect contact, or vehicle transmission. - Antonie van Leeuwenhoek (1676) observed bacteria and other microorganisms using a single-lens microscope of his own design.

Feature 1.2. Antonie van Leeuwenhoek and his microscope

Antonie van Leeuwenhoek was born in Delft, Dutch Republic, on October 24, 1632. He opened a draper's shop, which he ran throughout the 1650s. His status in Delft grew throughout the years. In 1660 he received a lucrative job for the Delft sheriffs' assembly chamber in the City Hall, a position which he would hold for almost 40 years. Raised in Delft, the Netherlands, Van Leeuwenhoek worked as a draper in his youth, and founded his own shop in 1654. He made a name for himself in municipal politics, and eventually developed an interest in lens making [3]. Using his handcrafted microscopes, he was the first to observe and describe single- celled organisms , which he originally referred to as animalcules, and which are now referred to as unicellular organisms. He was also the first to record microscopic observations of muscle fibers, bacteria, spermatozoa, and blood flow in capillaries (small blood vessels). Van Leeuwenhoek did not author any books; his discoveries came to light through correspondence with the Royal Society, that published his letters.

- Robert Hooke made the first microscopic observation of the fruiting bodies of molds in 1665. - Athanasius Kircher was the first to observe microorganisms. One of his books contains a chapter in Latin which reads in translation ”Concerning the wonderful structure of things in nature, investigated by Microscope”. - Ferdinand Cohn (in the 19th century) is considered the father of microbiology. He was a botanist who described several bacteria, including Bacillus and Beggiatoa genus. He also formulated a scheme for the taxonomic classification of bacteria and discovered endospores. - Louis Pasteur formulated the theory of spontaneous generation thereby solidifying microbiology’s identity as a biological science. He also designed methods for food preservation (pasteurization) and vaccines against several diseases such as anthrax, fowl cholera and rabies.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

- Robert Koch is best known for his contributions to thegerm theory of disease, proving that specific diseases were caused by specific pathogenic microorganisms. He developed a series of criteria that became known as the Koch’s postulates. - Winogradsky was the first to develop the concept of chemolithotrophy and thereby reveal the essential role played by microorganisms in geochemical processes. He was responsible for the first isolation and description of both nitrifying and nitrogen-fixing bacteria. - Felix d’Herelle discovered bacteriophages and was one of the earliest applied microbiologists. - Martinus Beijerinck and Sergei Winogradsky (in the late 19th century) were the founders of GENERAL MICROBIOLOGY. Beijerinck made two major contributions to microbiology: 1. The discovery of viruses; 2. The development of enrichment culture techniques.

1.4. IMPORTANCE OF MICROORGANISMS

 Throughout history, microorganisms have given formidable challenges to humans as infectious agents of disease.  The great majority of microorganisms benefit humans by recycling the elements of life, producing many foods and industrial products, and serving as research tools. - Microorganisms are nature’s great recyclers of elements, (some bacteria release nitrogen from animal waste). - Other microorganisms grow on the roots of pod-bearing plants and bring nitrogen back into the cycle of life by using nitrogen to make organic compounds. These compounds are released into the soil where they are utilized as nutrients by plants, such as potatoes. - The range of industrial products of microorganisms is broad and includes diverse products such as perfumes and antibiotic production; - Some bacteria produce enzymes that accelerate the transformation of organic compounds. For example: the enzymes called pectinase decomposes the pectin fibers that bind cellulose fibers in plants. Once the pectin is dissolved, the cellulose can be spun to form linen. Other bacteria are employed to produce enzymes to form a starch used in the sizing of porous material during paper processing. - Through fermentation, microorganisms transform the organic constituents of food. For example, bacteria naturally present in cucumbers grow in the cucumbers and partially digest the plant tissue into a flavorful pickle. Sausages are the product of microbial growth occurring within meat and spices. The baker puts yeast in the dough of baked bread and rolls, and so the dough rises.  Many dairy products are produced through the activity of microorganisms. For instance, cheese is manufactured by heating milk, adding enzymes to curdle the milk protein, and combining bacteria or molds with the milk curds. For example: cheddar cheese, takes its flavor from the acids produced by lactobacilli and streptococci growing within the ripening curd. Yogurt is a form of milk in which bacteria have produced acid from the milk sugar and made the milk sour [11].  Many microbes are responsible for numerous beneficial processes such as: - Industrial fermentation (e.g. the production of alcohol, vinegar and dairy products).

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

- Being vehicles for cloning in more complex organisms such as plants. - Producing important enzymes such as Taq polymerase, reporter genes for use in other genetic systems. - Novel molecular biology techniques such as the yeast two-hybrid system. - Bacteria can be used for the industrial production of aminoacids (e.g. Corynebacterium glutamicum is one of the most important bacterial species with an annual production of more than two millions tons of aminoacids, mainly L-glutamate and L-lysine. - A variety of biopolymers, such as polysaccharides, polyesters and polyamides, are produced by microorganisms. - Microorganisms are beneficial for microbial biodegradation or bioremediation. - Some benefit may be conferred by consuming fermented foods: o probiotics are bacteria potentially beneficial to the digestive system; o prebiotics are substances consumed to promote the growth of probiotic microorganisms. - The ways the microbiome influences human and animal health, as well as methods to influence the microbiome, are active areas of research [12]. - Research suggests that microorganisms could be useful in treating cancer. Various strains of non-pathogenic clostridia can infiltrate and replicate within solid tumors. - There are approximately ten times as many bacterial cells in the human flora as there are human cells in the body. The vast majority of the bacteria in the body are rendered harmless by the protective effects of the immune system, and some are beneficial. Some microorganisms are agents of disease. This plate briefly surveys some examples of how microorganisms influence the quality of our lives. The most common fatal diseases are digestive and respiratory infections. Diarrheal diseases are produced by bacteria like: Campylobacter, Escherichia coli O157 H7, Salmonella, Vibrio, Listeria and Yersinia. Tuberculosis kills about 2 million people per year, mostly in Sub Saharan Africa Countries.

Table 1.1. Some characteristics of major groups of microorganisms

Group Size Characteristics Practical significance Bacter 0,5-1,5 µ Prokaryotic: unicellular organisms grow Some are harmful and cause diseases ia by 1,0-3,0 on artificial laboratory media; (approximately 10%); the majority are µ; range: reproduction asexual by simple cell beneficial with important role in: (1) 0,2 by 100 division natural cycling of elements; (2) µ manufacturing industry; some spoil foods and some make foods edible Viruse Range: All are obligate parasites that do not Cause diseases in humans, animals, plants s 0,015-0,2 grow on artificial laboratory media; and microorganisms (bacteria) µ electron microscopy required to see viruses Yeasts Range: Eukaryotic: unicellular; grow on media Some cause diseases by mycotoxins; some 5,0-10,0 µ like bacteria; reproduction by asexual are used for production of beverages (budding) cell division or sexual process (alcoholic and nonalcoholic) or food supplement Molds Range: Eukaryotic: multicellular, with many Responsible for decomposition 2,0-10,0 µ distinctive structural features; cultivated (deterioration) of many materials; useful by several in laboratory much like bacteria; for industrial production of many mm reproduction by asexual and sexual chemicals (penicillin); cause diseases in processes animals and plants

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 1.3. Top Ten of Deadliest Infections of the World

10. Influenza (Flu) – The most recent flu to hit the headlines was Swine influenza. The alarm bells started ringing when it was found to be a new variant of the H1N1 strain. Perhaps the biggest reason to fear influenza is its ability to combine and mutate to form new strains. 9. HIV (AIDS) – Human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) works by effectively destroying the body’s defenses. More than 30 million people have died of AIDS, while nearly 40 million are currently infected.

8. TBC (Tuberculosis) – TB is a highly contagious disease because it easily spread through airborne droplets (for example: a sneeze). Around a third of the population is actually infected with TB. Tuberculosis kills around 1,5 million people worldwide every year, second only to malaria.

7. Anthrax – The active ingredient of Anthrax is the imaginatively named ”Lethal toxin”. It is not actually lethal until in combination with edema factor, and protective antigen. Together these cause wide scale tissue destruction and bleeding with dark, non-clotting blood oozing from bodily orifices. This deadly effect attracted the military and anthrax was weaponized by US and USSR. In the 1979, in Sverdlovsk, Russia, 68 civilians were killed. Bioterrorism is also a possibility with a series of fatal postal attacks carried out in the USA in 2001. 6. Cholera – It is easily passed through contaminated food and water. In the present it is estimated to kill 120,000 peoples per year. In the past epidemics and pandemics killed millions. The most virulent strains of cholera can kill within 2 hours if the patient is left untreated. 5. MRSA (Methicillin-resistant Staphylococcus aureus) – so called ”Superbug” is a skin infection and it has been known to kill within 24 hrs. The most virulent strains of MRSA are

ST1: USA 400 and ST8: USA 300. 4. Rabies – Worldwide rabies kills around 55,000 people, mostly in Africa and India, but it does still exist in the US and Europe. Rabies is invariably fatal if not treated immediately after bite. Rabies first infects the central nervous system and ultimately causes disease in the brain, leading to death.

3. Smallpox – The disease was responsible for 300 million death since 1800 alone. In hemorrhagic smallpox, the most serious form, there is no blistering of the skin, instead there is bleeding under skin causing it to turn black. ”Black pox” kills in about 6 days. The world has been free of smallpox since 1976 with the last recorded case two-year old Kahima Banu in Bangladesh. It was first used as a bioweapon by British as early as 1789 against Australian aborigines.

2. Bubonic Plague – Is responsible for the Black Death which swept Europe in the Middle Ages killing an estimated 100 million people. The plague is spread by a bacteria carried by rat fleas. Yersinia pestis still exists and sporadic cases occur even in the USA. There have been major outbreaks as recent as 1946, but nothing on the scale of the great plague in history. 1. EBOLA – Is actually a group of viruses, all of which are native of central Africa. The first reported cases in the mid – 1970s appear to have been related to the local taste for bush meat (for example, indigenous wildlife). It is highly contagious with evidence that it can be spread via air. There is no treatment or vaccine. It has been judged a category A bioterrorism agent, the largest number of the human flora being in the gut flora, and a large number on the skin [4].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

1.5. EUKARYOTIC AND PROKARYOTIC MICROORGANISMS (ms)

Both terms (eukaryotes and prokaryotes) are derived from the Greek world karyon, which means “nut” (nucleus of a cell). Prokaryotes are organisms whose cells lack a nucleus, while eukaryotes are organisms whose cells have a well-defined nucleus.

Table 1.2. Differences between prokaryotic and eukaryotic cells

Features Prokaryotic cells Eukaryotic cells Groups where found as unit of Bacteria Algae, fungi, protozoa, plants and structure animals Size: range of organisms 1-2 by 1-4 µ or less Greater than 5 µ in width or diameter Genetic system Nucleoid, chromatin body or nuclear Nucleus, mitochondria, chloroplasts Location material Structure of nucleus Not bounded by nuclear membrane; Bounded by nuclear membrane; one circular chromosome more than one chromosome Sexuality Zygote nature is merozygotic (partial Zygote is diploid diploid) Nature of cytoplasmic matrix Absent Present Pinocytosis Absent Present Gas vacuoles Can be present Absent Mesosomes Present Absent Ribosomes 70 S, distributed in the cytoplasm 80 S, arranged on membranes as in endoplasmic reticulum; 70 S in mitochondria and chloroplasts Mitochondria Absent Present Golgi apparatus Absent Present Endoplasmic reticulum Absent Present Membrane bound (true) Absent present vacuoles Other cell structures Cytoplasmic membranes Generally do not contain sterols; Sterols are present; do not carry out contain part of respiratory and, in respiration and photo-synthesis some, photosynthetic machinery Cell wall Peptidoglycan (murein or Absence of peptidoglycan mucopeptide) as component Locomotor organelles Simple fibril ”9+2” microtubules Metabolic mechanisms Wide variety, particularly that of Glycolysis pathway for anaerobic anaerobic energy; yielding reactions; energy some fix nitrogen gas; some accumulate poly-β-hydroxybutyrate as reserve material DNA base ratios as moles % of 28 to 73 About 40 guanine + cytosine (G+C%)

Eukaryotic microorganisms possess membrane bound cell organelles (suspended in the cytoplasm) and include fungi and protists. For example: mitochondria are sites of energy production for cellular work; ribosomes are mases of RNA and proteins that function as the site of protein synthesis; lysosomes are vesicles that contain enzymes for cellular digestive processes; the Golgi apparatus is a set of

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

tubules and vesicles in which protein is packaged for export; the endoplasmic reticulum consists of flattened tubules involved in the transport of newly-synthesized protein [9]. The cells of eukaryotic organisms possess well-defined nucleus separated from the cell cytoplasm by a nuclear membrane/ envelope. The nucleus contains the genetic information of the cell in multiple strands of DNA and protein called chromosomes. Many familiar organisms are eukaryotes. Animals, including humans and all plants are eukaryotic. The fungi, protozoa and one-celled algae are also eukaryotic. Prokaryotic microorganisms are conventionally classified as lacking membrane-bound organelles and include eubacteria and archaebacteria [1].

1.6. ACELLULAR AND UNICELLULAR ORGANISMS

A. Unicellular organisms (single-celled organism) are organisms that consist of only one cell. The main groups of unicellular organisms are: bacteria, archaea, protozoa, unicellular algae and unicellular fungi. Unicellular organisms are believed to be the oldest form of life, possibly dating back to 3.8 billion years ago. Prokaryotes (Protista) and some fungi areunicellular. Although some of these organisms live in colonies, they are still unicellular. The word prokaryote comes from the Greek (pro = before and karyon = nut or kernel). Prokaryotes can be divided into two domains: Archaea and Bacteria. B. Acellular cells/ Non-cellular life Is the type of life that exists without a cellular structure. This term presumes the phylogenetic scientific classification of viruses as lifeforms, which is a controversial issue. A virus is a small infectious agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea [7].

1.7. UNITSOF MEASUREMENTFOR MICROBES

All linear measurements in microbiology are expressed in metric units. The basic unit of the metric system is the meter (“m”). The equivalent length in the US system is about three feet. The centimeter (cm) is commonly the largest unit of length used for measuring microorganisms One inch = 2,5 cm. For example: Ascaris lumbricoides (a parasitic roundworm common to the intestines) can measure 10 cm or longer. The millimeter (mm) is 1/10th of a centimeter. For example a colony of bacteria growing in a Petri dish of nutrient medium. The micrometer (µ) or micron is the unit of the measurement most frequently used in microbiology and is visible only with a high powered microscope. An average epithelial cell of human skin has a length of about 10 nm. Most bacteria measure about 1 to 5 nm in length. The nanometer (nm) is the unit of length commonly used by microbiologists to measure the dimensions of viruses. The smallest viruses are about 20 nm across. The largest are about 300 nm across. The picometer (pm) is equivalent to a tenth of a nanometer. The thickness of the viral envelope is about 1 pm. It is rarely encountered in microbiology. It has replaced the equivalent of measurement known as the Ångstrom unit [11].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

1.8. SPECTRUM OF MICROORGANISMS

A common feature to most microorganisms is that we may use a microscope to observe their structural details. Microorganisms are grouped by structural, functional and biochemical qualities. A. Viruses are among the smallest microorganisms, and require electron microscopes in order to be observed. This infectious agent replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to microorganisms - bacteria [7].

Virus particles (virions) consist of two or three parts: 1. The genetic material made from either DNA or RNA; 2. A protein coat that protects these genes; 3. In some cases an envelope of lipids that surrounds the protein coat when they are outside a cell. Viruses occur in three major shapes: icosahedral, helical, and complex form.

The origins of viruses are unclear: 1. Some may have evolved from plasmids; 2. Others may have evolved from bacteria. The average virus is about one-one hundredth the size of the average bacterium.

Viruses spread in many ways: 1. Viruses in plants are transmitted from plant to plant by insects (alphids); 2. Viruses in animals can be carried by blood-sucking insects (which are known as vectors). Influenza viruses are spread by coughing and sneezing. Norovirus and Rotavirus causes viral gastroenteritis and are transmitted by the fecal-oral route, entering in the body by food or water. HIV is one of several viruses transmitted through sexual contact and by exposure to infected blood. The range of host cells that a virus can infect is called its host range. Viral infections in animals provoke an immune response, that usually eliminates the infecting virus. Immune responses can also be produced by vaccines which confer an artificially acquired immunity to the specific viral infection. Antibiotics have no effect on viruses.

B. Bacteria (singular = bacterium). Constitute a large domain of prokaryotic microorganisms. Bacteria were among the first life forms to appear on earth and are present in most of its habitats. The word bacteria derives from the Greek - bacteria, meaning “cane”, because the first areas to be discovered were rod-shaped. Bacteria inhabit soil, water, acidic hot springs, radioactive waste and the deep portions of earth’s crust. There are typically 40 million bacterial cells in a milliliter of fresh water. There are approximately 5 x 1030 bacteria on earth, forming a biomass, which exceeds that of all plants and animals. Bacteria are vitalin recycling nutrients such as hydrogen sulphide and methane to produce energy. Although the term bacteria, traditionally included all prokaryotes, the scientific classification changed after discovering in the 1990s that prokaryotes consist of two very

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

different groups of organisms that evolved from an ancient common ancestor. These evolutionary domains are called bacteria and archaea. Archaebacteria. The Archaea constitute a domain or kingdom of single-celled microorganisms. These microbes are prokaryotes, meaning that they have no cell nucleus, or any other membrane-bound organelles in their cells. The word Archaea comes from the Ancient Greek pxa alpha, meaning “ancient things”, as the first representatives of the domain Archaea were methanogens.

Table 1.3. Scientific classification of bacteria

Gram positive/no outer membrane Gram negative/outer membrane present Actinobacteria (high G+C) Aquificae Firmicutes (low G+C) Bacteroides/Fibrobacteres;- Chlorobi (FCB group) Tenericutes (no wall) Deinococcus - Thermus Fusobacteria/Proteobacteria Gemmatimonadetes/Spirochaetes Nitrospirae/Synergistetes Planctomycetes/Verrucomicrobia/Chlamydiae (PVC group) Unknown/ungrouped bacteria Acidobacteria Deferribacteres Chloroflexi Dictyoglomi Chrysiogenetes Thermodesulfobacteria Cyanobacteria Thermotogae

C. Fungi are complex microorganisms, that subdivide into two groups: 1. Molds; 2. Yeasts.

Molds are long, branching chains of cells called hyphae. With vigorous growth, hyphae may result in a visible mass, called a mycelium. Fungi commonly employ spores as a means of reproduction. Many molds prefer acidic environments such as citrus fruits, cheese, and bread. Yeasts are single-celled fungi, about the size of large bacteria. They are important in bread production and fermentation of juices to produce wine. Together with bacteria, the fungi are the prime decomposers of the world’s organic matter.

Scientific classification of molds and easts: 1. Molds: Common genera of molds include: Acremonium, Alternaria, Aspergillus, Cladosporium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma, Trichophyton.

Food production:

 Aspergillus species (A. oryzae and A. sojae) are koji molds which breakdown the starch in rice, barley, sweet potatoes, etc. This process is called saccharification, and is used in the production of sake, shochu and other distilled spirts. Koji molds are also used in the preparation of Katsuobushi.  Monascus purpureus is a red rice yeast and is common in Asia diets. This yeast contains monacolins which inhibit cholesterol synthesis.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 Penicillium nalgiovense improves flavor and reduce bacterial spoilage of dry-cured sausage (salami) and may appear as a powdery white coating.  Fusarium venenatum – quorn.  Geotrichum candidum – cheese.  Neurospora sitophila – oncom.  Penicillium species – various cheese including brie and blue cheese.  Rhizomucor miehei – microbial rennet for making vegetarian and other cheese.  Rhizopus oligosporus – tempeh. Pharmaceutical products from molds:  Penicillium notatum – antibiotic penicillin which adversely affects the growth of Gram positive bacteria, spirochetes and certain fungi.  Aspergillus terrens – Lovastatin.  Tolypocladium inflatum – immunosuppressant drug – Cyclosporine.

Mold health issues: The term “toxic mold” refers to molds that produce mycotoxins (such as Stachybotrys chartatum) including aflatoxins, ochratoxins, fumonisins, trichothecenes, citrinin and patulin.

2. Yeasts: Deak and Benchat have presented an excellent simplified key to foodborne yeasts.

Figure 1.1. Classification of foodborne yeasts

Division: Deuteromycotina Division: Ascomycotina Family: Saccharomycetaceae Family: Cryptococcaceae (the Subfamily: Nadsonioideae imperfects reproduce by budding) Genus: Hanseniaspora Genus: Brettanomyces Subfamily: Saccharomycotoideae Candida Genus: Debaryomycetes Cryptococcus Issatchenkia Rhodotorula Cluyveromyces Trichosoron

Pichia Saccharomyces Torulaspora Zygosaccharomyces Subfamily: Schizosaccharomycetoideae

Genus: Schizosaccharomyces

D. Prions. Are proteins that can fold in multiple, structurally distinct ways. The word prioncoined in 1982 by Stanley B. Prusiner, is derived from the words “protein” and „infection” [1]. The first prion protein was discovered in mammals and is referred to as the major prion protein (PrP). A protein as an infectious agent must contain nucleic acids (either DNA, RNA, or both).

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

All known prion diseases in mammals affect the structure of the brain or other neural tissue and are currently untreatable and universally fatal [10]. This infectious agent causes mammalian transmissible spongiform encephalopathies including:  Bovine spongiform encephalopathy (BSE, also known as “mad cow disease”);  Scrapie – in sheep. In humans PrP causes Creutzfeldt-Jakob Disease (CJD), variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Sträussler-Scheinker syndrome, Fatal Familial Insomnia (FFI) and Kuru.

Feature 1.4. Prion Diseases BSE

You are what you eat CJD and BSE

The term "kuru" derives Creutzfeldt –Jakob disease is a from the Fore word degenerative neurological disorder that is "kuria/guria" ("to shake"), a incurable and invariably fatal. CJD is at reference to the body tremors times called a human form of mad cow that are a classic symptom of the disease (bovine spongiform encephalopathy disease; it is also known among or BSE). the Fore as the "laughing However, given that BSE is believed to sickness" due to the pathologic be the cause of variant Creutzfeldt–Jakob bursts of laughter people would (vCJD) disease in humans, the two are often display when afflicted with the confused. disease. BSE may be most easily transmitted to

It is now widely accepted human beings by eating food contaminated that kuru was transmitted with the brain, spinal cord or digestive tract among members of the Fore tribe of infected carcasses. However, the infectious ofPapua New Guinea via agent, although most highly concentrated in funerary cannibalism [13]. nervous tissue, can be found in virtually all tissues throughout the body, including blood [5].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that it reads accurately.

___1. The branches of microbiology can be classified into pure and applied sciences. ___2. The branches of environmental microbiology are: medical microbiology, industrial microbiology and microbial biotechnology. ___3. Antonie van Leeuwenhoek was instrumental in the development of the oil immersion lens. ___4. The world would function more effectively in the absence of microorganisms. ___5. Microorganisms are called microbes because they are all microscopic. ___6. Prokaryotes differ from eukaryotes solely on the basis of the nucleus.

MULTIPLE CHOICE QUESTIONS

1. Which of the following pioneers of microbiology is credited with the discovery of microorganisms using high magnifying lenses (early microscopes): a. Antonie van Leeuwenhoek; b. Girolamo Fracastoro; c. Robert Hooke; d. Avicena. 2. Microorganisms are grouped by structural, functional and biochemical qualities in: a. viruses; b. bacteria; c. molds; d. yeasts. 3. Prions were discovered by: a. Stanley B. Prusiner; b. Louis Pasteur; c. Robert Hooke; d. Ferdinand Cohn.

CONCEPT QUESTIONS

What event, discovery, or invention would you You think that bacteria are a necessary consider the turning point that marks the evil? Explain why? birth of microbiology?

How do you view disease and germs? What do you suppose the world would be like if there were cures for all infections, diseases and means to destroy all microbes?

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Identify the groups of microorganisms included in the scope of microbiology, and explain the reason for encompassing such diversity.

COMPLETE THE FOLLOWING SENTENCES

 ______are the study of the pathogenic microbes.

 ______are the study of bacteria.

 ______are the study of fungi.

 ______are bacteria potentially beneficial to the digestive system.

 ______are substances consumed to promote the growth of probiotic microorganisms.

 The most common fatal diseases are ______and ______infections.

______made the first microscopic observation of the fruiting bodies of molds in 1665.

QUOTE

Mark with X if you like or dislike this quote.

(1) ”Never memorize something that you can look up”, (Albert Einstein). 1

BACTERIA JOKES

Give an explanation for the following jokes. You may find the explanation in the text of Chapter 1.

 (Q) What is bacteria? (A) The rear entrance to cafeterias.

Explanation:

 (Q) Why did the bacteria cross the playground? (A) To get to the other slide.

Explanation:

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. CTI Reviews (2016). Biology: Biology, Biology, Cram101 Textbook Reviews. ISBN: 1478448911, 9781478448914. 2. ”Commonly Asked Questions About BSE in Products Regulated by FDA's Center for Food Safety and Applied Nutrition (CFSAN)" (2008). Center for Food Safety and Applied Nutrition, Food and Drug Administration. 14 September 2005. Archived from the original on 9 May 2008. Retrieved 8 April 2008. 3. https://en.wikipedia.org/wiki/Antonie_van_Leeuwenhoek. 4. https://www.planetdeadly.com/nature/deadliest-infectious-diseases (2013). 5. I Ramasamy; M Law; S Collins; F Brook (2003). "Organ distribution of prion proteins in variant Creutzfeldt–Jakob disease". The Lancet Infectious Diseases. 3 (4): 214–222. doi:10.1016/S1473-3099(03)00578-4. PMID 12679264. 6. Jaini, Padmanabh S. (1998) [1979]. The Jaina Path of Purification, Delhi: Motilal Banarsidass, ISBN 81-208-1578-5. 7. Koonin EV, Senkevich TG, Dolja VV (2006). The ancient Virus World and evolution of cells. Biology Direct;1:29. doi:10.1186/1745-6150-1-29. PMID 16984643. 8. Madigan M., Martinko J. (2006).Brock Biology of Microorganisms (13th ed.). Pearson Education. p. 1096. ISBN 0-321-73551-X. 9. Michael J. Pelczar, Jr., E.C.S. Chan, Noel R. Krieg (1986). ”Microbiology”, ISBN 10: 0070492344 / ISBN 13: 9780070492349, Published by Mcgraw-Hill College. 10. Prusiner SB (Nov 1998). "Prions". Proceedings of the National Academy of Sciences of the United States of America. 95 (23): 13363–83. Bibcode:1998PNAS...9513363P. doi:10.1073/pnas.95.23.13363. PMC 33918 . PMID 9811807. 11. Simona Ivana (2016). General Microbiology, New Edition, Plasticine Collection, Editura Printech, ISBN: 978-606-23-0640-3, 194 pages. 12. Speedy Publishing LLC (2014). Microbiology: Speedy Study Guides, ISBN: 1634288947, 9781634288941. 13. Whitfield, Jerome T.; Pako, Wandagi H.; Collinge, John; Alpers, Michael P. (2008). "Mortuary rites of the South Fore and kuru". Philosophical Transactions of the Royal Society B: Biological Sciences. 363 (1510): 3721–3724. doi:10.1098/rstb.2008.0074. ISSN 0962-8436. PMC 2581657 . PMID 18849288.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 2 The Morphology of Bacteria

2.1. Shape of bacteria. Group patterns. (arrangement of bacterial cells) 2.2. Bacterial classification and identification systems 2.3. Identification of unknown bacteria

B. anthracis is a Gram- positive, endospore-forming, rod-shaped bacterium, with a width of 1.0–1.2 µm and a length of 3–5 µm. It is one of the few bacteria known to synthesize a protein capsule (poly-D-gamma- glutamic acid). Like Bordetella pertussis, it forms a calmodulin-dependent adenylate cyclase exotoxin known as (edema factor), along with a lethal factor. B. anthracis spores are extraordinarily well-suited to Bacillus anthracis use (in powdered and aerosol form) as biological weapons. nickname ”Teutonic Knight”

Learning objective

Basic morphological differences between bacteria

Key points

The Forms of bacteria and their associations

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

2.1. SHAPES OF BACTERIA

Among the major characteristics of bacterial cells are their size, shape, structure, and arrangement. These characteristics constitute the morphology of the cell. Depending on the species, individual cells are spherical, rod-like, or helical. Furthermore, in certain species of bacteria the cells are arranged in groups, the most common of which are pairs, clusters, chains, trichomes, and filaments. It is important to recognize these patterns of shape and arrangement, since they are often characteristic of a taxonomic group (for example: a genus). All of these morphological features are regarded as the gross morphological characteristics of bacterial cells [3]. A. The shape of bacterium – is governed by itsrigid cell wall. The three basic forms based on the shape of a single cell are: 1. Spheres (coccus). Cocci may be perfect spheres, but they may also be oval, bean- shaped, or even pointy. 2. Round (ended cylinders; bacillus or little rod) - Depending on the bacterial species they may be blocky, spindle-shaped, round-ended, long and threadlike (filamentous), or even clubbed or drumstick-shaped. When a rod is short and plump, it is called a coccobacillus, and if it is gently curved it is a vibrio. 3. Spirillum (coiled or helical) - A rigid helix, twisted twice or more along its axis. Another spiral cell are spirochetes which are helically twisted cylinders; selemonads cylinders curved in one plane [5].

Fig. 2.1. Shapes and aggregations of bacteria (A. cocci; B. bacilli)

Cocci (Sarcina ventriculi) Tetrad

Diplococci (Streptococcus pneumoniae)

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Cocobacillus (Salmonella)

Chain of bacilli (Bacillus cereus) Palisades

Enlarged rod (Fusobacterium) Diplobacillus

Chain of bacilli (Bacillus anthracis) Bacillus

It is rather common for cells of the same species to vary to some extend in shape and size. This phenomenon is called pleomorphism and these cells can exhibit a variety of shapes. For example, the cells of Corynebacterium diphtheriae are generally considered rod-shaped, in culture they display variations such as club-shaped, swollen, curved, filamentous and coccoid. Coccus (plural cocci) derived from the Greek “kokkos” means “berry”, are bacteria whose overall shape is spherical or nearly spherical.

Figure 2.2. Shapes of bacteria (Staphylococcus aureus)

Aggregations

A diplococcus (plural diplococci) is a round bacterium (a coccus) that typically occurs in pairs of two joined cells. Its name comes from “diplo” (meaning “double”) and “coccus” (meaning “berry”). Examples: Streptococcus pneumoniae, Moraxella catarrhalis, Neisseria gonorrheae, and Neisseria meningitides [2].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

The cocci may be single, paired (diplococci), in tetrads (groups of four), in irregular clusters (staphylococci and micrococci), or in chains of four to hundreds of cells (streptococci). A cubical pack of eight, sixteen, or more cells is called a sarcina.

Figure 2.3. Shapes of bacteria

streptococci tetrad coccus

diplococci

sarcina

Figure 2.4. Shapes of bacteria (diplococci encapsulated)

Coccobacillus (plural coccobacilli) is a type of rod-shaped bacteria. The term reflects an intermediate shape between coccus (spherical) and bacillus (elongated). Examples: Haemophilus influenzae, Chlamydia trachomatis, Coxiella burnetti. Bacillus (plural bacilli) is a rod-shaped bacterium. Bacilli usually divide in the same plane and are solitary, but can combine to form diplobacilli (pairs),streptobacilli (chains) and palisades (typical of thecorynebacteria) [6]. A palisades arrangement is formed when the cells of a chain remains attached, but only by a small hinge region at the ends. The cells tend to fold (snap) back upon each other forming a row of cells oriented side by side. Spiral bacteria form the third major bacterial morphology. Spiral bacteria can be subclassified as spirilla, spirochetes, or vibrios based on the number of twists per cell, cell

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

thickness, cell flexibility and motility. Spirillum (plural Spirilla) refers to rigid spiral bacteria that are Gram negative, and are frequently amphitrichous or lophotrichous. Examples: Spirillum spp.,Campylobacter jejuni (the main food borne pathogen), Helicobacter pylori (a cause of peptic ulcers and gastric cancer). Spirochete (plural Spirochetes) refers to very thin, elongate, flexible, spiral bacteria that are motile via endoflagella. Spirochetes may be observed using dark field microscopy or Warthin-Starry stain [1]. Examples: Spirochaetes, Leptospira species (which cause Leptospirosis), Borrelia burgdorferi (a tick-borne bacterium that causes Lyme disease), Treponema pallidum (which cause treponematoses). Vibrio (plural vibrious) refers to Gram negative, comma-shaped rods with a partial twist.

Figure 2.5. Shapes of bacteria

filamentous bacteria

Mycobacteria spirochete

Corynebacteria (palisades arrangement)

Figure 2.6. Borrelia burgdorferi

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

RESEARCH NEWS

If we can prevent Vibrio cholerae from becoming curved we limit their ability to make people sick

Princeton University researchers discovered the protein that allows the bacterium Vibrio cholerae to morph into a corkscrew shape that likely helps it twist into and then escape, the protective mucus that lines the inside of the gut. The researchers report in the Journal Cell that expression of the shape (changing protein) which they named CrvA, is activated through the process of quorum sensing in which bacteria communicate with one another to coordinate an infection. "We know that if V. cholerae can't be curved, it can't make animals as sick" (Gitai et. all). The bacteria may use quorum sensing to alert one another that their environment has changed from water, where the rod shape is advantageous to a host's gut.

Shape has selective value

 Shape has a vector through evolutionary time: rod-like organisms first appeared, and coccoid forms being derivatives at the end of evolutionary lines.  Prokaryotes with different genealogies may converge morphologically, indicating that a similar shape may confer advantages in certain environments. How does the morphology of bacteria contribute to natural selection? Simply put bacteria with different shape present different physical features to the outside world, and these features help cells cope with and adapt to external conditions [4].

Shape contributes

A measure of survival value in the face of three primary selective pressures: 1. Nutrient acquisition; 2. Cell division; 3. Predators. A measure of optimizing in the face of five secondary mechanism: 1. Attachment to surfaces;

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

2. Passive dispersal; 3. Active motility; 4. Internal differentiation; 5. External differentiation. Bacteria respond to predation by developing means of escape, thereby initiating a familiar arms race between predator and prey [7]. This contributes to bacterial diversity and illustrates how cell shape plays a role in three basic defensive strategies: 1. Escaping capture, by being too small or too fast; 2. Resisting ingestion by becoming too large or too long; 3. Making themselves inaccessible, by growing in aggregates or biofilms.

2.2. BACTERIAL CLASSIFICATION AND IDENTIFICATION SYSTEMS

A scheme of classification must make use of current knowledge and show natural relationships, flexible enough to include new information on species and relationships, and complement the several disciplines in microbiology. No one system of classification has been permanent or universally accepted. Of the current proposed systems, we will present. The general scheme of classification of Bergey’s Manual of Systematic Bacteriology, a manual of bacterial descriptions and classification published continuously since 1923, and more detailed system that emphasizes the major, medically important bacterial families. The ninth edition of Bergey’s Manual organizes the Prokaryote Kingdom into four major divisions: The Gracilicutes have Gram negative cell walls and thus are thin-skinned; The Firmicutes have Gram positive cell walls that are thick and strong; The Tenericutes lack a cell wall, and, therefore, are soft; The Mendosicutes are primitive bacteria with unusual cell walls and nutritional habits. The system used in Bergey’s Manual further organizes bacteria into subcategories such as classes, orders and families, but these are not available for all groups. An example of the entire classification of one bacterial species is shown in Table 2.1.

Table 2.1. Taxonomic Rankings for Staphylococcus aureus

Scientific classification Includes Domain: Bacteria Any of various prokaryotic microorganisms such as an archaeon Kingdom: Eubacteria All bacteria Phylum (division): Firmicutes All bacteria with Gram positive cell wall Class: Coccus Coccal bacterium Order: Bacillales Facultative anaerobic, reproduces asexually by binary fission Family: Staphylococcaceae Coccal bacterium found in the respiratory tract and on the skin Genus: Staphylococccus Staph infections such as pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, abcesses, endocarditis, toxic shock syndrome, bacteremia, sepsis Species:S. aureus – Specialty: ICD-9-CM; 041.11 Cause of skin infections, respiratory infections and food poisoning. Also known as “Golden Staph” and Orostaphira

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

aureus Staphylococcus

The individual members of certain species can show variations. These variations are called strains and types. A strain or variety of bacteria is a culture that appears or behaves differently in some way from other cultures of that species. Types are subspecies that may also show differences in antigenic makeup (serotypes), in phage (virus) susceptibility (phage types), and in pathogenicity (pathotypes) [5].

2.3. IDENTIFICATION OF UNKNOWN BACTERIA

The methods that the microbiologist uses to identify bacteria according to genus and species fall into the categories of morphology (microscopic and macroscopic), bacterial physiology or biochemistry, serological analysis and genetic techniques. Final differentiation of the unknown species is accomplished by comparing its profile to profiles of known bacteria in tables, charts and keys. Some bacteria may be identified by a few physiological tests, and Gram stain. Others may require a whole spectrum of tests.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Study of unknown bacteria

UNKNOWN BACTERIA

STAINING

Simple basic staining (Shape of bacteria)

Gram staining (Gram positive/Gram negative) Acid fast staining (Acid fast/Non acid fast)

MOBILITY TEST

Hanging drop preparation (Motile/non-motile)

CULTURAL CHARACTERISTIC

Cultivation of bacteria in nutrient broth (Characteristics of growth in broth) Cultivation of bacteria on agar plates (Characteristics of colonies on plates/of

Cultivation of bacteria on agar slants growth on slants) BIOCHEMICAL TESTS

Series of biochemical tests (Types of biochemical reactions)

IDENTIFICATION

Reffering to “Bergey’s Mannual of (Finding the name of the unknown Determinitative Bacteriology” bacteria upto genus and species level)

FURTHER CONFIRMATION

SEROLOGICAL PCR TEST TESTS

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. The shape of bacterium is governed by its rigid cytoplasmic membrane (plasma membrane). ___2. A diplococcus is a round bacterium that typically occurs in pairs of two joined cells. ___3. Spirillum spp. is the main foodborne pathogen. ___4. Vibrio refers to comma-shaped rods. ___5. The Terenicutes are primitive bacteria with unusual cell walls and nutritional habits.

MULTIPLE CHOICE QUESTIONS

1. The Gracilicutes: a. have Gram negative cell walls, and thus are thin-skinned; b. have Gram positive cell walls that are thick and thin-skinned; c. lack a cell wall and so are soft; d. are primitive bacteria with unusual cell walls.

2. A measure of survival value in the face of three primary selective pressures: a. nutrient acquisition; b. cell division; c. competition; d. active motility.

CONCEPT QUESTIONS

Construct the scientific name of a newly discovered species of bacterium, using your name, a pet's name, a place, or a unique characteristic. Be sure to use proper notation and endings:

If you performed a microscopic examination of an appropriately stained preparation of Staphylococcus aureus, would you expect all the cells to be arranged in clusters?

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Explain why some species of cocci appear as Draw the three bacterial shapes. How are chains but others appear in a cuboidal spirochetes and spirilla different? What is a arrangement: Vibrio? A cocobacillus? What is pleomorphism?

What characteristics are used to classify Demonstrate how cocci may divide in several bacteria? What are the most useful planes, and show the outcome of this division. characteristics for categorizing bacteria into Show how the arrangements of bacilli occur, families? What is the species level in bacteria? including palisades: What are subspecies?

COMPLETE THE FOLLOWING PROPOSITIONS

 ______lack a cell wall and so are soft.

 ______have Gram positive cell walls that are thick and strong.

 ______derived from the Greek kokkos that means berry.

 ______is a rod-shaped bacterium.

QUOTE

Mark with X if you like or dislike these quotes.

(1) ”A desk is a dangerous place from which to view the world” (John le Carré).

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of the Chapter 1.

 (Q) Did you hear about the famous microbiologist who visited 30 different countries, and spoke 6 languages? (B) He was a men of many cultures.

Explanation:

 Hey son don't eat so much, we are rods and you are looking like a coccus!

Explanation:

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Humphrey, Peter A.; Dehner, Louis P.; Pfeifer, John D., eds. (2008). "Chapter 53: Histology and histochemical stains". The Washington Manual of Surgical Pathology. Philadelphia: Lippincott Williams & Wilkins. p. 680. ISBN 9780781765275. 2. https://en.wikipedia.org/wiki/Bacterial_cellular_morphologies. 3. Michael J. Pelczar, Jr., E.C.S. Chan, Noel R. Krieg (1986). ”Microbiology”, ISBN 10: 0070492344 / ISBN 13: 9780070492349, Published by Mcgraw-Hill College. 4. Mitchell J.G. (2002). The energetics and scaling of search strategies in bacteria. American Naturalist. 160:727–740. An exquisitely detailed, far-ranging discussion of how the physical requirements imposed by motility influence and constrain bacterial shape [PubMed]. 5. Simona Ivana (2016). General Microbiology, New Edition, Plasticine Collection, Printech Publishing House, ISBN: 987-606-23-0640-3, 194 pages. 6. "The Size, Shape, And Arrangement Of Bacterial Cells" (2016). Midlands Technical College. Archived from the original on 9 August 2016. Retrieved 8 August 2016. 7. Young K.D. (2007). The selective value of bacterial shape. Microbiol Mol Biol Rev. 2006;70:660–703. A comprehensive recent attempt to compile, describe and classify the ways in which bacteria may utilize morphology to cope with a variety of evolutionary pressures. [PMC free article] [PubMed].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 3 Prokaryotic cell structure

3.1. Extracellular (External) structures 3.1.1. Bacterial flagellum 3.1.2. Bacterial fimbriae 3.1.3. Pilus 3.2. Bacterial conjugation

L. monocytogenes is a Gram- positivebacterium, named after Joseph Lister. Motile via flagella at 30 °C and below, but usually not at 37 °C, L. monocytogenes can instead move within eukaryotic cells by explosive polymerization of actin filaments (known as comet tails or actin rockets). Listeria monocytogenes can cause meningitis in newborns. Pregnant mothers are often advised not to eat soft cheese such as Brie, Camembert, feta, and queso blanco fresco, which may be Listeria monocytogenes contaminated. nickname ”Russian Girl”

Learning objective

Bacterial cytology: The study of microscopic details of bacteria Structures external to the cell wall

Key points

Ultrastructure of a typical bacterial cell

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

There are two major kinds of prokaryotes: 1. Bacteria 2. Archaea (single-celled organisms) Some prokaryotic cells also have other structures like the cell wall, pili (singular pilus) and flagella (singular flagellum). Each of these structures and cellular components plays a critical role in the growth, survival and reproduction of prokaryotic cells [10].

Appendages Flagella/ Axial filaments Pili, fimbriae

Glycocalyx (capsules, slime layers) Cell envelope Cell wall Cell membrane

Cell pool Ribosomes Protoplasm Mesosomes Granules Nuceloid/ chromatin bodies

Figure 3.1. Structure of a bacterial cell

flagellum Describe my anatomy

cytoplasm ribosomes cell membrane cell wall

nucleoid

mesosome plasmid

fimbriae inclusion bodies

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

3.1. EXTRACELULAR (EXTERNAL) STRUCTURES

3.1.1. Bacterial flagellum (Appendages: structures for swimming)

The flagellum (plural: flagella) is an appendage of truly amazing construction, unique in the biological world. A flagellum is a lash-like appendage with role in locomotion. The word flagellum in Latin means “whip”. There are large differences between prokaryotic and eukaryotic flagella in the composition of the protein, and in the structure and mechanism of propulsion. However, both are used for swimming. Prokaryotic flagella run in a rotary movement while eukaryotic flagella run in a bending movement. The prokaryotic flagella uses a rotary motor, and eukaryotic flagella uses a complex sliding filament system. Eukaryotic flagella is ATP driven, while prokaryotes are protein driven. A flagellum is composed of three parts: a basal body associated with the cytoplasmic membrane and cell wall, a short hook, and a helical filament which is usually several times as long as the cell. Some Gram negative bacteria have a sheath surrounding the flagellum. The chemical composition of the basal body is unknown, but the hook and filament are composed of protein subunits (monomers) arranged in a helical fashion. The protein of the filament is known as flagellin. Unlike a hair, a flagellum grows at its tip rather than at the base. Flagellin monomers synthesized within the cell are believed to pass along the hollow center of the flagellum and are added to the distal of the filament.

Figure 3.2. Bacterial flagellum

hook

basal body

flagellin

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Structure and composition

 The bacterial flagellum is made up of the protein flagellin.  Its shape is 20 nm in diameter and varies from 1 to 70 μm in length.  It is helical and has a sharp bend just outside the outer membrane. This hook allows the axis of the helix to point directly away from the cell. The mechanism of the hook/basal body articulation is very much like a ball in a socket. This arrangement permits the hook and its filament to rotate 3600, rather than undulating back and forth like a whip, as was once thought. Gram positive organisms have two of these basal body rings (S ring and M ring), one in the peptidoglycan layer and one in the plasma membrane. Gram negative organisms have four such rings: the L ring associates with the lipopolysaccharides (outer membrane), the P ring associates with peptidoglycan layer, the M ring is embedded in the plasma membrane, and the S ring is directly attached to the plasma membrane. The filament ends with a capping protein [3]. The flagellar filament is the long helical screw that propels the bacterium when rotated by the motor through the hook. In most bacteria, the filament is made up of eleven protofilaments approximately parallel to the filament axis. Each protofilament is a series of tandem protein chains. The bacterial flagellum is driven by a rotary engine (the Mot complex) made up of protein located at the flagellum’s anchor point on the inner cell membrane. The engine is a powered by proton motive force (hydrogen ions) across the bacterial cell membrane due to a concentration gradient set up by the cell’s metabolism [10]. The rotor transports protons across the membrane and is turned in the process. The rotor alone can operate 6,000 to 17,000 rpm, but with the flagellar filament attached usually only reaches 200 to 1000 rpm. The direction of rotation can be switched almost instantaneously, caused by a slight change in the position of a protein, FliG, in the rotor. The flagellum is highly energy efficient and uses very little energy. The rotational speed of the flagella varies in response to the intensity of the proton motive force, thereby permitting certain forms of speed control and attaining remarkable speeds in proportion to their size. In comparison to macroscopic life forms it is very fast. For example, a cheetah only achieves about 25 body lengths/sec., while a bacterial flagella can achieve. Through the use of their flagella, bacterium are able to move rapidly towards attractants and away from repellents. They do this by means of a biased random walk, with runs and tumbles brought about by rotating the flagellum counterclock-wise and clockwise respectively. This phenomenon is called bacterial chemotaxis. Swimming toward a chemical is termed positive chemotaxis while swimming away is called negative chemotaxis. Although chemicals may act as attractants or repellents, the stimulus is in fact not the chemical itself but rather a change in the concentration of the chemical with time, a temporal gradient. Such gradients are sensed by means of protein chemoreceptors which are located on the cytoplasmic membrane and are specific for various attractants and repellents. Flagella are left-handed helices and bundle and rotate together only when rotating counterclock-wise. When some of the rotors reverse direction, the flagella unwind and the cell starts tumbling [6].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 3.3. Flagellar movement

Flagellar arrangement

Different species of bacteria have different numbers and arrangements of flagella. A. Polar organelle:  Monotrichous bacteria have a single flagellum (for example: Vibrio cholerae);  Lophotrichous bacteria have multiple flagella located at the same spot on the bacteria’s surfaces;  Amphitrichous bacteria with flagella at both poles of the cell. B. Peritrichous bacteria have flagella projecting in all directions (for example: Escherichia coli).

Figure 3.4. Flagellar arrangement - monotrichous -

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 3.5. Flagellar arrangement - lophotrichous -

Figure 3.6. Flagellar arrangement - amphitrichous –

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Other forms of flagella

Selemonas form a thick structure called a fascicle. The flagellum was found to be quite unrelated to the flagellum of ciliate protozoa, instead consisting of a fascicle of numerous bacterial-type flagella twisted just outside the cell body into helical bundles to form strong organs of propulsion.

Figure 3.7. Spirochetes

DNA in nucleoid inner (plasma membrane)

cell wall (peptidoglycan)

Spirochetes have two or more specialized flagella (endoflagella) arising from opposite poles of the cell which together constitute the so-called axial filament that is located within the periplasmic space between the flexible cell wall and an outer sheath [4]. Flagella are left-handed helices and bundle and rotate together only when turning counterclock-wise. When some of the rotors reverse direction, the flagella unwind and the cell starts tumbling.

Figure 3.8. Gliding motility

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Gliding motility. Some bacteria (for example: Cytophaga spp.) are motile only when they are in contact with a solid surface. As they glide they exhibit a sinuous, flexing motion. This kind of movement is comparatively slow, only a few micron per second.

3.1.2 Bacterial fimbriae (non-locomotor appendages for clinging; sometimes called attachment pili)

Fimbria (plural fimbriae) is an appendage composed of curling proteins. A fibria is a short pilus that is used to attach the bacterium to a surface (they are some times called “attachment pili”). Fimbria ranges from 3-10 nm in diameter and can be up to several micrometers long. A bacterium can have as many as 1,000 fimbriae. They may be straight or flexible. Fimbriae are only visible with the use of an electron microscope. Theyare either located at the poles of a cell, or are evenly spread over its entire surface. Fimbria are found in Gram negative bacteria. Some fimbriae can contain lectins. These are necessary to adhere to target cells because they can recognize oligosaccharide units on the surface of these target cells. Some aerobic bacteria form a thin layer at the surface of a broth culture. This layer, called a pellicle, consists of many aerobic bacteria that remain on the broth, from which they take nutrients while they congregate near the air [5]. Fimbriae carry adhesins by which they attach themselves to the substratum (Escherichia coli uses them to attach itself to mannose receptors) so that the bacteria can withstand shear forces and obtain nutrients [10]. Fimbriae are composed of a protein called fimbrillin (or pilin), they are 3 to 25 nm in diameter and 10 to 20 μm in length. Fimbrillin produced in Gram negative bacteria is a subunit protein with a molecular weight of 17 to 20 k Da. Fimbriae function as cellular organelles for attachment to cells and/or mucosal surfaces. Fibriae that use this attachment function are often referred to as adhesins. For example, the adherence of enteric bacteria to mucosal surfaces that is mediated by type 1 (type-specific) can be inhibited by preincubation of the bacteria with mannose. Among the Gram positive bacteria, only a limited number of species express cell surface fimbriae including some streptococci, corynebacteria and Actinomyces species (A. viscosus and A. naeslundii). The role of fimbriae as virulence factors in Gram negative bacteria has been studied extensively in Neisseria gonorrhaeae and Neisseria meningitidis. Virulent strains of N. gonorrhaeae are able to adhere avidly to mucosal cells in the genital tract. This is also the means by which the gonococcus (agent of gonorrhea) invades the genitourinary tract and Escherichia coli invades the intestine. Unlike the fimbriae from Gram negative bacteria, fimbriae of Actinomyces species are covalently linked to the cell wall of the peptidoglycan layer. The ability of the oral Actinomyces species to adhere to buccal mucosal cells and to congregate with cariogenic oral streptococci facilitates biofilm formation and the initiation of the dental plaque. Fimbria clearly play a key role in the ability of these periodontal pathogens to colonize and initiate infection.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 3.9. Bacterial fimbriae, pili and flagella

flagella

fimbriae

pilus

Table 3.1. Bacterial fimbriae and their adhesins

Bacteria Target structure Adhesin Oral Lis-gingipain Porphyromonas Mucosal Arg-gingipain gingivalis Cells Hemagglutinin Gonococci Urothelial cells Pilin Steptococcus pyogenes Fibronectin Protein F Bordetella pertussis Bronchial mucosal cells Pertactin Streptococcus mutans Dental enamel Adhesin P1

3.1.3 Pilus (sometimes called sex pili)

A pilus is a hair-like appendage found on the surface of many bacteria. All pili are primarily composed of oligomeric pili proteins. Pili are antigenic. They are fragile and constantly replaced with pili of different composition, resulting in altered antigenicity [5]. Conjugative pili allow for the transfer of DNA between bacteria in the process of bacterial conjugation. During conjugation, a pilus emerging from donor bacterium ensnares the recipient bacterium, draws it in close, and eventually triggers the formation of a mating bridge, which establishes direct contact and the formation of a controlled pore that allows transfer of DNA from the donor to the recipient [9]. Pili are longer than fimbriae and there are only a few per cell. Present in both Gram negative and Gram positive bacteria, pili are involved in many processes such as conjugation, adherence, motility, biofilm formation and immunomodulation.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

3.2. BACTERIAL CONJUGATION

Luria and Delbruck demonstrated in 1943 that bacteria have a stable hereditary system. Conjugation represents the direct transfer of DNA from one bacterial cell to another bacterial cell. The transferred DNA is a plasmid, a circle of DNA that is distinct from the main bacterial chromosome. The F plasmid is similar to a virus or a transposon in its ability to move independently of the main chromosome. The transfer of the plasmid takes advantage of the complementary nature of double stranded DNA. One strand of the plasmid is transferred and the other remains in the original cell. Both strands have the complementary stranded added so that each ends up with a complete plasmid [1]. Cells carrying F plasmid are designed F+ and those lacking it are F-. The F plasmid contains approximately 100 genes, which give the plasmid several important properties:  Cells carrying the F plasmid promote the synthesis of pili (singular pilus) on the bacterial cell surface. Pili are minute proteinaceous tubules that allow the F+ cells to attach to other cells and maintain contact with them that is, to conjugate.  F+ and F- cells can conjugate. When conjugation occurs, the F+ cells can act as F donors. The transfer of the F plasmid from F+ to F- is rapid, so the F plasmid can spread like wildfire through a population from strain to strain.  Sometimes F carries within its genome one or more of its (insertion-sequence) elements. A crossover between the two circular DNAs leads to the integration of the plasmid into the bacterial chromosome. When this integration occurs, F can drive the transfer of the entire host chromosome into the recipient cell, along with its own integrated F DNA. Therefore, strains with an integrated F factor are termed high frequency of recombination (Hfr) strains. Because they transfer chromosomal markers efficiently, Hfr strains are the ones used for genetic mapping [2].  The integrated F factor occasionally leaves the chromosome of an Hfr by itself and moves back to the cytoplasm. This modified F, called F' (pronounced F prime), can now transfer these specific host genes to a recipient (F-) cell in an infectious manner, in the same way that F spread. Thus the recipient cell now contains two copies of the same gene-one resident copy on its bacterial chromosome and one copy of the newly transferred cytoplasmic F' factor.

Figure 3.10. Bacterial conjugation

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. There are four major kinds of prokaryotes. ___2. The word “flagellum” in Latin means “whip”. ___3. The chemoreceptors are located in the cell wall. ___4. The fimbria is used to attach the bacterium to a surface. ___5. In general, rods are motile and cocci are non-motile.

MULTIPLE CHOICE QUESTIONS

1. Polar organelle are: a. monotrichous; b. lophotrichous; c. amphitrichous; d. peritrichous.

2. Other forms of flagella are: a. Gram positive bacteria; b. spirochetes; c. gliding bacteria; d. Gram negative bacteria.

3. Fimbria are found in: a. Gram negative bacteria; b. Gram positive bacteria; c. gliding bacteria; d. selemonas bacteria.

CONCEPT QUESTIONS

Fill the blanks with the cellular components of Draw the structure of a flagellum and how it prokaryotic cell: operates. What are the four main types of flagellar arrangement?

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

COMPLETE THE FOLLOWING SENTENCES

 ______organisms have two basal body rings (S ring and R ring).

 ______organisms have four basal body rings (L, P, M, S).

 ______bacteria have a single flagellum.

 ______bacteria have flagella at both poles of the cell.

 Strains with an integrated F factor are called ______.

QUOTES

Mark with X if you like or dislike these quotes.

(1) ”Always trust in microbiologist because they have the best chance of predicting when the world will end” (Teddie O. Rahube). (2) ”Chance favors the prepared mind” (Louis Pateur). (1) (2)

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of Chapter 1.

 (Q) What is the fastest way to determine the sex of a chromosome? (A) Pull down its genes.

Explanation:

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Amin Elsersawi (2016). Gene Editing, Epigenetic, Cloning and Therapy. Editor: Author House. ISBN: 1524621986, 9781524621988. 2. Anthony JF Griffiths, William M Gelbart, Jeffrey H Miller, and Richard C Lewontin (1999). Modern Genetic Analysis. New York: W. H. Freeman; 1999. ISBN: 10: 0-7167-3118-5. 3. Diószeghy Z., Závodszky P., Namba K., Vonderviszt F. (2004). "Stabilization of flagellar filaments by HAP2 capping". FEBS Lett. 568 (1–3): 105–9. doi:10.1016/j.febslet.2004.05.029. PMID 15196929. 4. https://en.wikipedia.org/wiki/Flagellum. 5. https://en.wikipedia.org/wiki/Pilus. 6. Kim M., Bird J.C., Van Parys A.J., Breuer K.S., Powers T.R. (2003). "A macroscopic scale model of bacterial flagellar bundling". Proc. Natl. Acad. Sci. U.S.A. 100 (26): 15481–5. doi:10.1073/pnas.2633596100. PMC 307593 . PMID 14671319. 7. Macnab R.M. (1977). "Bacterial flagella rotating in bundles: a study in helical geometry". Proc. Natl. Acad. Sci. U.S.A. 74 (1): 221–5. doi:10.1073/pnas.74.1.221. PMC 393230 . PMID 264676. 8. Macnab R.M. (2003). "How bacteria assemble flagella". Annu. Rev. Microbiol. 57: 77–100. doi:10.1146/annurev.micro.57.030502.090832. PMID 12730325. 9. Mattick J.S. (2002). "Type IV pili and twitching motility". Annu. Rev. Microbiol. 56 (1): 289–314. doi:10.1146/annurev.micro.56.012302.160938. PMID 12142488. 10. Simona Ivana (2016). General Microbiology, New Edition, Plasticine Collection, Printech Publishing House, ISBN: 987-606-23-0640-3, 194 pages.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 4 The cell envelope: The outer wrapping of bacteria

4.1. Bacterial capsule 4.2. The cell wall 4.2.1. The Gram positive cell wall 4.2.2. The Gram negative cell wall 4.2.3. Exceptions in the cell wall

Bacillus cereus is a Gram- positive , rod-shaped, motile, beta hemolytic bacterium. Some strains are harmful to humans and cause foodborne illness, while other strains can be beneficial as probiotics for animals. It is the cause of "fried rice syndrome", as the bacteria are classically contracted from fried rice dishes that have been sitting at room temperature for hours. B. cereus bacteria are facultative anaerobes, and Bacillus cereus produce endospores. Its virulence nickname ”Rice Man” factors include cereolysin and phospholipase C.

Learning objective

Structure and chemical composition of cell envelope

Key points

The three basic layers that can be identified in electron micrographs are the capsule, the cell wall and the cell membrane

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

4.1. BACTERIAL CAPSULE

Some bacterial cells are surrounded by a viscous substance forming a covering layer or envelope around the cell wall. The capsule is a gelatinous layer covering the entire bacterium composed of polysaccharide (i.e. poly = many; saccharide = sugar). The capsule is located immediately to exterior of the murein layer of Gram positive bacteria and the outer membrane of Gram negative bacteria (exception: the capsule of Bacillus anthracis that is composed entirely of a polymer of glutamic acid). The sugar component of polysaccharidae varies within the species of bacteria. This determines their serologic types. Example: Streptococcus pneumoniae has 84 different serologic types discovered so far. Capsules composed of a single kind of sugar are termed homopolysaccharides and are usually synthesized outside the cell from disaccharides by exocellular enzymes. The synthesis of glucan (a polymer of glucose) from sucrose by S. mutans is an example. Other capsules are composed of several kinds of sugars and are termed heteropolysaccharides. The capsule of Klebsiella pneumoniae is an example [8]. If the layer is too thin to be seen by light microscopy it is called a microcapsule. If it is so thick that many cells are embedded in a common matrix, the material is called slime.

Importance of bacterial capsule

 Virulence determinants - Capsules are antiphagocytic. If pathogenic bacteria lose a capsule (by mutation), they won't be able to cause disease.  Identification of bacteria - (a) Using specific antiserum against capsular polysaccharide; (b) Colony characteristics in culture media; capsulated organisms form mucoid colonies.  Development of vaccines - Capsular polysaccharides are used as antigens in certain vaccines. For example: the purified capsular polysaccharides of 23 types of S. pneumoniae are present in current vaccine.  Initiation of infection – Capsules help the organism to adhere to host cells. The capsule also facilitates and maintains bacterial colonization of biologic and inanimate (prosthetic heart valves) surfaces through formation of biofilms [9].

Examples of capsulated bacteria

Bacteria Yeasts Streptococcus pneumoniae Klebsiella pneumoniae Haemophilus influenzae Criptococcus neoformans Pseudomonas aeruginosa Neisseria meningitidis Mneomonics to remember capsulated bacteria and yeasts: Some KillersHavePreetyNice Capsule Capsulated Gram negative bacteria– Escherichia coli (some strains), Neisseria meningitidis, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Salmonella spp. Capsulated Gram positive bacteria(glycocalyx, slime layers): Bacillus megaterium (synthesizes a capsule composed of polypeptides and polysaccharides), Streptococcus pyogenes (synthesizes a hyaluronic acid capsule), Streptococcus pneumoniae, Streptococcus agalactiae (produces a polysaccharide capsule of nine antigenic types that all contain sialic acid: Ia, Ib, II, III, IV, V, VI, VII, VIII), Staphylococcus epidermidis. Capsules too small to be seen with an ordinary microscope are called microcapsules. For example: M protein of Streptococcus pyogenes

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Demonstration of capsule

 India ink staining – the capsule appears as a clear halo around the bacterium;  Serological methods – capsular material is antigenic and can be demonstrated by mixing it with a specific anticapsular serum. This phenomenon is the basis of Quellung reaction (a method used to visualize capsule under a microscope). It is used in vaccination. Vaccination using capsular material is effective against some organisms. For example: Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitides [1].

Figure 4.1. Bacterial capsule

bacterial capsule

4.2. THE CELL WALL

The cell wall is a very rigid structure that gives shape to the cell and it is placed external to the cytoplasmic membrane. Its main function is to prevent the cell from expanding and bursting because of intake of water, since most bacteria live in hypotonic environments [8].

Structure and chemical composition

The bacterial cell wall differs from that of all other organisms by the presence of peptidoglycan (sometimes called murein), an insoluble, porous, cross-linked polymer of enormous strength and rigidity. It is located immediately outside of the cytoplasmic membrane.  Peptidoglycan is made up of a polysaccharide backbone consisting of alternating N-acetyl muramic acid (NAM) and N-acetylglucosamine (NAG) residues in equal amounts.  Peptidoglycan is responsible for the rigidity of the bacterial cell wall and for establishing the cell shape (Mre B protein has been identified as a homologue of actin and facilitates cell shape).  Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 It is relatively porous and is not considered to be a permeability barrier for small substrates.  The peptidoglycan layer is thicker in Gram positive bacteria (20 to 80 nm) than in Gram negative bacteria (7 to 8 nm) with the attachment of the S-layer.  Peptidoglycan forms around 90% of the dry weight of Gram positive bacteria, but only 10% of Gram negative cells.  For both Gram positive and Gram negative bacteria, particles of approximately 2 nm can pass through the peptidoglycan.  There are two main types of bacterial cell walls: - Gram positive bacteria, Gram negative bacteria.  If the bacterial cell wall is entirely removed it is called a protoplast, while if it’s partially removed it is called a spheroplast. A protoplast is that portion of a bacterial cell consisting of the cytoplasmic membrane and the cell material bounded by it. Protoplast can be prepared from Gram positive bacteria by treating the cells with an enzyme such as lysozyme, which selectively dissolves the cell wall or by culturing the bacteria in the presence of an antibiotic such as penicillin, which prevents the formation of the cell wall. The treated cell has two membranes, the cytoplasmic membrane of the protoplast plus the outer membrane of the cell wall, (the cell is called a spheroplast).

Feature 4.1. “The man behind the Gram stain”

Those who work in diagnosis bacteriology labs will hear his name mentioned every day, probably several times, but how many of us know about where the name Gram came

from? [2].

Hans Christian Gram (September 13, 1853 - November 14, 1938) was a Danish bacteriologist noted for his development of the Gram stain. The work that gained Gram an international reputation was his development of a method of staining bacteria, to make them more visible under a microscope. The stain later played a major role in classifying bacteria. Gram was a modest man, and in his initial publication he stated “I have therefore published the method, although I am aware that as yet it is very defective and imperfect; but I hope that in the hands of other investigators it will turn out to be useful”.

Gram staining is a technique used to identify bacteria into two different groups, Gram positive and Gram negative. The staining technique has been widely used in medical diagnosis. In 1884, Hans Christian Gram noticed that certain strains were preferentially taken up by bacteria from post mortem lung tissue samples. He used crystal violet as the initial stain and fixed the stain with potassium triiodide (Lugol’s solution). After this he used ethanol to wash the stain away. He did this with both Streptococcus pneumoniae and Klebsiella pneumoniae bacteria observing that Streptococcus pneumonia retained the stain after washing with alcohol (Gram positive) whereas Klebsiella pneumoniae did not (Gram negative ). Today, Hans Christian Gram would be 161 years old [8].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 4.2. Gram stain

Gram positive bacteria have a thick mesh -like cell wall made of peptidoglycan (50- 90% of cell envelope) and, as a result, are stained purple by crystal violet, whereas Gram negative bacteria have a thinner layer (10% of the cell envelope), so they do not retain the purple stain and are counter-stained red by the safranin [3]. There are four basic steps of the Gram stain: 1. Applying a primary stain (crystal violet - CV) to a heat-fixed smear of a bacterial culture. CV dissociates in aqueous solutions into CV+ and chloride - (Cl ) ions. These ions penetrate through the cell wall and cell membrane of both Gram positive and Gram negative cells and the bacteria cells are all purple. 2. The addition of iodide binds to crystal violet and traps it in the cell. Iodide interacts with CV+ and forms large complexes of crystal violet and iodine within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but it is a trapping agent that prevents the removal of the CV- I complex and, therefore, color the cell. 3. Rapid decolorization with ethanol or acetone. A Gram negative cell loses its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. Furthermore, when the alcohol decolorizer is applied, the alcohol dissolves lipids in the outer membrane of Gram negative cells, permitting the dye to be removed from the thin cell wall. By contrast the crystals of dye are trapped in the cell walls of Gram positive bacteria, where they are relatively inaccessible and resistant to removal. The decolorization step is critical and must be timed correctly; the crystal violet stain is removed from both Gram positive and negative cells if the decolorizing agent is left on too long (a matter of seconds). 4. Counterstaining with safranin (carbol fuchsin). After decolorization, the Gram positive cell remains purple and the Gram negative cell loses its purple color. Counterstain (which is usually positively charged safranin or basic fuchsine), is applied last to give decolorized Gram negative bacteria a red color[5]. This century-old method remains the universal basis for bacterial taxonomy and identification. It allows differentiation of four major categories based up on color reaction and shape: Gram positive rods, Gram positive cocci, Gram negative rods, Gram negative cocci [16]. Even in this day of elaborate and expensive medical technology, the Gram stain

remains an important and unbeatable first tool of diagnosis.

4.2.1. The Gram positive cell wall

 Gram positive cell walls are thick and the peptidoglycan (murein) layer constitutes almost 95% of the cell wall.  They have O-acetylgroups on carbon-6 of some MA residues.  The matrix substances in the walls of Gram positive bacteria may be polysaccharides or teichoic acids (TA).

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 TA (Greek – teikhos “wall”= specific fortification wall) are bacterial polysaccharides of glycerol phosphate or ribitol phosphate linked via phosphodiester bonds.  TA are found in bacteria from the genera Staphylococcus, Streptococcus, Bacillus, Clostridium, Corynebacterium and Listeria.  TA that remain anchored to lipids are referred to as lipoteichoic acids.  The main function of TA is to provide rigidity to the cell wall by attracting cations such as magnesium and sodium.  TA assist in regulation of cell growth by limiting the ability of autolysins to break the β(1-4) bond between the N-acetylglucosamine and the N-acetylmuramicacid.  Zwitterionic TA are suspected ligands for toll-like receptors 2 and 4. Zwitterionic TA (derived from German “Zwitter”= Hibrid and for merely called dipolar ion) is a neutral molecule with positive and negative electrical charges. TLRs (tool-like receptors autolysin) is an enzyme that hydrolyzes the components of a biological cell or a tissue in which it is produced.

Figure 4.2. Gram negative bacterial cell wall

outer lipid membrane

peptidoglycan

plasma membrane

4.2.2. The Gram negative cell wall

Is more complex in morphology because it contains an outer membrane (OM), a thin shell of peptidoglycan, and an extensive space between the PG and the cell membrane. The outer membrane is somewhat similar in construction to the cell membrane, except that it contains additional types of lipids, polysaccharides and proteins. The outer leaf of the OM consists of lipopolysaccharide (LPS) integrated into a layer of lipid. The LPS has toxic properties

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

and is also known as composed of three covalently linked parts: (1) Lipid A, firmly embedded in the membrane; (2) Corepolysaccharide, located at the membrane surface; (3) Polysaccharide O antigens, extend like whiskers from the membrane surface into the surrounding medium. The outer membrane serves as a partially chemical sieve by allowing only relatively small molecules to penetrate. Access is provided by special membrane channels, the porin proteins, that span completely across the outer membrane. The size of these porins can be altered so as to block the entrance of chemicals, making them a defense of Gram negative bacteria against certain antibiotics. Between the peptidoglycan and the cell membrane is a well-developed region called the periplasmic space. This space is an important receptacle and reaction site for a large and varied pool of substances that are entering and leaving the cell. It contains enzymes that are involved indigesting molecules entering the cell and special binding proteins that function in cell transport.

Figure 4.3. Outer lipid membrane

lipolysaccharides

O-specific polysaccharide chain integral (3-8 units) protein

corepolysaccharide

porin Peripheral protein

Gram negative cell walls are thin and, unlike Gram positive cell walls, they contain a thin peptidoglycan layer adjacent to the cytoplasmic membrane. LPS, also called endotoxins, are composed of polysaccharides and lipid A which are responsible for much of the toxicity of Gram negative bacteria.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

4.2.3. Exceptions in the cell wall

Figure 4.4. Mycobacterium tuberculosis - Ziehl-Neelsen stain

Mycobacterium and Nocardia contain peptidoglycan and stain Gram positive, but the bulk of their cell wall is comprised of unique types of lipids. One of these is a very long chain fatty acid called mycolic acid or cord factor. The thick, waxy nature imparted to the cell wall by these lipids is also responsible for a high degree of resistance to certain chemicals and dyes [7]. Such resistance is the basis for the acid- fast stain used to diagnose tuberculosis and leprosy.

Archaebacteria possess cell walls that do not contain peptidoglycan, and they are usually composed of proteins, glycoproteinsor polysaccharides. Since a few archaebacteria and all mycoplasmas lack a cell wall entirely, their cell membrane must serve the function of both protection and transport. These forms have a cell membrane stabilized and reinforced by special types of lipids.

Figure 4.5. Mycoplasma hyopneumoniae.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 L forms or L phase variants (for the Lister Institute where they were discovered) are wall - deficient forms. L forms arise naturally from a mutation in the wall – forming genes, or they may be induced artificially by treatment with a chemical such as lysozyme or penicillin that disrupts the cell wall (for example: protoplast and spheroplast) [4].

RESEARCH NEWS

Protein Mur J and new antibiotics

Duke University researchers have now provided the first close-up glimpse of a protein, called Mur J, which is crucial for building the bacterial cell wall and protecting it from outside attack (nature structural and molecular biology-Mur J's molecular structure).

The study could provide insight into the development of broad spectrum antibiotics, because nearly every type of bacteria needs this protein action (Seok Yong Lee, Duke University).

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. The bacterial cell wall differs from that of all other organisms by the presence of peptidoglycan (murein). ___2. Gram positive cell walls have a thick layer of peptidoglycan. ___3. Gram negative cell walls have an outer membrane. ___4. Capsules composed of a single kind of sugar are called homopolysaccharides. ___5. The cell wall is a very rigid structure that gives shape to the cell.

MULTIPLE CHOICE QUESTIONS

1. The importance of bacterial capsule is: a. antiphagocytic role; b. identification of bacteria; c. development of vaccines; d. initiation of infection.

2. Capsulated Gram negative bacteria are: a. Escherichia coli; b. Neisseria meningitidis; c. Bacillus megaterium; d. Staphylococcus epidermidis.

CONCEPT QUESTIONS

Compare the structure and chemistry of What is the function of peptidoglycan? the cell wall of Gram positive eubacteria To which part of the cell envelope does it versus those of Gram negative eubacteria: belong? What happens to a cell that has its peptidoglycan disrupted or removed?

What is Gram stain? What is there in What would happen if one stained a the structure of bacteria that causes some to Gram positive cell with safranin? A Gram- stain purple and others to stain red? How negative one with crystal violet? What would does the structure of the cell walls differ in happen to the two types if the mordant was Gram positive and Gram negative bacteria? left out?

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

COMPLETE THE FOLLOWING SENTENCES

 ______has 84 different serologic types of bacterial capsule.

 ______is a very rigid structure that gives shape to the cell.

 ______contain peptidoglycan.

 LPS are also called ______.

 There are two main types of bacterial cell walls:______.

QUOTES

Mark with X if you like or dislike these quotes.

(1) ”Let me tell you the secret that has led me to my goal; my strength lies solely in my tenacity” (Louis Pasteur). (2) ”Our greatest responsibility is to be good ancestors” (Jonas Salk).

(1) (2)

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of the Chapter 1.

 (Q) What do you call the leader of a biology gang? (A) The nucleus.

Explanation:

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Goldblatt D. (2000). "Conjugate vaccines". Clinical and Experimental Immunology. 119 (1): 1–3. doi:10.1046/j.1365-2249.2000.01109.x. ISSN 0009-9104. PMC 1905528 . PMID 10671089. 2. http://microbiologymatters.com/?p=941. 3. https://en.wikipedia.org/wiki/Gram_stain. 4. Krishna Prakashan Media (2003). Microbiology. ISBN: 8187224665, 9788187224662. 5. Microbiology (1993): Principles and Explorations, p 65; Jacquelyn.G. Black Prentice Hall. 6. "Medical Chemical Corporation" (2016). med-chem.com. Retrieved 9 March 2016. 7. Rex Bookstore (2007). Inc.Foundations in Microbiology' 2007. Ed.(sixth Edition)2007 Edition. ISBN: 0071262326, 9780071262323. 8. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages. 9. https://microbeonline.com/bacterial-capsule-structure-and-importance-and- examples-of-capsulated-bacteria.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 5

Structures internal to the cell wall

5.1. The cytoplasmic membrane (plasma membrane) 5.2. Intracellular (internal) structures 5.2.1. The prokaryotic cytoplasm 5.2.2. Nuclear material (the bacterial DNA and plasmids)

Escherichia coli also known as E. coli) is a Gram-negative, facultatively anaerobic, rod- shapedbacterium of the genus Escherichia. Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in their hosts. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and preventing colonization of the Escherichia coli intestine with pathogenic

nickname ”Vamp” bacteria.

Learning objective

The structure of cytoplasmic membrane and nuclear material

Key points

Cytoplasmic inclusions and vacuoles and nuclear material

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

5.1. THE CYTOPLASMIC MEMBRANE (PLASMA MEMBRANE)

Immediately beneath the cell wall is the cytoplasmic membrane. Plasma membrane is approximately 7.5 nm (0.0075 micron) thick and is composed primarily of phospholipids (about 20-30 percent) and proteins (about 60 to 70 percent). The phospholipids form a bilayer in which most of the proteins are tenaciously held (integral proteins). These proteins can be removed only by destruction of the membrane, as by treatment with detergents. Other proteins are only loosely attached (peripheral proteins) and can be removed by mild treatments such as osmotic shock. The lipid matrix of the membrane has fluidity, allowing the components to move around laterally. The phospholipid bilayer structure has both hydrophilic (water-loving) and hydrophobic (water-fearing) components. The hydrophilic components line up on the external and internal surface of the membrane in contact with the environment on the outside and the internal contents of the cell on the inside. The hydrophobic parts of the membrane orient towards the interior of the bilayer, stabilizing and contributing to the structure. The bacterial cell membrane is a highly selective barrier. This barrier prevents materials from simply diffusing into and out of the cell. This allows the cell’s components to be separated from the environment [8].

Fluid mosaic model

 According to the fluid mosaic model of S.J. Singer and G.L. Nicolson (1972) biological membranes can be considered as a two-dimensional liquid in which lipid and protein molecules diffuse more or less easily.  Lipid bilayers (LB) form through the process of cell-assembly. The cell membrane consists primarily of a thin layer of amphipathic phospholipids which spontaneously arranges so that the hydrophobic “tail” regions are isolated from the surrounding polar fluid, causing the more hydrophilic “head”regions to associate with the intracellular (cytosolic) and extracellular faces of the resulting bilayer [3]. This forms a continuous, spherical lipid bilayer. Forces such as van der Waals, electrostatic, hydrogen bonds, and noncovalent interactions contribute to the formation of the lipid bilayer.  LB are generally impermeable to ions and polar molecules. The arrangement of hydrophilic heads and hydrophobic tails of the lipid bilayer prevent polar solutes from diffusing across the membrane but generally allows for the passive diffusion of hydrophobic molecules.  The phospholipid bilayer structure (fluid mosaic model) with specific membrane proteins accounts for the selective permeability of the membrane, passive and active transport mechanisms.  Cell membranes contain a variety of biological molecules, notably lipids and proteins. Material is incorporated into the membrane, or deleted from it, by a variety of mechanisms:  fusion of intracellular vesicles with the membrane (exocytosis);  the membrane may form blebs around extracellular material that pinch off to become vesicles (endocytosis).  The cell membrane consists of three classes of amphipathic lipids:  phospholipids;  glycolipids;  sterols.  The fatty chains in phospholipids and glycolipids usually contain an even number

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

of carbon atoms, typically between 16 and 20.  Lipid vesicles or liposomes are circular pockets that are enclosed by a lipid bilayer.  Plasma membrane also contain carbohydrates,predominantly glycoproteins, but with some glycolipids (cerebrosides and gangliosides).  Plasma membrane has large content of proteins, typically around 50% of the membrane’s volume.  The cell membrane, being exposed to the outside environment, is an important site of cell-to-cell communication. As such, a large variety of protein receptors and identification proteins, such as antigens, are present on the surface of the membrane.  Types of proteins:  integral proteins (IP) – transmembrane proteins;  peripheral proteins (PP);  lipid anchored proteins (LAP).  The permeability of a membrane is the rate of passive diffusion of molecules through the membrane.  The cytoplasmic membrane also contains various enzymes involved in respiratory metabolism and in synthesis of capsular and cell-wall components.  Proteins are synthesized within the cell, but some can pass across the cytoplasmic membrane barrier to the outside. Examples of such exported molecules are the protein components of cell walls (porins or lipoproteins) or the exocellular enzymes that are secreted by many bacteria into their culture medium, such as penicillinases, proteinases and amylases.

A related question is: how does a cell:

“Know which of the many kinds of proteins within the cell to transport out of the cell?”. This question has been partially answered: The genes that code for these proteins carry a message that results in the addition of a sequence of about 20 extra amino acids (the signal peptide) to the proteins during their synthesis within the cell.

Figure 5.1. Fluid mosaic model

peripheral protein phospholipid glycolipid integral protein glycoprotein

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

5.2. INTRACELLULAR (INTERNAL) STRUCTURES

5.2.1.The prokaryotic cytoplasm

The cell material bounded by the cytoplasmic membrane may be divided into: 1. The cytoplasmic area, granular in appearance and rich in the macromolecular RNA- protein body as known as ribosomes on which proteins are synthesized. 2. The chromatin area, rich in DNA. 3. The fluid portion with dissolved substances. Unlike animal or plant cells, there is no endoplasmic reticulum to which ribosomes are bound. We keep saying that the cell membrane keeps the “inside” of the cell apart from the “outside”. So what is actually on the inside of the cell and why is it so important? Cytosol is the water-like fluid found in bacterial cells. It contains all the other internal compounds and components the bacteria needs for survival. The fluid and all its dissolved or suspended particles is called the cytoplasm of the cell. Proteins, aminoacids, sugars, nucleotides, salts, vitamins, enzymes, DNA, ribosomes, and internal bacterial structures, all float around the cell in the cytoplasm. All these components are vital to the life of the cell and are contained by the cell membrane. In prokaryotes that carry on photosynthesis, the necessary pigments are suspended in the cytoplasm. Prokaryotic cells contain ribosomes and other multiprotein complexes, intracellular membranes (mesosomes), cytoskeleton, nutrient storage structures, inclusions, gas vacuoles, carboxysomes, magnetosomes, etc. The cytoplasm has a gel-like consistency, with rather different properties than the simple solutions that we typically make up in the laboratory. It is a bag of proteins and other macromolecules, each coated with a layer of water. There is so little free water in the cell that one-third of all water molecules are making hydrophilic contacts with the macromolecules in the cell. The bacterial DNA is enclosed inside the bacterial cytoplasm. This means that the transfer of cellular information through the processes of translation, transcription, and DNA replication can interact with other cytoplasmic structures, for example: ribosomes. Most bacterial DNA are circular although some examples of linear DNA exists (e.g. Borrelia burgdorferi). Plasmids are small independent pieces of DNA and it can be described as an extrachromosomal DNA in a bacterial cell.

Ribosomes

Ribosomes are important cellorganelles. A ribosome is a large complex of RNA and protein. It does RNA translation, building proteins from amino acids using messenger RNA as a template.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 5.2. Bacterial ribosomes

newly born protein

aminoacids tRNA

Large subunit

small subunit

mRNA aminoglycoside

 All prokaryotes have 70 S (where S= Svedberg units) ribosomes made up of a 50 S and 30 S subunits.  The 50 S subunit contains the 23 S and 5 S rRNA while the 30 S subunit contains 16 S rRNA.  They consists of two major components:  the small ribosomal subunit which reads the RNA;  the large subunit which joins aminoacids to form a polypeptide chain.  Each subunit is composed of one or more ribosomal RNA ( rRNA) molecules and a variety of proteins.  A ribosome is made of complexes of RNAs and proteins and is, therefore, a ribonucleoprotein.  Ribosomes were first observed in the mid-1950s by Romanian cell biologist George Emil Palade using an electron microscope with dense particles or granules. In 1974, he would win a Nobel Prize for this discovery.  By convention, all sedimentation coefficients are expressed in the Svetberg units (a measure of the rate of sedimentation and centrifugation).  For bacterial ribosomes ultracentrifugation yields intact ribosomes (70 S) as well as separated ribosomal subunits, the large subunit (50 S) and the small subunit (30 S). The largest particles (whole ribosomes) sediment near the bottom of the tube, whereas the smaller particles appear in upper fractions. Their small subunit has a 16 S RNA subunit (consisting of 1540 nucleotides bounded to 21 proteins) [9].  The large subunit (120 nucleotides), a 23 S RNA subunit (2900 nucleotides) and 31 proteins.  Prokaryotic ribosomes are around 20 nm (200 A) in diameter and are composed of 65% rRNA and 35% ribosomal proteins;  Bacterial ribosomes are composed of one or two rRNA strands.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 Ribosomes are the workplaces of protein biosynthesis, the process of translating mRNA into protein.  Ribosomes are classified as being either free or membrane-bound. An individual ribosome might be membrane-bound when it makes one protein, but free in the cytosol when it makes another protein.  Ribosomes may sometimes be described as non-membranous organelles (ribosomes have no phospholipid membrane).  When a ribosome begins to synthesize proteins that are needed in some organelles, the ribosome making this protein can become membrane-bound.  In bacterial cells, ribosomes are synthesized in the cytoplasm through the transcription of multiple ribosome gene operons.  Some bacteria contain intracellular membranes in addition to their cytoplasmic membranes.  Examples of bacteria containing intracellular membranes are phototrophs, nitrifying bacteria and methane-oxidizing bacteria.

Figure 5.3. Structure of ribosomal subunit

smaller subunit 30s

larger subunit 30s

Inclusions (granules)

Inclusions are considered to be nonliving components of the cell that do not possess metabolic activity and are not bounded by membranes. The most common inclusions are glycogen, lipid droplets, crystals, pigments and poly beta-hydroxybutyrate (PHB). Granules usually contain crystals or inorganic compounds and are not enclosed by membranes. Examples are the sulfur granules of photosynthetic bacteria and the polyphosphate granules of Corynebacterium and Mycobacterium. These are often termed metachromatic granules because they stain a contrasting color (red, purple) in the presence of certain blue dyes such as methylene blue. A unique type of inclusion found insome aquatic bacteria are gas vesicles that provide buoyancy and flotation [6].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Volutin granules are an intracytoplasmic storage form of complexed inorganicpolyphosphate, the production of which is used as one of the identifying criteria when attempting to isolate Corynebacterium diphtheriae on Löffler's medium. Polyphosphate granules are called metachromatic granules due to their ability to display the metachromatic effect. They appear red when stained with methylene blue [10].

Gas vacuoles

Gas vacuoles are membrane-bound, spindle-shaped vesicles, found in some planktonic bacteria and Cyanobacteria. They are made up of a shell of protein that has a highly hydrophobic inner surface, making it impermeable to water but permeable to most gases. Gas vacuoles provide buoyancy to the bacterial cells by decreasing their overall cell density. Positive buoyancy is needed to keep the cell in the upper parts of the water column, so that they can continue to perform photosynthesis [4].

Microcompartments

Bacterial microcompartments are membrane-bound organelles that are made of a protein shell that surrounds and encloses various enzymes.

 Carboxysomes – are bacterial microcompartments found in many autrotophic bacteria, such as Nitrobacteria. They are proteinaceous structures that morphologically resemble phage heads and contain the enzymes of carbon dioxide fixation in these organisms [1].  Magnetosomes – are bacterial microcompartments found in magnetotactic bacteria that allow them to sense and align themselves along a magnetic field (magnetotaxis). They are composed of the mineral magnetite or greigite and are surrounded by a lipid bilayer membrane. The ecological role of magnetotaxis is unknown, but is though to be involved in determiningthe optimal oxygen concentrations.

Figure 5.4. Bacterial inclusions

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

5.2.2 Nuclear material (the bacterial DNA and plasmids)

In contrast to eukaryotic cells, bacterial cells contain neither a distinct membrane enclosed nucleus nor a mitotic apparatus. The heredity material of bacteria exists in the form of a single circular strand of DNA designated as the chromatin body or bacterial chromosome. Because it is not a discrete nucleus, this nebulous structure has been designated by such terms as the nucleoid, the chromatin body, the nuclear equivalent and even the bacterial chromosome [7]. The nucleoid can be made visible under the light microscope by Feulgen staining, which is specific for DNA. Bacterial chromosomes are generally ~ 1000 times longer than the cells in which they reside. The DNA molecule is a remarkably simple and elegant storage medium for genetic information. Watson and Crick published a companion paper to their 1953 DNA structure, in which they pointed out that the elegant simplicity of the copying mechanism they proposed was complicated by the fact that the two strands wind around each other [2]. The very first investigation into the structure of the bacterial nucleoid came in the form of light microscopy of stained specimens and electron microscopy of thin sections. These studies showed that although the nucleoid is mostly separate from the rest of the cytoplasm, it is not bound by a nuclear membrane. The circular chromosome exists within the cell as a compact structure that is organized into separate superhelical domains [2]. DNA is kept highly compacted at the local level by a balance of forces, including supercoiling, compaction by proteins, transcription, transection and perhaps even RNA-DNA interactions. In conclusion, the DNA of most bacteria is contained in a single circular molecule called the bacterial chromosome. The chromosome forms an irregular shaped structure called the nucleoid. This sits in the cytoplasm of the bacterial cell [8].

Plasmids

The plasmids are tiny circular extrachromosomal strands, which often confer protective traits upon bacteria, such as resisting drugs and radiation and producing toxins and enzymes. Every plasmid has its own origin of replication - a stretch of DNA that ensures it gets replicated (copied) by the host bacterium. For this reason plasmids can copy themselves independently of the bacterial chromosome, so there can be many copies of a plasmid (even hundreds) within one bacterial cell [5]. Many plasmids contain genes that, when expressed, make the host bacterium resistant to an antibiotic. Other plasmids contain genes that help the host to digest unusual substances, or to kill other types of bacteria. Under stressful conditions, bacteria with the plasmid will live longer and have more opportunity to pass on the plasmid to daughter cells or to other bacteria. Plasmids have been a key factor to the development of molecular biotechnology. They act as delivery vehicles, or vectors, to introduce foreign DNA into bacteria. The use of plasmids for DNA delivery began in the 1970s when DNA from other organisms was first cut and pasted into specific sites within the plasmid DNA. The modified plasmids where then reintroduced into bacteria [5].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. A ribosome is made from complexes of RNA, and proteins. It is a ribonucleoprotein. ___2. Inclusions are not bounded by membranes. ___3. Volutin granules are also named methachromatic granules. ___4. Gas vacuoles provides buoyancy to the bacterial cell by decreasing their overall cell density.

MULTIPLE CHOICE QUESTIONS

1. Examples of inclusions (granules): a. volutin granules; b. ribosome granules; c. cytosol granules; d. gas vacuoles.

2. Examples of microcompartments: a. carboxysomes; b. magnetosomes; c. ribosomes; d. nucleosomes.

CONCEPT QUESTIONS

List five functions that the cytoplasmic What are the chromatin in body and membrane performs in bacteria: plasmids made of? What are their functions?

What is unique about the structure of Explain the structure and function of bacterial ribosomes? How do they function? inclusions and granules. What are metachromatic granules, and what do they contain?

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

COMPLETE THE FOLLOWING SENTENCES

 ______are bacterial microcompartments that contain the enzymes of carbon dioxide fixation.

 Magnetosomes are bacterial microcompartments composed of the mineral ______.

 Every plasmid has its own ______.

 Ribosomes may sometimes be described as ______.

QUOTES

Mark with X if you like or dislike these quotes.

(1) ”A good teacher is a master of simplification and an enemy of simplest” (Louis Berman). (2) ”A good teacher must know the rules; a good pupil, the exceptions” (Martin Fischer).

1 2

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of the courses.

I just found Bacteria growing on my chocolate bar. I guess there is Life on Mars.

Explanation:

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Badger MR, Price GD (2003). "CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution". J. Exp. Bot. 54 (383): 609–22. doi:10.1093/jxb/erg076. PMID 12554704. 2. Esteban Toro and Lucy Shapiro (2010). Bacterial Chromosome Organization and Segregation. Cold Spring Harbor Laboratory Press; all rights reserved. doi: 10.1101/cshperspect.a000349. 3. https://en.wikipedia.org/wiki/Cell_membrane. 4. https://en.wikipedia.org/wiki/Bacterial_cell_structure. 5. https://www.sciencelearn.org.nz/resources/1900-bacterial-dna-the-role-of-plasmids. 6. Rex Bookstore, Inc. (2007). Foundations in Microbiology' 2007 Ed.(sixth Edition)2007 Edition. ISBN: 0071262326, 9780071262323. 7. Pelczar, Chan and Noel (2001). Edition-5; Microbiology. Part 2: Bacteria, Chapter 5 (Morphology and fine structure of bacteria).Published by Mc Graw Hill India (2001). ISBN 10: 0074623206; ISBN 13: 9780074623206. 8. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages. 9. The Molecular Biology of the Cell (2002). Fourth edition. Bruce Alberts, et al. Garland Science (2002) pg. 342 ISBN 0-8153-3218-1. 10. Willey, J.M., Sherwood, L.M. and Woolverton, C.J. (2011). Prescott's Microbiology, 8th Ed. McGraw Hill.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 6 Bacterial endospores

6.1. General characterisation 6.2. Endospore structure 6.3. The position of the endospore in the bacterial cell 6.4. Endosporulation 6.5. Germination 6.6. Endospore resistance 6.7. The importance of the bacterial endospore

Clostridium botulinum is a Gram- positive, rod-shaped, anaerobic, spore-forming, motile bacterium with the ability to produce the neurotoxin botulinum. The botulinum toxin can cause a severe flaccid paralytic disease in humans and animals and is the most potent toxin known to humankind, natural or synthetic, with a lethal dose of 1.3-2.1ng/kg in humans. Depending on their ability to produce botulinum toxin there are four distinct groups, (I-IV). Clostridium botulinum nickname ”Sea wolf”

Learning objective

Certain species of bacteria produce spores, either within the cell (endospores) or external to the cell (exospores).

Key points

The spore is a metabolically dormant form which under appropriate conditions can undergo germination and outgrowth to form a vegetative cell

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

6.1. GENERAL CHARACTERISATION

Bacterial endospores = outer layer of keratin resistant to chemicals, staining and heat; the bacterium is able to stay dormant for years, protected from temperature differences, absence of air, water and nutrients. Endospores are structures unique to bacteria.They are thick-walled, highly refractive bodies that are produced (one per cell) by Bacillus, Clostridium, Sporosarcina, Thermoactinomyces, and a few other genera. The shapes of endospores also show their location within the vegetative cell vary depending on the species [7]. An endospore is a dormant, tough and non-reproductive structure produced by Gram positive bacteria. The endospore consists of the bacterium DNA, ribosomes and large amounts of dipicolimic acid (DA). DA is a spore-specific chemical that appears to help in the ability of endospores to maintain dormancy. This chemical comprises up to 10% of the spore’s dry weight.  Endospores can survive without nutrients.  They are resistant to ultraviolet radiation, desiccation, high temperature, extreme freezing and chemical disinfectants.  Thermoresistant endospores were first hypothesized by Ferdinand Cohn after studying Bacillus subtilis growth on cheese after boiling the cheese.  Astrophysicist Steinn Sigurdsson said “there are viable bacterial spores that have been found on Earth and that are 40 million years old on earth and we know they’re very hardened to radiation”.  Endospores are commonly found in soil and water, where they may survive for long periods of time. For example: they can survive at a temperature of 800 C, but spores can remain alive for up to two hours in water that is boiling (1000C).  Bacterial spores have also been recovered alive from the intestines of Egyptian Mummies!  When the environment becomes favorable, the spore can convert to a vegetative, reproducing cell. Until that occurs, the spore is dormant, it has no metabolic activity.

6.2. ENDOSPORE STRUCTURE

Figure 6.1. Bacterial endospore structure

coat

core

inner membrane

cortex

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Endospores are round to oval bodies existing free: - in the environment; - within the swollen bacterial cells that produced them. Seen under the transmission electron microscope, spore structure can be appreciated in detail. The core of the spore consists of DNA and dehydrated cytoplasm taken from the parent cell. The core is surrounded by a core membrane or wall taken from the ingrown bacterial cell membrane (spore septum). Outside of and adjacent to the core wall is the cortex. The cortex is composed of an unusual loose arrangement of peptidoglycan. Surrounding the cortex, inner and outer membranes of proteins that make up the spore coat. This layer confers chemical resistance to the endospore. The outermost layer is the exosporium, basal layer composed of glyco and lipoproteins. The spore coat acts like a sieve that excludes large toxic molecules like lysozyme. It is resistant to many toxic molecules and may also contain enzymes that are involved in germination. Dipicolinic acid represents 20% of the dry weight of the endospore and it stabilizes the DNA [6].

Staining techniques for endospores

Endospore staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample which can be use in classifying bacteria [2]. The techniques for staining endospores are direct or indirect methods. One of the indirect methods is Gram stain and one of the direct methods is malachite green stain. Mueller and Schaeffer-Fulton staining there are special techniques used for visualizing endospores.

Figure 6.2. Malachite green stain

endospore

vegetative cell

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

6.3. THE POSITION OF THE ENDOSPORE IN THE BACTERIAL CELL

The arrangement of spore layers is as follows: 1. Exospore; 2. Spore coat; 3. Spore cortex; 4. Core wall. The position of the endospore differs among bacterial species and is useful in identification.

The main types within the cell are terminal, subterminal and central endospores:

1. Terminal endospores are seen at the poles of the cells. For example: Clostridium tetani. 2. Central endospores are more or less in the middle of the cells. For example: Bacillus spp., Clostridium spp. 3. Subterminal endospores are usually seen far enough towards the poles, but close enough to the center (so as not to be considered either terminal or central). For example: Clostridium botulinum. 4. Lateral endospores are seen occasionally. For example: Bacillus laterosporus [7].

6.4. ENDOSPORULATION

The progressive development of the spore within the parent cell is called sporulation (Kaiser, 2011). When a bacterium detects that environmental conditions are becoming unfavorable, it may start the process of endosporulation, which takes about eight hours [3].

The process begins when: (1) the bacterial DNA replicates and half of the DNA gathers at one end with a small amount of cytoplasm; (2) the cell membrane turns inward (spore septum) to form a double membrane enclosing the DNA and cytoplasm. This double membrane becomes the core wall; (3) cortex material develops within the core wall and thickens externally. As the spore forms, the parent cell swells at the spore end. The protein of inner and outer membranes or spore coat appear to be followed by the appearance of the exosporium basal layer. Sporulation is now complete and the mature endospore will be released when the surrounding vegetative cell is degraded [3].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 6.3. Endosporulation

Stages VI and VII Spore maturation septum forespore and mother cell lysis

Stages IV and V Stage II Stage III Spore cortex and vegetative cell Asymmetric cell Forespore coat synthesis division engulfment

6.5. GERMINATION

When the external environment is favorable, spores revert back to vegetative bacilli in a process called germination. In this process the endospore's layers break down and the vegetative bacillus emerges. Reactivation of the endospore involves activation, germination and outgrowth. Germination involves the dormant endospore starting metabolic activity and thus breaking hibernation. It is commonly characterized by rupture or absorption of the spore coat, swelling of the endospore, an increase in metabolic activity, and loss of resistance to environmental stress [3]. Germination is followed by outgrowth and involves the core of the endospore manufacturing new chemical components. The old spore coat is developed into a fully functional vegetative cell.

6.6. ENDOSPORE RESISTANCE

 Endospores possess five times more sulfur than vegetative cells concentrated in spore coats as cystine (an aminoacid) stabilized by S-S linkages.  In 1995 bacterial spores were found in the gut of a fossilized bee trapped in amber from a tree in the Dominican Republic by Raul Cano. After the spores were analyzed by microscopy it was determined that the cells were very similar to Bacillus sphericus which is found in bees of the Dominican Republic today [5].  Endospores are resistant to most agents that would normally kill the vegetative cells. They are resistant to alcohols, quaternary ammonium compounds and detergents.  Endospores can be destroyed by: (1) sterilant alkylating agents (e.g. ethylene oxide) and 10% bleach are effective against endospores; (2) burning or by autoclaving at a temperature exceeding the boiling point of water (1000 C); (3) tyndalization; (4) ionising radiation, such as x-rays and gamma-rays.  The heat resistance of endospores is due to a variety of factors:  calcium dipicolinate may stabilize and protect the endospores DNA;  small acid-soluble proteins (SASPs) saturate the endospores DNA and protect it from heat, dryness, chemicals and radiations; they also function as a carbon and energy source for the development of a vegetative bacterium during

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

germination [3].  Dehydration of the endospore is thought to be very important in the endospore’s resistance to heat and radiation.  DNA repairing enzymes contained within the endospore areable to repair damaged DNA during germination.

6.7. THE IMPORTANCE OF THE BACTERIAL ENDOSPORE

 The molecular details of endospore have been studied in the model organism Bacillus subtilis. This studies contributed to our understanding of the regulation of gene expression, transcription factors and the sigma factor subunits of RNA polymerase [4].  Endospores of the bacterium Bacillus anthracis were used in the 2001 anthrax attack. The powder of extracellular anthrax endospores was found in contaminated postal letters.  Geobacillus stearothermophilus endospores are used as biological indicators when an autoclave is used in sterilization procedures.  In biotechnology, Bacillus subtilis endospores are useful for the expression of recombinant proteins and for the surface display of peptides and proteins as a tool for fundamental and applied research in the fields of microbiology, biotechnology and vaccination[1].

Feature 6.1. Bacterial survival outside host

FAVOURABLE CONDITIONS GROWTH

VEGETATIVE CELL

3.Journalist dies Anthrax attacks from respiratory anthrax and

1. Two cases of colleague contractsthe cutaneous anthrax. 2. Letter disease. containing anthrax 4. Microsoft Office sent to senator Tom receives letter from Daschle. Malaysia containing anthrax.

ENDOSPORE

UNFAVOURABLE CONDITIONS DORMANCY Nutrient starvation, temperature or pH extremes, cell crowding, antibiotic exposure

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. Endospores can survive without nutrients. ___2. Thermoresistant endospores were first hypothesized by Martin Fischer after studying Bacillus anthracis growth on meet. ___3. One of the direct methods for identification of endospore is Gram staining.

MULTIPLE CHOICE QUESTIONS

1. Examples of terminal endospores are: a. Clostridium tetani; b. Clostridium botulinum; c. Bacillus anthracis; d. Bacillus laterosporus.

2. Examples of central endospores are: a. Clostridium botulinum; b. Clostridium perfringens; c. Bacillus anthracis; d. Bacillus cereus.

CONCEPT QUESTIONS

What is the vegetative stage of a bacterial cell? Is spore formation in bacteria a method of What is an endospore, and how does it reproduction or a means of multiplication? function? Explain: Why are endospores so difficult to destroy? Describe the endospore forming cycle:

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

COMPLETE THE FOLLOWING SENTENCES

 ______of the endospore is thought to be very important in the endospore's resistance

to heat and radiation.

 ______endospores are used as biological indicators when an autoclave is

used in sterilization procedures.

 Central endospores are more or less in the middle of the cells. For example: ______.

 Lateral endospores are seen occasionally. For example: ______.

QUOTE

Mark with X if you like or dislike this quote.

(1) Whether our efforts are or not, favored by life, let us be able to say, when we come near the great goal, ”I did what I could”.

(1)

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of the courses.

 A couple of biologists had twins. They named one Jessica and the other Control.

Explanation:

 (Q) How did the English major define microtome on his biology exam? (A) An itsy-bitsy book.

Explanation:

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Abel-Santos E. (2012). Bacterial Spores: Current Research and Applications. Caister Academic Press. ISBN 978-1-908230-00-3. 2. Hussey Marise; Zayaitz Anne (2011). "Endospore Stain Protocol". American Society for Microbiology. Archived from the original on 2012-06-01. Retrieved 2012-03-06. 3. https://en.wikipedia.org/wiki/Endospore. 4. Makino K., Amemura M., Kim S.K., Nakata A., Shinagawa H. (1993).Role of the sigma 70 subunit of RNA polymerase in transcriptional activation by activator protein PhoB in Escherichia coli.Genes Dev. 1993 Jan;7(1):149-60. 5. Pommerville Jeffrey C. (2014). Fundamentals of microbiology (10th ed.). Burlington, MA: Jones & Bartlett Learning. ISBN: 1449688616. 6. Prescott L. (1993). Microbiology, Wm. C. Brown Publishers, ISBN 0-697-01372-3. 7. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 7 Reproduction and Growth

7.1. Reproduction (modes of cell division) binary fission 7.2. Some unusual forms of reproduction in bacteria 7.3. Sexual reproduction of bacteria 7.3.1. Bacterial transformation 7.3.2. Bacterial transduction 7.3.3. Bacterial conjugation 7.4. Normal growth cycle (growth curve) of bacteria

Salmonella is a genus of rod- shaped bacteria of the Enterobacteriaceae family. There are two species of Salmonella: Salmonella bongori and Salmonella enterica. Salmonella enterica is further divided into six subspecies and over 2500 serovars. Salmonellae are found worldwide in both cold-blooded and warm-blooded animals, and in the environment. Salmonella spp. nickname ”Black Widow”

Learning objective

Distinguish among the reproductive types in prokaryotes

Key points

Binary fission is a reproductive type in which the chromosome is replicated and the resultant prokaryotic is an exact copy of the parental prokaryote, thus leaving no opportunity for genetic diversity

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

7.1. REPRODUCTION (MODES OF CELL DIVISION) BINARY FISSION

Definition

Binary fission is a type of reproduction in which the chromosome is replicated and the resultant prokaryote is an exact copy of a parental prokaryote, thus leaving no opportunity for genetic diversity [14]. The most common mode of cell division in the usual growth cycle of bacterial populations is transverse or binary fission, in which a single cell divides after developing a transverse septum. Binary fission is an asexual reproductive process. During binary fission the parent cell enlarges, duplicates its chromosome, and forms a central transverse septum that divides the cell into two daughter cells. Binary means that one cell becomes two, and transverse refers to the position of the division plane forming across the width of the cell [9]. Conceptually this is a simple process; a cell just needs to grow to twice its starting size and then split in two. The main structures to observe during binary fission are the cell wall, the cell membrane, the nucleoid that contains DNA and the cytoplasm of the cell. The process of binary fission begins as the bacterium slightly elongates. While this is taking place, the DNA of the cell replicates to form twice the normal amount of DNA. Then the many types of proteins that comprise the cell division machinery assembly at the future division site. A key component of this machinery is the FtsZ protein. FtsZ Protein monomers assemble into a ring-like structure at the center of a cell. Other components of the division apparatus then assemble at the FtsZ ring [15]. As division occurs the cytoplasm is cleaved in two, and, in many bacteria a new cell wall is synthesized. Most bacteria including Salmonella spp. and Escherichia coli reproduce by binary fission. During this type of asexual reproduction, the single DNA molecule replicates and both copies attach to the cell membrane. The cell membrane begins to grow between the two DNA molecules. Once the bacterium is close to double its original size, the cell membrane begins to pinch inward. A cell wall then forms between the two DNA molecules dividing the original cell into two identical daughter cells. If conditions are just right, one bacterium could become a billion (1,000,000,000) bacteria in just 10 hours through binary fission. It is often said that a bacterium can divide every 20 or 30 minutes.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 7.1. Binary fission of bacteria

cell elongates and

DNA is replicated

cells separate bacterial cell cell wall and plasma membrane begin to divide

7.2. SOME UNUSUAL FORMS OF REPRODUCTION IN BACTERIA

There are groups of bacteria that use unusual forms of patterns of cell division to reproduce. Some of these bacteria grow to more than twice their starting cell size and then use multiple divisions to produce multiple offspring cells. Some other bacterial lineages reproduce by budding. Still, others form internal offspring that develop within the cytoplasm of a larger mother cell [3].  Cyanobacterium stanieria never undergoesbinary fission. It starts out as a small, spherical cell approximately 1 to 2 micron in diameter.  This cell is referred to as a baeocyte (which literally means “small cell”). The baeocyte begins to grow forming a vegetative cell up to 30 micron in diameter. As it grows, the cellular DNA is replicated over and over, and the cell produces a thick extracellular matrix. The vegetative cell transitions into a reproductive phase where it undergoes a rapid succession of cytoplasmic fissions to produce dozens or even hundreds of baeocytes [2].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 7.2. Budding in bacteria

bud becomes a bud begins to separate daughter form on parent cell cell bud

Budding produces chains nucleus

new bacterial cell

 Budding in bacteria - Budding involves a cell forming a protrusion that breaks away and produces a daughter cell. Genome replication follows and one copy of the genome gets into the bud.  Then the bud enlarges, eventually becoming a daughter cell and finally gets separated from the parent cell. Budding has been observed in some members of the Planctomycetes, Cyanobacteria, Firmicutes and Proteobacteria [4]. Fragmentation - Some bacteria form more complex reproductive structures that help disperse the newly formed daughter cells. Mostly during unfavorable conditions, bacterial protoplasm undergoes compartmentalization and subsequent fragmentation, forming minute bodies called gonidia. Under favorable conditions each gonidium grow to a new bacterium [6]. It becomes apparent that prior to fragmentation the bacterial genome has to undergo repeated replication so that each fragment gets a copy of it. Examples include fruiting body formation by Myxobacteria and aerial hyphae formation by Streptomyces. Bacteria produce extensive filamentous growth, such as the Nocardia spp. which reproduces by fragmentation of the filaments into small bacillary or coccoid cells, each of them giving rise to a new growth. Species of the genus Streptomyces and related bacteria produce many spores per organism by developing crosswallsof the hyphal tips; each spore gives rise to a new organism [13].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 7.3. Fragmentation

Figure 7.4. Fragmentation

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 Intracellular offspring production by some Firmicutes - Epulopiscium spp.,Metabacterium polyspora and the Segmented Filamentous Bacteria (SFB) form multiple intracellular offspring. In these bacteria shares characteristics with endospore formation in Bacillus subtilis. The internal offspring grow within the cytoplasm of the mother cell. Once offspring development is complete the mother cell dies and releases the offspring [7].

7.3. SEXUAL REPRODUCTION OF BACTERIA

7.3.1. Bacterial transformation

Definition

Transformation represents the alteration of a bacterial cell caused by the transfer of DNA from another cell, especially if it is pathogenic. In 1928, a British medical officer, Frederick Griffith reported the curious results of a set of experiments with Streptococcus pneumoniae, the “so-called pnumococcus”. Griffith found that by injecting deadly smooth strain bacteria into healthy mice, this resulted into the pneumonia and death. When he injected harmless rough strain bacteria into healthy mice no sickness or death resulted from the injections. In a third experiment Griffith injected heat-killed “S” strain bacteria into healthy mice and these animals did not become sick or died. Continuing with his experiments, Griffith injected into o group of mice a mixture of harmless “R” strain bacteria and harmless heat killed “S” strain bacteria. To his complete surprise, the mice died of pneumonia. Taking blood samples from the dead mice, he found only “S” strain bacteria-living encapsulated bacteria. Something caused the live, harmless “R” strain bacteria to transform into live, deadly “S” strain bacteria. In transformation, genetic material of one bacterial cell goes into another bacterial cell by some unknown mechanism and it coverts one type of bacterium into another type (non capsulated to capsulated form). The process of transformation is widely employed in genetic engineering to transform organisms. Bacterial cells are often made competent by treating them with calcium chloride followed by heat shock treatment. In short, transformation is the process by which DNA fragments are taken up and incorporated by a cell.

Figure 7.5. Bacterial transformation

bacterial cell

bacterial chromosome

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

7.3.2. Bacterial transduction

Definition

Transduction represents horizontal gene transfer mechanism in prokaryotes where genes are transferred using the virus. In this method, genetic material of one bacterial cell goes to other bacterial cell by using bacteriophages or phages (viruses infecting bacteria). It was first reported in Salmonella typhimurium by Zinder and Lederberg (1952). Bacteriophages are widely used as vectors in recombinant DNA technologies. Bacteriophages are viruses that attack bacteria [14]. They are involved in transfer of genetic material from one bacterium to the other. Such virus mediated gene transfer is termed as transduction. The carrier phage is the transducer or vector.

Figure 7.6. Bacterial transduction

byrecombination

phageits injects DNA

enzymesdegrade host DNA

injects donor injects DNA

transducing phage

donor incorporated DNAis

cell synthesizes cell phages new

intochromosome recipient’s phage

Transduction may be generalized (random) and specialized(specific DNA fragments). Generalized transduction is mediated by lytic phages where any DNA segment can be transferred by the virus [5]. A portion of the donor bacterial DNA accidently gets enclosed in a capsid. Upon lysis and further infection of this virus particle to another bacterium, the genetic material of the donor is released and recombination occurs between the injected DNA segments and homologous part of the recipient chromosome, forming a rDNA[5]. Generalized transduction can be either complete or abortive. In complete transduction, the exogenote or the transduced DNA fragment gets integrated within the recipient bacterial chromosome (endogenote), forming a recombinant chromosome. In abortive transduction exogenote does not get integrated with the endogenote and remains in the cytoplasm as a free particle. These fragments cannot undergo replication. Specialized transduction is mediated by lysogenic phages. Here specific DNA fragments are integrated into the host chromosome. Phage DNA gets integrated with the bacterial chromosome, viral genome gets integrated into the bacterial genome called prophage. The prophage undergoes replications along with bacterial genome replication. Upon induction, the prophage detaches from the bacterial chromosome. At times, a portion of the bacterial DNA remains attached to the detached phage DNA. This excised phage DNA undergoes lytic cycle and infects another bacterium and transfers the bacterial genes from the donor bacterium.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

At this stage only the bacterial genes that are close to the site of prophage are transferred. Lysogenic phages like lambda phages are widely used as vectors in rDNA technology.

7.3.3. Bacterial conjugation

Definition

Conjugation represents the temporary fusion of organisms, especially as part of sexual reproduction. Bacterial conjugation is the unidirectional transfer of genetic material from a donor cell to a recipient by cell to cell contact or through conjugation tube. The process was first described by Lederberg, Hayes and Woolman in Escherichia coli. The bacterium with the F plasmid is the donor, F+ or male. Fertility factor genes confer bacteria the ability to transfer genetic material to the recipient cell [5]. In Escherichia coli, both these strains are present, one with F factor, F+ or male and other without F plasmid, F- female. In bacterial conjugation F plasmids are the ones generally transferred, not the entire bacterial genome.

The conjugation has the following steps:

1. F+ cells produces hair like appendages called sex pili which facilitates cell to cell with F- strain by forming a conjugation tube. The formation of sex pili is governed by genes of Ffactor. 2. Replication of F factor making a copy. 3. Transfer the copy of F plasmid to the recipient cell via conjugation tube. 4. Conjugation tube dissolves. Now the F- strain is also F+. In short: F+ cell+ F- cell= F+cell+ F+ cell.

Sexduction or Education represents conjugation between F' cell and F- cell. What is F' cell? F' cell has an independent detached F plasmid with some bacterial genes attached to it. This plasmid is called F- plasmid. The integration of F plasmid in the formation of Hfr strains is a reversible process. Sexduction is the conjugation between F' with F- recipients. Sexduction offers a high rate of recombination. After conjugation, F cell receive F plasmid diploid for genes (partial diploids). Sexduction or F- duction represents the transfer of F factors to receipt occur in mating between F1 and F-. The transfer of F1 to recipient produces partial diploids or heterogenotes. Recombination may occur between the recipient’s chromosome and F1 producing recombinants. Recombination of this type, mediated by F1 factors, is called sexduction or F- duction [5].

Sexduction has the following steps:

1. Formation of F' cells or F' plasmids. A cell that has detached F plasmid with some bacterial genes attached to it is referred as F' cells. 2. F' cells produces hair like appendages called sex pili which facilitates cell to cell contact with F- strain by forming a conjugation tube. The formation of sex pili is governed by genes of F factor. 3. Replication of F' plasmid making a copy. 4. Transfer the copy of F' plasmid to the recipient cell via conjugation tube. 5. Conjugation tube dissolves. Now F- cell is diploid for few bacterial genes.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

7.4. NORMAL GROWTH CICLE (GROWTH CURVE) OF BACTERIA

In the laboratory bacteria are usually grown using solid or liquid media. Solid growth media such as agar plates are used to isolate pure cultures of a bacterial strain. The use of selective media (media with specific nutrients added or deficient, or with antibiotics added) can help identify specific organisms.

Feature 7.2. Frau Hesse's medium to acknowledge her forgotten ”service to science and tohumanity”

In microbiology labs, feeding bacteria is a major preoccupation, and preparing the proper growth medium in a lab's ”kitchen” is often the first step of any experiment. In the earliest days of microbiology, scientists were stumped about how to isolate bacteria. Robert Koch used thin slices of potatoes as naturally occurring ”petri dishes” when he began his studies of bacterial pathogens. He tried gelatin as a solidifying agent. This had the desirable

feature of being a transparent gel, but it had the serious disadvantage 0 of becoming liquid above 25 C, thick is below the optimum temperature for the growth of human disease-producing bacteria. But behind the talented laboratory technicians that supported Robert Koch's genius was an even more unsung heroine of microbiology. It was Walther Hesse's wife (who was often an assistant and scientific illustrator for the lab) Angelina Fanny Hesse who made the isolation of bacteria possible [11].

The time required for a complete fission cycle-from parent cell to two new daughter cells-is called the generation or doubling time. In bacteria, the generation time is a doubling process in which each new fission cycle or generation increases the population by a factor of 2. So, the parent stage consists of 1 cell, the first generation consist of 2 cells, the second of 4 cells, the third of 8, then 16, 32, 64 and so on. As long as the environment remains favorable, this doubling effect can continue at a constant rate [9]. The length of the generation time is a measure of the growth rate of an organism. This growth pattern is termed exponential. Population growth also conforms to a geometric progression, expressed on a graph as a constantly increasing slope, and defined as a sequence of numbers in which each pair of adjacent

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

numbers has the same ratio throughout the sequence. An easier way to calculate the size of a population over time is to use an equation such n [12] as: Nf = (Ni) 2 . For example: Staphylococcus aureus in an burger after it sits in warm for 4 hours. We will assume that Ni is 10 (number of cells deposited in the sandwich while being prepared). To derive n, we need to divide 4 hours (240 minutes) by the generation time (we will use 20 minutes). This comes out to 12, so 2n is equal to 212. Referring to a table on the powers of numbers 212 is found to be 4,096. Final number = 10 x 4,096 = 40,960 cells in the sandwich [12].

Aspects of population growth

Quantitative laboratory studies indicate that a population typically displays a predictable pattern or growth curve [12]. The method commonly used to observe the population growth pattern is a viable counting technique, in which the total number of live cells is counted over a period of time.

This method entails the following sequence of events:

1. Placing a tiny number of cells into a sterile liquid medium. 2. Incubating this culture over a period of several hours. 3. Sampling the broth at regular intervals during incubation. 4. Plating each sample onto solid media. 5. Counting the number of colonies present after incubation.

Stages in the normal growth curve

Bacterial growth follows four phases:

1. The lag phase is a relatively “flat” period on the graph when the population does not appear to be growing. This is a period of slow growth when the cells are adapting to the high nutrient environment and preparing for fast growth. During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and are not yet able to divide. 2. The second phase of growth is the log phase, also known as the logarithmic or exponential phase. The log phase is marked by rapid exponential growth. The rate at which cells grow during this phase is known as the growth rate (k), and the time it takes for the cells to double is known as the generation time (g). The log phase is a period characterized by cell doubling. The number of the new bacteria appearing per unit time is proportional to the present population.Under controlled conditions, Cyanobacteria can double their population four time a day. Exponential growth cannot continue indefinitely, however, because the medium is soon depleted of nutrients and enriched with wastes [10]. 3. The third phase of growth is the stationary phase and is caused by depletion of nutrients. The cells reduce their metabolic activity and consume non-essential cellular proteins. This phase is a transition from rapid growth to a stress response state and there is increased expression of genes involved in DNA repair, antioxidant metabolism and nutrient transport. In stationary phase the number of new cells created is limited by the growth factor and as a result the rate of cell growth matches the rate of cell death. The result is a “smooth” horizontal linear part of the curve during the stationary phase [1].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

4. The phase of decline or death in which bacteria die. This could be due to lack of nutrients, a temperature which is too high or low, or the wrong living conditions. Some species of Gram negative cocci die very rapidly, so that there may be very few viable cells left in a culture after 72h or less. Other species die so slowly that viable cells may persist for months or even years. In biology,cells multiply in number when one cell divides into two.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. Most bacteria reproduce by binary fission. ___2. Escherichia coli never undergoes binary fission. ___3. Segmented filamentous bacteria form multiple intracellular offspring. ___4. Transduction represents the alteration of a bacterial cell caused by the transfer of DNA from another, especially if it is pathogenic.

MULTIPLE CHOICE QUESTIONS

1. Generalized transduction is mediated by: a. lytic phages; b. enzymes; c. bacteria; d. viruses.

2. Examples of forms of reproduction by budding in bacteria are: a. Planctomycetes; b. Cyanobacteria; c. Proteobacteria; d. Gracilicutes.

CONCEPT QUESTIONS

Describe the differences between the Would you expect the generation time to various modes of cell division in bacteria. be a constant characteristic of a bacterial species? Explain:

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Compare the growth curve of bacterial In the lag phase of growth the number of population with growth curve of human bacteria remains constant. Does this mean the population! What lies ahead? Which of the cells are dormant and inert? Explain: two populations will survive longer on earth? Explain why and which are your predictions for the future?

COMPLETE THE FOLLOWING SENTENCES

 ______never undergoes binary fission.

 ______represents conjugation between F' cell and F- cell.

QUOTES

Mark with X if you like or dislike this quotes.

(1) ”When I approach a child, he inspires in me two sentiments; tenderness for what he is, and respect for what he may become!” (Louis Pasteur). (2) ”Science knows no country, because knowledge belongs to humanity, and is the torch which illuminates the world science; is the highest personification of the nation because that nation will remain the first which carries the furthest the works of though and intelligence.” (Henry Broks).

1 2

BACTERIA JOKE

Give a explanation for the following joke. You may find the explication in the text of the courses.

 Biology is the only science in which multiplication is the same thing as division.

Explanation:

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Bridges B.A., Foster P.L., Timms A.R. (2001). "Effect of endogenous carotenoids on "adaptive" mutation in Escherichia coli FC40". Mutat. Res. 473 (1): 109–19. PMC 2929247 . PMID 11166030. 2. D. S. Weiss (2004). Bacterial cell division and the septal ring. Molecular Microbiology, vol. 54, pp. 588-597. 3. E. R. Angert (2005). Alternatives to binary fission in bacteria. Nature Reviews Microbiology, vol. 3, pp. 214-224. 4. https://micro.cornell.edu/research/epulopiscium/binary-fission-and-other-forms- reproduction-bacteria. 5. http://www.biologyexams4u.com/2012/11/bacterial-transduction-generalized- and.html. 6. http://www.biologyexams4u.com/.../reproduction-in-bacteria-vegetative.html. 7. J.B. Waterbury and R.Y. Stanier (1978). Patterns of growth and development in pleurocapsalean cyanobacteria. Microbiological Reviews (1978) vol. 42, pp. 2-44. 8. K. Gerdes, J. Møller-Jensen, G. Ebersbach, T. Kruse and K. Nordström (2004). Bacterial mitotic machineries. Cell, vol. 116, pp. 359-366. 9. Krishna Prakashan Media (2003). Microbiology. ISBN: 8187224665, 9788187224662. 10. "Marshall T. Savage - An Exponentialist View". 11. Popular Science (2014). https://www.popsci.com/.../forgotten-woman-who-made- mic... 12. Rex Bookstore Inc., 2007. Foundations in Microbiology' 2007 Ed.(sixth Edition) 2007 Edition. ISBN: 0071262326, 9780071262323. 13. S. K. Soni (2007). Microbes: A Source of Energy for 21st Century. New India Publishing, 2007. ISBN: 8189422146, 9788189422141. 14. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages. 15. William Margolin (2005). FTSZ AND THE DIVISION OF PROKARYOTIC CELLS AND ORGANELLES. Nat Rev Mol Cell Biol. Author manuscript; available in PMC 2016 Feb 18. Published in final edited form as:Nat Rev Mol Cell Biol. 2005 Nov; 6(11): 862– 871. doi: 10.1038/nrm1745.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 8 Bacterial nutrition

8.1. Mode of nutrition in bacteria 8.2. Nutritional types of bacteria 8.3. Symbiotic bacteria 8.4. Saprobes and parasitic bacteria 8.5. Absorption of nutrients: transport mechanisms 8.5.1. Passive transport 8.5.2. Active transport 8.5.3. Bulktransport

Clostridium perfringens is a Gram-positive, rod-shaped, anaerobic, spore- formingbacterium of the genus Clostridium. C. perfringens is everpresent in nature and can be found as a normal component of decaying vegetation, marine sediment, the intestinal tract of humans and other vertebrates, insects, and soil. C. perfringens is the third Clostridium perfringens most common cause of food

nickname ”Lemon Face” poisoning in the United Kingdom and the United States.

Learning objective

The essential nutrients: macronutrients and micronutrients Sources of essential nutrients/Nutritional types of bacteria

Key points

Autotrophs and heterotrophs

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

8.1. MODE OF NUTRITION IN BACTERIA

Definition

Nutrition is a process by which chemical substances called nutrients are acquired from the environment and are used for growth and metabolism. The essential nutrients do not vary extensively from microbe to microbe at the level of the bioelements. There are two categories of essential nutrients:  Macronutrients;  Micronutrients.

Macronutrients are required in relatively large quantities and play principal roles in cell structure and metabolism. Examples of macronutrients are molecules that contain carbon, hydrogen, oxygen and nitrogen. Micronutrients or trace elements are needed in a lot smaller amount for enzyme and pigment structure and function. They include elements such as zinc, manganese, and nickel. The source of common essential nutrients are CHNOPS: Carbon; Hydrogen; Nitrogen; Oxygen; Phosphorous; Sulfur [1].

Another way to categorize nutrients is according to their carbon content: 1. Inorganic nutrient:  is a simple atom or molecule that is composed of some other combination of atoms besides carbon and hydrogens;  natural reservoirs are mineral deposits in the crust of the earth, bodies of water and the atmosphere. Examples include:  metals and their salts (magnesium sulfate, ferric nitrate, sodium phosphate);  gases (oxygen, carbon dioxide) and water. 2. Organic nutrients:  contain carbon atoms in combination with hydrogen atoms;  are usually the products of living things;  they range from the simplest organic molecule, methane (CH4), to large polymers (carbohydrates, lipids, proteins and nucleic acids).

Sources of essential nutrients

Carbon sources

Microorganisms need carbon for all cellular structures and processes. They are dependent on other life forms. Common organic molecules that satisfy heterotrophs requirements are proteins, carbohydrates, lipids and nucleic acids [15]. Facts about some organic nutrients available to heterotrophs:  Exist in a form that is simple enough for absorption (monosaccharides and amino acids).  Larger molecules must be digested by the cell prior to absorption.  Not all heterotrophs can use the same carbon sources, some are restricted to a few substrates, whereas others are so versatile that they can metabolize more than 100 different substrates (e.g. Pseudomonas spp.) [4].

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Autotrophs useCO2 (an inorganic gas) as their carbon source. Because autotrophs have the special capacity to convert CO2 into organic compounds, they are not dependent on other livings things and are considered self-sustaining. These organisms form the basis of the food chain that all other life forms depend on. Carbon dioxide makes up about 0,35% of the earth’s atmosphere. The percentage of this gas is gradually increasing due to the burning of hydrocarbons (gasoline and other fuel) which contributes to the gradual warming of the earth also known as the “greenhouse effect”. Examples of autotrophs are photosynthetic microbes and plants.

Figure 8.1. Greenhouse effect

Chemoautotrophs: These organisms require neither sunlight not organic nutrients.

Hydrogen sources

Hydrogen is a major element in all organic compounds and in several organic ones, including water (H2O), salts (H2PO4) and certain naturally occurring gases (H2S, CH4 and H2). These gases are both used and produced by microbes. Hydrogen performs the following overlapping roles in the biochemistry of cells:  maintaining pH;  forming hydrogen bonds in macromolecules;  acting as a prime force in the oxidation-reduction reactions of respiration.

Nitrogen sources

The main reservoir of nitrogen is nitrogen gas (N2) which makes up about 79% of the earth’s atmosphere.

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Chart 8.1. The procentage of oxygen and nitrogen sources

Oxygen Other Nitrogen

This element is indispensable to the structure of proteins, DNA, RNA and ATP. Such compounds are the primary nitrogen source for heterotrophs, but, to be useful, they must first be degraded into their basic building blocks (proteins into amino acids; nucleic acids into nucleotides) [5]. Some bacteria and algae utilize inorganic nitrogenous nutrients (NO3, NO2 or NH3).

Nitrate Nitrite Ammonia

In prokaryotes a small number of bacteria can change N2 (nitrogen gas) into compounds usable by other organisms through the process of nitrogen fixation. Regardless of the initial form in which the inorganic nitrogen enters the cell, it must first be converted into NH3 - the only form that can be directly combined with carbon to synthesize aminoacids and other compounds [9].

Oxygen sources

Oxygen plays an important role in the structural and enzymatic functions of the cell. It is a major component of organic compounds such as carbohydrates, lipids and proteins. Free gaseous oxygen (O2) makes up 20% of the atmosphere. It is absolutely essential to the metabolism of many organisms. Oxygen is likewise a common component of inorganic salts such as sulfates, phosphates and nitrates [6].

Phosphorous (phosphate) sources

Phosphate is a key component of nucleic acids and is thereby essential to the genetics of cells and viruses. It also servesin cellular energy transfers. Phosphate (PO4),that is derived from phosphoric acid, (H3PO4) can be found in rocks and oceanic mineral deposits. Some compounds are phospholipids (cell membrane) or coenzymes. The lack of phosphate will limit the ability of some organisms to grow.

100

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Sulfur sources

Sulfur is widely distributed throughout the environment in mineral form. It is an essential component for some vitamins (B1) and aminoacids. It contributes to the structural stability and shape of proteins by forming unique linkages called disulfide bonds. Rocks and sediments (gypsum) may contain sulfate (SO4), sulfides (FeS), hydrogen sulfide gas (H2S) and elemental sulfur (S).

PABA (Para-Amino-Benzoic-Acid) was once a key component in sun screen lotions. It is similar to folic acid. It stopped being added to sunscreen when they realized that it was more important for bacteria then for us.

Other nutrients important in microbial metabolism

The rest of the elements that are used in microbial nutrition are called mineral ions. Potassium is essential to protein synthesis and membrane function. Sodium is important for some types of cell transport. Calcium is a stabilizer of the cell wall and endospores of bacteria. Magnesium is a component of chlorophyll; a stabilizer of membranes and ribosomes; a participant in cell energetics. Iron is an important component of the cytochrome pigments. Some microbes need ions such as zinc, copper, cobalt, nickel, molybdenum, manganese, silicon, iodine, boron and metal ions which can influence certain diseases by their effects on microorganisms [15].

The contents of Escherichia coli bacterium is:  water 70%;  proteins;  a cell as „simple” as E.coli contains about 5,000 different compounds, yet it needs to absorb only eight basic types of compounds to synthesize this great diversity;  about 97% of the dry cell weight is composed of organic macronutrients;  about 96% of the cell is composed of the six bioelements (CHNOPS);  bioelements are needed in the overall scheme of cell growth, but most of them are available to the cell as compounds and not as pure elements.

Growth factors: essential organic nutrients

An organic compound such as an aminoacid or a vitamin that cannot be synthesized by an organism and must be provided as a nutrient is a growth factor. A notable example of the need for growth factors occurs in Haemophilus influenzae, a bacterium that causes meningitis and respiratory infections in humans. It requires hemin (factor X), NAD (factor V), thiamine and pantothenic acid (vitamins), uracil and cysteine. Microbes that require complex nutrients growth factors or other special conditions are termed ”fastidious”.

101

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

8.2. NUTRITIONAL TYPES OF BACTERIA

Nutritional types of bacteria

Autotrophs Heterotrophs (Prepare own food) (Depend on Autotrophs)

1. Autotrophs are bacteria which obtain their nutrition from inorganic compounds. Carbon Photo- Photo- dioxide is typically the sole source of cellular autotrophs heterotrophs carbon. Autotrophs will use hydrogen sulfite, (trap solar (trap solar ammonia or hydrogen gas to reduce carbon into energy) energy) necessary sugars. Nitrifying bacteria, which oxidize ammonia to create nitrites and nitrates, are an example of bacteria which use autotrophic nutrition.

2. Heterotrophs bacteria require organic sources of carbon such as sugars, fats and amino acids, and are called heterotrophs. Saprophytic bacteria are an example. They feed on their nutrition from dead organic matter. Using enzymes, these bacteria will breakdown complex compounds and use the

nutrients to release energy. Saprophytic bacteria Chemo- are essential decomposers and play an important Chemo- autotrophs role in ecosystem by releasing simpler products heterotrophs (energy from which plants and animals can use [2]. (energy from chemical) chemical)

3. Phototrophs absorb light energy, then utilize it in photosynthesis to create cellular energy. There are two types of phototrophs; those which do not produce oxygen as a byproduct are called anaerobicphototrophs, while those which do produce oxygen are termed aerobic phototrophs. Both autotrophs and heterotrophs can be phototrophs. Cyanobacteria are an example of bacteria which execute photoautotrophic nutrition. 4. Chemotrophs are bacteria that obtain chemical energy from their surroundings and convert it into adenosine triphosphate (ATP) for cellular use. Chemotrophs obtain energy from oxidation-reduction reactions of inorganic compounds such ammonia, hydrogen sulfide and iron. For instance, sulfur bacteria is a chemoautotroph which produces energy by oxidizing hydrogen sulfide into sulfur and water. 5. Litotrophs are bacteria which use reduced inorganic compounds as the electron donor (H- donor) in anaerobic or aerobic respiration.

102

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 8.1. Heterotrophic and Autotrophic bacteria

Heterotrophic bacteria Autotrophic bacteria

The property of Are organisms that can produce their own

decomposition of organic food from the substances available in their

compounds is employed in surroundings using light (photosynthesis) or chemical energy (chemosynthesis). several industrial processes Autotrophs are fundamental to the food such as ripening of cheese, in the retting of fibers and in chains of all ecosystems in the world. the curing of tobacco [7]. They take energy from the sunlight or The aerobic breakdown of from inorganic chemicals and use it to create organic matter is called energy- rich molecules such as carbohydrates. “decay” as “decomposing”. It This mechanism is called primary production. is usually complete and not Most ecosystems are supported by the accompanied by the release autotrophic primary production of plants that [14] of foul gases. capture photons initially released by the sun .

Anaerobic breakdown of The process of photosynthesis splits a water molecule, releasing oxygen into the organic matter is called atmosphere, and reducing carbon dioxide to “ferme ntation”. It is usually incomplete and is always release the hydrogen atoms that fuel the accompanied by the release metabolic process of primary production. of foul gases. Anaerobic breakdown of proteins is called putrefaction.

Are bacteria heterotrophic or autotrophic?

Most bacteria are heterotrophs (like us).Heterotrophs must get their food from another source (such as in our gut or on our skin). Heterotrophs cannot synthesize their own food and rely on other organisms (plants and animals) for nutrition. Some bacteria are autotrophs, which means they make their own food from performing either photosynthesis (using the sun) or chemosynthesis (using inorganic compounds).

Autotrophs produce their own energy by one of the following two methods:

 Photosynthesis Photo(auto)trophs use energy from the sun to convert water from the soil and carbon dioxide from the air into glucose. Glucose provides energy to plants and is used to make cellulose which is then used to build cell walls. The bacterial photosynthesis is different from that of green plants since water is not used as a hydrogen donor. Hence oxygen is not released as

103

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

a byproduct. For this reason, the process is described as anoxygenic photosynthesis.

light CO2+H2O sugars+sulphur+water energy

Technically, the definition is that autotrophs obtain carbon from inorganic sources like carbon dioxide (CO2), while heterotrophs get their reduced carbon from other organisms. Autotrophs are usually plants; they are also called “self feeders” or “primary producers”.

 Chemosynthesis Chemoautotrophs use energy from chemical reactions to make food. The chemical reactions are usually between hydrogen sulfide/methane with oxygen. Carbon dioxide is the main source of carbon for chemoautotrophs. For example: bacteria found inside active volcanoes, inside hydrothermal vents in sea floor, or in hot water springs. Chemosynthetic bacteria can be classified in two categories: 1. Nitrifying bacteria which derive energy by oxidizing ammonia into nitrates. For example: Nitrosomonas, Nitrobacter. 2. Sulphur bacteria which derive energy by oxidizing.

Heterotrophs take in autotrophs as food to carry out functions necessary to keep them alive. Thus, heterotrophs (all animals, fungi, bacteria and protozoa) depend on autotrophs, or primary producers, for the energy and raw materials they need. Heterotrophs obtain energy by breaking down organic molecules (carbohydrates, fats and proteins) obtained in food. Carnivorous organisms rely indirectly on autotrophs, as the nutrients obtained from their heterotroph prey come from autotrophs they have consumed [8]. Organotrophs exploit reduced carbon compounds as energy sources, like carbohydrates, fats and proteins from plants and animals. Photo(organo)heterotrophs such as Rhodospirillaceae and purple non sulfur bacteria synthetize organic compounds by utilization of sunlight coupled with oxidation of inorganic substances, including hydrogen sulfide, elemental sulfur, thiosulfate and molecular hydrogen. They use organic compounds to build structures. They do not fix carbon dioxide and apparently do not have the Calvin cycle. Chemo(litho)heterotrophs can be distinguished from mixotrophs (or facultative chemolithotroph) carbon as the carbon source. Litotrophs use in organic compounds, such as hydrogen sulfide, elemental sulfur, ammonium and ferrous iron, as reducing agents for biosynthesis and chemical energy storage. Photo(auto)trophs and litho(auto)trophs use a portion of the ATP produced during photosynthesis or the oxidation of inorganic compounds to reduce NADP+ to form organic compounds. Some organisms rely on organic compounds as a source of carbon, but areable to use light or inorganic compounds as a source of energy. Such organisms are not defined as autotrophic but rather as heterotrophic. An organism that obtains carbon from organic compounds but obtains energy from light is called a photo(hetero)troph while an organism that obtains carbon from organic compounds but obtains energy from the oxidation of inorganic compounds is termed a chemo(hetero)troph, chemo(litho)heterotroph or lithoherotroph. In conclusion: Organotrophs are organic compounds that used as electron donor. Lithotrophs are inorganic compounds that are used as electron donor.

104

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Organotrophic organisms are often also heterotrophic, using organic compounds as sources of electrons and at the same time carbon . Heterotrophs: Organic compounds are metabolized to get carbon for growth and development. Autotrophs: Carbon dioxide (CO2) is used as source of carbon.

Table 8.1. Comparison table

Type of nutrition Autotroph Heterotroph Produce own food Yes No Food chain level Primary Secondary and tertioary Types Photo(auto)troph Photo(hetero)troph Chemo(auto)troph Chemo(hetero)troph Examples Plants, algae and some bacteria Herbivores, omnivores and carnivores

Table 8.2. Classification of organisms based on their metabolism

Energy Sunlight photo- source (preformed molecules) chemo- Electron Organic compound organo- donor Inorganic compound litho- -troph Carbon Organic compound hetero- source Carbon dioxide auto-

Typical examples are as follows:

 Chemolitho(auto)trophs obtain energy from the oxidation of inorganic compounds and carbon from the fixation of carbon dioxide [9]. In this category we can find: nitrifying bacteria, sulfur-oxidizing bacteria, iron-oxidizing bacteria, Knallgas-bacteria (hydrogen oxidizing bacteria). Knallgas is “bang-gas” (a mixture of hydrogen and oxygen - oxyhydrogen).  Photolitho(auto)trohps obtain energy from light and carbon from the fixation of carbon dioxide, using reduced equivalents from inorganic compounds. Examples: Cyanobacteria (water as reducing equivalent donor); Chlorobiaceae, Chromatiaceae (hydrogen sulfide as reducing equivalent donor); Chloroflexus(hydrogen as reducing donor).  Chemolitho(hetero)trophs obtain energy from the oxidation of inorganic compounds, but cannot fix carbon dioxide. Examples: Thiobacillus, Beggiatoa, Nitrobacter, Wolinella, Knallgas-bacteria, sulfate-reducing bacteria.  Chemo(litho)heterotrophs obtain energy from the oxidation of inorganic compounds, but cannot fix carbon dioxide. Examples: Thiobacillus, Beggiatoa, Nitrobacter, Wolinella, (with hydrogen as reducing equivalent donor), Knallgas-bacteria, sulfate-reducing bacteria.  Chemo(organo)heterotrophs obtain energy, carbon and reducing equivalents for biosynthetic reactions from organic compounds. Examples: most bacteria (Escherichia coli, Bacillus, Actinobacteria).  Photo(organo)heterotrophs obtain energy from light, carbon and reducing equivalents for biosynthetic reactions from organic compounds. Examples: Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodomicrobium, Rhodocyclus, Heliobacterium, Chloroflexus.

Mixotrophs

Some organisms usually unicellular, can switch between different metabolic modes, for example between phototrophy, photoheterotrophy and chemoheterotrophy in Chroococcales.

105

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Such mixotrophic organisms may dominate their habitat due to their capacity to use more resources than either photoautotrophic or organoheterotrophic organisms. Examples: Most cyanobacteria are photoautotrophic, since they use light as an energy source, water as electron donor and carbon dioxide as a carbon source. Fungi are chemoorganotrophic since they use organic carbon as both an electron donor and carbon source [10]. Prokaryotes show a great diversity of nutritional categories. For example: purple sulfur bacteria and cyanobacteria are generally photoautotrophic whereas purple non-sulfur bacteria are photoorganotrophic. Some bacteria are limited to only one nutritional group, whereas other are facultative and switch from one mode to the other, depending on the nutrient sources available [10].

8.3. SYMBIOTIC BACTERIA

These are bacteria which live in a mutually beneficial association with other organisms. Such bacteria derive the essential nutrients from their host organisms and in that process help the host through some of their biological activities. The bacteria found in the human alimentary canal Escherichia coli are nonpathogenic. These bacteria check the growth of harmful putrefying bacteria. In addition, these bacteria release vitamins K and B12 which are necessary for blood components. The human host provides shelter and food for these bacteria. A similar example is that of cellulose digesting bacteria, which occur in the alimentary canal of ruminant mammals such as cows and goats [15]. The most familiar example of symbiotic bacteria are the nitrogen fixing bacteria found in the root nodules of leguminous plants. Bacteria such as Rhizobium and Pseudomonas reside in the root nodules and reduce atmospheric nitrogen directly to ammonia. This becomes the source of nitrogen for the host plants. In return, the plant provides nutrients and protection to the bacteria.

8.4. SAPROBES AND PARASITIC BACTERIA

Saprobes (saprophyte) are free living microorganisms that feed on organic detritus from dead organisms, and parasites derive nutrients from the cells ortissues of a living host. Most microbes of biomedical importance belong to these two categories. Saprobes occupy a niche as decomposers of plant matter, animal carcasses and even dead microbes. If not for the work of decomposers, the earth would gradually fill up with dead plant and animal material, and the nutrients they contain would not be recycled. They release enzymes to the extracellular environment and digest the food particles into smaller molecules that can pass freely into the cell [13]. Parasitic bacteria are bacteria which occur in the body of animals and plants, obtaining their organic food from there. Most of these bacteria are pathogenic, causing serious diseases in the host organisms either by exploiting them or by releasing poisonous secretions called toxins.

106

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 8.2. Kingdom system

There are six kingdoms recognized separated (somewhat imperfectly) by form

(morphology) and mode of nutrition [11].

The six kingdoms are: Archaebacteria, Eubacteria, Protista, Fungi, Plantae, Animalia. 1. Arhaebacteria – Prokaryotic, unicellular, nutrition by absorption (heterotrophic). This group of bacteria-like organisms live in harsh (high temperature, salinity, etc.) environments similar to that of ancient earth. Their cell wall structure is different from that of typical bacteria. A recent study revealed that their genome is 44% unique from that of other prokaryotes or eukaryotes. 2. Eubacteria – Prokaryotic, unicellular, nutrition mainly by absorption (heterotrophic) with some photo or chemosynthesis (autotrophic), all single celled organisms with no membrane surrounding the genetic material (Bacteria, Blue-green

algae) and circular DNA. This organisms play a major role in global recycling,

breaking down dead organisms to nutrients. Eubacteria often form mutualistic rela tionships (both benefit) with many other organisms including corals and humans. Many human diseases are caused by Eubacteria: bubonic plague, typhus, syphilis, gonorrhea, botulism and most other forms of food poisoning.

8.5. ABSORPTION OF NUTRIENTS: TRANSPORT MECHANISMS

Three general types of transport are:

1. Passive transport, which follows physical laws, that are not unique to living systems and do not generally require the work of the cell. 2. Active transportwhich requires the activities of living membranes and consuming energy. 3. Bulk transportthe movement of large masses of material across membranes. Remember that membranes have two major components: phospholipids arranged in a bilayer and membrane proteins.

8.5.1. Passive transport

Based on the thermodynamics of the system, particles will move from an area of high concentration to an area of low concentration in order to increase the entropy of the cell. G=H-TS  G= Gibbs free energy (Joules)  H= Enthalpy (Joules)  T= Absolute temperature (◦K)  S= Entropy (Joules/◦K) Passive transport is independent from membrane proteins and from the catabolism of biological molecules for energy.

107

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 8.2. Active and passive transport

Simple diffusion (SD)

In SD, small noncharged molecules or lipid soluble molecules pass between the phospholipids to enter or leave the cell, moving from areas of high concentration to areas of low concentration (they move down their concentration gradient). The existence of this motion is evident in Brownian movement of small particles suspended in liquid. It can be sensed in a variety of other simple observations. A drop of perfume released into one part of a room is soon smelled in another part, or a lump of sugar in a cup of tea is soon spread through the whole cup without stirring. Oxygen and carbon dioxide and most lipids enter and leave cells by simple diffusion [12].

Osmosis

Is a physical phenomenon that represents the diffusion of water through a membrane. In an osmotic system, the membrane is selectively or differentially permeable, having openings that allow the free movement of water but block the movement of larger molecules. It provides a model of how a cell deals with water.

Some major examples of osmosis:  absorption of water by plant roots;  reabsorption of water by the proximal and distal convoluted tubules of the nephron;  reabsorption of tissue fluid into the venule ends of the blood capillaries;  absorption of water by the alimentary canal-stomach, small intestine and colon.

This osmotic relationship is determined by the relative concentrations of the solutions on either side of the cell membrane (the protoplasm of the cell versus its external environment).

108

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

There are 3 types of situations in which this could vary:

1. Isotonic – the environment is equal in concentration to the cell’s internal environment, and since diffusion of water proceeds at the same rate in both directions, there is no net change (here the external solution concentration and the internal concentration of the organism are the same). 2. Hypotonic – here the external solution concentration is less than the concentration of the organism. In this case the water will rush into the organism. In such systems, the net direction of osmosis is into the cell, and cells without walls swell and may burst. 3. Hypertonic – Here the external solution concentration is greater than the concentration of the organism. In this case the water will rush out of the organism. Hypertonic conditions are also out of balance with the tonicity of the cell’s protoplasm, but in this case, the environment is a stronger solution than the protoplasm. Salt water and concentrated sugar solutions are examples of such solutions, and food preservation with salt and sugar takes advantage of their hypertonic effect. In general, isotonic conditions pose little stress on cells, so survival depends on counteracting the adverse effects of hypertonic and hypotonic environments. The majority of bacterial cells have compensated this by developing a cell wall that protects them from bursting even as the protoplast becomes turgid with water. A microbe living in a highly salty environment (hypertonic) has the opposite problem and must either restrict its loss of water of the environment or increase the salinity of its internal environment. Halobacteria living in the Great Salt Lake and the Dead Sea actually concentrate salt in their cells to become isotonic with the environment, thus they have a physiological need for high salt concentrations.

Facilitated diffusion (FD)

Is a form of passive transport mediated by transport proteins imbedded within the cellular membrane. FD allows the passage of lipophobic molecules through the cell membrane’s lipid bilayer. Only molecules carried by specialized membrane proteins are transported. FD uses channel proteins to facilitate solute movement. Channel proteins are pores immersed in the lipid bilayer membrane. They facilitate a thermodynamically favorable net movement of particles and demonstrate an affinity and specificity for the particle being transported. Each protein channel contains a selectivity filter which is a collection of aminoacid residues concentrated in the interior of the protein channel. Potassium channels select K+ over Na+ by a factor of over one thousand despite differing in atomic radius by a mere 0.38 Ǻ.

8.5.2. Active transport (AT)

AT (simply put) is the movement of the particles through a transport protein from low concentration to high concentration at the expense of metabolic energy. The most common energy source used by cells is adenosine triphosphate or ATP, though other sources such as light energy or the energy stored in an electrochemical gradient are also utilized. In the case of ATP, energy is chemically harvested through hydrolysis. Active transport is classified as either Primary Active Transport (PAT) or Secondary Active Transport (SAT).

Features that characterize active transport systems are:  The transport of nutrients against the natural diffusion gradient or in the same direction as the natural gradient, but at a rate faster than by diffusion alone.

109

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 Special membrane proteins (permeases and pumps).  The expenditure of energy. Example of substances transported actively are monosaccharides, aminoacids, organic acids, phosphates and metal ions. The most universal example of ATP hydrolysis driving primary active transport in cells is the sodium-potassium pump. The sodium-potassium pump is responsible for controlling both sodium and potassium concentrations inside the cell. The sodium-potassium pump is extremely important in maintaining the cell’s resting potential.

Group translocation

Is a special type of active transport. It combines the transport of a nutrient with its conversion to a substance that is immediately useful to the cell.

8.5.3. Bulk transport (BT)

Some cells actively transport large molecules, particles, liquids, or even other cells across the cell's membrane. BT is the movement of substances across a membrane within a small vacuole (vesicles are small vacuoles; vacuoles, including vesicles, are enclosed by a single layer of membrane). Exocytosis: Is the process by which a cell moves the contents of secretory vesicles out of the cell via the cell'smembrane. Endocytosis: Is the opposite process by which the contents of secretory vesicles are moved into the cell via the cell's membrane. There are also other bulk transport mechanisms (for example: phagocythosis and pinocytosis).

Revision

Types of movement across membranes:  Passive Transport Mechanisms 1. Simple diffusion; 2. Facilitated diffusion; 3. Osmosis.  Transport Mechanisms – requiring energy from cells 1. Active transport; 2. Bulk transport.

Definitions

Diffusion = The movement of particles such as molecules or ions, from a region of higher concentration to a region of lower concentration (down a concentration gradient). Channel proteins have hollow cores that enable ions and small polar molecules to cross the membrane by passing through channel proteins. Carrier proteins bind to a particle, e.g. a large polar sugar molecule, then move it across the membrane releasing it on the other side of the phospholipid bilayer (carriers particle). Osmosis is the diffusion of wateracross membranes (active transport and bulk transport mechanisms). Phagocytosis = Eating (solid molecules are engulfed by the cell). Pinocytosis = Drinking (liquid molecules are engulfed by the cell).

110

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. The source of common essential nutrients are CHNOPS. ___2. Examples of inorganic nutrients are: metals, gases and water. ___3. Potassium is an important component of the cytochrome pigments. ___4. Bacterium Escherichia coli contain 70% water.

MULTIPLE CHOICE QUESTIONS

1. Some major examples of osmosis are: a. absorption of water by plant roots; b. absorption of water by small intestine and colon; c. reabsorption of water by blood capillaries; d. reabsorption of water by worms.

2. Which are the types of movement across membranes by passive transport mechanisms: a. simple diffusion; b. facilitated diffusion; c. osmosis; d. bulk transport.

CONCEPT QUESTIONS

Differentiate between micronutrients and Briefly describe the general function of the macronutrients. bio elements CHNOPS in the cell.

Compare autotrophs and heterotrophs: Describe the nutritional strategy of two types of lithotrophs described in the course:

111

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Describe how one might determine the Using the concept of synergy, can you nutrient requirements of a microbe from Mars. describe a way to grow a fastidious microbe? If, after exhausting the nutrients schemes, it still does not grow, what other factors might one take into accounts?

COMPLETE THE FOLLOWING SENTENCES

 ______is the movement of substances across a membrane within a small vacuole.

 ______is the process by which a cell moves the contents of secretory

vesicles out of the cell via the cell membrane.

 Active transport is classified as either ______

QUOTE

Mark with X if you like or dislike this quote.

(1) ”Intuition will tell the thinking mind where to look next” (Jonas Salk).

BACTERIA JOKE

Give a explanation for the following joke. You may find the explication in the text of the courses.

 BBC Sci-Tech News: bacteria have a sense of smell. So, there's no Bacteria in France then.

Explanation:

112

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Education (2010). The acronym "S.P. Cohn" was also used in high school biology classes to represent the six chemical elements. "CHNOPS: The Six Most Abundant Elements of Life". Pearson Education. Pearson BioCoach. Retrieved 2010-12-10. Most biological molecules are made from covalent combinations of six important elements, whose chemical symbols are CHNOPS. ... Although more than 25 types of elements can be found in biomolecules, six elements are most common. These are called the CHNOPS elements; the letters stand for the chemical abbreviations of carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur. 2. Hogg Stuart (2013). Essential Microbiology (2nd ed.). Wiley-Blackwell. p. 86. ISBN 978-1-119-97890-9. 3. H., Schurr, Sam (2011). Energy, ecnomic growth, and the environment. New York. ISBN 9781617260209. OCLC 868970980. 4. http://microbiowiki.wikifoundry.com/page/Microbial+Nutrition+and+Growth. 5. https://quizlet.com/49460935/microbiology-exam-2-ch-7-flash-cards/. 6. https://www.coursehero.com/file/p4chk10/Oxygen-Because-oxygen-is-a-major- component-of-organic-compounds-such-as/. 7. https://www.tutorvista.com/content/...iii/.../nutrition-bacteria.php. 8. https://en.wikipedia.org/wiki/Autotroph. 9. https://quizlet.com/20902073/microbial-metabolism-flash-cards/. 10. https://en.wikipedia.org/wiki/Primary_nutritional_groups. 11. https://www.hinsdale86.org/staff/kgabric/6kingdom.html. 12. http://www2.yvcc.edu/Biology/109Modules/Modules/MembraneTransport/membran etransport.htm. 13. Krishna Prakashan Media (2003). Microbiology. ISBN: 8187224665, 9788187224662. 14. Prakash S. Bisen, Mousumi Debnath, G. B. Prasad (2012). Microbes: Concepts and Applications. John Wiley & Sons, 2012. ISBN: 1118311892, 9781118311899. 15. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages.

113

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 9 Microbial metabolism and enzymes

9.1. Heterotrophic microbial metabolism 9.2. Fermentation 9.3. Special metabolic properties 9.1.1. Methylotrophy 9.1.2. Syntrophy 9.4. Anaerobic respiration 9.5. Enzymes

A. flavus is complex in its morphology and can be classified into two groups based on the size of sclerotia produced. Group I consists of L strains with sclerotia greater than 400 μm in diameter. Group II consists of S strains with sclerotia less than 400 μm in diameter. Both L and S strains can produce the two most common aflatoxins (B1 and B2). Unique to the S strains is the production of aflatoxin G1 and G2 which typically are not produced by Aspergillus flavus A. flavus. nickname ”Sunny Boy”

Learning objective

The term metabolism denotes all the organized chemical activities performed by a cell, which comprise two general types, energy production and energy use

Key points

A cell must be capable of performing a multitude of chemical changes in order to stay alive, grow, and reproduce

114

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

9.1. HETEROTROPHIC MICROBIAL METABOLISM

The term metabolism derived from the Greek “metabollein” meaning “change”, pertains to all chemical reactions and physical functioning of the cell.

The metabolism has two components:  Anabolism (also called biosynthesis) is any process that results in synthesis of cell molecules and structures;  Catabolism is the opposite, or the complement of anabolism. Catabolic reactions are degradative. They break bonds, convert larger molecules into smaller components, and often produce energy [4].

Metabolites (compounds released by the complex networks of metabolism) often serve multipurpose roles in the economy of the cell. Anaerobic respiration or glycolysis is the degradation of glucose to pyruvic acid, a process that does not require oxygen. Pyruvic acid is processed in aerobic respiration via the tricarboxylic acid (TCA) cycle and its associated respiratory chain. While oxygen is the final electron acceptor in aerobic respiration, other acceptors like sulfate, nitrate, or nitrite are also employed inanaerobic respiration. Important intermediates in glycolysis are glucose-6-phosphate, fructose-1,6-diphosphate, glycerol-3-phosphate, diphosphoglyceric acid, phosphoglyceric acids, phosphoenolpyruvate, and pyruvic acid [10]. Fermentation: Anaerobic respiration in both the electron donor and final electron acceptors are organic compounds. The phosphogluconate pathway is an alternate anaerobic pathway for hexose oxidation that also provides for the synthesis of NADPH and pentoses. Acetylcoenzyme A is a product of pyruvic acid processing. The net effect of pyruvic acid oxidation in the TCA cycle is the generation of ATP, some GTP, CO2, and H2O. The respiratory chain completes energy extraction. Important redox carriers of the electron transport system are NAD, FAD, coenzyme Q, and cytochromes. Glycolysis and the TCA cycle are bidirectional of amphibolic pathways. In addition to its use as a fuel, some metabolites of these pathways double as building blocks. Intermediates that are convertible into aminoacids through amination contribute to peptide synthesis. The same aminoacids from peptide degradation can be deaminated and used as a fuel. Components for purines and pyrimidines are derived from aminoacid pathways. Prokaryotic heterotrophic metabolism is much more versatile than that of eukaryotic organisms. Many prokaryotes using glycolysis (also called EMP pathway) for sugar metabolism and the citric acid cycle to degrade acetate, produce energy in the form of ATP and reduce power in the form of NADH or quinols. However, many bacteria and archaea utilize alternative metabolic pathways other than glycolysis and the citric acid cycle. A well-studied example is sugar metabolism via the keto- deoxy-phospho-gluconate pathway (also called ED pathway) in Pseudomonas. There is a third alternative sugar-catabolic pathway used by some bacteria, the pentose phosphate pathway.

115

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

The mitochondria (the small membrane -bound intracellular organelle) is the site of eukaryotic energy metabolism arising from the endosymbiosis of a bacterium related to obligate intracellular Rickettsia, and also the plant-associated Rhizobium and Agrobacterium [1].

9.2. FERMENTATION

Definition

Fermentation is a specific type of heterotrophic metabolism that uses organic carbon instead of oxygen as a terminal electron acceptor [5]. Many organisms can use fermentation under anaerobic conditions and aerobic respiration when oxygen is present. These organisms are facultative anaerobes. To avoid the overproduction of NADH, obligately fermentative organisms usually do not have a complete citric acid cycle. Instead of using an ATP synthase as in respiration, ATP in fermentative organisms is produced by substrate-level phosphorylation where a phosphate group is transferred from a high-energy organic compound to ADP to form ATP. As a result of the need to produce high energy, phosphate-containing organic compounds (generally in the form of Coenzyme A-esters) fermentative organisms use NADH and other cofactors to produce many different reduced metabolic by-products, often including hydrogen gas. These reduced organic compounds are generally small organic acids and alcohols derived from pyruvate, the finite product of glycolysis. Examples include ethanol, acetate, lactate and butyrate. Industrially, fermentative organisms are very important and are used to make many different types of food products [2]. Not all fermentative organisms use substrat-level phosphorylation. Instead, some organisms areable to couple the oxidation of low-energy organic compounds directly to the formation of a proton (sodium) motive force and therefore ATP synthesis. Examples: succinate fermentation by Propionigenium modestum and oxalate fermentation by Oxalobacter formigenes. These reactions yield extremely low-energy.

9.3. SPECIAL METABOLIC PROPERTIES

9.3.1. Methylotrophy

Methylotrophy refers to the ability of an organism to use C1-compounds as energy sources. These compounds include methanol, methylamines, formaldehyde and formate. Examples of methylotrophs include the bacteria Methylomonas and Methylobacter. Methanogenesis is the biological production of methane. It is carried out by methanogens, strictly anaerobic Archaea such as Methanococcus, Methanocaldococcus, Methanobacterium, Methanothermus, Methanosarcina, Methanosaeta and Methanopyrus[6].

9.3.2. Syntrophy

The biochemistry of methanogenesis is unique in nature in its use of a number of unusual cofactors to sequentially reduce methanogenic substrates to methane, such as coenzyme M and methanofuran. These methanogens can often be found in environments containing fermentative organisms. The tight association of methanogenes and fermentative bacteria can be considered to be syntrophic because the methanogens which rely on the fermentors for hydrogen, release

116

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

feedback inhibition of the fermentors by the build-up of excess hydrogen that would otherwise inhibit their growth [5]. Syntrophy refers to the pairing of multiple species to achieve a chemical reaction that would be energetically unfavorable. The best studied example is Syntrophomonas. These reactions help prevent the excess sequestration of carbon over geologic times scales, releasing it back to the biosphere in usable forms such as methane and carbon dioxide.

9.4. ANAEROBIC RESPIRATION

Anaerobic organisms have a lower reduction potential than oxygen, meaning that respiration is less efficient in these organisms and leads to slower growth rates than aerobes. Many facultative anaerobes can use either oxygen or alternative terminal electron acceptors for respiration depending on the environmental conditions. Denitrification – nitrate as electron acceptor is the utilization of nitrate (NO3) as a terminal electron acceptor. Many facultative anaerobes use denitrification because nitrate, like oxygen, has a high reduction potential. Many denitrifying bacteria can use Ferric Iron (Fe3+) and some organic electron acceptors [2]. Denitrification involves the step wise reduction of nitrate to nitrite (NO2), nitric oxide (NO), nitrous oxide (N2O) and dinitrogen (N2) by the enzymes nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase, respectively. Escherichia coli produces only nitrate reductase and therefore can accomplish just the first reduction leading to the accumulation of nitrite. Paracoccus denitrificans or Pseudomonas stutzeri reduce nitrate completely. Complete denitrification is an environmentally significant process because some intermediates of denitrification (nitric oxide and nitrous oxide) are important greenhouse gases that react with sunlight and ozone to produce nitric acid, a component of acid rain [1]. Denitrification is also important in the biological treatment of wastewater where it is used to reduce the amount of nitrogen released into the environment thereby reducing eutrophication.

Other inorganic electron acceptors

Ferric iron (Fe3+) is a widespread anaerobic terminal electron acceptor both for autotrophic and heterotrophic organisms.Model organisms include Shewanella putrefaciens and Geobacter metallireduncens. There is significant interest in using these organisms as bioremediation agents in ferric iron rich contaminated aquifers. These bacteria are of considerable interest for bioremediation, especially when heavy metals or radionuclides are used as electron acceptors.

Organic terminal electron acceptors

A number of organisms, instead of using inorganic compounds as terminal electron acceptors, are able to use organic compounds to accept electrons from respiration. Examples include:  fumarate reduction to succinate;  trimethylamine N-oxide (TMAO) reduction to trimethylamine (TMA);  dimethyl sulfoxide (DMSO) reduction to dimethyl-sulfide (DMS);  reductive dechlorination (RD). Commonly TMAO is chemical produced by fish, and when reduced to TMA produces a strong odor.

117

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

DMSO is a common marine and fresh water chemical which is also odiferous when reduced to DMS [5]. RD is the process by which chlorinated organic compounds are reduced to form their non-chlorinated endproducts. As chlorinated organic compounds are often important environmental pollutants, reductive dechlorination is an important process in bioremediation.

Chemolitothotrophy (Clt)

Clt is a type of metabolism where energy is obtained from the oxidation of inorganic compounds. Most chemolithotrophic organisms are also autotrophic. There are two major objectives to Clt:  the generation of energy (ATP);  the generation of reducing power (NADH). Many organisms are capable of using hydrogen as a source of energy. Hydrogen-oxidizing organisms, often inhabit oxic-anoxic interfaces in nature to take advantage of the hydrogen produced by anaerobic fermentative organisms, while still maintaining a supply of oxygen (for example: Cupriavidus necator).

Sulfur oxidation (So)

So involves the oxidation of reduced sulfur compounds (such as sulfide), inorganic sulfur 2- (So)and thiosulfate (S2O3 ) to form sulfuric acid (H2SO4). A classic example of a sulfur-oxidizing bacterium is Beggiatoa, a microbe originally described by Sergei Winogradski, one of the founders of environmental microbiology. Another example is Paracoccus.

Ferrous iron (Fi)

Fi is a soluble form of iron that is stable at extremely low pHs or under anaerobic conditions. There are three distinct types of ferrous iron-oxidizing microbes:  The first are acidophiles such as the bacteria Acidithiobacilus ferrooxidans and Leptospirillum ferrooxidan, Ferroplasma. These microbes oxidize iron in environments that have a very low pH and are important in acid mine drainage.  The second type of microbes oxidize ferrous iron at circum-neutral pH. These microorganisms live at the oxic-anoxic interfaces and are microaerophiles.  The third type of iron-oxidizing microbes are anaerobic photosynthetic bacteria which use ferrous iron to produce NADH for autotrophic carbon dioxide fixation [7].

Nitrification

- Nitrification is the process by which ammonia (NH3) is converted to nitrate (NO 3). Nitrification is the net result of two distinct processes: -  oxidation of ammonia to nitrite (NO 2) by nitrifying bacteria (Nitrosomonas);  oxidation of nitrite to nitrate by the nitrite-oxidizing bacteria (Nitrobacter).

Anammox

Anammox stands for anaerobic ammonia oxidation. This form of metabolism occurs in members of the Planctomyces and involves the coupling of ammonia oxidation to nitrite reduction.

118

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Anammox bacteria contain a hydrazine-containing intracellular organelle called the anammoxasome, surrounded by a highly compact ladderane lipid membrane. These lipids are unique in nature. These organisms could be used to remove nitrogen in industrial wastewater treatment processes [12]. It has also been shown that anammox has a widespread occurrence in anaerobic aquatic systems and has been speculated to account for approximately 50% of nitrogen gas production in the ocean.

Phototrophy

Phototrophs are capable of using light as a source of energy to produce ATP and organic compounds such as carbohydrates, lipids and proteins. Phototrophic bacteria are found in the phyla Cyanobacteria, Chlorobi, Proteobacteria, Chloroflexi and Firmicutes. Along with plants these microbes are responsible for all biological release of oxygen on earth. The photosynthetic bacteria contain different photosynthetic pigments such as chlorophyllus and carotenoids, allowing them to take advantage of different portions of the electromagnetic spectrum and thereby inhabit different niches.

Nitrogen fixation

Nitrogen is an element required for growth by all biological systems. These prokaryotes are very important from an ecological point of view and are often essential for the survival of entire ecosystems. This is especially true in the ocean, where nitrogen-fixing cyanobacteria are often the only sources of fixed nitrogen and in soils, where specialized symbioses exist between vegetables and their nitrogen-fixing partners to provide the nitrogen necessary for the growth of these plants.

9.5. ENZYMES

A microbial cell could be viewed as a microscopic factory complete with basic building materials a source of energy, and a “blueprint” for running its extensive network of metabolic reactions [8]. A cell must be capable to perform a multitude of chemical changes in order to stay alive, grow and reproduce. The chemical changes involved are exceedingly complex considering the diversity of materials used as food on the one hand, and the variety of substances synthesized into cell constituents on the other [11]. How does the cell accomplish these changes? They are made possible through the action of hundred of different enzymes (the biological catalysts). Enzymes are a remarkable example of catalysts, chemicals that increase the rate of a chemical reaction without becoming part of the products. Do not make the mistake of thinking that an enzyme creates a reaction. There can be no life without enzymes. For example: a single Escherichia coli cell dividing every hour synthesizes 4,000 molecules of lipid, almost 1,000 protein molecules (each containing about 300 aminoacids) and 4 molecules of RNA/second [3]. How does an enzyme act? An understanding of enzyme action requires some simple, yet essential knowledge of the kinetics of chemical reactions.

119

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

All reactions whether or not catalyzed by an enzyme, represent a rearrangement of molecules. Any rearrangement requires the breaking of old bonds and the formation of newones (for example: the formation of water from oxygen and hydrogen).

Feature 9.1. Checklist of

enzymecharacteristics  Act as organic catalysts to speed up the rate of cellular reaction;  Are composed of protein and may require co-factors;  Enable metabolic reactions to proceed at a speed compatible with life, and also help regulate them;

 Are not used up or permanently changed by the reaction;  Lower the activation energy required for a chemical reaction to proceed;  Provide a reactive site for target molecules called substrates;  Work rapidly;

 Can be regulated by feedback and genetic mechanisms;  Have unique characteristics such as shape, specificity and function;  Associate closely with substrates but do not become integrated into the reaction products;

 Are limited by particular conditions of temperature and pH;  Are reusable, thus functioning in extremely low concentrations.

Certain substances in small amounts have the unique capacity of speeding up the chemical reactions without themselves being altered by the reaction. They accelerate the velocity of the reaction without necessarily initiating it. Substances that behave in this manner are called catalysts or catalytic agents [9]. For example, hydrogen and oxygen do not combine to any appreciable extend under normal atmospheric conditions. The platinum greatly increases the speed at which this reaction takes place without being used up in the reaction. We may define an enzyme as an organic catalyst produced by a living cell.

Enzymes may be classified in two types on the basis of the place of action:  Intracellular enzymes or endoenzymes – functioning in the cell. They synthesize cellular material and also perform catabolic reactions which provide the energy required by the cell. In terms of their presence in the cell, enzymes are not all produced in equal amounts or at equal rates and may be classified in:  Constitutive enzymes that are always present and are in relatively constant amounts, regardless of the amount of substrate. Some of the enzymes (glycolysis or sugar) breakdown;they called constitutive enzymes.  Induced (inducible) enzymes are not constantly present and are produced only when their substrate is present. The process is referred to as enzyme induction and the substrate responsible for evoking the formation of the enzyme is an inducer. An example of an inducible enzyme is beta-galactosidase. Its inducer is sugar lactase.

120

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 9.2. The origin of the word

enzyme

The word enzyme was coined in 1878 by Kuhne from a Greek term meaning “In yeast” because their actions were similar to yeast fermentation.

Liebig claimed that fermentation was caused by chemical substances not associated with living cells, but Pasteur stated that the fermentation process was inseparable from living cells. As we know today, neither position was strictly wrong.The Pasteur-Liebig controversy was resolved in 1872 by Buchner, who demonstrated that a cell-free juice, prepared from yeasts by filtration, contained active enzymes.

In any cell there are a thousand or more different enzymes. The enzymes present in a microbial cell are determined by the environmental conditions and by the cell’s genetic constitution. This means that at any given moment, the enzyme content of a microbe is a reflection of the manner in which that cell copes with the environment [9].

Feature 9.3. The role of microbial enzymes in disease

Many bacterial pathogens secrete unique exoenzymes that help them avoid host defenses or promote their multiplication in tissues. They are so called virulence factors or toxins.  Streptococcus pyogenes produces streptokinase that digests blood clots and

apparently assists in the invasion of wounds. S. pyogenes produces throat and skin infections.  Staphylococcus aureus produces lipases that increase the virulence of this species by promoting its invasion of oil-producing glands on the skin.  Pseudomonas aeruginosa is a respiratory pathogen that produces elastase which digests elastin (a protein common in many tissues).  Clostridium perfringens is an agent of gas gangrene, synthesized lecithinase C, a lipase that profoundly damages cell membranes and accounts for the death of the tissue

associated with this disease. Not all enzymes digest tissues; some, like penicillinase,

inactivate penicillin.  Viruses do not synthesize enzymes independently, they instruct the host cell to synthesize a small but impressive group of viral enzymes. Most of this are involved in viral replication, but some contribute to cell damage and viral escape. Influenza virus contains spikes of neuraminidase that help liberate the virus from an infected cell. Retroviruses contain reverse transcriptase. For example: AIDS virus promotes conversion of viral RNA to viral DNA, thereby allowing the virus genes to be inserted into a host chromosome.  Bacteriophages instruct the synthesis of lysozyme which digests and weakens the bacterial cell wall and facilitates lysis and release.

Enzymes are proteins or proteins combined with other chemical groups. They are denaturated by heat, are precipitated by ethanol or high concentrations of inorganic salts like

121

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

ammonium sulfate, and do not dialyze (pass through semipermeable membranes). Many enzymes consist of a protein combined with a low-molecular-weight organic molecule called a coenzyme. Coenzymes are organic compounds that work in conjunction with an apoenzyme to perform a necessary alteration of a substrate [10]. The general function of a coenzyme is to remove a functional group from one substrate molecule and add it to another, thereby serving as a transient carrier of this group. The protein portion in this instance is referred to as the apoenzyme. When united, the two form the complete enzyme, identified as the holoenzyme.

Feature 9.4. Apoenzime and Coenzime

APOENZYME + COENZIME HOLOENZYME

Inactive protein Inactive organic

High molecular molecule weight Low molecular nondialyzable weight Active dialyzable

The integral part of some coenzymes is a vitamin. The metallic cofactors (iron, copper, magnesium, manganese, zinc, cobalt, selenium, etc.) participate in precise functions between the enzyme and its substrate. They are also called enzyme activators.

Table 9.1. Some vitamins and their coenzymes

Vitamin Coenzyme

Thiamine (B1) Cocarboxylase Riboflaxin (B2) Riboflavin adenine dinucleotide Niacin Nicotinamide adenine dinucleotide Pyridoxine (B6) Pyridoxal phosphate Folic acid Tetrahydrofolic acid

122

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 9.5. The catalytic Major classes of enzymes

antibodies (abzymes)

Class No Class Catalytic reaction The immunologists are already 1 Oxidoreductases Electron-transfer well acquainted with the concept of “a reactions Tranfer of functional lock-and-key specific fit because of 2 Transferases groups the way that antibodies lock into their target. This knowledge led to the 3 Hydrolases Hydrolysis reactions inevitable question: can antibodies, Lyases 4 Two double bonds which are also proteins, act as

enzymes too? The answer is a 5 Isomerases Isomerization reactions qualified yes-if the antibody is carefully chosen. 6 Ligases Breakage of ATP These catalytic antibodies, or abzymes, have extreme specificity for their substrate and can speed up a reaction, form an abzyme-substrate complex . Antibodies are very easy to fine-tune to fit almost any possible substrate configuration and can be

manufactured in large amounts using monoclonal methods. The applications for medicine and industry are

considerable. This technology has the potential for treating cancers, Many enzymes occur in different structural forms but possess identical catalytic infections and other medical problems. The possible use of abzymes to purify properties. Such enzymes are called isozymes drugs and inactivate viruses is already or isoenzymes. Most enzyme reactions may be being explored. represented by the following overall reaction:

Enzyme E+Substrate S Enzyme-substrate complex ES Product P+Enzyme E. There is a high chemical affinity of the substrate for certain areas of the enzyme surface, called the active sites. A strain or distortion is produced at some linkage in the substrate molecule, making it labile (unstable) and it undergoes a change determined by the particular enzyme. The enzyme is then free to combine with more substrate to repeat the action. The main function of an enzyme is to lower the activation energy barrier to a chemical reaction.Activation energy refers to the amount of energy required to bring a substance to the reactive state. The active area on an enzyme surface is actually a very small area, which means that large regions of the enzyme protein do not contribute to enzyme specificity or enzyme action. Among the conditions affecting the activity of an enzyme are the following:  concentration of enzyme;  concentration of substrate;  pH;

123

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 temperature. Each enzyme functions optimally at a particular pH and temperature. Extreme variations in pH can even destroy the enzyme, as can high temperatures; boiling for a few minutes will denature (destroy) most enzymes. Extremely low temperatures, for all practical purposes, stop enzyme activity, but do not destroy the enzymes. Many enzymes can be preserved by holding them at temperatures around 00 Cor lower. The activity of an enzyme can be inhibited by chemical agents in several different ways. Enzyme inhibition may be classified as either nonreversible or reversible. There are two major types of reversible inhibition namely, competitive inhibition and noncompetitive inhibition. Competitive inhibition can be reversed by increasing the substrate concentration, where as noncompetitive inhibition cannot. There are generally two types of control proteins, namely: allosteric enzymes and regulatory proteins. Allosteric enzymes are so called because the site on the enzyme molecule where an effector molecule acts is different from the catalytic site. The enzymatic reaction rate can be controlled by substrate concentration. As the substrate concentration increases, the reaction rate increases too until a limiting value is reached when all the enzymes are saturated. Insome microbes, highly specific proteolytic enzymes (protein-degrading), or proteases breakdown other enzymes which are no longer required for metabolic reactions. In a few instances, alteration in enzyme activity is brought about by a phenomen called covalent modification of the regulatory enzyme molecule itself so that it can switch back and forth from an active to an inactive form.This modification is accomplished by the action of other enzymes.

Induction

Is the process that occurs when an inducer (the effector molecule) which is either the substrate or a compound related to the substrate of the enzyme-catalyzed reaction, is required for enzyme synthesis to occur.

Repression

Is the process that takes place when a regulatory protein, the repressor, binds to a specific segment of the DNA called the operator. Effector molecules act as corepressors in preventing synthesis of the enzyme. Corepressors function by combining with the repressor to form an active complex which combines with the operator gene to prevent messenger ribonucleic acid (mRNA) synthesis by the structural genes. The operator gene is one of the regulator genes on a deoxyribonucleic acid (DNA) chromosome. The operator gene prevents gene expression by negative control. In other cases, it can enhance gene expression by positive control. In this case, the repressor binds to the inducer, undergoes a conformational change, and is converted into an activator, which triggers gene expression. In many bacteria the structural genes governing the biosynthesis of proteins are positioned in the exact order of the sequence of reactions in the particular metabolic pathway. A group of such consecutive genes forming an operational unit was named an operon by Francois Jacob and Jackques Monod. The operon includes both the structural and associated operator genes. The regulator genes function primarily at the level of transcription and not at the level of translation.

124

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

In many bacteria, the addition of Regulator genes such an end produc. For example: an amino acid - to the culture medium results in the

Repressor- Operator inhibition of the synthesis of the producing gene gene enzymes. This process is termed end- product repression or feedback repression. DNA Induction and end-product molecule repression of enzyme synthesis are specific responses to a particular Inducer Repressor Corepressor Structural genes metabolite or to a closely related group for messenger of metabolites which we have called RNA synthesis effector molecules.

Inactive Active complex complex

These controls allow cells to use the substrate that supports the most rapid rate of growth in the presence of several others. A good example of this is the glucose effect. In a medium containing both glucose and lactose, Escherichia coli preferentially uses glucose (“preferred” substrate). Lactose is not metabolized until the glucose is used up. This type of regulation is called catabolite repression of enzyme synthesis.

125

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. Metabolism is derived for the Greek term metabollein meaning change. ___2. Anabolism is also called biosynthesis. ___3. Anaerobic respiration is the degradation of glucose to pyruvic acid. ___4. Fermentation is a specific type of autotrophic metabolism that uses hydrogen instead of oxygen as a terminal electron acceptor.

MULTIPLE CHOICE QUESTIONS

1. Which are the types of ferrous iron-oxidizing microbes? a. acidophiles; b. anaerobic photosynthetic bacteria; c. aerobic photosynthetic bacteria; d. barophiles.

2. Which of these bacteria are considered methanogens? a. Methanococcus; b. Methanobacterium; c. Methanothermus; d. Methanophera.

CONCEPT QUESTIONS

What is the definition of heterotrophic What is the definition of the microbial metabolism? Explain: fermentation? Explain:

Which are the special metabolic properties? Describe glucose effect:

126

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

What is chemolitothotrophy? What is sulfur oxidation?

How does an enzyme act? Which are the classification of enzymes on the basis of the site of action?

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of the courses.

 I wish I was adenine, then I could get paired with U.  Girl you're so hot you denaturate my proteins.

Explanation:

127

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Firdos Alam Khan (2011). Biotechnology Fundamentals, CRC Press. ISBN: 1439897123, 9781439897126. 2. Firdos Alam Khan (2015). Biotechnology Fundamentals, Second Edition, CRC Press. ISBN: 1498723446, 9781498723442. 3. Eugene W. Nester (1978). Holt, Rinehart and Winston. Experiments in microbiology. ISBN-10: 0030393361, ISBN-13: 978-0030393365. 4. https://quizlet.com/93684895/micro-chapter-7-flash-cards/. 5. https://en.wikipedia.org/wiki/Microbial_metabolism. 6. https://ipfs.io/ipfs/QmXoypizjW3WknFiJnKLwHCnL72vedxjQkDDP1mXWo6uco/ wiki/Microbial_metabolism.html. 7. Jiao Y, Kappler A, Croal LR, Newman DK (2005). "Isolation and Characterization of a Genetically Tractable Photoautotrophic Fe(II)-Oxidizing Bacterium, Rhodopseudomonas palustris Strain TIE-1". Appl Environ Microbiol. 71 (8): 4487–96. doi:10.1128/AEM.71.8.4487- 4496.2005. PMC 1183355 . PMID 16085840. 8. Kathleen Park Talaro, Barry Chess (2012). Foundations in Microbiology, Eighth Edition. McGraw-Hill. ISBN: 978-0-07-337529-8,0-07-337529-2. 9. P. M. Swamy (2008). Laboratory Manual on Biotechnology, Rastogi Publications. ISBN: 8171339182, 9788171339181. 10. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages. 11. SYED TANVEER AHMED INAMDAR (2012). BIOCHEMICAL ENGINEERING: PRINCIPLES AND CONCEPTS. PHI Learning Pvt. Ltd. ISBN: 8120345851, 9788120345850. 12. Zhu G, Peng Y, Li B, Guo J, Yang Q, Wang S (2008). "Biological removal of nitrogen from wastewater". Rev Environ Contam Toxicol. Reviews of Environmental Contamination and Toxicology. 192: 159–95. doi:10.1007/978-0-387-71724-1_5. ISBN 978-0- 387-71723-4. PMID 18020306.

128

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 10 The influence of environmental factors on microbes (Part I)

10.1. Temperature 10.2. Gas requirements 10.3. Osmotic pressure 10.4. Radiation

Stachybotrys chartarum, also called Stachybotrys atra, Stachybotrys alternans or Stilbospora chartarum, is a black mold that produces its conidia in slime heads. S. chartarum was originally discovered on the wall of a house in Prague in 1837 by Czech mycologist August Carl Joseph Corda. It requires high moisture content in order to grow and is associated with wet gypsum material and wallpaper. Stachybotrys chartarum nickname ”Gavroche”

Learning objective

The growth of the microorganisms is greatly affected by the chemicaland physical nature of their surroundings

Key points

Prokaryotes are present and grow anywhere life can exist

129

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

10.1. TEMPERATURE

The environments in which some prokaryotes grow would kill most of the other organisms. For example: Bacillus infernus is able to live over 1,5 miles bellow the earth's surface without oxygen and at 600C temperature. These microorganisms which can thrive and grow in such harsh conditions are often called extremophiles. Microbial cells assume the ambient temperature of their natural habitats and adapt to growth within a certain range of temperatures encountered in that habitat, called cardinal temperatures. The minimum temperature is the lowest temperature that permits a microbe's continued growth and metabolism. At very low temperatures membranes solidify and enzymes don't work rapidly. The optimum temperature encompasses a small range, intermediate between the minimum and maximum, which promotes the fastest rate of growth and metabolism. This optimum temperature usually varies, from 00C to as high as 750C, whereas microbial growth occurs at temperature extending from -200C to over 1200C [8]. The maximum temperature is the highest temperature at which growth and metabolism can occur. Microorganisms are classified into five classes based on their temperature ranges for growth.

Feature 10.1 Did you know?

Prokaryotes have been found growing at or close to 1000C. Now thermophilic prokaryotes have been reported growing in surface of chimneys or black smokers located along rifts and ridges on the ocean floor that spew sulphide-rich super-heated vent water with temperatures above 3500C. These microbes can grow and reproduce at or above 1120C. Some thermostable enzymes from these organisms have important industrial and

scientific uses. For example: the Taq polymerase from the thermophilic Thermus aquaticus is used extensively in the polymerase chain reaction.

Microorganisms are classified into five classes based on their temperature ranges for growth. 1. Psychrophiles or cryophiles are extremophilic organisms that are capable of growing and reproducing in cold temperatures, ranging from -200 C to +100 C. Temperature as low as -150 C are found in pockets of very salty water (brine) surrounded by sea ice. They are particularly interesting to astrobiology, in the theory about the possibility of extraterrestrial life, and to geomicrobiology, the study of microbes active in geochemical processes [3]. In an experimental research performed at University of Alaska Fairbanks, a 1000-litre biogas digester using psychrophiles harvested from ”mud from a frozen lake in Alaska” has produced 200-300 liters of methane/day [3].

130

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 10.1. Psychrophiles

These microorganisms are isolated from Arctic and Antarctic habitats and the deep ocean. To this group belong microorganisms such as Vibrio, Alcaligenes, Pseudomonas, Arthrobacter, Moritella, Bacillus, Photobacterium. The psychrophilic Chlamydomonasnivalis turns a snowfield or glacier pink with its bright red spores (watermelon snow). This type of snow is common during the summer in alphine, and coastal polar regions worldwide, such as the Sierra Nevada of California. Compressing the snow by stepping on it or making snow balls leaves it looking red [4]. 2. True psychrophiles must be distinguished from facultative psychrophiles or psychrotrophs which are mesophilic organisms that grow slowly in cold (00C-70C) but have an optimum temperature above 200C - 300C. The spoilage of refrigerated food is mainly caused by microorganisms belonging to this group. 3. Mesophiles are organisms that grow at moderate temperatures. They grow optimally around 200C to 450C, and an individual species may grow at the extremes of 100C or 500C. Most of the organisms fall under or within this category including human pathogens (human body temperature is 370C). Organisms in this group inhabit animals and plants, as well as soil and water in temperate subtropical and tropical regions.

131

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Thermoduric microbes can survive short exposure to high temperatures, but are normally mesophiles. They are common contaminants of heated or pasteurized food and often they are, spore-forming microbes. Examples of such bacteria are: Bacillus, Clostridium, Enterococci. 4. Thermophiles are microorganisms that can grow optimally at temperatures higher than 450C. Such heat-living microbes live in soil and water associated with volcanic activity and in habitats directly exposed to the sun (composts, self-heating haystacks, hot water lines and hot springs). Thermophiles are found in hot springs like those in Yellowstone National Park and deep sea hydrothermal vents, as well as decaying plant matter such as peat bogs and compost. Thermophiles produce some of the bright colors of Grand Prismatic Spring, Yellowstone National Park [5]. Thermophiles vary in heat requirements, with a general rate of growth rangingbetween 450C-800C (a few thermophilic bacteria grow around 2500C). Microorganisms have more heat-stable enzymes and proteins synthesis systems, which function at high temperature. Aminoacids like proline make the polypeptide chain less flexible and chaperones also aid in folding of proteins to stabilize them. Many thermophiles are spore-forming species of Bacillus and Clostridium. A small number of them are pathogens. 5. Hyperthermophiles are microorganisms that can grow at 960C or above and have a maximum of 1000C. They optimally grow at temperatures between 800C and about 1130C. For example: marine hyperthermophiles found in hot floors of the sea floor such as Pyrococcus and Pyrpdictiumoccultum. An extraordinary heat-tolerant hyper-thermophile is the Strain 121 which has been able to double its population during 24 hours in an autoclave at 1210 C (hence its name); the current record growth temperature is 1220 C for Methanopyrus kandleri. The cell membrane contains high levels of saturated fatty acids to retain its shape at high temperature.

Examples of hyperthermophiles bacteria:

1. Strain 121 (that lives in the Pacific Ocean); 2. Pyrolobus fumarii (living at 1130 C in Atlantic hydrothermal vents); 3. Pyrococcus furiosus (first discovered in Italy near a volcanic vent); 4. Archaeoglobus fulgidus; 5. Methanococcus jannaschii; 6. Methanopyrus kandleri strain 116 (between 80-1220 C in a Central Indian Ridge); 7. Geothermobacterium ferrireducens (which thrives between 65-1000 C in Obsidian Pool, Yellowstone National Park); 8. Thermotoga maritime.

(1) The symbol for Potassium on the periodic table is ”K”. (2) Polyethylene is the most common type of plastic.

132

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 10.2. Extremophiles microbes survive on Mars?

Tardigrades (also known as water bears or moss piglets) are water- [10] dwelling, segmented micro-animals, with eight legs . The name Tardigrada (meaning "slow stepper") was given by the Italian biologist Lazzaro Spallanzani [1]. Tardigrades can survive in extreme environments. For example: they can withstand temperatures from just above absolute zero to well above the water boiling point (100 °C), pressures about six times greater than those found in the deepest ocean trenches,

ionizing radiation at doses hundreds of times higher than the lethal dose for a human, and the vacuum of outer space. They can go without food or water for more than 10 years, drying out to the point where they are 3% or less water, only to rehydrate, forage, [9] and reproduce .

10.2. GAS REQUIREMENTS

The three atmospheric gases that most influence microbial growth are O2, CO2,N2. An aerobe is an organism able to grow in the presence of atmosphere; the one that grows in its absence is an anaerobic. With respect to oxygen requirements, several general categories of microbes are recognized. An aerobe (aerobic organism) grows well in the presence of normal atmospheric oxygen and possesses the enzymes needed to process toxic oxygen products. An organism that cannot grow without oxygen is an obligate aerobe. A facultative anaerobe is a microorganism which does not require O2 for growth but grows better in its presence. Aerotolerant anaerobes do not utilize oxygen, but can survive in its presence such as Enterococcus faecalis. An anaerobe does not grow in normal atmospheric oxygen, and it lacks the metabolic enzyme systems for using oxygen in respiration. Many anaerobes also lack the enzymes for processing toxic oxygen and they cannot tolerate any free oxygen in the immediate environment. Strict or obligate anaerobes are killed or inhibited by oxygen. They live in highly reduced habitats such as deep muds, lakes, oceans, soil, and even the bodies of animals. They are represented by Fusobacterium, Bacteroides, Clostridium pasteurianum, Methanococcus, Neocallimastix. A microaerophile does not grow at normal atmospheric tensions, but requires a small amount of oxygen in metabolism. Actinomyces israellii and Treponema pallidum are examples of microaerophiles.These microorganisms require O2 levels between the range of 2% and 16% for growth.

133

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

The different relationships with O2 appear due to several factors, including the inactivation of proteins and the effect of toxic O2 derivatives. Enzymes can be inactivated when sensitive groups like sulfhydryls are oxidized. A notable example is the nitrogen fixation carried out by enzymes nitrogenase which are very O2 sensitive oxygen accepts electrons and is readily reduced because its two outer orbital electrons are unpaired. The reduction products such as superoxide radical, hydrogen peroxide and hydroxyl radical can result from flavo-proteins, several other cell constituents and radiation. - - O2+e O2 They are extremely toxic because they are powerful oxidizing agents and rapidly destroy cellular constituents. Microorganisms possess enzymes that give protection against toxic O2 products. Obligate aerobes and facultative anaerobes usually contain the enzymes superoxide dismutase (SOD) and catalase which catalyze the destruction of superoxide radical and hydrogen peroxide. Peroxidase can also be used to destroy peroxide. - - 2O2 +2H O2+H2O2 (superoxide dismutase) 2H2O2 2H2O+O2 (catalase) + - N2O2 + NADH + H 2H2O + NAD (peroxidase) The capnophiles grow best at a higher CO2 tension that is normally present in the atmosphere. Incubation is carried out in a system that provides 3% to 10% CO2 (a CO2 incubator, special plastic bag, or candle jar). Examples of such bacteria are: Neisseria, Brucella, and Streptococcus pneumoniae.

Figure 10.2. Test tubes of thioglycollate broth

Oxygen concentration High

Loose – fitting cap

Low

(a) (b) (c) (d) obligate obligate facultative aerotolerant aerobes anaerobess anaerobes anaerobes

high high high high 134

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Aerobic and anaerobic bacteria can be identified by growing them in test tubes of thioglycollate broth:  Obligate aerobes need oxygen to grow. They need oxygen because they cannot ferment or respire anaerobically. They gather at the top of the tube where the oxygen concentration is highest [6].  Facultative anaerobes use oxygen if it is available, but also have anaerobic methods of energy production. They can metabolize energy aerobically or anaerobically. They gather mostly at the top because aerobic respiration generates more ATP than either fermentation or anaerobic respiration.  Microaerophiles require oxygen for energy production, but are harmed by atmospheric concentrations of oxygen (21% O2). They need oxygen because they cannot ferment or respire anaerobically. They are poisoned by high concentrations of oxygen. They gather in the upper part of the test tube, but not the very top.  Aerotolerant organisms do not use oxygen but are not harmed by it. They do not require oxygen as they metabolize energy anaerobically. They can be found equally spread throughout the test tube.

10.3. OSMOTIC PRESSURE

The hydrostatic pressure can reach 600 to 1100 atm in the deep sea with temperature between 20C and 30C. Barophiles are bacteria so strictly adapted to high pressures that they will rupture when exposed to normal atmosphere pressure [7]. The barophilic organisms are those growing in the guts of deep sea invertebrates such as amphipods and holothurians and grow more rapidly at high pressures. These bacteria may play an important role in nutrient recycling in the deep sea. They are found on ocean floors (380 atm) and at the bottom of the Pacific Ocean [2]. Examples of barophiles are some bacteria such as Colwellia, Photobacteria, Shewanella. Obligate barophiles are not able tosurvive without high pressures. Most piezophiles grow in darkness and are usually very UV-sensitive. Examples of such bacteria are: Halomonas salaria, requires a pressure of 1000 atm and a temperature of 30 C. Xenophyophores have been found in the deepest ocean trench, 6.6 miles (10,541 meters) below the surface. Many bacteria are barotolerant. Although most microbes exist under hypotonic or isotonic conditions, a few, called the halophiles or osmophiles live in solutions that have a high solute concentration. Obligate halophiles abundantly inhabit strongly saline lakes and ponds. Examples of such bacteria are Halobacterium and Halococcus. They optimally grow in solutions of 25% NaCl, but require at least 15% NaCl for their growth. However, it is common to use high osmotic pressures preserving food (jams, jellies, syrups, and brines) and preventing the growth of contaminants).

10.4. RADIATION

Various forms of electromagnetic radiation stream constantly onto the earth from the sun. Phototrophs may use visible light rays as an energy source. Non-photosynthetic microbes tend to be damaged by the toxic oxygen products produced by contact with light. Staphylococcus aureus, can produce a yellow carotenoid pigment that protects against the damaging effect of light by absorbing and dismantling toxic oxygen.

135

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Other types of radiation that can damage microbes are ultraviolet and ionizing rays. Sunlight is the major source of radiation on earth. It includes visible light, ultraviolet radiation, infrared rays and radio waves. Many forms of electromagnetic radiation are very harmful to microorganismslike some prokaryotes (Deinococcus radiodurous), and bacterial endospores that are resistant to the radiation. Destruction of DNA is the most important cause of death of microorganisms. Ultraviolet radiation kills all kind of microorganisms. The most lethal UV radiation has a wavelength of 260 nm, and is the one most effectively absorbed by DNA. Most phototrophic organisms are also autotrophs, obtaining carbon from atmospheric carbon dioxide in a process called photosynthesis. An example of such bacteria are Cyanobacteria that are responsible both for the oxygenation of the earth in what is now known as the Great Oxygenation Event. UV formation of thymine dimmers in DNA which inhibits DNA replication and function.

Feature 10.3. The GreatOxygenation Event

The Great Oxygenation Event (GOE), also called the Oxygen Catastrophe, Oxygen Crisis, Oxygen Holocaust, Oxygen Revolution, or Great Oxidation, was the biologically induced appearance of dioxygen (O ) in Earth's atmosphere. Geological, isotopic, and 2 chemical evidence suggest that this major environmental change happened around 2.3 billion years ago (2.3 Ga). Cyanobacteria, which appeared about 200 million years before the GOE, began producing oxygen by photosynthesis. Before the GOE, any free oxygen they produced was chemically captured by dissolved iron or organic matter. Free oxygen is toxic to obligateanaerobic organisms, and the rising concentrations may have wiped out most of the Earth's anaerobic inhabitants at the time. Cyanobacteria were therefore responsible for one of the most significant extinction events in Earth's history. Additionally, the free oxygen reacted with atmospheric methane, a greenhouse gas, greatly reducing its concentration and triggering the Huronian glaciation, possibly the

longest snowball Earth episode in the Earth's history. Eventually, aerobic organisms began to evolve, consuming oxygen and bringing about an equilibrium.

Cyanobacteria

136

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. Bacillus infernus is able to live over 3.5 miles below the earth's surface without oxygen, and 900 C. ___2. Microorganisms are classified into seven classes based on their temperature ranges for growth. ___3. Mesophiles are organisms that grow at moderate temperatures. ___4. Microorganisms possess enzymes that afford protection against toxic oxygen products. ___5. Phototrophs may use visible light rays as an energy source.

MULTIPLE CHOICE QUESTIONS

1. Which are the microorganisms which do not require oxygen for growth but do grow better in its presence? a. a facultative anaerobic microorganism; b. a microaerophilic microorganism; c. an aerobic microorganism; d. an anaerobic microorganism.

2. Examples of microaerophilic bacteria are: a. Actinomyces israellii; b. Treponema pallidum; c. Bacteroides spp.; d. Fusobacterium spp.

3. Examples of anaerobic bacteria are: a. Clostridium pasteurianum; b. Bacteroides spp.; c. Enterococcus faecalis; d. Treponema pallidum.

CONCEPT QUESTIONS

What might be the habitat of an Describe an extremophil that can live aerotolerant anaerobe (facultative on Mars: anaerobe)?

137

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Construct a mnemonic with the following hyperthermophilic bacteria: Strain 121; Pyrolobus fumarii; Archaeoglobus fulgidus; Methanococcus jannaschii, Geothermobacterium ferrireducens.

COMPLETE THE FOLLOWING SENTENCES

 ______are killed or inhibited by oxygen.

 ______are examples of microaerophilic.

 ______grow best at a higher CO2 tension that is

normally present in the atmosphere.

QUOTE

Mark with X if you like or dislike this quote.

(1) ”Come forth into the light of things, Let Nature be your Teacher” (William Wordsworth). 1

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of the courses.

 (Q) Y'all want to hear a Potassium joke? (A) :K

 Did you hear the one about the recycling triplets? Their names are Polly, Ethel and Ian.

Explanation:

138

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. BordensteinSarah (2014). "Tardigrades (Water Bears)". Microbial Life Educational Resources. National Science Digital Library. Retrieved 2014-01-24. 2. CTI Reviews (2016). Microbiology for the Health Sciences: Biology, Microbiology, Cram101 Textbook Reviews. ISBN: 1478435771, 9781478435778. 3. Gupta Sujata (2010). "Biogas comes in from the cold". New Scientist. London: Sunita Harrington. p. 14. Retrieved 2011-02-04. 4. https://en.wikipedia.org/wiki/Watermelon_snow. 5. https://en.wikipedia.org/wiki/Thermophile. 6. https://simple.wikipedia.org/wiki/Anaerobic_organism. 7. https://quizlet.com/212385696/exam-2-flash-cards/. 8. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages. 9. Simon Matt (2014). "Absurd Creature of the Week: The Incredible Critter That's Tough Enough to Survive in the vacuum of Space". Wired. Retrieved 2014-03-21. 10. William Miller (2013). "Tardigrades". American Scientist. Retrieved 2013-12-02.

139

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 11 The influence of environmental factors on microbes (Part II)

11.1. Microbial interactions

Vibrio parahaemolyticus is a curved, rod-shaped, Gram- negativebacterium found in brackishsaltwater, which, when ingested, causes gastrointestinal illness in humans. This bacterium is motile, with a single, polar flagellum. While infection can occur by the fecal-oral route, ingestion of bacteria in raw or undercooked seafood, usually oysters, is the predominant cause of acute Vibrio parahaemolyticus gastroenteritis caused by V. nickname ”Seagull” parahaemolyticus.

Learning objective

Microbes live in direct proximity to other life forms, and these mixed populations give rise to interrelationships of elaborate and fascinating scope

Key points

Symbiosis, mutualism, synergism, commensalism, antagonism, parasitism

140

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

11.1. MICROBIAL INTERACTIONS

Some generalities that apply to such relationships are: 1. They may involve multicellular organisms such as animals and plants; 2. They may occur between microbes; 3. They can have beneficial, neutral, or harmful effects on the organisms involved.

Ecological interrelationships of microorganisms

Equal Unequal relationship relationship

Inter- Noninter- Neither member One member dependent dependent harmed, one harmed, one benefitted benefitted (commensalism) (antagonism, parasitism)

Microbial symbiosis can occur through interactions between host and microbial organisms and are beneficial to both parties that maintain a shared habitat or interchange nutrients.This kind of relationship is also called mutualism [25].

Microbial symbiosis in the gastrointestinal tract

Symbiotic microorganisms reside in the gastrointestinal tract of humans. Imbalances in the bacterial composition are called dysbiosis (e.g. Crohn's disease in humans). Bacteroides fragilis is a human symbiotic microorganisms that protect animals from experimental colitis induced by Helicobacter hepaticus. The gut is home to microbiome who is formed of healthy and unhealthy bacteria. The immune system's primary home is in the gut. A great diversity of symbiotic bacteria is necessary for animals to have fundamental nutrients, digest certain compounds, protect against outside pathogens, and create a healthy intestinal structure [24]. An equilibrium between symbionts and pathobionts is critical to fight against outside pathogens (e.g. inflammatory bowel disease). Probiotics increase intestinal health’ improve the immune system and prevent diseases like allergies, autoimmune disease, and cancer. Lactobacillus reuteri help us understand how changes in the gastrointestinal microbiome can promote health. Staphylococcus epidermidis is important for the skin microflora. It produces antimicrobial peptides (AMPs) that maintain an inflammatory homeostasis by reducing the release of extra cytokine and supports homeostasis and general health in areas such as oral cavity, gastrointestinal tract and oropharynx.

141

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Synergism

Is a cooperative relationship between organisms that is beneficial to both members, but not obligatory. Both organisms benefit from the relationship, but the association is not obligatory.

11.1.1. Neutralism

Represents a lack of interaction between two populations. It can occur if populations are far apart from each other. It can be found most frequently in very dilute populations where competition is minimal or when the organisms use different substrates and need no completion. Neutralism has been observed in a chemostat culture of Lactobacillus and Streptococcus yoghurt starter cultures [25]. The individual population sizes in the chemostat were found to be the same and the separate mono-cultures under the same conditions.

11.1.2. Amensalism

Amensalism is any relationship between organisms of different species in which one organism is inhibited or destroyed while the other organism remains unaffected. There are two types of amensalism: competition and antibiosis. The bread mold penicillium commonly grows on any bread that has passed its shelf life. This mold is capable of producing penicillin, which destroys many of the forms of bacteria that would also like to grow in the bread. The Penicillium does not benefit from the death of the other bacteria, making this an example of antibiosis amensalism [9].

11.1.3. Competition

An interactive association between two species both of which need some limited environmental factor for growth and thus grow at suboptimal rates because they must share the growth limiting resource [19]. Competition is the single most important interaction in nature. The competing populations can be stabilized by feedback control. Symbiosis: any relationship in which two or more individuals of different species coexist and mutually benefit each other. A prominent example is the relationship between the termite which cannot digest the wood (cellulose) it consumes and the intestinal resident protist that can digest the cellulose. The termite provides protection and food for the flagellated protist Trychonympha, while the protist digests the cellulose so that the termite can utilize the glucose produced for energy. In synergistic infectious, a combination of organisms (sometimes as many as 10 species) can produce tissue damage that a single organism could not cause alone. Gum disease, dental caries, tetanus, and gas gangrene all have a synergistic component.

11.1.4. Cooperation

A cooperative behavior is one that benefits an individual (the recipient) other than the one performingthe behavior (the actor). A cooperative interaction benefits a recipient, and is selected on that basis [28].

Table 11.1. Hamilton's classification of the four types of social behaviors

142

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Effect on recipient + - Effect + Mutual benefit Selfishness On - Altruism Spite actor

Based on Hamilton's definition, thereare four unique types of social interactions:  mutualism (+/+)  selfishness (+/-)  altruism (-/+)  spite (-/-)

11.1.5. Mutualism

Obligatory relationship between two populations that are beneficial for both of them. In mutualism, the interaction is necessary for survival. Symbiosis describes specific interactions that cannot be performed alone, but aren't necessary for survival. In microbial systems, this is most often seen as the production of public foods. Bacteria produce numerous factors that are released into the environment beyond the cell membrane. One very popular example of mutually beneficial microbial interactions involves the production of siderophores. This are iron-scavenging molecules produced by many microbial taxa including bacteria and fungi [22]. Iron is a major limiting factor for bacterial growth because most iron in the environment is in the insoluble Fe (III) form. In order for bacteria to access this limiting factor, cells will manufacture these enzymes, and then secrete them into the extracellular space. Once released, the siderophore will sequester the iron, making it metabolically accessible for the bacteria investigated the social nature of the production of siderophores in Pseudomonas aeruginosa [8; 21]. When cells were grown in pure culture, they were placed in an iron-limiting environment, populations of cells that secreted siderophores (wild-type) outcompeted a population of mutant non-secretors. Therefore, siderophore production is beneficial when iron is limiting. When the same population was placed in an iron-rich environment, the mutant population outcompeted wild-type population, demonstrating that siderophore production is metabolically costly [10]. When both wild type and mutant bacteria were placed in the same mixed population, the mutants can gain the benefit of siderophore production without paying the cost and hence increase in frequency. This concept is commonly referred to astragedy of the commons. This is a term originally used by Garret Hardin, to denote a situation where individuals acting independently and rationally according to each's self-interest behave contrary to the best interests of the whole group by depleting some common resource. ”Commons” in this sense has come to mean such resources as atmosphere, oceans, rivers, fish stocks, the office refrigerator, energy or any other shared resource which is not formally regulated; no common land in its agricultural sense [2]. In the bacteria Escherichia coli a Prisoner Dilemma situation can be observed when mutants exhibiting a Grow Advantage in Stationary Phase (GASP) phenotype complete with a wild type (WT) strain in batch culture. In such batch culture settings, where the growth environment is homogenized by shaking the cultures, WT cells cooperate by arresting bacterial growth in order to prevent ecological collapse while the GASP mutans continue to grow by defecting to the wild type regulatory

143

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

mechanism [27; 30]. Saccharomyces cerevisiae possesses multiple genes. Each produce invertase, an enzyme that is secreted to digest sucrose outside of the cell. This public good production creates the potential for individual cells to cheat by stealing the sugar digested by their neighbors without contributing with the enzyme themselves. Another example of mutualism is the relationship between bovines and bacteria within their intestines. The ungulates benefit from the cellulose produced by the bacteria, which facilitates digestion. The bacteria benefit from having a stable supply of nutrients in the host environment. Mutualistic interactions are vital for terrestrial ecosystem function as more than 48% of land plants rely on mycorrhizal relationships with fungi to provide them with inorganic compounds and trace elements [26]. Humans also engage in mutualisms with other species, including their gut flora without which they would not be able to digest food efficiently.

11.1.6. Quorum sensing (QS)

QS is a system of stimulate and response correlated to population density. One way that microbes communicate and organize with each other in order to partake in more cooperative interactions is through quorum sensing [3]. Quorum sensing describes the phenomenon in which the accumulation of signaling molecules in the surrounding environment enables a single cell to assess the number of individuals (cell density) so that the population as a whole can make a coordinated response. These bacteria also have a receptor that can specifically detect the signaling molecule. When the inducer binds the receptor, it activates transcription of certain genes, including those for inducer synthesis [6]. There is a low likelihood of a bacterium detecting its own secreted inducer.

Examples of bacteria who form quorum sensing

Figure 11.1. Aliivibrio ( Vibrio) fischeri( luciferase)

1. In many situations the cost bacterial cells pay in order to coordinate behaviors outweighs the benefits unless there is a sufficient number of collaborators. For instance, the bioluminescent luciferase produced by Aliivibrio (Vibrio) fischeri would not be visible if it was produced by a single cell [11]. It is a bioluminescent bacterium that lives as a mutualistic symbiont in the photophore (or light producing organ) of the Hawaiian bobtail squid.

2. The opportunistic bacteria Pseudomonas aeruginosa also uses quorum sensing to coordinate the formation of biofilms, swarming motility, exopolysaccharide production, virulence and cell aggregation [23].

144

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

These bacteria can grow within a host without harming it, until they reach a certain concentration. Then, they become aggressive, their number is sufficient to overcome the host's immune system, and form a biofilm, leading to disease within the host. Garlic and Ginseng experimentally block quorum sensing in Pseudomonas aeruginosa.

11.1.7. Parasitism or predation

Is any relationship in which one organism captures, subdues and feeds upon another organism. Predation is an interaction between organisms in which one benefits and one is harmed based on the ingestion of the smaller sized organism, the prey, by the larger organism, the predator. The end result is an oscillation between predator and the prey. Examples of this behavior are phagocytosis as in an amoeba capturing a bacterium or paramecium or an African Lion subduing a zebra. Parasitism is when the predator is smaller than the prey. A wide range of microbial groups contain parasitic members: viruses, bacteria and amoeba.

11.1.8. Bacterial intelligence

Is the intelligence shown by microorganisms. The concept encompasses complex adaptive behavior showed by single cells, and altruistic and/or cooperative behavior in populations of like or unlike cells mediated by chemical signaling that induces physiological or behavioral changes in cells and influences colony structures [5]. It has been suggested that a bacterial colony loosely mimics a biological neural network. The bacteria can take inputs in form of chemical signals, process them and then produce output chemicals to signal other bacteria in the colony [1].

Examples of microbial intelligence

 The formation of biofilms requires joint decision by whole colony.  Bacteria reorganize themselves under antibiotic stress.  Individual cells of myxobacteria and cellular slime molds coordinate to produce complex structures or move as multicellular entities.  Populations of bacteria use quorum sensing to judge their own densities and change their behavior accordingly. This occurs in the formation of biofilms, infectious disease processes and the light organs of bobtail squid [12].  Some strains of bacterium Escherichia coli are able to internalize themselves into a host's cell even without the presence of specific receptors as they bring their own receptor to which they then attach and enter the cell.  Under rough circumstances, some bacteria transform into endospores to resist heat and dehydration.  Individual cells of myxobacteria and cellular slime molds coordinate to produce complex structures or move as multicellular entities.  Bacteria reorganize themselves under antibiotic stress.

145

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

11.1.8.1. Social IQ score of bacteria

Is a recently proposed quantitative score devised as a comparative genomic tool to assess the genome potential of bacteria to conduct successful cooperative and adaptable behaviors (or social behaviors) in complex adverse environments. The IQ score of humans is supposed to reflect their mathematical, analytical and logical capabilities. Social intelligence is an individual's capacity to perceive and understand the environment from local surroundings to what is happening in the world-and to respond to that understanding in a personally and socially effective manner [7].

Feature 11.1. Bacteria's Social - IQ

The score is based on the number of genes which afford bacteria abilities to communicate and process environmental information (two-component system and transcription factor genes) to make decisions and to synthesize offensive (toxic) and defensive (neutralizing) agents as those needed during chemical warfare other microorganisms. Three bacteria species stand out with significantly high social-IQ score among all sequenced bacteria, indicating a capacity for exceptionally brilliant social skills. ”Ordinary” ”Gifted” ”Brilliant”

P. vortex P. curdlanolyticus P. larvae P.Y412MC10 P.polymiyxa

B. subtilis S. cellulosum P. JDR-2 M. xanthus P. denitriformis

M. tuberculosis E.coli B. anthracis

Pseudomonas Staphylococcus

Hitectures The Paenibacillus genus bacteria, to which the three smartest bacteria belong are known to be a rich source for industrial, agricultural and medical applications. P. vortex possesses advanced social motility employing cell-cell attractive and repulsive chemotactic signaling and physical links. When grown on soft surfaces P. vortex colonies behave much like a multicellular organism by the formation of foraging swarms that act as arms sent out in search for food.

These swarms have an aversion to crossing each other's trail and collectively

change direction when food is sensed. The ”swarming intelligence” P vortex, is further marked by the fact that of the swarms can even split and reunite when detecting scattered patches of nutrients.

The Social IQ provide a measure of the genome's capacity for social intelligence. The score is based on the number of genus that afford bacteria abilities to communicate and process environmental information to make decisions and to

synthesize offensive (toxic) and defensive (neutralizing) agents as needed during chemical warfare with other microorganisms.

146

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 11.1. Types of social interactions based on Hamilton's definition

Parasitism or predation

+ -

Commensalism + - 0 - Amensalism

Mutualism + + 0 0 - - Competition

Commensalism 0 + - 0 Amensalism - +

Parasitism or predation

+ Positive (win) - Negative (loss) 0 Neutral Species 1

Species 2

11.1.9. Commensalism

Association in which one organism is benefitted and the other organism is neither benefitted no harmed. An example is Saccharomyces cerevisiae releasing riboflavin for use by Lactobacillus casei growth. Another example is metabiosis process. It is used for the productions of the Japanese drink sake In commensalism one member (A) is neither harmed nor benefitted, yet A provides benefits to the other member (B). In this situation B is called the commensal of A. The word is derived from the ”commensal” meaning ”eating at the same table” in human social interactions.Pierre-Joseph van Beneden introduced the term ”Commensalism” in 1876.

Examples of commensalism:

 The remora which eats leftover food from a whale and ”hitches a ride”.  The relationship between Cacique birds and the wasp Polybia rejecta.  The best example is the vulture and the lion. Once the lion has finished its meal, the vulture swoops down and finished off the carcass. The lion is not affected by this while the vulture gets to eat [13]. A classic example of commensalism between microorganisms is satellitism, a phenomenon based on nutritional or protective factors. In nutritional satellitism, microbe A provides a growth factor that microbe B needs. For example: Staphylococcus aureus provides growth factors for Haemophilus influenzae, which grows as tiny satellite colonies near the streak of Staphylococcus. By itself, Haemophilus could not grow on blood agar. Bacterial culture of Haemophilus influenzae is performed on agar plates, the one preferred being chocolate agar with added X (hemin) and V (nicotinamide adenine dinucleotide)

147

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

0 factors at 37 C in a CO2 enriched incubator. Colonies of Haemophilus influenzae appear as convex, smooth, grey or transparent. Haemophilus influenzae grows in the hemolytic zone of Staphylococcus aureus on blood agar plates, the hemolysis of cells by S. aureus releases factor V which is needed for its growth. Haemophylus influenzae will not grow outside the hemolytic zone of S. aureus due to the lack of nutrients such as factor V in these areas. Fildes agar is the best for isolation [14].

11.1.10. Satelitism

Is the phenomenaby which certain bacterial species grow more vigorously in the immediate vicinity of colonies of other unrelated species. This happens a result of the production of an essential metabolite by the latter species [15].

Figure 11.2. Haemophilus influenzae and Staphylococcus aureus – satellitism

Staphylococcus aureus

Haemophilus influenzae

11.1.11. Selfishness

1. Soil bacteria are extremely resistant to antimicrobials in more ways than scientists can count. Yet, for some reason, these bacteria have refused to share these defensive traits with other more dangerous bacteria, or even one another. Scientists claim that understanding why this occurs may be an important step in understanding how to prevent the world's next ”superbug” from ever evolving [16]. Superbug is strain of bacteria that is resistant to one or more antibiotics that would normally treat the bacteria. Among some of the more common superbugs are methicillin- resistant Staph aureus (MRSA) and multiple-drug or extensively drug resistant tuberculosis (MDR-TB and XDR-TB). 2. ”Selfish” bacteria link IBD (Inflammatory Bowel Disease) and gut microbiota changes. IBD affects 1 in every 250 people in the UK, but its causes are unknown.

148

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Studies have shown that IBD patients have a different profile of gut microbes, which is called dysbiosis [20].

11.1.12. Altruism – ”Charitable” behavior found in bacteria”

Altruistic behavior (AB) is most common between individuals with high genetic relatedness, though this it is not completely necessary. AB can also be evolutionarily beneficial if the cooperation is directed towards individuals who share the gene of interest, regardless of whether this is due to coancestry or some other mechanism [29]. Programmed cell death (PCD) (also known as apoptosis or autolysis). Several altruistic possibilities have been suggested for PCD, such as providing resources that could be used by other cells for growth and survival in Saccharomyces cerevisiae. Researchers of Boston University have discover ”charitable” behaviour in bacteria populations, where individuals with the highest antibiotic resistance sacrifice so the whole population can better fight off medication. This bacterial altruism results where the most resistant isolates produce a small molecule called indole [17]. Indole acts something of a steroid, helping the strain's more vulnerable members bulk up enough to fight off the antibiotic onslaught. But while indole may save the grow, its production takes a toll on the fitness level of the individual isolates that produce it. Some strains of Escherichia coli harbor genes that trigger cell death upon infection by bacteriophage T4. These may provide examples of the evolution of altruistic behavior in bacteria[4].

11.1.13. Antagonism

In this unequal interaction, one microbe secretes antimicrobial chemicals that inhibit or destroy another microbe in the same habitat. This gives the first microbe a competitive advantage.

149

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 11.3. Microbial antagonism

An example of microbial antagonism in the human body is the resistance of established mouth bacteria to new strains that can be introduced via mouth-to- mouth contact. After a kiss, for example, new bacteria are introduced into the hostile [18] environment of a foreign mouth . Once there, the invasive bacteria's growth is inhibited by antimicrobial compounds secreted by the native flora, as well as by the fight competition for resources.

The most prominent examples are seen in the antibiosis (production of antibiotics) by fungi and bacteria, and in the production of bacteriocins by bacteria:  Antibiosis is an association between two or more organisms that is detrimental to

at least one of them.  Antibiosis is the antagonistic association between an organism and the metabolic substances produced by another. Many antimicrobial agents affect more than one

cellular target and may infect both primary and secondary damages that lead to cell death.

Agents can be classified in following groups: (1) Agents that are less selective in their scope of destructiveness severe damage on many cell pacts, and are generally very biocidal (heat, radiation, some

disinfectants); (2) Moderately selective agents with intermediate specificity (certain disinfectants and antiseptics);

(3) More selective agents (drugs) whose target is usually limited to specific cell structure or function and whose effectiveness is restricted only to certain microbes.

150

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. ”Superbug” is a strain of bacteria that is resistant to one or more antibiotics that would normally treat the bacteria. ___2. In Levinthal medium, capsulated strains show distinctive iridescence. ___3. The bioluminescent luciferase is produced by Pseudomonas aeruginosa. ___4. An example of commensalism is L. lactis who produced the milk fermentation. ___5. Based on Hamilton's definition there are five unique types of social interactions.

MULTIPLE CHOICE QUESTIONS

1. Obligatory relationship between two populations that benefits both populations is: a. mutualism; b. neutralism; c. amensalism; d. commensalism.

2. Association in which one organism is benefitted, and the other organism is neither benefitted no harmed is represented by: a. commensalism; b. competition; c. parasitism; d. mutualism.

CONCEPT QUESTIONS

What is symbiosis? Give some examples Describe a cooperative behavior based on of symbiotic relationships: Hamilton's classification:

Explain what is mutualism. Give some What is Quorum sensing? Give some examples: examples:

151

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Give some example of bacterial What is satellitism? Explain this intelligence. Who is the smartest bacteria in phenomenon: the world?

COMPLETE THE FOLLOWING SENTENCES

 ______is the intelligence shown by microorganisms.

 Individual cells of ______and ______coordinate to produce complex

structures or move as multicellular entities.

 ______is the association in which one organism

is benefitted, and the other organism is neither benefitted nor harmed.

QUOTE

Mark with X if you like or dislike this quote.

(1) ”A school teacher or professor cannot educate individuals, s (he) educates only species. A thought that deserves taking to heart” (George Lichtenberg).

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of the courses.

 Over 40,000 parasites and 250 bacteria are shared in a typical French kiss.  Two thirds of the World is made up of water, yet 80% of that water is undrinkable, due to Bacteria build up and salt water. Only 18% of the water on the Earth is used for drinking, in the continents of Europe, North America, South America, Asia, Antarctica and Oceania, the other 2% used commercially for reasons such as car washes and window cleaning usage. Unlucky Africa!

Explanation:

152

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Cohen Inon, et al. (1999). "Continuous and discrete models of cooperation in complex bacterial colonies" (PDF). Fractals. 7.03 (1999):: 235–247. 2. Charles Anukwonke (2015). The Concept of Tragedy of the Commons: Issues and Applications. Research. DOI: 10.13140/RG.2.1.4977.9362. 3. Czaran T., Hoekstra R.F. (2009). Microbial Communication, Cooperation and Cheating: Quorum Sensing Drives the Evolution of Cooperation in Bacteria. PLoS One 4:6655. 4. David A. Shub (1994). Bacterial Viruses: Bacterial altruism? Volume 4, Issue 6, p555–556, June 1994. Current Biology. DOI: http://dx.doi.org/10.1016/S0960-9822(00)00124- X. 5. Ford, Brian J. (2004). "Are Cells Ingenious?" (PDF). Microscope. 52 (3/4): 135–144. 6. G. N. Cohen (2014). Microbial Biochemistry, Springer. ISBN: 9401789088, 9789401789080. 7. Gordon Richard, Seckbach Joseph (2016). Biocommunication: Sign-mediated Interactions Between Cells And Organisms, Vol. 1 din Astrobiology : exploring life on earth and beyond. World Scientific. ISBN: 1786340461, 9781786340467. 8. Griffin AS, West SA, Buckling A. (2004). Cooperation and competition in pathogenic bacteria. Nature 430:1024–27. 9. https://study.com/academy/lesson/amensalism-examples-definition-quiz.html. 10. https://en.wikipedia.org/wiki/Microbial_cooperation. 11. https://www7.dict.cc/wp_examples.php?lp_id=1&lang=en&s=luciferase. 12. https://en.wikipedia.org/wiki/Microbial_intelligence. 13. https://quizlet.com/95120149/parasitism-and-other-relationships-flash-cards/. 14. https://en.wikipedia.org/wiki/Haemophilus_influenzae. 15. https://quizlet.com/19638115/microbiology-vocabulary-chap-7-flash-cards/. 16. http://www.natureworldnews.com/articles/7196/20140522/selfishness-soil-bacteria- saving-catastrophe.htm. 17. https://wyss.harvard.edu/new-research-finds-evidence-of-charitable-behavior-in- bacteria/. 18. https://www.reference.com/science/microbial-antagonism-a21dd5830ad9ae0c. 19. Lawrence K. Wang, Volodymyr Ivanov, Joo-Hwa Tay, Yung-Tse Hung (2010). Environmental Biotechnology, Vol. 10 din Handbook of Environmental Engineering. Springer Science & Business Media. ISBN: 1603271406, 9781603271400. 20. Louise E. Tailford, C. David Owen, John Walshaw, Emmanuelle H. Crost, Jemma Hardy-Goddard, Gwenaelle Le Gall, Willem M. de Vos, Garry L. Taylor & Nathalie Juge (2015). “Discovery of intramolecular trans-sialidases in human gut microbiota suggests novel mechanisms of mucosal adaptation.” Nature Communications. 8 July 2015 doi: 10.1038/ncomms7624. 21. Miethke, M., Marahiel M. A. (2007). Siderophore-Based Iron Acquisition and Pathogen Control. Microbiol. Mol. Biol. Rev. 71:413-451. doi: 10.1128/MMBR.00012-07. 22. Neilands JB. Siderophores (1995). Structure and function of microbial iron transport compounds. J. Biol. Chem. 270:26723–6. 7. doi: 10.1074/jbc.270.45.26723.

153

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

23. Qing Wei, Luyan Z. Ma (2013) . Biofilm Matrix and Its Regulation in Pseudomonas aeruginosa. Int J Mol Sci. 2013 Oct; 14(10): 20983–21005. Published online 2013 Oct 18. doi: 10.3390/ijms141020983. 24. Round June L., and Sarkis K. Mazmanian (2009). "The gut microbiota shapes intestinal immune responses during health and disease." Nature Reviews Immunology 9, no. 5 (2009): 313-323. 25. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages. 26. Thompson, J. N. (2005). The geographic mosaic of coevolution. Chicago, IL: University of Chicago Press. 27. Vulic M, Kolter R. (2001). Evolutionary Cheating in Escherichia coli Stationary Phase Cultures. Genetics 158: 519–526 . 28. West SA, Griffin AS, Gardner A. (2007). Social semantics: altruism, cooperation, mutualism, strong reciprocity and group selection. Eur. Soc. for Evol. Biol. 20:415–432. 29. West SA, et al. (2006). Social evolution theory for microbes. Nat. Rev. Microbiol. 4:597–607. 30. Zinser E., Kolter R. (2004).Escherichia coli evolution during stationary phase. Res. Microbiol. 155:328–336.

154

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 12 An introduction to the viruses

12.1. History 12.2. Structure and composition 12.3. Virus replication 12.4. Effect of virus infection on cells 12.5. Medical importance of viruses 12.6. Epidemiology

Yellow fever, known historically as yellow jack, yellow plague, or bronze john, is an acuteviral disease. The disease is caused by the yellow fever virus and is spread by the bite of the female mosquito. It infects only humans, other primates, and several species of mosquitoes. In cities, it is spread primarily by mosquitoes of the Aedes aegypti species. The virus is an RNA virus of Yellow Fever Virus the genus Flavivirus. nickname ”Yellow Jack”

Learning objective

Viruses exist that infect other living cells Animal and plant viruses vary greatly in size and shape

Key points

Virions range in size from 20 to 350 nm and represent the smallest and simplest infectious agents

155

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

12.1. HISTORY

Definition (by Michael J. Pelczar, Jr., et al., 1986) referred to viruses are noncellular infectious entities whose genomes are a nucleic acid, either DNA or RNA, which reproduce only in living cells; and which use the cells biosynthetic machinery to direct the synthesis of specialized particles (virions), which contain the viral genomes and transfer them efficiently to other cells. Definition (by Wikipedia) - a virus is a small infectious agent that replicates only inside the living cells of other organisms. The word is from the Latin virus referring to poison, first attested in English in 1398 in John Trevisa's translation of Bartholomeus Angliens's De Proprietatibus Rerum. Virulent comes from Latin virulentus (poisonous). The English plural is viruses. The term virion (plural virions) dates from 1959 and is used to refer to stable infective viral particle that is fully capable of infecting other cells of the same type. Viruses are infections agents so small that they can only be seen at magnifications provided by the electron microscope [36]. They are 10 to 100 times smaller than most bacteria, with an approximate size range of 20 to 300 nm. Thus they pass through the pores of filters which do not permit the passage of most bacteria.

Figure 12.1. Tobacco mosaic virus

The first substantial revelations about the unique characteristics of viruses occurred in 1892, when D. Ivanovski isolated the tobacco mosaic virus in a culture dish. Tobacco mosaic virus was precipitated from a suspension by using ethyl alcohol and would still remain infective, while the same treatment destroyed bacteria and other cells.

In 1935 tobacco mosaic virus was crystallized by Dr. Wendell Stanley of the University of California. Crystals are formed only by purified chemical compounds and not by cells or by mixtures of dissimilar molecules. Early in the twentieth century F.W.Twort in England and F. d'Hérelle in France, working in independently, demonstrated the existence of filterable agents which infected and destroyed bacteria. These bacterial viruses become known as bacteriophages, or simply phages. In 1898, Friedrich Loeffler and Paul Frosch isolated the virus that causes foot-and-mouth disease in cattle. Foot-and-mouth disease (FMD) has severe implications for animal farming, since it is highly infectious and can be spread by infected animals through aerosols, through contact with contaminated farming equipment, vehicles, clothing, or feed, and by domestic and wild predators[3]. By the 1950's, virology had grown into a multifaceted discipline that promised to provide much information on disease, genetics and even life itself.

156

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

The main characteristics of viruses

1. Viruses are incapable of independent growth in artificial media. They can grow only in animalor plant cells or in microorganisms. They reproduce in these cells by replication. 2. Viruses are obligate intracellular parasites. If the least requirement for life is that an organism duplicates itself, then viruses may be viewed as microorganisms. 3. Viruses lack a metabolic machinery of their own to generate energy or to synthesize proteins. Viruses have information in their genes for usurping the host cell's energy-generating and protein-synthesizing systems. 4. Viruses are small packets of genes. The viral genetic material is either DNA or RNA, but the virus lacks both (host cells have both DNA and RNA). 5. The nucleic acid is enclosed in a highly-specialized protein coat of varying design. 6. The coat of the viruses protects the genetic material when the virus is outside of any host cell. It serves as a vehicle for entry into another specific host cell. The structurally complete mature and infections virus is called virion. 7. During reproduction in the host cells viruses may cause diseases. 8. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archea. 9. Viral infections in animals provoke an immune responsesthat usually eliminates the infecting virus. Some viruses (e.g. AIDS, viral hepatitis) evade these immune responses and result in chronic infections. 10. Antibiotics have no effect on viruses, only several antiviral drugs that don't destroy their target (viruses) but inhibit their development. They are one class of antimicrobials based on monoclonal antibodies. Antivirals can be found in essential oils of some herbs, such as eucalyptus oil.

History

1884 – the French microbiologist Charles Chamberland invented a filter with pores smaller than bacteria. 1892 – the Russian biologist Dmitri Ivanovsky used this filter to study tobacco mosaic virus. 1898 – the Dutch microbiologist Martinus Beijerinck repeated the experiments and become convinced that the filtered solution contained a new form of infectious agent that he called “contagium vivum fluidum” [6]. Beijerink was convinced that the viruses were liquid in nature. The theory was later discredited by Wendell Stanley, who proved that they were particles. 1898 – Friedrich Loeffler and Paul Frosch passed the first animal virus-agent of foot-and- mouth disease (aphthovirus) through a similar filter. By the end of the 19th century, viruses were defined in terms of their infectivity, their ability to be filtered and their requirements for living hosts [37]. In the early 20 century – the English bacteriologist Frederich Twort discovered a group of viruses that infect bacteria, now called bacteriophages (phages). The French-Canadian microbiologist Felix d'Hérelle described viruses that, when added to bacteria on agar, would produce areas of dead bacteria. Phages were heralded as a potential treatment for diseases such as typhoid and cholera [6]. In 1908 Ross Grouville Harrison invented a method for growing tissue in lymph.

157

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

In 1913 E. Steinhart, C. Israeli and R.A. Lambert uses this method to grow vaccinia virus in fragments of corneal tissue of guinea pigs . In 1928 N.B. Maitland and M.C. Maitland grew vaccinia virus in suspensions of minced hens' kidneys. In 1950s their method was used for vaccine production of poliovirus on a large scale. In 1931 – the American pathologist Ernest William Goodpasture grew influenza and several other viruses in fertilized chickens' eggs. In 1949, John Franklin Enders, Thomas Weller and Frederick Robbins grew poliovirus in cultured human embryo cells. Jonas Salk made an effective polio vaccine. In 1931 the German engineers Ernst Ruska and Max Knoll invented electron microscopy. In 1935, the American biochemist and virologist, Wendell Meredith Stanley examined the tobacco mosaic virus and found that it was mostly made of protein. In 1941, Bernal and Fankuchen obtained the first X-ray diffraction pictures of the crystallized virus and in 1955 Rosalind Franklin discovered the full structure of the virus. In the same year,Heinz Fraenkel-Conrat and Robley Williams showed that purified tobacco mosaic virus RNA and its protein coat can assemble by themselves to form functional viruses [16]. The second half of the 20th century was the golden age of virus discovery and most of over 2,000 recognized species of animal, plant, and bacterial viruses were discovered during these years. In 1957 equine arterivirus and the cause of Bovine Virus diarrhea (a pestivirus) were discovered. In 1963 the hepatitis B virus was discovered by Baruch Blumberg. In 1965, Howard Termin described the first retrovirus. In 1990 Howard Martin Temin and David Baltimore described reverse transcriptase the enzyme that retroviruses use to make DNA copies of their RNA. In 1983 Luc Montagnier's team isolated the retrovirus HIV.

The position of viruses in the biological world

The exceptional and curious nature of viruses spurs numerous questions:

1. Are they organisms – that is, are they alive? 2. What are their distinctive biological characteristics? 3. How can particles so small, simple and seemingly insignificant be capable of causing disease and death? 4. What is the connection between viruses and cancer?

The structure and behavior of viruses have invariably led to debates about their position in the microbial world. The viruses have been described as ”organisms at the edge of life”. Although they have genes, they do not have a cellular structure and their own metabolism and cannot naturally reproduce outside a host cell [36]. In keeping with their special position in the biological spectrum, it is best to describe viruses as infectious particles (rather than organisms) and as either active or inactive (rather than living or dead).

Paleovirologists affirm that are three main hypothesis that aim to explain the origins of viruses:

158

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

1. Regressive hypothesis (also called the degeneracy hypothesis or reduction hypothesis). This theory states that viruses may have once been small cells that parasitized larger cells and over time, genes not required by their parasitism were lost. 2. Cellular origin hypothesis claims that some viruses may have evolved from bits of DNA or RNA plasmids that ”escaped” from the genes of a larger organismor transposons [6]. 3. Coevolution hypothesis (also called the virus first hypothesis) proposes that viruses may have evolved from complex molecules of protein and nucleic acid at the same time as cells first appeared on earth. Viroids are molecules of RNA that are not classified as viruses because they lack a protein coat [7].

Viral architecture

Is observed through special stains in combination with electron microscopy:

1. Negative staining uses very thin layers of an opaque salt to outline the shape of the virus against a dark background and to enhance textural features on the viral surface; often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. 2. Electron dense ”stains”: these are solutions of heavy metals salts, such as tungsten (wolfram, meaning heavy stone), that scatter the electrons from regions covered with the stain.

12.2. STRUCTURE AND COMPOSITION

Capsid

Coverning Envelope (not found in all viruses)

Virus particle Nucleic acid molecule Central core (DNA or RNA) Various proteins (enzymes)

A complete virus particle, known as a virion consists of nucleic acid surrounded by a protective coat of protein called a capsid or shell. The capsid and the nucleic acid strand taken as a whole are referred to as the nucleocapsid. In some animal viruses the nucleocapsid (nucleic acid and capsid) is covered by an outer membrane like structure called the envelope, which is made of lipoproteins. Virions, that have envelops are sensitive to lipid solvents such as ether and chloroform. Nonenveloped viruses are referred to as naked virions.

The viral capsid

The capsid and the entire structure of the virus can be mechanically probed through atomic force microscopy. The capsomeres consist of a number of protein subunits or molecules called protomers. When a virus particle is magnified several hundred thousand times, the capsid is a prominent feature, often having the exact and geometric perfection of a crystal. In general, each capsid is constructed from smaller, identical building blocks called capsomeres.

159

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Each capsomer is a cluster of identical protein molecules with a configuration that allows it to interlock with other capsomers, depending on how the capsomers are shaped and arranged. This binding results in two different capsid types: helical and icosahedral.

Figure 12.2. Viral capsid

genome glycoprotein I

capsid glycoprotein III

coat

Helical capsids

Have the simpler structure. These viruses are composed of a single type of capsomer stacked around a central axis to form a helical structure, which may have a central cavity, or tube (Collier, p. 37; Common Viral Infections, Pedia Press). In electron micrographs the appearance of the helical capsid varies according to the type of virus. The nucleocapsids or naked helical viruses are very rigid and tight, thereby creating a gross cylindrical appearance. Plant viruses with helical symmetry are typically rod-shaped. One of the first viruses studied by electron microscopy was the tobacco mosaic virus. Its nucleic acid core is covered by a capsid consisting of closely packed capsomeres arranged in a regular helix. Animal viruses with capsids displaying helical symmetry include measles, mumps, influenza and rabies. In these viruses, the nucleocapsid is a flexible structure packed within a fringed lipoprotein envelope.

160

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

12.3. Helical capsid

Coat protein subunit

capsid

DNA

Icosahedral capsids (symmetry)

The capsids are arranged in a three-dimensional, many-sided figure generally known as a polyhedron. The minimum number of identical capsomers required is twelve, each composed of five identical sub-units [39]. An icosahedral virus is made of triangular capsomers that are very uniform and symmetrical and can become attached to one another along their three sides is results, according to the rules of geometry and crystallography, a hollow icosahedral shell. The icosahedrons are regular polyhedron with 20 triangular facets and 12 vertices. This means that the capsid has 20 facets, each of thembeing an equilateral triangle. These facets come together and form 12 corners. In the simplest capsid, there is a capsomere at each of the 12 vertices [2]. Hexons are in essence flat.Pentons, which form the 12 vertices, are curved. The same protein may act as the subunit of both the pentamers and hexamers or they may be composed of different proteins [39]. Some bacteriophages, such as the T-even coliphages (T2, T4 and T6) have very complex structures, including a head and a tail. They are said to have binal symmetry because each virion has both an icosahedral head and a hollow helical tail [38]. For example: the phage phi X174 exhibits the simplest capsid. In larger and more complex capsids, the triangular facets are subdivided into a progressively larger number of equilateral triangles. Thus, a capsid may be composed of hundreds of capsomeres, but it is still based on the simple icosahedrons model [29].

161

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 12.1. – R. Buckminster Fuller – The domes

R. Buckminster Fuller an American architect, engineer and inventor, discovered that an icosahedral shell is easy to assemble and provides on enclosure possessing minimum stress [11]. This is the idea behind Fuller's geodesic domes, the design of which he patented in 1947. These domes usually look spherical and cover more space with less material than any other buildings ever designed. These domes are actually subtriangulated icosahedra constructed of almost identical triangular units in clusters of fives and sixes [13].

Spherical viruses have in reality icosahedral symmetry. In photomicrographs the capsid may appear spherical or cubical and individual capsomers may look either ring-or dome-shaped. Viruses of the common cold and polio (picornoviruses) have naked icosahedral nucleocapsids. Examples of icosahedral viruses are polioviruses (picornaviruses) and adenoviruses which cause poliomyelitis and respiratory infections.

Figure 12.4. (1) Helical, (2) Spherical and (3) Complex (Bacteriophage)

3 1

162

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 12.5. Polyhedral (Adenovirus)

spikes

capsid DNA

Viral envelope

The envelopes are derived from portions of the host cell membranes (phospholipids and proteins), but include some viral glycoproteins. During development inside the host cell, enveloped viruses replace some or all of the regular membrane proteins with special viral proteins [17]. Some proteins form a binding layer between the envelope and the capsid of the virus, and others (glycoproteins) remain exposed on the outside of the envelope. These protruding molecules, called spikes function by attaching themselves to another host cell, or by invading it. The lipid bilayer envelope of this viruses is sensitive to desiccation, heat and detergents. Enveloped viruses possess great adaptability and can change in a short time in order to exude the immune system. Enveloped viruses can cause persistent infections (infectious disease, also known as transmissible disease or communicable disease, is a illness resulting from an infection. Because the envelope is supple, this kind of viruses may take on shapes ranging from a sphere to a bullet or a filament. Examples of enveloped viruses: Influenza virus and HIV use this strategy.

163

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 12.6. Inluenza virus (1) genome, (2) envelope, (3) capsid, (4) spikes

3 2 2 1 4 4

Envelopes are acquired from the membrane of the host cell

Complex viruses

While some viruses have symmetrical shapes, another have asymmetrical structures. They are called ”complex viruses”. These viruses possess a capsid that is neither purely helical nor purely icosahedral, and may possess extra structures such as protein tails or a complex outer walls [33]. 1. The poxviruses (including the agent of smallpox) are typical in that they contain a nucleic acid core, but they lack a regular capsid and in its place have several layers of lipoproteins and coarse fibrils. 2. Large bacteriophages (such as Enterobacteriaphage T4) which are viruses, that parasitize bacteria, have a complex structure with a cubical head, a helical tail, and fibers for attaching themselves to the host cell and injecting the viral genome. T4 bacteriophage infects Escherichia coli. It has an icosahedral head, its tail makes it asymmetrical, or ”complex” in terms of structure. The viral genome's of poxviruses is associated with proteins within a central disk structure known as a nucleoid, which is surrounded by a membrane and two lateral bodies of unknown function [39]. The virus has an outer envelope with a thick layer of protein. The whole virion is slightly pleiomorphic, ranging from ovoid to brick shape. Mimivirus is the largest characterized virus, with a capsid diameter of 400 nm. Protein filaments measure 100 nm. The capsid appears hexagonal but, isin reality is probably icosahedral[18]. In 2011, researchers discovered a larger virus on the ocean floor of the coast of Las Cruces, Chile. This virus is called Megavirus chilensis.

164

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

In 2013, the Pandoravirus genus was discovered in Chile and Australia, and has genomes about twice as large as Megavirus and Mimivirus. Some viruses infect Archaea and have complex structures unrelated to any other form of viruses. These include a wide variety of usual shapes, ranging from spindle-shaped structures, to viruses that resemble hooked rods, teardrop or even bottles [31].

RESEARCH NEWS

Viral life cycle could be a target for drug development

Dengue and Zika Virus are both positive strand RNA flaviviruses, which

means that once a virus particle infects a cell, its RNA genome can be

immediately translated by cellular machinery into viral proteins to make new

virus particles and spread the infection.

Dengue Virus block the initiation of cellular translation. This step is when the

ribosome machinery, which reads the RNA genetic code and converts it into an

aminoacid protein chain, attaches onto the start of the RNA transcript. This virus

does not block the two known pathways that suppress translation initiation for

cellular RNAs, but how this occurs is stil a mystery.

Zika Virus also follows the some pattern of cellular behavior of repressing the

cell's translation and stress

response while promoting its own

protein translation. In this way,

the flaviviruses clamp down on

the cell's responses to thwart

infection, including deploying

antiviral proteins.

"How the virus escapes

global translation repression by

switching on its own translation

is a crucial step in the viral life

cycle that could be a target for

drug development" (Alessia

Ruggieri et. all, University of

Heidelberg, Germany).

Dengue virus can cause febrile illness, hemorrhagic fever, and even death,

and infects an estimated 390 million people worldwide each year.

Zika Virus can also cause fevers and has been linked to severe neurological

birth defects in newborns born to infected mothers.

165

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Nucleic acid

The viral genome, containing all the genetic information,is composed of nucleic acids. The genome of higher organisms consists of double-stranded DNA (dsDNA), but the genome of a virus can consist of DNA or RNA that is either double-stranded or single-stranded. All four types of genome have been found in bacterial, animal and plant viruses. There are millions of different viruses, although only about 5,000 of them have been described in detail. The vast majority of viruses have RNA genomes. The content of genetic information per virion varies from about 3 to 300 kilobases per strand of nucleic acid. Virions contain only a single copy of nucleic acid and they are haploid. Retroviruses are an exception. They are diploid virions because they contain two identical single-stranded RNA genomes [10]. Plant viruses tend to have single-stranded RNA genome and bacteriophages tend to have double-stranded DNA genomes.The structure of the nucleic acid in the virion may be either linear or circular. The DNA of most animal viruses is a linear molecule of either dsDNA or ssDNA. The RNA in animal viruses exists only as linear double-stranded or single-stranded molecules.The RNA genome, unlike the DNA genome, may exist as a segmented genome (divided into several units). Single-stranded viral RNA molecules which function as mRNA in the host cells has been designated as positive or (+) strands [28]. Viruses with negative, or minus-strand (-) RNA molecules must first replicate their RNA (using RNA transcriptase carried within the virion) to form a complementary strand which then acts as the mRNA.RNA tumor viruses have two equal positive strand RNA molecules.

Examples: Polyomoviruses have circular viral genomes. Adenoviruses have linear genomes. Examples of such viruses in which all segments are not required to be in the some virion for the virus to be infections.

Brome Mosaic Virus (BMV) infects Bromus inermis - the genome is divided into separate parts and it is called segmented. Hepadnaviridae contains a genome that is partially double-stranded and partially single- stranded [19]. Several types of ssDNA and ssRNA viruses have genomes that are ambisense, meaning that transcription can occur on both strands in a double-stranded replicative intermediate. Example of such viruses are geminiviruses (ssDNA plant viruses) and arenoviruses (ssRNA viruses of animals). The smallest viral genomes have the ssDNA circoviruses (family Circoviridae) code for only two proteins and have a genome size of only two kilobases (1 kilobase is considered the size of an average gene). The largest virus – pandoravirus – have genome sizes of about two megabases which code for about 2500 proteins [30].

166

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Table 12.1. Genome

Nucleic Acid RNA DNA Virus single-stranded Double stranded S5 ds

Nucleic Acid DNA RNA Both DNA and RNA (at different stages in the life cycle) Shape Linear Circular Segmented Strandedness Single-stranded Double-stranded Double-stranded with regions of single-strandedness Sense Positive sense (+) Negative sense (-) Ambisense (+/-)

Other chemical components in the virus particle.

Proteins

Proteins are found in the structure of the capsid, virions also contain internal proteins; some are basic proteins bound to the nucleic acid. In papovaviruses these basic proteins are regular cellular histones. In adenoviruses they are histonelike, but are specified by the viruses. Viruses may contain enzymes for specific operations within their host cell.The most common viral enzyme is an RNA polymerase. This transcribes the viral RNA, allowing viral replication to proceed. The RNA tumor viruses contain an enzyme (RNA-dependent DNA polymerase, or reverse transcriptase) that synthesized a DNA strand using the viral RNA genome as a template[22]. Phages contain small peptides and polyamines.

Lipids (phospholipids)

A wide variety of lipid (fatty) compounds have been found in viruses. Examples: Phospholipids, glycolipids, neutral fats, fatty acids, fatty aldehydes, and cholesterol. The predominant lipid substance is the phospholipid, and is found in the viral envelope. Some enveloped animal viruses have spikes made of glycoprotein on the envelope (for example influenza virus and other myxoviruses). All viruses contain carbohydrate. Nucleic acid itself contains ribose or deoxyribose [29].

12.3. VIRUS REPLICATION (MODES OF VIRAL MULTIPLICATION)

The process of viral multiplication is an extraordinary biological phenomena. Multiplication takes place by replication, in which the viral protein and nucleic acid components are reproduced within susceptible host cells. Viruses are minute parasites that appropriate the synthetic and genetic machinery of cells.

167

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

The steps of virus infection and replication are therefore:

(1) absorption that consists in virus attachment to the external surface of the cell; (2) penetration and component uncoating entrance of the virion into the host cell; (3) replication and biosynthesis expression of the viral genome at the expense of the host's synthetic equipment, resulting in the production of the various virus components; (4) maturation, assembly of these individual viral parts into whole, intact virions; (5) release, escape from the host cell of the active, infectious viral particles.

(1) Adsorption (Attachment)

The processoccurs in two steps:

 The first step – involves preliminary attachment by ionic bonds or charges and is easily reversed by a shift in pH or salt concentration.  The second step – appears to involve firmer, more specific attachment and is irreversible. For example: HIV infects a limited range of human leucocytes. This is because its surface protein, gp 120, specifically interacts with the CD4 molecule – a chemokine receptor – [24] which is most commonly found on the surface of CD4+T-cells .

(2) Penetration/Uncoating of animal viruses

Animal viruses exhibit some impressive mechanism for entering a host cell.Virions enter the host cell through receptor-mediated endocytosis or membrane fusion. This is often called viral entry. Instead, the flexible cell membrane of the host is induced to admit the viruses or its nucleic acid by one of the following means:  In the case of endocytosis, the entire virus is engulfed by the cell and enclosed in a vacuole or vesicle. One mechanism consists of engulfment of whole virions by the cells in a phagocytic process called viropexix followed by uncoating or removal of the capsid. This takes place in the phagocytic vacuoles and is due to the action of enzymes called lysosomal proteases. When enzymes in the vacuole dissolve its coating (capsid, envelope) the virus is said to be uncoated, a process that releases the viral nucleic acid into the cytoplasm.  By direct fusion of certain enveloped viruses with the cell membrane of the host cell. This fusion results in the release of the viralnucleocapsid material into the cytoplasm of the host cell. Examples: influenza virus and mumps viruses.

(3) Replication and biosynthesis of virus-specific molecules

Shortly after penetration it, follows an interval of time called the latent period. The viral nucleic acid is released from the capsid and is accessible to the enzyme required to translate, transcribe, or replicate it. Some viruses are uncoated in the cytoplasm,while others are uncoated in the nucleus. Replication involves synthesis of viral messenger RNA (mRNA from ”early” genes viral protein synthesis possible assembly of viral proteins, then viral genome replication mediated by early or regulatory protein expression.

168

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

(4) Assembly and Release of mature viruses

When a critical number of various viral components has been synthesized, the components (virus specific molecules) are assembled into mature virus particles in the nucleus (the DNA viruses with the exception of poxviruses) and/or cytoplasm (the RNA viruses such as poxviruses) of the infected cell [15].

(5) Release

Release of completed virions from the host cell is the final step in virus multiplication. Viruses can be released from the host cell by lysis, a process that kills the cell by bursting its membrane and cell wall if present. Some viruses undergo a lysogenic cycle where the viral genome is incorporated by genetic recombination into a specific place in the host's chromosome when integrated in a host cell this way, the viral genome is known as a ”provirus” or, in case of bacteriophages as a ”prophage”. Enveloped viruses (e.g. HIV) are released from the host cell by budding.

Reverse transcribing viruses

With RNA genomes (retroviruses) use a DNA intermediate to replicate whereas those with DNA genomes (pararetroviruses) use an RNA intermediate during genome replication. Both types use a reverse transcriptase, or RNA-dependent DNA polymerase enzyme, to carry out the nucleic acid conversion. They are susceptible to antiviral drugs that inhibit the reverse transcriptase enzyme (e.g.: zidovudine and lamivudine) [34; 35]. These have (1) ssRNA in their particles – Retroviridae (HIV virus), Metoviridae, Pseudoviridae; (2) dsDNA – Canlimoviridae, Hepadnaviridae – which includes hepatitis B virus.

12.4. EFFECT OF VIRUS INFECTION ON CELLS

The short and long – term effects of viral infections on animal cells are well document.Cytopathic effect or cytopathogenic effect (a breviated PE). 1. Was defined as virus-induced damage to the cell that alters its microscopic appearance[21]. 2. Refers to structural changes in the host cells that are caused by viral invasion. The infecting virus causes lysis of the host cell, or when the cell dies without lysis due to an inability to reproduce. Individual cells may become disoriented, undergo gross changes in shape or size, or develop intracellular changes. Typically, the first sign of viral infections is the rounding of cells. Then Inclusion Bodies (IB) often then appear in the cell nucleus and/or cytoplasm of the host cell, and are aggregates of stainable substances, usually proteins. They typically represent sites of viral multiplication in a bacterium or a eukaryotic cell and usually consist of viral capsid proteins [5]. Examples of viral inclusion bodies in animals are: Negri bodies in rabies Guarnieri bodies in vaccinia, variola, Bollinger bodies in fowlpox. Some viral infections cause a strange CPE, the formation of Syncytia that are large cytoplasmic masses that contain many nuclei. Viral infections may have clinically relevant phenotypical CPEs.

169

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

For example: with the hepatitis C virus (HCV), liver steatosis is characteristic enough of the virus that it may be used to help identify the genotype (the genetic composition of the virus). The causes of death of the host cell include cell lysis, alterations to the cell's surface membrane and apoptosis. This is the process of programmed cell death (PCD) that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. The most serious persistent viruses remain in a chronic latent state, becoming periodically reactivated [4]. Examples of this are herpes simplexviruses (fever blisters and genital herpes) and herpes zoster virus (chickenpox and shingles) which can go into latency in nerve cells and later emerge under the influence of various stimuli to cause recurrent infections. Some viruses, such as Epstein-Barr virus can cause cells to proliferate without causing malignancy while others, such as papilloma viruses, are established causes of cancer [1].

12.5. MEDICAL IMPORTANCE OF VIRUSES

The number of viral infections that occur on a worldwide basis is almost impossible to accurately predict. Certainly, viruses are the most common cause of acute infections that do not result in hospitalization, especially when one considers widespread diseases such as colds, hepatitis, chickenpox, influenza, herpes and warts. Many serious diseases such as Ebola virus disease, AIDS, influenza and SARS are caused by viruses. There is a possible connection between human herpesvirus 6 (HHV6) and neurological diseases such as multiple sclerosis and chronic fatigue syndrome [23]. There is a controversy over whether the bornavirus which causes neurological disease in horses, could be responsible for psychiatricillnesses in humans. These latent viruses might sometimes be beneficial, as the presence of the virus can increase immunity against bacterial pathogens, such as Yersinia pestis.

12.6. EPIDEMIOLOGY

Transmission of viruses can be vertical (it occurs from mother to the offspring) and horizontal (from person to person). Examples: For vertical transmission: hepatitis B, HIV, varicella zoster virus; For horizontal transmission through: - body fluids – HIV, EBOLA; - blood by contaminated transfusion or needle sharing – hepatitis C; - exchange of saliva by mouth – Epstein Barrvirus; - ingestion of contaminated food and water Norovirus; - inhalation of aerosols containing virions which influenza virus; - insect vectors such as mosquitoes, which penetrate the skin of the host, e.g. dengue. Epidemiology is used to break the chain of infection in populations during outbreaks.

Viral diseases

It is important to find the source, or sources of the outbreak and to identify the virus. Once the virus has been identified, the chain of transmission can sometimes be broken by vaccines.

170

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Often, infected people are isolated from the rest of the community, and those that have been exposed to the virus are placed in quarantine. In 2001, an outbreak of food-and-mouth disease in the United Kingdom led to the slaughter of thousands of cattle.

Feature 12.2. Epidemics and pandemics

When outbreaks cause an unusually high proportion of cases in a population, they are called epidemics. If outbreaks spread worldwide, they are called pandemics. An epidemic is the rapid spread of infectious disease to a large number of people in a given population within a short period of time usually two weeks or less[14; 32]. For example, in meningococcal infections an attack rate in excess of 15 cases per 100,000 people for two consecutive weeks is considered an epidemic.

A pandemic is an epidemic of infectious disease that has spread through human populations across a large region. [20] For example: multiple continents, or even worldwide . Recent pandemics include the HIV pandemic as well as the 1918 and 2009 H1N1 pandemic. The Black Death was a devastating pandemic killing over 75 million people. Native American populations were devastated by the contagious diseases, in

particular smallpox brought to the Americas by European colonists. The number of death have been estimated to approximately to 70% of the indigenous populations. The damage done by this disease aided European attempts to

displace and conquer the native population. The 1918 flu pandemic, which lasted until 1919 was a category 5 influenza pandemic caused by an unusually severe and deadly influenza A virus. Older estimates

say it killed 40-50 million people, while more recent research suggest that it may have killed as many as 100 million people, or 5% of the world's population in 1918. The Joint United Nations Programme on HIV/AIDS (UNAIDS) and the World

Health Organization (WHO) estimate that AIDS has killed more than 25 million people since it was first recognized on 5 June 1981, making it one of the most destructive epidemics in recorded history. In 2007 there were 2.7 million new HIV infections and 2

million HIV-related deaths. Marburg virus, first discovered in 1967, attracted widespread press attention in April 2005 for an outbreak in Angola.

Ebola virus disease has also caused intermittent outbreaks with, high mortality rates since 1976 when it was first identified. The worst and most recent one is the West Africa epidemic [9].

171

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 12.3. Viral cancers

Viral cancers occur only in a minority of infected persons or animals.

Cancer viruses come from a range of virus families, including both RNA and DNA

viruses, and so there is no single type of oncovirus . Viruses accepted to cause human cancers include some genotypes of human papillomavirus, hepatitis B virus, hepatitis C virus, Epstein-Barr virus, Kaposi's [27] Sardoma-associated herpesvirus and human T-lymphotropic virus . The most recently discovered human cancer virus is a polyomavirus (Merkel cell polyomavirus) that caused a rare form of skin cancer called Merkel cell carcinoma. Hepatitis viruses can develop into a chronic viral infection that leads to liver cancer. Infection by human T-lymphotropic virus can lead to tropical spastic poroporesis and adult T-cell leukemia. Human papillomaviruses are an established cause of cancers of cervix, skin).

Kaposi's sarcoma-associated herpesvirus causes Kaposi's sarcoma and body cavity [8] lymphoma . Epstein-Barr virus causes Burkitt's lymphoma Hodgkin's lymphoma, B [25] lymphoproliferate disorder and nasopharyngeal carcinoma . Merkel cell polyomavirus closely related to SV40 and mouse polyomaviruses have been used as animal models for cancer viruses for over 50 years [12].

Animal viruses

Viruses are important pathogens of livestock. Diseases such as bluetongue or foot-and- mouth disease are caused by viruses. Pets if not vaccinated, are susceptible to serious viral infections. Canine parvovirus is caused by a small DNA virus and infections are often fatal in pups. Cleavage is the name of the indentation in a cell's surface created when it is about to divide.

172

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

RESEARCH NEWS

Do ERV make us smarter?

Retroviruses including some viruses which are dangerous (HIV), while others are believed to be harmless (ERV = endogenous retroviruses). ERV existed in the human genome for millions of years and they can be found in a part of DNA, so called "Junk DNA". The genes that control the production of various proteins in the body represent a smaller proportion of our DNA than

endogenous retroviruses.

If it turns out that they are able to influence the production of proteins, this will provide us with a huge new source of information about the human brain (Johan Jakobsson). This makes it a possible tool for evolution, and even a possible underlying cause of neurological diseases. These studies indicate a deviating regulation of ERV in several

neurological diseases such as ALS,

schizophrenia and bipolar disorder. Several thousands of the retroviruses that have established themselves in our genome may serve as "docking platforms" for a protein called "TRIM 28". This protein has the ability to "switch off" not only viruses but also the standard genes adjacent to them in the DNA helix, allowing the presence of ERV to affect gene expression.

173

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

RESEARCH NEWS

LET’S TALK ABOUT VACCINES (PRESERVATIVES AND STABILIZERS)

Tragic consequences have followed the use of multi-dose vials that did not contain a preservative and have served as the driving force for this requirement. 1. Thimerosal – (An ethylmercury based preservative) was phased out of vaccines in the late 1990s. Numerous studies have shown that autism rates are no lower in children who received vaccines without thimerosal than those who did. Today, influenza is the only vaccine that still contains thimerosal as a preservative. Scientists performed several studies, all of which showed that thimerosal at the level contained in vaccines hadn’t cause harm. Today, breastfed infants ingest 15 times more mercury in breast milk than is contained in the influenza vaccine. 2. Formaldehide – May be used as an antimicrobial and inactivates the microbes and biological substances used in vaccines. It is a by product of protein and DNA synthesis, so it is commonly found in the blood stream. The quantity of formaldehyde found in blood is 10 times greater than that found in any vaccine. 3. Aluminum – Is an adjuvant which improves the vaccine’s performance by helping to stimulate the body’s immune system to produce antibodies. Aluminum is commonly found in air, food, water, infant formula and breast milk. The quantity of aluminum in vaccines is small. Typically, infants have between 1 and 5 nanograms of aluminum in each milliliter of blood. Researchers have shown that after vaccines are injected, the quantity of aluminum detectable in an infant’s blood does not change and that about half of the aluminum from vaccines is eliminated from the body within one day. 4. Monophosphoryl lipid A – This adjuvant has been tested for safety in tens of thousands of people and was isolated from the surface of bacteria and detoxified, so that it cannot cause harm. 5. Egg protein – If your child has had an allergic reaction to eggs products, you should be sure to discuss this with your child’s doctor. 6. Polyethylene glycol – In addition to these ingredients you may have heard that vaccines contain products such as antifreeze and other outrageous components. This is not true. The claim of antifreeze being in vaccines comes from the use of polyethylene glycol in one brand of the flu vaccine. It is used to inactivate the virus, and to purify certain vaccines. But, it is not antifreeze and has a low toxicity. It is the basis of a number of laxatives and skin creams, and an irrigating solution in surgical procedures and in drug overdoses. 7. Fetal cells – Are used to make five vaccines: rubella, chickenpox, hepatitis A, shingles, and rabies. Viruses (unlike bacteria) require cells to grow and human cells are often better than animal cells at supporting the growth of human viruses. Fetal cells are virtually immortal, meaning they can reproduce many, many times before dying. 8. Gelatin – Is used in some vaccines as a stabilizer. Vaccines are injected, not ingested (except rotavirus vaccine, which does not contain gelatin). Gelatin in vaccines has been highly purified and hydrolyzed (broken down by water), so that it is much smaller than that found in nature.

174

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. The viral genome, containing all the genetic information, and is composed of nucleic acids. ___2. Virions contain two copy of the nucleic acid and they are diploid. ___3. Retroviruses are haploid virions. ___4. The DNA genome may exist as a segmented genome. ___5. Proteins are found in the structure of the capsid.

MULTIPLE CHOICE QUESTIONS

1. Virus particle contain small peptides and polyamines to: a. phages; b. adenoviruses; c. papovaviruses; d. pandoraviruses.

2. The largest characterized virus is: a. Poxvirus; b. Mimivirus; c. Pandoravirus; d. Archaeaviruses.

CONCEPT QUESTIONS

What association did the following people Draw a simple virion, identifying the have with Virology? Dmitri Ivanovsky, following structures: capsid, capsomere, nucleic Martinus Beijerinck, Wendell Stanley, acid core, and envelope: Frederich Twort, Bernal and Fankuchen, Baruch Blumberg, Howard Termin.

Compare the mode of multiplication of Describe the mechanisms of penetration of viruses with that of some representative animal viruses into host cells: bacteria:

175

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

What are viruses? Are they alive? What How can particles so small, simple and are their distinctive biological characteristics? seemingly insignificant be capable of causing disease and death? What is the connection between viruses and cancer?

COMPLETE THE FOLLOWING SENTENCES

 ______discovered a group of viruses that infect bacteria, called

bacteriophages.

 ______grew influenza and several other viruse in fertilized chickens'eggs.

 ______was discovered by Baruch Blumberg.

QUOTE

Mark with X if you like or dislike this quote.

(1) ”I cannot however, but think that the world would be better and brighter if our teachers would dwell on the Duty of Happiness as well as the Happiness of Duty; for we ought to be as cheerful as we can, if only because to be happy our selves in a most effectual contribution to the happiness of others.” (John Lubbock).

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of the courses.

 A virus walks into a bar. The bartender says: - We don’t serve viruses in this bar! The virus replaces the bartender and says: - Well, now we do! -

Explication:

 (Q) What did the conservative biologist says? (A) The only cleavage I want to see is at the cellular level.

Explanation:

176

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Andrew Vecsey (2010). Simplification of Everything. ISBN: 132986462X, 9781329864627 2. B.V. Venkataram Prasad, Michael F Schmid (2012). Principles of Virus Structural Organization. Adv Exp Med Biol. Author manuscript; available in PMC 2013 Sep 9.Published in final edited form as:Adv Exp Med Biol. 2012; 726: 17–47. doi: 10.1007/978-1-4614-0980-9_3. 3. "Canadian Food Inspection Agency – Animal Products – Foot-and-Mouth Disease Hazard Specific Plan" (2008) . Archived from the original on June 5, 2008. 4. Cotran RS, Kumar V, Collins T. Cellular pathology I: cell injury and cell death. In: Cortan RS, Kumar V, Collins T (1999). Robbins Pathologic Basis of Disease. 6. W.B. Saunders Co; Philadelphia, PA: 1999. pp. 1–29. 5. Cruts M, Gijselinck I, van der Zee J, Engelborghs S, Wils H, Pirici D, Rademakers R, Vandenberghe R, Dermaut B, Martin JJ, van Duijn C, Peeters K, Sciot R, Santens P, De Pooter T, Mattheijssens M, Van den Broeck M, Cuijt I, Vennekens K, De Deyn PP, Kumar- Singh S, Van Broeckhoven C (2006). "Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21". Nature. 442 (7105): 920–4. doi:10.1038/nature05017. PMID 16862115. 6. Dennis Holley (2017). General Biology II: Organisms and Ecology. Dog Ear Publishing. ISBN: 1457554046, 9781457554049. 7. Dimmock p. 55. 8. Dharam V. Ablashi, Louise G. Chatlynne, James E. Whitman, Jr., Ethel Cesarman (2002). Spectrum of Kaposi's Sarcoma-Associated Herpesvirus, or Human Herpesvirus 8, Diseases. Clin Microbiol Rev. 2002 Jul; 15(3): 439–464. doi: 10.1128/CMR.15.3.439-464.2002. 9. "2014 Ebola Virus Disease (EVD) outbreak in West Africa". WHO. 21 April 2014. Archived from the original on 29 July 2014. Retrieved 3 August 2014. 10. Ellen G. Strauss, James H. Strauss (2007). Viruses and Human Disease. Elsevier, 2007. ISBN: 0080553168, 9780080553160. 11. Encyclopædia Britannica (2007). "Fuller, R. Buckminster". Encyclopædia Britannica Online. Archived from the original on October 21, 2007. Retrieved April 20, 2007. 12. Feng H., Shuda M., Chang Y., Moore P.S. (2008). Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science. 2008 Feb 22;319(5866):1096-100. doi: 10.1126/science.1152586. Epub 2008 Jan 17. 13. Fuller, Buckminster (1983). Inventions, The Patented Works of R. Buckminster Fuller. St. Martin's Press. pp. vii. 14. Green MS; Swartz T; Mayshar E; Lev B; Leventhal A; Slater PE; Shemer Js (2002). "When is an epidemic an epidemic?". Isr. Med. Assoc. J. 4 (1): 3–6. PMID 11802306. 15. Harvey Lodish, Arnold Berk, S Lawrence Zipursky, Paul Matsudaira, David Baltimore, James Darnell (2000). Molecular Cell Biology, 4th edition. New York: W. H. Freeman. ISBN-10: 0-7167-3136-3 16. https://en.wikipedia.org/wiki/Tobacco_mosaic_virus. 17. https://en.wikipedia.org/wiki/Viral_envelope. 18. https://en.wikipedia.org/wiki/Mimivirus.

177

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

19. https://en.wikipedia.org/wiki/Hepadnaviridae . 20. https://en.wikipedia.org/wiki/Pandemic. 21. Kathleen P. Talaro (2001).Foundations in Microbiology. ISBN: 0072320427. 22. Koonin EV, Gorbalenya AE, Chumakov KM (1989). "Tentative identification of RNA-dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases". FEBS Lett. 252 (1–2): 42–6. doi:10.1016/0014-5793(89)80886-5. PMID 2759231. 23. Konstantine I. Voumvourakis, Dimitrios K. Kitsos, Sotirios Tsiodras, George Petrikkos, Eleftherios Stamboulis (2010). Human Herpesvirus 6 Infection as a Trigger of Multiple Sclerosis. Mayo Clin Proc. 2010 Nov; 85(11): 1023–1030. doi: 10.4065/mcp.2010.0350. 24. Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendrickson WA (June 1998). "Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody". Nature. 393 (6686): 648–59. Bibcode:1998Natur.393..648K. doi:10.1038/31405. PMID 9641677. 25. Matthew P. Thompson, Razelle Kurzrock (2004). Epstein-Barr Virus and Cancer. Clinical Cancer Research. DOI: 10.1158/1078-0432.CCR-0670-3 Published February 2004. 26. Michael J. Pelczar, Jr., E.C.S. Chan, Noel R. Krieg (1986). ”Microbiology”, ISBN 10: 0070492344 / ISBN 13: 9780070492349, Published by Mcgraw-Hill College. 27. Moore P.S., Chang Y. (2010). Why do viruses cause cancer? Highlights of the first century of human tumour virology. Nat. Rev. Cancer. 2010;10:878–889. [PMC free article] [PubMed] 28. Patton JT (editor). (2008). Segmented Double-stranded RNA Viruses: Structure and Molecular Biology. Caister Academic Press. ISBN 978-1-904455-21-9. 29. PELCZAR. MICROBIOLOGY:APPLICATION BASED APPROACH. Tata McGraw-Hill Education. ISBN: 1259081753, 9781259081750. 30. Philippe N, Legendre M, Doutre G, Couté Y, Poirot O, Lescot M, Arslan D, Seltzer V, Bertaux L, Bruley C, Garin J, Claverie JM, Abergel C (2013). "Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes". Science. 341 (6143): 281–6. Bibcode:2013Sci...341..281P. doi:10.1126/science.1239181. PMID 23869018. 31. Prangishvili D, Forterre P, Garrett RA (2006). Viruses of the Archaea: a unifying view. Nature Reviews Microbiology. 2006;4(11):837–48. doi:10.1038/nrmicro1527. PMID 17041631. 32. Principles of Epidemiology, Third Edition (2012). Atlanta, Georgia: Centers for Disease Control and Prevention. 2012. 33. Rossmann M.G., Mesyanzhinov V.V., Arisaka F., Leiman P.G. (2004). The bacteriophage T4 DNA injection machine. Curr. Opin. Struct. Biol. 14(2): 171-80. Doi: 10.1016/j.sbi.2004.02.001. http://dx.doi.org/10.1016%2Fj.sbi.2004.02.001. PMID 4322005. 34. Sarafianos, SG; Das, K; Clark Jr, AD; Ding, J; Boyer, PL; Hughes, SH; Arnold, E (1999). "Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids". Proceedings of the National Academy of Sciences of the United States of America. 96 (18): 10027–32. Bibcode:1999PNAS...9610027S. doi:10.1073/pnas.96.18.10027. PMC 17836 . PMID 10468556.

178

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

35. Shafer, RW; Kozal, MJ; Winters, MA; Iversen, AK; Katzenstein, DA; Ragni, MV; Meyer Wa, 3rd; Gupta, P; et al. (1994). "Combination therapy with zidovudine and didanosine selects for drug-resistant human immunodeficiency virus type 1 strains with unique patterns of pol gene mutations". The Journal of Infectious Diseases. 169 (4): 722–9. doi:10.1093/infdis/169.4.722. PMID 8133086. 36. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages. 37. Steinhardt E, Israeli C, Lambert R.A. (1913). Studies on the cultivation of the virus of vaccinia. The Journal of Infectious Diseases. 1913;13(2):294–300. doi:10.1093/infdis/13.2.294. 38. Tata McGraw-Hill Education (2009). Principles of Microbiology. ISBN: 0070141207, 9780070141209. 39. Z. R. RATHER (2009). SUCCESSIVE BOTANY: FOR B. Sc Part 1 - Based on Kashmir University Syllabus, As Per Single Paper Scheme, 2 nd. Zahoor Rashid Rather.

179

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 13 Bacterial viruses and prions

13.1.Bacterial viruses (bacteriophages) 13.2. Prions

Enterobacteria phage T4 is a bacteriophage that infects Escherichia colibacteria. The T4 phage is a member of the T-even phages, a group including enterobacteriophages T2 and T6. T4 is capable of undergoing only a lytic lifecycle and not the lysogenic lifecycle. The T4 phage's double- stranded DNAgenome is Bacteriophage T about 169 kbp long and 4 encodes 289 proteins. nickname ”Buddha Man”

Learning objective

Bacterial viruses, or bacteriophages have provided the microbiologist with a model for Virology and Molecular Biology

Key points

Bacteriophages, viruses that infect bacteria, were discovered independently by Frederick Twort in England in 1915 and by Felix D'Herelle at the Pasteur Institute in Paris in 1917

180

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

13.1. BACTERIAL VIRUSES (BACTERIOPHAGES)

Bacteriophages are the most abundant form of biological entity in aquatic environments, reaching levels of 250,000,000 bacteriophages per milliliter of seawater [26]. These viruses infect specific bacteria by binding to surface receptor molecules and then entering the cell. In minutes, bacterial polymerase starts translating viral mRNA into protein [8]. These proteins go on to become either new virions within the cell, helper proteins which help assembly of new virions, or proteins involved in cell lysis [18].

Figure 13.1. Bacteriophage T4

protein coat

DNA sheath

core

cell wall

181

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

The major way bacteria defend themselves from bacteriophages is by producing enzymes that destroy foreign DNA. These enzymes, called restriction endonucleases, cut up the viral DNA that bacteriophages inject into bacterial cells [9].

Figure 13.2. Bacteriophage (litic cycle)

The steps of replication of T4 phage in Escherichia coli are:

1. Adsorption – is the first step in infection of a host bacterial cell. Adsorption of phage T4 to its host cell by means of the interaction of the tip of the phage core(tail tube within the sheath) and the cytoplasmic membrane of the host spheroplast. 2. Penetration - in the T4-even phage penetration is achieved when: a. the tail fibers of the virus attach to the cell and hold the tail firmly against the cell wall; b. the heat contracts, driving the tail core into the cell through the cell wall and membrane; c. the virus injects its DNA the way a syringeinjects a vaccine. The protein coat, which forms the phage head, and the tail structure of the virus remain outside the cell. 3. Transcription – occurs in several stages leading to the formation of immediate early, delayed early, and late gene products, so named on the basis of their time of appearance. Immediate early phage genes are transcribed using the existing bacterial RNA polymerase [23]. 4. Assembly and release – about 25 minutes after initial infection, some 200 new bacteriophages have been assembled and the bacterial cell bursts, releasing the new phages to infect other bacteria and begin the cycle over again [3]. Bacteria also contain a system that uses CRISPR sequences to retain fragments of the genomes of viruses. Addgene provides Cas 9 and gDNA expression plasmids that have been

182

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

designed for use in bacteria. Since 2013, the CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life [13]. This is a metaphor used to describe the relationships between organisms, both living and extinct. Archaean viruses. These viruses are double-stranded DNA viruses with unusual and sometimes unique shapes. They have been studied in most detail in the thermophilic archaea, particularly the orders Sulfalobales and Thermoproteales.

RESEARCH NEWS

Could this be how multicellular organisms evolved?

Joe Pogliano found that shortly after bacteriophages infect bacteria they destroy much of the existing architecture of the bacterial cells, including bacterial DNA, then hijack the remaining cellular machinery. The viruses then reorganize the entire cell into an efficient, centralized factory to produce the next generation of viruses. Using fluorescent microscopy, Vorrapon Chaikeeratison and Katrina Nguyen, found that invading viruses organize the structures within bacteria to mimic those found the eukaryotic cells. Elizabeth Villa (UC San Diego) used cryo-electron tomography and observed that the viral offspring being assembled around the nucleus-like compartment in the bacterium. Could this be how multicellular organisms evolved? The theory called "viral eukaryogenesis" suggests that the first eukaryotic cell was created when a large virus took over a bacterium.

183

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Role in aquatic ecosystems

A teaspoon of seawater contains about one million viruses. Most of these are bacteriophages. They infect and destroy bacteria in aquatic microbial communities, and are the most important mechanism of recycling carbon in the marine environment [20]. Viral activity also contribute to the biological pump, the process whereby carbon is sequestered in the deep ocean [22]. The biological pump is the ocean's biologically driven sequestration of carbon from the atmosphere to the deep sea [17]. Microorganisms constitute more than 90% of the biomass in the sea. It is estimated that viruses kill approximately 20% of this biomass each day and that there are 15 times as many viruses in the oceans as there are bacteria and archaea. Viruses are the main agents responsible for the rapid destruction of harmful algal blooms[21] which often kill other marine life [7]. Marine mammals are susceptible to viral infections. In 1988 and 2002, thousands of harbor seals were killed in Europe by phocine distemper virus [6]. Many other viruses circulate in marine mammal populations such as caliciviruses, herpesviruses, adenoviruses and parvoviruses [22].

Feature 13.1. 1918 Influenza virus

Biological Weapons

The smallpox virus, was one of the world’s most devastating virus the infamous 1918 influenza virus was a virus recreated in a laboratory. There are two centers in the world that are authorized by the WHO to keep stocks of smallpox virus. The Vector Institute in Russia and the Centers for Disease Control and Prevention in the United States.

lipid envelope neuraminidase (sialidase)

capsid hemagglutinin

184

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 13.2. Cowpea Mosaic Virus

Materials science and nanotechnology

Viruses are commonly used in materials science as scaffolds for covalently linked surface modifications. A particular quality of viruses is that they can be tailored by directed evolution. Cowpea mosaic virus (CPMV) particles are used at the Naval Research Laboratory in Washington, D.C., to amplify signals in DNA microarray based sensors. In this application, the virus particles separate the fluorescent dyes used for signaling to prevent the formation of non-fluorescent dimmers that act as quenchers. Another examples is the use of CPMV as a nanoscale bread board for molecular electronics.

Oncolytic viruses

Virotherapy. Viruses have been modified by scientists to reproduce in cancer cells and destroy them but not infect healthy cells. Talimogene laherparepvec (T-VEC) is a modified herpes simplex virus that has had a gene, which is required for viruses to replicate in healthy cells, deleted and replaced with a human gene (GM-CSF) that stimulates immunity. When this virus infects cancer cells, it destroys them and, in doing so, the presence of GM-CSF gene attracts dendritic cells from the surrounding tissues of the body which process the dead cancer cells and present components of them to other cells of the immune system.

Figure 13.3. Talimogene laherparepvec (T-VEC)

185

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Clinical trials are experiments done in clinical research.This virus had a great success in clinical trials and is expected to be approved for the treatment against skin cancer called melanoma in late 2015[4].These viruses which are reprogrammed to kill cancer cells are called oncolytic viruses. Geneticists use viruses as vectors to introduce genes into cells that they are studying. This is useful for making the cell produce a foreign substance, or to study the effect of introducing a new gene into the genome. Eastern European scientists have used phage therapy as an alternative to antibiotics for some time, and the interest in this approach is increasing, because of the high level of antibiotic, that causes resistance.

Antiviral drugs

These drugs are often nucleoside analogues which viruses mistakenly incorporate into their genomes during replication.

Examples of nucleoside analogues are: - acyclovir for Herpes simplex virus infections; - lamivudine for HIV and Hepatitis B virus infections; - protease inhibitors for HIV; - ribavirin combined with interferon for hepatitis C. The treatment of chronic carriers of the hepatitis B virus by using a similar strategy using lamivudine has been developed.

Genetic mutation

Antigenic drift is a mechanism for variation in viruses that involves the accumulation of mutations within the genes that code for antibody-binding sites [25]. This results in a new strain of virus particles which cannot be inhibited by the antibodies that were originally targeted against previous strains, making it easier for the virus to spread throughout a partially immune population [5]. Antigenic drift occurs in influenza A, influenza B and influenza C viruses. Antigenic shift occurs when there is a major change in the genome of the virus. This can be a result of recombination or reassortment. When this happens with influenza viruses pandemics might result [18]. Genetic recombination is the process by which a strand of DNA is broken and then placed at the end of a different DNA molecule. Recombination is common in both RNA and DNA viruses.

186

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 13.4. Antigenic shift and antigenic drift

human H2N2 human H3N2

genetic reassortment Antigenic Shift avian H3N8

Point mutation of hemagglutinin and neuraminidase gene Antigenic Drift

Antigenic shift is the process by which two or more different strains of a virus combine to form a new subtype having a mixture of the surface antigens of the two or more original strains [9]. Antigenic shift is a specific case of reassortment or viral shift that confers a phenotypic change. Antigenic shift is contrasted with antigenic drift, which is the natural mutation over time of known strains of influenza which may lead to a loss of immunity. It can also be found in vaccine mismatch [9]. Antigenic shift occurs only in influenza virus A because it infects more than just humans. Is important in the emergence of new viral pathogens as it is a pathway that viruses may follow to enter a new niche.

13.2. PRIONS

A prion is a protein that can fold in multiple, structurally distinct ways, at least one of which is transmissible to other prion proteins. The word prion, coined in 1982 by Stanley B. Prusier, is derived from the words protein and infection. While several yeast proteins have been identified as having prionogenic properties, the first prion protein was discovered in mammals and is referred to as the major prion protein (Prp). Prp (prion protein or protease-resistant protein) also known as CD 230 (cluster of differentiation 230) is the only known example pf prion protein in animals [19]. In humans, it is encoded by the PRNP gene (PRioN Protein). Expression of the protein is most predominant in the nervous system but occur in many other tissues throughout the body. All known prion diseases in mammals affect the structure of the brain or other neural tissue.

187

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

This infectious agent causes mammalia n Transmissible Spongiform Encephalopathies that include:  bovine spongiform encephalopathy (BSE, also known as ”mad cow disease”);  scrapie in sheep.

13.5. The prion theory

normal PrP proteins

prion version of PrP invades, forcing PrP to nucleus refold into a prion form

unlike normal Prp

In humans PrP causes

046.1 – Creutzfeldt-Jakob Disease; A81.0 – Subacute spongiform encephalopathy (CJD); 046.11 – Variant Creutzfeldt-Jakob Disease (VCJD); 046.71 – Gerstmann-Sträussler-Scheinker syndrome; 046.72 – Fatal Familial Insomnia; 046.0 – Kuru (046.0); A81.1 – Subacute sclerosing panencephalitis: - Dawson inclusion body encephalitis; - Van Bogaert Sclerosing leukoencephalopathy. A81.2 – Progressive multifocal leukoencephalopathy; - Multifocal leukoencephalopathy NOS. A81.8 – Other atypical virus infections of central nervous system; A81.9 – Atypical virus infection of central nervous system, unspecified.

046 – Slow virus infection and prion diseases of central nervous system ICD – 10 version 2015 A81 – Atypical virus infections of central nervous system (prion diseases of the central nervous system)

188

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Prion disease of central nervous system (NOS)

Prions are not considered living organisms because they are misfolded protein molecules which may propagate by transmitting a misfolded protein state. The correct three – dimensional structure is essential to its functioning, although some parts of functional proteins may remain unfolded. Failure to fold into native structure generally produces inactive proteins, but in some instances misfolded proteins have modified or toxic functionality. Several neurodegenerative and other diseases are believed to result from the accumulation of amyloid fibrils formed by misfolded proteins [16]. Many allergies are caused by incorrect folding of some proteins, because the immune system does not produce antibodies for certain protein structures. Aggregated proteins are associated with prion-related illnesses such as CJD, bovine spongiform encephalopathy, mad cow disease. The recent European Medicines Agency approved the use of Tafamidis (Vyndagel, a kinetic stabilizer of tetrameric transthyretin) used in the treatment of the transthyretin amyloid diseases.

13.6. Prion protein structure

Amyloids are insoluble fibrous protein aggregates sharing specific structural traits. Their misfolded structures alter their proper configuration in a way that they erroneously interact with one another or other cell components forming insoluble fibrils (such as neurofilaments – neurofibril, that are about 10 nanometers in diameter).

189

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

These refolded prions can then go on to convert more proteins themselves, leading to a chain reaction in large amounts of the prion form [1].

13.7. Prion protein

Amino acids in normal beta helix

amino acids in alpha helix

diseased prion

Amino acids in sheet form

The incubation period (the time elapsed between exposure to a pathogenic organism and when symptoms first appear) of prion diseases is determined by the experimental growth rate associated with prion replication, which is a balance between the linear growth and the breakage of aggregates [12]. Prion aggregates are extremely stable and accumulate inside the infected tissue, causing tissue damage and cell death. This structural stability means that prions are resistant to denaturation by chemical and physical agents [11]. The endogenous properly folded form is denoted PrPc (for Common or Cellular), whereas the disease-linked misfolded form is denoted PrPsc (for Scrapie). They can be formed by combining PrPc, polyadenylic acid and lipids in a Protein Misfolding Cyclic Amplification (PMCA) reaction. PMCA is an amplification technique to multiply misfolded prions originally developed by Soto and colleagues. It is a test for spongiform encephalopathies like BSE [15]. Stanley B. Prusiner (1982 of the University of California, San Francisco),won the Nobel Prize for his research intoprions and the Prion Protein (PrP) in Physiology of Medicine (1997). The protein that prions are made of (PrP) is found throughout the body, even in healthy people and animals. The normal form of the protein is called PrPc and the infectious form is called PrPsc. PrPc is the normal protein found on the membranes of cells. PrP play important roles in cell-cell adhesion and intracellular signaling in vivo, and is involved in cell-cell communication in the brain. PrPres (isoform of PrP) may be infectious. 190

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

PrPsc is the infectious isoform of PrP and is able to convert normal PrPc proteins into the infections isoform by changing their conformation or shape and alters the way the proteins interconnect. PrPsc always causes prion disease. In these proteins there is a higher proportion of β sheet structure instead of the normal Δ helix structure.Aggregations of these abnormal isoforms form highly structured amyloid fibers which accumulate to form plaques. The end of each fiber acts as a template onto which free protein molecules may attach, allowing the fiber to grow. Prion replication mechanism – heterodimer model of prion propagation. This model assumed that a single PrPsc molecule binds to a single PrPc moleculeand catalyzes its conversion into PrPsc. The two PrPsc molecules then come apart and can go on to convert more PrPc. PrPsc exists only in aggregated forms such as amyloid, where cooperatively may act as a barrier to Spontaneous conversion [27].

Table 13.1. Diseases caused by prion

Animal species Disease Sheep, goat Scrapie Cattle (BSE) Mink Transmissible mink encephalopathy (TME) White-toiled deer, elk, mule deer, moose Chronic wasting disease (CWD) Cat Feline spongiform encephalopathy (FSE) Nyala, oryx greater kudu Exotic ungulate encephalopathy (EUE) Ostrich Spongiform encephalopathy (has not been shown to be transmissible) Human CJD Iatrogenic CJD (ICJD) Variant CJD (vCJD) Familial CJD (fCJD) Sporadic CJD (sCJD) Gerstmann-Sträussler- Scheinker Syndrome (GSS) Fatal familial insomnia (FFI) Kuru

Prion causes neurodegenerative diseases be aggregating extracellularly within the central nervous system to form plaques known as amyloid, which disrupt the normal tissue structure. This disruption is characterized by ”holes” in the tissue with resultant spongy architecture due to the vacuole formation in the neurons [24]. Incubation period is relatively long (5 to 20 years). All known prion diseases, collectively called transmissible spongiform encephalopathies (TSEs) are untreatable and fatal. In 2010 a team from New York described detection of PrPsc by Surround Optical Fiber Immunoassay (SOFIA). This method was combined with amplification. In 2011, it was discovered that prions could be degraded by lichens. In 2013, a study revealed that 1 in 2,000 people in the United Kingdom might harbor the infections prion protein that causes vCJD. Transmission of these diseases can arise in three different ways: acquired, familial or sporadic. Current research suggests that the primary method of infection in animals is through ingestion. Prions may be deposited in the environment such as the remains of dead animals and via urine, saliva and other body fluids.

191

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

In 2014, within the collaboration between Rocky Mountain Laboratories, USA and the University of Verona, Italy it was developed for the first time a protocol for the detection of CJD in living patients. The RT-QuIC assay, a microplate reader-based prion detection method, was applied to nasal brushing obtained from the olfactory epithelium of living patients affected with CJD. The assay had a sensitivity of 97% and specificity of 100% for the detection of CJD. Astemizole, brand name Hismanal has been found to have anti-prion activity. It is a second-generation antihistaminic drug that has a long span action. A gene for the normal protein has been identified: the PRNP gene. In all inherited cases, the prion disease is a mutation in the PRNP gene. Many different PRNP mutations have been identified and these proteins are more likely to fold into abnormal prion. Prions can aggregate only proteins of identical amino acid make-up.

M Heavy metal poisoning

Recent reports suggest that imbalance of brain metal homeostasis is a significant cause of PrPsc – associated neurotoxicity. This hypothesis include a functional role for PrPc in metal metabolism, and loss of this function due to aggregation to the disease-associated PrPsc form as the cause of brain metal imbalance.

Viral hypothesis

The virion hypothesis states that TSEs are caused by a replicable informational molecule (nucleic acid) bound to PrP. Evidence in favor of a viral hypothesis:  Strain variation: differences in prion infectivity, incubation, symptomatology, and progression among species resembles that seen between RNA viruses.  The long incubation and rapid onset of symptoms resembles lentiviruses, such as HIV- induced AIDS [10].  Viral like particles that do not appear to be composed of PrP have been found in some of the cells of scrapie-or CJD-infected cell lines.Gregor Mendel genetic experiments were done with pea plants.

192

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Feature 13.3. (D)evil inserts itself like Prions

Virulence

HIGH

avoid attack A two- dimensional

model of responses ENGAGEMENT

to evil ignore embrace

LOW

“We are living in the era of premeditation and perfect crimes. Our criminals are no longer those helpless children who pleaded love as their excuse. On the contrary, they are adults and have the perfect alibi: philosophy which can be used for anything even for transforming murders into judges.” (Albert Camus, the Rebel)

Mass evil requires that the collective, quite literally, loses its mind, loses its ability to see the world it is making even as it is making it-to see its own present, and no longer just to remain hidden from its future [14].

Taking with me from Dante's phrase that hell is populated by ”Those who have lost the benefit of the intellect” I argue that ”Earthly hells” are the result of a certain kind of mindlessness on the part of people entrusted to be the brains of their respective collective.

Dante's concept of the intellect is shaped by an ontology in which love is the source of all. Evil inserts itself into every body of believers, because there is a willingness by the ”Brains-trust” of the collective to accept the inferior or inadequate for the substance it is seeking [19]. That inferior can be the reward of certitude, of status, of belonging. It can be any number of things, but it is always love's surrogate rather than love itself [14]. And the very desire to maintain that surrogate, to serve it, to stabilize it, to keep feasting off it, when it is so empty intensifies the corruption of collective intelligence. Corrupted intelligence creates false complexities where are clear lines of true simplicities and false simplicities where there are thickets of reasons that require much labor and time to disentangle [14]. Hence when love is absent, or targeting the wrong objects, the intellect does not fulfil its real function, and doing evil is bound up with the intellect not being guided by love's light. Hell becomes inevitable when a group becomes sufficiently steeped in its illusions that it does not see the awaiting terror. Concomitantly, when a group reaches this stage they cannot be touched by true words: the group, then, literally belongs to the (D)evil [14]. ”To think what we are doing by thinking over from the very beginning everything we ever thought we were doing”. (Bernard Bergen)

193

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. Bacteriophages are the most abundant form of biological entity in aquatic environments. ___2. A teaspoon of seawater contains about one million viruses. ___3. Microorganisms constitute more than 40% of the biomass in the sea. ___4. Antigenic drift occurs when there is a major change in the genome of the virus. ___5. Antigen drift is the process by which two or more different strains of a virus, combine to form a new subtype having a mixture of the surface antigens of the two or more original strains.

MULTIPLE CHOICE QUESTIONS

1. Examples of antiviral drugs are: a. acyclovir; b. lemivudine; c. penicilin; d. kanamicin.

2. In the treatment of hepatitis C is used: a. ribavirin + interferon; b. lamivudine; c. protease inhibitors; d. acyclovir.

CONCEPT QUESTIONS

Draw and describe the bacteriophage T4: Draw and describe the lytic cycle:

194

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Compare the antigenic drift with the What are prions? Describe and give some antigenic shift: examples of prion diseases:

COMPLETE THE FOLLOWING SENTENCES

 ______is used in the treatment of herpes simplex virus infections.

 ______is used in the treatment of HIV.

 ______is used in the treatment of Hepatitis C.

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of the courses.

 (Q) What did Gregor Mendel say when he founded genetics? (A) Woopea!

Explication:

 25 years ago, it took 6 million dollars to rebuild a man. It now takes 40 million dollars to create bacteria… Damn inflation, you scary!!!

Explanation:

195

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Aguzzi A (Jan 2008). "Unraveling prion strains with cell biology and organic chemistry". Proceedings of the National Academy of Sciences of the United States of America. 105 (1): 11–2. Bibcode:2008PNAS..105...11A. doi:10.1073/pnas.0710824105. PMC 2224168 . PMID 18172195. 2. Bergh O., Børsheim K.Y., Bratbak G., Heldal M. (1989). High abundance of viruses found in aquatic environments. Nature. 1989;340(6233):467–8. doi:10.1038/340467a0. PMID 2755508. Bibcode: 1989Natur.340..467B. 3. bio scholar series. 4. "Cancer Virotherapy Journal" (2015). Retrieved 23 November 2015. 5. CTI Reviews (2016). Prescott's Principles of Microbiology: Biology, Microbiology. Cram101 Textbook Reviews. ISBN: 1490281266, 9781490281261. 6. Hall A.; Jepson P.; Goodman S.; Harkonen T. (2006). "Phocine distemper virus in the North and European Seas – Data and models, nature and nurture". Biological Conservation. 131 (2): 221–229. doi:10.1016/j.biocon.2006.04.008. 7. "Harmful Algal Blooms: Red Tide: Home CDC HSB" (2009). www.cdc.gov. Retrieved 23 August 2009. 8. https://en.wikipedia.org/wiki/Virus. 9. https://en.wikipedia.org/wiki/Introduction_to_viruses. 10. https://en.wikipedia.org/wiki/Prion. 11. Li, J.; Browning, S.; Mahal, S. P.; Oelschlegel, A. M.; Weissmann, C. (2010). "Darwinian Evolution of Prions in Cell Culture". Science. 327 (5967): 869–72. Bibcode:2010Sci...327..869L. doi:10.1126/science.1183218. PMC 2848070 . PMID 20044542. Lay summary – BBC News (January 1, 2010). 12. Masel J, Jansen VA, Nowak MA (Mar 1999). "Quantifying the kinetic parameters of prion replication". Biophysical Chemistry. 77 (2–3): 139–52. doi:10.1016/S0301- 4622(99)00016-2. PMID 10326247. 13. Mohanraju P., Makarova K.S., Zetsche B., Zhang F., Koonin E.V., van der Oost J. (2016). "Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems". Science. 353 (6299): aad5147. doi:10.1126/science.aad5147. PMID 27493190. 14. Nancy Billias (2008). Territories of Evil, Volumul 45 din At the interface, Volumul 45 din At the interface/probing the boundaries, ISSN 1570-7113, EBSCO ebook academic collection. Rodopi, 2008. ISBN: 9042023694, 9789042023697. 15. Saborio G.P., Permanne B., Soto C. (2001). Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature, 411, 810-813. 16. Selkoe D.J. (December 2003). "Folding proteins in fatal ways". Nature. 426 (6968): 900–4. Bibcode:2003Natur.426..900S. doi:10.1038/nature02264. PMID 14685251. 17. Sigman D.M. & G.H. Haug (2006). The biological pump in the past. In: Treatise on Geochemistry; vol. 6, (ed.). Pergamon Press, pp. 491-528 18. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages.

196

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

19. Simona Ivana, Nicolae Starciuc, Delia Costache, Cristea Costache (2017). (D)evil inserts itself like prions. Bio-Innovations in Translational Immunology and Applied Microbiology (BITIAM). December 2017 Vol: 1(1): 14-16. http://indigoroyal.com/devil-inserts- itself-like-prions/. 20. Shors p. 593. 21. Suttle CA (September 2005). "Viruses in the sea". Nature. 437 (7057): 356–61. Bibcode:2005Natur.437..356S. doi:10.1038/nature04160. PMID 16163346. 22. Suttle CA (October 2007). "Marine viruses—major players in the global ecosystem". Nature Reviews Microbiology. 5 (10): 801–12. doi:10.1038/nrmicro1750. PMID 17853907. 23. Tata McGraw-Hill Education (2009). Principles of Microbiology. ISBN: 0070141207, 9780070141209. 24. Takayuki Shibamoto, Leonard F. Bjeldanes (2009). Introduction to Food Toxicology, Food Science and Technology. Academic Press. ISBN: 0080921531, 9780080921532. 25. Wang-Shick Ryu (2016). Molecular Virology of Human Pathogenic Viruses. ISBN: 0128009993, 9780128009994. 26. Wommack K.E., Colwell R.R. (2000). Virioplankton: viruses in aquatic ecosystems. Microbiology and Molecular Biology Reviews. 2000;64(1):69–114. doi:10.1128/MMBR.64.1.69-114.2000. PMID 10704475. 27. Z. R. RATHER (2009). SUCCESSIVE BOTANY: FOR B. Sc Part 1 - Based on Kashmir University Syllabus, As Per Single Paper Scheme, 2 nd. Zahoor Rashid Rather , 2009.

197

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 14 Fungi – molds and yeasts

14.1. The Kingdom of the Fungi 14.2. Organization of microscopic fungi 14.3. Asexual and sexual reproduction 14.4. Physiology 14.5. Molds 14.6. Yeasts

The genus name is derived from the Latin root penicillium, meaning "painter's brush", and refers to the chains of conidia that resemble a broom. The genus includes a wide variety of species molds that are the source molds of major antibiotics. Penicillin, a drug produced by P. chrysogenum (formerly, P. notatum), was accidentally discovered by Alexander Fleming in 1929, and found to inhibit the Penicillium notatum growth of Gram-positive nickname ”Brush Man” bacteria.

Learning objective

The fungi (singular, fungus) are a group of eukaryotic organisms that are of great practical and scientific interests to microbiologists

Key points

The science of study of fungi is called Micology

198

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

14.1. The Kingdom of the Fungi – Molds and yeasts

The fungi (singular, fungus) are a group of eukaryotic organisms that are of great practical and scientific interest to microbiologists. The approximately 100,000 species of fungi can be divided for practical purposes into the macroscopic fungi (mushrooms, puffballs gill fungi) and the microscopic fungi (molds, yeasts).  Cells of the microscopic fungi exist in two basic morphological types: yeasts and hyphae (molds).Whereas molds are filamentous and multicellular, yeasts are usually unicellular.  Fungi are eukaryotic spore-bearing protists that lock chlorophyll.  They generally reproduce both sexually and asexually.  A yeast is any unicellular fungus with round to oval cells that reproduces asexually by giving off small cells called buds.  Some species of yeasts may form pseudohyphae, a string of yeast cells that have not separated after each new bud is formed [17].  A true hypha is a long, tubular or thread-like cell typical of the filamentous fungi or molds.  Some fungal cells exist only in a yeast form, others occur primarily as hyphae; and a few, called dimorphic, can exist as either, depending upon growth conditions. This particularity is characteristic for some pathogenic molds [31].  The fungi are heterotrophic organisms – they require organic compounds for nutrition. All fungi acquire nutrients from preformed organic materials called substrates.  Most fungi are saprobes (saprophytes) because they decompose complex plant and animal remains, breaking them down into simpler chemical substances that are returned to the soil, thereby increasing its fertility.  Saprophylic fungi are also important in industrial fermentations. For example the brewing of beer, the making of wine, and the production of antibiotics such as penicillin.  Fungi have enzymesfor digesting an incredible array of substrates including feathers, hair, cellulose, petroleum, products, and rubber [29].  Various fungi thrive in substrates with high salt or sugar content, at relatively high temperatures, and even in snow and glaciers.  As parasites (less than 300 species) fungi cause diseases in plants, humans and other animals.  Fungi often have bright colors, but these colors are not due to photosynthetic pigments.  The science or study of fungi is called mycology.

14.2. ORGANIZATION OF MICROSCOPIC FUNGI

 The cells of most microscopic fungi grow in discrete and distinctive colonies.  The colonies of yeasts are much like those of bacteria in that they have a soft uniform texture and appearance [29].

199

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 14.1. Yeast reproduction

yeast cell

new bud chain of buds

 The colonies of filamentous fungi are noted for the striking, cottony, hairy, or velvety textures that arise from their microscopic organization and morphology.

Figure 14.2. Colonies of filamentous fungi

sporangium spores columella

stolon

hyphae mycelium

rhizoids

200

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 Fungi are eukaryotic chemoorganotrophic organisms that have no chlorophyll.  The thallus (plural thalli) or body of a fungus may consist of a single cell as in the yeasts.  The thallus consists of filaments, 5 to 10 µm across, which are commonly branched.  The yeast cell or mold filament is surrounded by a true cell wall. The slime molds are the exception to this rule, which have a thallus consisting of a naked amoeboid mass of protoplasm [5].  Yeasts vary considerably in size, ranging from 1 to 5 µm in width and from 5 to 30 µm or more in length.  They are commonly ”egg-shaped”, but some are elongated or spherical.  Yeasts have no flagella or other organelles of locomotion.  The thallus of a mold consists essentially of two parts: (1) the mycelium (plural, mycelia) and (2) the spores (resistant, resting or dormant cells).

Figure 14.3. Thallus of a mold

Growth of a hypha from a spore

cell wall nuclei spore

septum

Coenocytic hypha Septate hypha

 The mycelium is a complex of several filaments called hyphae (singular, hypha).  Each hypha is about 5 to10 µm wide (bacterial cell is usually 1 µm in diameter).  Hyphae occur in three forms: 1. Nonseptate or coenocytic. Such hyphae have no septa and consist of one long, continuous cell not divided into individual compartments. 2. Septate with uninucleate cells. The hyphae are divided into segments by cross walls. 3. Septate with multinucleate cells. Each cell has more than one nucleus in each compartment.  Hyphae can also be classified according to their particular function. Mycelia can be either vegetative or reproductive: - vegetative hyphae (mycelia; aerial hyphae) penetrate into the medium in order to obtain nutrients. Soluble nutrients are absorbed through the walls; - reproductive mycelia (fertile)are responsible for spores production.  Hyphae are composed of:

201

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

1. The lumen which is an outer wall-like tube which is filled or lined by protoplasm; 2. The plasmalemma is located between the protoplasma and the wall, and is a double- layered membrane.  Growth of a hypha is distal, near the tip.  The young hypha may become divided into cells by crosswalls which are formed by centripetal invagination from the existing cell wall [22].  These crosswalls constrict the plasmalemma and grow inward to form an incomplete septum (plural, septa) that has a central pore which allows for protoplasmic streaming. Even nuclei may migrate from cell to cell in the hypha [22].  Normal outdoor air may contain 105 fungal spores per cubic meter, and moist air containing nutrients may have as many as 109 spores per cubic meter.  A sexual reproduction (spores) are the products of mitotic division of a single parent cell also called somatic or vegetative reproduction.  Sexual (spores) reproduction is formed through a process of two parental nuclei fusing, followed by meiosis.

14.3. ASEXUAL AND SEXUAL REPRODUCTION

There are many kinds of asexual spores: 1. Sporangiospores – these single-celled spores are formed within sacs called sporangia (singular sporangium) at the end of special hyphae (sporangiophores) [22].

Sporangium (filled with sporangiophores)

sporangiophore

202

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Aplanospores are nonmotile sporangiospores. Zoospores are motile sporangiospores, their motility being due to the presence of flagella. 2. Conidiospores or conidia (singularconidium) are free spores not enclosed by a special sporebearing sac. They develop by being pinched off the tip of a special fertile hypha, or by the segmentation of a preexisting vegetative hypha [29]. Small single-celled conidia are called microconidia and large multicelled conidia are called macroconidia. Conidia are formed at the tip or side of a hypha [22]. Conidia are the most common asexual spores, and they come in the following varieties. - Arthrospores or oidia (singular, oidium) are formed by disjointing of hyphal cells. - Chlamydospores are formed from cells of the vegetative hypha. These spores are highly resistant to adverse conditions [22]. - Blastospores (also called abud). These are spores formed by budding from a parent cell. - Phialospore is a conidium that is budded from the mouth of a vase-shaped sporangenous cell called a phialide or sterigma, leaving a small collar. - Paraspore is a conidium that grows out through small pores in the sporangenous cell; some are composed of several cells.

Figure 14.5.Conidiospores (Conidia)

Aspergillus Rhizopus

Sexual reproduction is carried out by fusion of the compatible nuclei of two parent cells.

Four different types of sexual spores have been identified, but we will consider the three most common: zigospores, ascospores, and basidiospores [31]. 1. Zygospores are large, thick-walled spores formed when the tips of two sexually compatible hyphae, or gametangia, of certain fungi fuse together. They are sturdy diploid spores formed by the meeting of the hyphae of two opposite strains (called the plus and minus strains). Meiosis of diploid cells of the sporangium results in haploid nuclei that develop into sporangiospores [29].

203

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 14.6. Zygospores

zygospore

conidiophore

2. Ascospores are haploid spores, and are created in a sac called an ascus (plural asci). There are usually eight ascospores in each ascus. The ascospores are formed when two different strains or sexes conjugate. In general, the male sexual organ (the antheridium) fuses with the female sexual organ (the ascogonidium). The end result is a number of terminal cells, each containing a diploid nucleus. Late in the cycle, the asci burst and release the ascospores.

Figure 14.7. Ascospores

204

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

3. Basidiospores are haploid sexual spores that are borne on a club-shaped structure called a basidium (plural, basidia). A basidium begins with one nucleus from each parent. The basidium assumes the characteristic shape of that species and generally produces four tapering projections the sterigmata. The four nuclei produce nuclear fission from meiosis and then move toward the sterigmata and form the basidiospores.

Figure 14.8. Basidiospores and basidium

Basidiospores

Basidium

4. Oospores are formed within a special female structure, the oogonium. Fertilization of the eggs, or oospheres, by male gometes formed in an antheridium givesrise to oospores. There are one or more oospheres in each oogonidium. Asexual and sexual spores may be surrounded by highly organized protective structures called fruiting bodies [34]. Asexual fruiting bodies have names such as acervulus and pycnidium. Sexual fruiting bodies have names such as perithecium and apothecium. Some fungi are dimorphic and they could exist in two forms.

205

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Some pathogenic fungi have a unicellular and yeast-like form in their host, but when growing in soil, or in a laboratory they have a filamentous mold form. Thus, a fungi colony may be a mass of yeast cells, or it may be a filamentous mass of mold.

14.4. PHYSIOLOGY

 Yeasts and molds can grow in a substrate (medium) containing concentrations of sugars that inhibit most bacteria this is why jams and jellies may be spoiled by molds but not by bacteria.  Yeasts and molds can generally tolerate more acidic conditions than most other microbes.  Some yeasts are facultative and they can grow under both aerobic and anaerobic conditions.  Molds and yeasts are usually aerobic microorganisms.  Fungi grow over a wide range of temperature, with the optimum for (1)most saprophytic species from 22 to 300C; (2) pathogenic species have a higher temperature optimum, generally 30 to 370C; (3) some fungi will grow at or near 00C and thus can cause spoilage of meat and vegetables in cold storage.  Fungi are heterotrophic and are capable of using a wide variety of materials for nutrition.  They cannot use inorganic carbon compounds (carbon dioxide) as their only carbon source. Carbon must come from an organic source, such as glucose.  Some species can use inorganic compounds of nitrogen, such as ammonium salts, but all fungi can use organic nitrogen (media for fungi usually contain peptone, a hydrolyzed protein product) [22].

Table 14.1. Comparative: physiology of fungi and bacteria

Characteristic Fungi Bacteria Cell type Eukaryotic Prokaryotic Optimum pH 3,8-5,6 6,5-7,5 Optimum temperature 22-300C (saprophytes) 20-370C 30-370C (parasites) (mesophiles) Oxygen requirement Strictly aerobic (molds) Aerobic to anaerobic Facultative (some yeasts) Light requirement None Some photosynthetic groups occus Sugar concentration in laboratory 4-5% 0,5-1% media Carbon requirement Organic Inorganic ad/or organic Cell-wall structural components Chitin, cellulose, hemicelluloses Peptidoglycan Antibiotic susceptibility Resistant to penicillins, Resistant to tetracyclines chloromphenicol griscofulvin, sensitive to p,t,c sensitive to griseofulsin

206

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

KINGDOM MYCOTA

SEXUAL SEXUAL REPRODUCTION REPRODUCTION IDENTIFIED Fungi imperfecti or DEUTEROMYCETES e.g. Cercospora Primitive fungi Advanced fungi Fusarium OOMYCOTA EUMYCOTA (mycelium (mycelium aseptate) septate)

PHYCOMYCETES ZYGOMYCETES ASCOMYCETES BASIDIOMYCETES Algal Fungi Conjugation fungi (sac fungi) (club fungi) e.g. Phytophthora e.g. Mucor rhizopus e.g. Yeast candido e.g. Puccinia agaricus albugo

14.5. MOLDS (MOULDS)

A mold or mould is a fungus that grows in the form of multicellular filaments called hyphae[18; 20]. Fungi that can adopt a single celled growth habit are called yeasts. Molds are considered to be microbes and can be found in the divisions Zigomycoto and Ascomycoto. Molds have diverse life-styles including saprotrophs, mesophiles, psichrophiles and thermophiles. Molds play a major role in decomposition. They secrete hydrolytic enzymes that degrade complex biopolymers such as starch, cellulose and lignin into simpler substances which can be absorbed by the hyphae. Many molds also synthesize mycotoxins and siderophores which inhibit the growth of competing microorganisms. A mycotoxin is a secondary metabolic toxinproduced by organisms of the fungi kingdom, commonly known as molds. Food-based mycotoxins were studied extensively worldwide throughout the 20th century. In Europe, statutory levels of a range of mycotoxins permitted in food and animal feed are set by a range of European directives and Commission regulations [8]. The US Food and Drug Administration has regulated and enforced limits and concentrations of mycotoxins in foods and feed industries since 1985.

Major groups of mycotoxins

1. Aflatoxins are a type of mycotoxin produced by Aspergillus species of fungi, such as Aspergillus flavus and Aspergillus parasiticus. There are four different types of mycotoxins produced, which are B1, B2, G1 and G2. Aflatoxin B1, the most toxic of them is a potent carcinogen (may produce liver cancer in many animal species).

207

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

2. Ochratoxin is a mycotoxin that comes in three secondary metabolite forms, A, B and C. All are produced by Penicillium and Aspergillus species. Aspergillus ochraceusis a contaminant of a wide range of commodities including beverages (drinks) such as beer or wine [2]. Aspergillus carbonarius is the main species found on vine fruit, which releases its toxin during the juice making process [2]. 3. Citrinin was first isolated from Penicillium citrinum, but has been identified in many species of Penicillium and several species of Aspergillus. Some of these species are used to produce cheese (P. camemberti), sake, miso, and soy sauce (A. oryzae). Citrinin is associated with yellowed rice disease in Japan and acts as a nephrotoxin in all animal species tested. Citrinin can also act synergistically with ochratoxin A to depress RNA synthesis in murine kidneys [30]. 4. Ergot Alkaloidsare compounds produced as a toxic mixture of alkaloids in the sclerotia of species of Claviceps. The ingestion of ergat sclerotia from infected cereals (bread produced by contaminated flour, cause ergotism Saint Anthony's fire) with two forms: gangrenous (affecting blood supply of extremities), and convulsive (affecting the central nervous system). It is still an important veterinary problem. Ergot alkaloids have been used pharmaceutically. 5. Patulin is a toxin produced by the Patulin expanseum Aspergillus, Penicillium, and Paecilomyces fungal species. It has been reported to damage the immune system in animals. In 2004, the European Community set limits to the concentrations of patulin in food products. They currently stand at 50 µg/kg in all fruit juice concentrations, at 25 µg/kg in solid apple products used for direct consumption, and at 10 µg/kg for children's apple products, including apple juice [21]. 6. Fusarium toxins are produced by over 50 species of Fusarium and have a history of infecting the grain developing cereals such as wheat and maize. They include a range of mycotoxins, such as: the fumonisins, which affect the nervous systems of horses and may cause cancer in rodents; the trichothecenes, which are associated with chronic and fatal toxic effects in animals and humans; and zearalenone, which is not correlated to any fatal toxic effects in animals or humans. In animal food: there have been outbreaks of dog food containing aflatoxin in North America in late 2005/ early 2006 and again in late 2011. Mycotoxins in animal fodder, particularly silage, can decrease the performance of farm animals and potentially kill them. Several mycotoxins reduce milk yield when ingested by dairy cattle. Few molds can begin growth at temperatures of 40C (390F) or below, that’s way food is typically refrigerated at this temperature. Certain molds can survive in harsh conditions such as the snow-covered soils of Antarctica, refrigeration, highly acidic solvents, anti-bacterial soap and even petroleum products such as jet fuel. Xerophilic molds are able to grow in relatively dry, salty, or sugary environments where water activity (aw) is less than 0.85 (aw is the partial vapor pressure water in a substance divided by the standard state partial vapor pressure of water). Verophile is an extremophilic organism that can grow and reproduce in conditions with a low availability of water. Common genera of molds include: Acremonium, Alternaria, Aspergillus, Cladosporium, Fusarium, Mucor, Penicillum, Rhizopus, Stachybotrys, Trichoderma, Trichophyton.

208

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Food production

The koji molds are a group of Aspergilus species. Aspergillus oryzae and Aspergillus sojae, have been cultured in eastern Asia for many centuries. They are use to ferment soybean and wheat mixture to make soybean paste and soy sauce [19]. Koji molds break down the starch in rice, barley,sweet potatoes, etc. (a process called saccharification) in the production of sake, shochu, katsuobushi. Red rice yeast is a product of the mold Monoscus purpureus grown on rice, and is common in Asian diets. The yeast contains several compounds, so called monocolins which are known to inhibit cholesterol synthesis [28]. Some sausages (salami) incorporate starter cultures of molds to improve flavor and reduce bacterial spoilage during curing. For example: Penicillium nalgiovense may appear as a powdery white coating on some varieties of dry – cured sausage.

Other molds that have been used in food production

 Fusarium venenatum– quorn;  Geotrichum candidum– cheese;  Neurospora sitophila– oncom;  Penicillium spp. – various cheeses including Brie and Blue cheese;  Rhizomucor miehei– microbial rennet for making vegetarian and other cheeses;  Rhizopus oligosporus– tempeh.

Pharmaceuticals from molds

 Medicinal fungi produce medically significant metabolites. The range of medically active compounds include antibiotics, anti-cancer drugs, cholesterol inhibitors, psychotropic drugs, immunosuppressant and fungicides [10]. 1. Antibiotics:  Beta-lactam antibiotics: penicillins, cephalosporins (derived from the fungus Acremonium), carbapenems (a naturally derived product of Streptomyces cattleya.  Aminoglycotide (oxazolidinones, sulfonamides, quinolances) – traditional Gram negative antibacterial therapeutic agents that inhibit protein synthesis. They include streptomycin derived from Streptomyces grisens, the earliest modern agent used against tuberculosis.  Anti cancer drugs (chemotherapeuticagents).  Antibiotic penicillin (involved by Penicillum notatum) was discovered by Alexander Fleming.

209

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 14.1. Penicillin and Sir Alexander Fleming

The story of Penicillin provides an elegant example of the benefits gained from collaboration of scientists from government agencies industry and universities. It was a discovery that would change the course of history. In 2002, Fleming was named in the BBC's list of the 100 Greatest Britons following a [32] nationwide vote . Sir Alexander Fleming was a Scottish biologist, pharmacologist and botanist. His best known discoveries [1; 4] are the enzyme-lysozyme and the antibiotic penicillin G (substance benzyl penicillin) . Fleming was conducting experiments in search of new antibacterial agents, particularly ones that would be effective against wound infections. In the course of these experiments, he observed a plate culture of [23] Staphylococcus aureus that had been contaminated by a mold . The area around the edges of the mold colony was clear-no bacterial colonies. Further studies of this phenomenon revealed that it was a mold of the genus Penicillium that produced a substance which was very potent against Staphylococcus aureus, and hence [23] very attractive as a potential chemotherapeutic agent . Fleming named the substance Penicillin. Penicillin was effective against bacteria in laboratory cultures, but was it effective in the human body?

The first clinical trial with a crude penicillin preparation was conducted on February 12, 1941. The patient, an Oxford police man, was dying from a Staphylococcus infection. The administration of penicillin resulted in an initial dramatic improvement, but five days later, when the supply of penicillin was exhausted, the staphylococci reemerged, the infection spread, and the patient died. This was a tragic and to a trial that did not succeed only because there was not enough penicillin available to treat the patient [23]. Britain at this point (1940-1941), was engaged in a grim war. Fortunately, the British reports of penicillin attracted the attention of Americans. As a result, the Rockefeller Foundation invited Harold W. Florey, a professor of pathology at Oxford University and N. G. Heatley, his colleague, to the United States to explore means of large-scale production of penicillin. They arrived in the United States on July of 1941. Meetings were arranged with members of the National Research Council, Charles Thom, a world class mycologist with the US Department of Agriculture and others. Work on penicillin production began immediately at the US Department of Agriculture's Northern Regional Research Laboratory in Peoria, Illinois. Major pharmaceutical companies and universities were called in to cooperate the research and development of penicillin production. The results proved dramatic Fleming's original mold cultures produced 2 units/ml.; within a matter of months improvements in technology increased the yield to 900 units/ml.; today the yield is approximately 50,000 units/ml.! [23]. In the autumn of 1941, there was little penicillin available in the US for treatment of patients. One year later, as a result of the collaborative efforts of governments, universities, and industry, appreciable quantities were available [23]. Following World War I, Fleming actively searched for anti-bacterial agents, having witnessed the death of many soldiers from sepsis resulting from infected wounds. ”When I woke up just after dawn on September 28, 1928, I certainly didn't plan to revolutionized all Medicine by discovering the world's first antibiotic or bacteria killer”. Fleming would later say: ”But I suppose that was exactly what I did”. Fleming noticed that one culture was contaminated with a fungus, and that the colonies of staphylococci immediately surrounding the fungus had been destroyed, whereas other staphylococci colonies farther away [3] were normal, famously remarking: ”That's funny” . Fleming showed the contaminated culture to his former assistant Merlin Price, who reminded him ”That's how you discovered lysozyme” [6]. The laboratory in which Fleming discovered and tested penicillin is preserved as the Alexander Fleming Laboratory Museum in St. Mary's Hospital, Paddington. Fleming finally abandoned penicillin, and not long after he did, Howard Florey and Ernst Boris Chain at the Radcliffe Infirmary in Oxford took up researching and mass-producing it with funds from the US and British Governments. They started mass production after the bombing of Pearl Harbor. By D-Day in 1944, [9] enough penicillin had been produced to treat all the wounded with the Allied forces . Fleming was modest about his part in the development of penicillin, describing his fame as the ”Fleming

Myth” and he praised Florey and chain for transforming the laboratory curiosity into a practical drug. But, sir Henry Harris said in 1998: ”Without Fleming, no Chain; without Chain, no Florey; without Florey, no Heatley; [7] without Heatley, no penicillin” .

210

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

 Several statin cholesterol -lowering drugs (such as lovastatin), fromAspergillus terreus are derived from molds.  Cyclosporine (a immunosuppressant drug) is used to suppress the rejection transplanted organs, is derived from the mold Tolypochadium inflatum. Mold growth in buildings can lead to a variety of health problems. The most important practices that can be followed to mitigate mold issues in buildings, is to reduce moisture levels that can facilitate mold growth. Daniele Del Nero, constructs scale models of houses and office buildings and then induces mold to grow on them, giving them a spooky, reclaimed-by-nature look [35]. Staci Levy sandblasts enlarged images of mold onto glass, then allows mold to grow in the crevasses she has made, creating a macro-micro portrait.

14.6. YEASTS

The word ”yeasts” comes from old English gist and from the Indo-European root yes, meaning ”boil”, ”foam”, or ”bubble”. In 1680, the Dutch naturalist Anton van Leeuwenhoek was the first to microscopically observe yeast, and he considered that it wasn't a living organism, but rather a globular structure. Louis Pasteur (1857) showed that by bubbling oxygen, cell growth could by increased, but fermentation was inhibited and this effect was latter called the ”Pasteur effect” [27]. By the late 18th century, two yeasts strains were used in brewing:  Saccharomyces cerevisiae – top fermenting yeast;  Saccharomyces carlsbergensis – bottom – fermenting yeast.

Nutrition and growth

Yeasts are chemoorganotrophs, meaning they use organic compounds as a source of energy and do not require sunlight to grow.  Carbon is obtained from hexose sugars, such as glucose and fructose, or disaccharides such as sucrose and maltose.  Yeast species either require oxygen for aerobic cellular respiration (obligate aerobes) or are anaerobic, but also aerobic methods of energy production (facultative anaerobes).  Yeasts grow best in a neutral or slightly acidic pH environment.  The temperature in which grow best can vary. For example, Leucosporidium frigidum grows at ~2 to 200C (28 to 680F); Saccharomyces telluris at 5 to 350C (41 to 950F), and Candida slooffi at 28 to 450C (82 to 1130F).  Yeasts are grown in the laboratory on solid growth media or in liquid broths.  Common media used for the cultivation of yeasts include potato dextrose agar or potato dextrose agar/broth, Wallerstein Laboratories nutrient agar peptone dextrose agar yeasts mold agar broth [24]. The antibiotic cycloheximide is sometimes added to yeast growth media to inhibit the growth of Saccharomyces yeasts and select for wild yeast species.  Yeasts are very common in the environment and are often isolated from sugar-rich materials.  Candida albicans, Rhodotorula rubra, Tomlopsis and Trichosporon eutaneum have been found living in between people's toes, as part of their skin flora. Yeasts are also present in the gut flora of mammals and some insects and even in hosts found in deep-sea environments.  Certain strains of some species of yeasts produce proteins called yeast killer toxins that allow them to eliminate competing strains.

211

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Killer yeast such as S. cervisiae is able to secrete toxic proteins which are lethal to receptive cells. The phenomena was first observed by Louis Pasteur (1877). The two most studied variant toxins in S. cerevisiae are K1 and K28.

The yeast cell's life cycle: 1. Budding; 2. Conjunction; 3. Spore.

 Yeasts, like fungi, may have asexual and sexual reproductive cycles.  The most common mode of vegetative growth in yeast is asexual reproduction by budding.  Some yeasts, including Schizosaccharomyces pompe, reproduce by haploid fission thereby creating two identically sized daughter cells. It is a facultative sexual microorganisms that can undergo mating when nutrients are limited.  The budding yeast S. cerevisiae reproduces by mitosis (like diploid cells do) when nutrients are abundant, but when starved, this yeast undergoes meiosis to form haploid spores. Some pucciniomycete yeasts such as Sporidiobolus and Sporobolomyces produce aerially dispersed asexual ballistoconidia a spore that is discharged into the air from a living being, usually a species of fungus. Most types of basidiospores formed on basidia are discharged into theair from the tips of sterigmata. Pucciniomycotais a subdivision of fungus within the division Basidiomycota.

The useful physiological properties of yeast have led to their use in the field of biotechnology: 1. Fermentation of sugars; 2. For making foods: a. baker's yeast in bread production; b. brewer's yeast in beer fermentation; brewer's yeast is also very rich in essential minerals and the B vitamins (except B12); c. yeast in wine fermentation; d. for xylitol production.  Dekkera/Brettanomyces is a genus of yeastknown for its important role in the production of ”lambic” and specially “sour alles”, along with the secondary conditioning of particular Belgian Trappist beer [31]. Trappist beer is brewed by Trappist breweries. Eleven monasteries — six in Belgium, two in the Netherlands and one each in Austria, Italy and United States — currently brew beer and sell it as Authentic Trappist Product. In most beer styles Brettanomyces is viewed as a contaminant and the characteristics it imparts are considered unwelcome ”off-flavors”. Auto-brewery syndrome or Gut fermentation syndrome is a syndrome in which intoxicating quantities of ethanol are produced through endogenous (Ancient Greek – inside), fermentation within the digestive system. Auto-brewery syndrome is a rare medical condition where the stomach houses brewer's yeast that breaks down starches into ethanol which enters the blood stream. Yeasts are used in winemaking, where they convert the sugars present in grape juice into ethanol.Most added wine yeasts are strains of Saccharomyces cerevisiae. Some yeasts, such as Zygosaccharomyces and Brettanomyces in wine can produce wine faults and subsequent spoilage.

212

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Brettanomyces produces an array of metabolites when growing in wine, some of which are volatile phenolic compounds (Brettanomyces character) [27]. Researchers from University of British, Columbia, Canada, have found a new strain of yeast that had reduced amines in red wine and Chardonnay. The amines produce off-flavors and cause headaches and hypertension in some people (about 30% of people are sensitive to biogenetic amines, such as histamines) [33].  Baker's yeast – Saccharomyces cerevisiae is the common name for the strains of yeast commonly used as a leaving agent in baking and bakery products, where it converts the fermentable sugars present in the dough into carbon dioxide and ethanol[10; 36]. Saccharomyces eviguous (S. minor) is used for baking in sourdough starter. Sourdough is a bread product made via a long fermentation of dough using Lactobacillus in symbiotic combination of yeasts.  Sourdough bread is made by the fermentation of dough using naturally- occurring lactobacilli and yeast. Compared to breads made with baker's yeast, sourdough bread has a mildly sour taste because of the lactic acid produced by the lactobacilli [11]. It is important in baking rye-based breads, where yeast does not produce comparable results.The most commonly used yeast species in the production of sourdough are Kazanchastania exigua (S. exiguans), S. cerevisiae,Candida milleri, Candida humilis [25]. Baker's yeast (S. cerevisiae) is the first eukaryote that has its entire genome sequenced. It has over 12 million base pairs and around 6000 genes. It contains enzymes that can reduce a carbonyl group into a hydroxyl group in fairly high yield, thus making it: useful bio-reagent in chemical synthesis.Baker's yeast reduce organometallic carbonyl compounds in very high yield[10].

Synthesis

Bioremediation  Yarrowia lipolytica degrade:  palm oil mill effluent;  TNT (an explosive material);  other hydrocarbons such as alkanes, fatty acids, fats and oils;  heavy metal biosorbent.  Baker's yeast is also used in the bio-remediation of Arsenic (III) from the ground water that contains toxic pollutants.  Different yeasts from Brazilian gold mines bioaccumulate free and complex silver ions.

Nonalcoholic beverages (carbonated beverages): 1. Root beer – a dark brown sweet beverage; 2. Kvass – a fermented drink made from rye (popular in Eastern Europe); 3. Kombucha – a fermented sweetened tea. In its preparation yeasts in symbiosis with acetic acid bacteria. Species of yeasts found in the tea are: Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pompe, Tomlospora delbrueckii and Zyosaccharomyces bailii. It is popular in Eastern Europe and some former soviet republics under the name ”chajnyi grib” which means ”tea mushroom”. Kefir and Kumis are made by fermenting milk yeasts and bacteria. Mauby (Spanish: mabi) made by fermenting sugar with yeasts naturally present on the bark of the Colubrina eliptica tree, (popular in Caribbean).  Root beer is a dark brown sweet beverage traditionally made using the root or bark of the Sassafras albidum or Smilax ornata (sarsaparilla) tree as the primary flavor. Root

213

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

beer may be alcoholic or non-alcoholic , and may be carbonated or non-carbonated. Modern, commercially produced root beer is generally sweet, foamy, carbonated, and non-alcoholic [12].  Kvass is a traditional Slavic fermented beverage commonly made from black or regular rye bread [16]. It is classified as a non-alcoholic drink by Russian and Ukrainian standards, as the alcohol content from fermentation is typically less than 1.2%.Generally, the alcohol content is low (0.5–1.0%) [13]. It may be flavored with fruits such as strawberries and raisins, or with herbs such as mint.  Kombucha refers to any of a variety of fermented, lightly effervescent sweetened black or green teadrinks that are commonly used as functional beverages for their unsubstantiated health benefits. Kombucha is produced by fermenting tea using a "symbiotic 'colony' of bacteria and yeast" (SCOBY). Actual contributing microbial populations in SCOBY cultures vary, but the yeast component generally includes Saccharomyces and other species[14; 15].  Kefir is a fermented milk drink made with kefir "grains" (a yeast/bacterial fermentation starter) and has its origins in the north Caucasus Mountains. Traditional kefir was made in skin bags that were hung near a doorway; the bag would be knocked by anyone passing through the doorway to help keep the milk and kefir grains well mixed [26].

Nutritional supplements

Yeast is used in nutritional supplements popular with health – conscious individuals and those following vegan diets (vegetarians). S. cerevisiae is an excellent source of protein and vitamins, especially the B-complex vitamins, minerals and cofactors.

Probiotics

S. boulardii maintain and restore the natural flora in the gastrointestinal tract: - reduce the symptoms of acute diarrhea; - reduce the chance of infection by Clostridium difficile; - reduce bowel movements in diarrhea predominant IBS (Irritable Bowel Syndrome) patients; - reduce the incidence of antibiotic, traveler's and HIV/AIDS – associated diarrheas.

Pathogenic yeasts

1. Opportunistic pathogens like Cryptococcus neoformans and Cryptococcus gatti cause infection in people with compromised immune systems. They are the species primarily responsible for cryptococcosis, a fungal disease that occurs in about one million HIV/AIDS patients, causing over 600,000 deaths annually. 2. Yeasts of the Candida genus, another group of opportunistic pathogens, cause oral and vaginal infections in humans, known as candidiasis. Candida is commonly found as a commensal yeast in the mucous membranes of humans and other warm-blooded animals [27].

214

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

TRUE-FALSE QUESTIONS

Determine whether the following statements are true (T) or false (F). If you consider a statement is false, explain why and reword the sentence so that is reads accurately.

___1. A yeast is any pluricellular fungus. ___2. The fungi are organotrophic organisms. ___3. Fungi have enzymes for digesting of substrates. ___4. Mycelia can be either vegetative or reproductive. ___5. Reproductive mycelia are responsible for spores production.

MULTIPLE CHOICE QUESTIONS

1. Examples of asexual spores are: a. sporangiospores; b. zigospores; c. ascospores; d. oospores.

2. Sexual fruiting bodies have names such as: a. acervulus; b. pycnidium; c. phialospores; d. blastospores.

CONCEPT QUESTIONS

Which kingdoms contain eukaryotic Differentiate between the yeast and microorganisms? Differentiate between the hypha types of fungal cell. What is a mold? concepts of unicellular, colonial, and What does it mean if a fungus is dimorphic? multicellular levels of fungi organization. Give examples:

How does a fungus feed? Where would Explainhow sexual spores are important one expect to find fungi? to fungi. Give the three main types, and explain how each is formed:

215

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Fill in the blanks: Conidia, Phialides, Fill in the blanks: Rhizoids; Metules, Vesicle, Conidiophore, Hyphae Sporangiophore, Sporangium, Sporangiospores, (Aspergillus spp.): Columella, Nonseptate vegetative hyphae (Stolon), Sporangiospores, Collapsed sporangium, Sporangiophore (Rhizopus nigricans):

Fill in the blanks: Conidiophore (aerial Fill in the blanks: Sporangiospores, hyphae), Conidiophore, Aerial hypha, Hyphae, Sporangium, Sporangioles, Columella, Subsurface hypha, Germination, Conidia Sporangiophore, Apophysis, Merosporangium, (Spores): Merospores:

COMPLETE THE FOLLOWING PROPOSITIONS

 ______are free spores not enclosed by a special sporebearing sac.

 ______or oidia are formed by disjointing of hyphal cells.

 ______are formed from cells of the vegetative hypha.

BACTERIA JOKES

Give a explanation for the following jokes. You may find the explication in the text of the courses.

 In other news, DNA helicase was arrested this morning for unzipping his genes in public!

Explication:

216

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. "Alexander Fleming Biography" (1945). Les Prix Nobel. The Nobel Foundation. 1945. Retrieved 27 March 2011. 2. Anil Aggrawal (2014). APC Essentials of Forensic Medicine and Toxicology. Avichal Publishing Company. ISBN: 817739441X, 9788177394412. 3. Brown, K. (2004). Penicillin Man: Alexander Fleming and the Antibiotic Revolution. 320 pp. Sutton Publishing. ISBN 0-7509-3152-3. 4. Colebrook, L. (1956). "Alexander Fleming 1881-1955". Biographical Memoirs of Fellows of the Royal Society. 2: 117–126. doi:10.1098/rsbm.1956.0008. JSTOR 769479. 5. David R. Boone, Richard W. Castenholz (2012). Bergey's Manual of Systematic Bacteriology: Volume One : The Archaea and the Deeply Branching and Phototrophic Bacteria. Springer Science & Business Media, 2012. ISBN: 038721609X, 9780387216096. 6. Hare, R. The Birth of Penicillin (1970). Allen & Unwin, London, 1970. 7. Henry Harris, Howard Florey and the development of penicillin, a lecture given on 29 September 1998, at the Florey Centenary, 1898–1998, Sir William Dunn School of Pathology, Oxford University (sound recording). 8. http://www.wikidoc.org/index.php/Mycotoxin. 9. https://en.wikipedia.org/wiki/Alexander_Fleming. 10. https://en.wikipedia.org/wiki/Baker%27s_yeast. 11. https://en.wikipedia.org/wiki/Sourdough. 12. https://en.wikipedia.org/wiki/Root_beer. 13. Ian Spencer Hornsey (2003). A history of beer and brewing, page 8. Royal Society of Chemistry, 2003. "A similar, low alcohol (0.5–1.0%) drink, kvass … may be a 'fossil beer'". 14. Jarrell J., Cal T., Bennett J.W. (2000). "The Kombucha Consortia of yeasts and bacteria". Mycologist. Elsevier. 14 (4): 166–170. doi:10.1016/S0269-915X(00)80034-8. 15. Jonas Rainer, Farah Luiz F. "Production and application of microbial cellulose". Polymer Degradation and Stability. 59 (1-3): 101–106. doi:10.1016/s0141-3910(97)00197-3. 16. Julia Volhina (3 December 2011). "Kvass (Russian Fermented Rye Bread Drink)". EnjoyYourCooking. 17. Kurtzman C.P., Fell J.W. (2005). in: The Yeast Handbook, Gábor P, de la Rosa CL, eds. Biodiversity and Ecophysiology of Yeasts. Berlin: Springer. pp. 11–30. ISBN 978-3-540- 26100-1. 18. Madigan M, Martinko J, eds. (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall. ISBN 0-13-144329-1. OCLC 57001814. 19. Microbiology Laboratory Theory and Application.' Michael Leboffe and Burton Pierce, 2nd edition. pp.317 20. Moore D, Robson GD, Trinci AP, eds. (2011). 21st Century Guidebook to Fungi (1st ed.). Cambridge University Press. ISBN 978-0521186957. 21. Mohamed Abou-Donia (2015). Mammalian Toxicology. John Wiley & Sons, 2015. ISBN: 1118682858, 9781118682852. 22. Naveen Kango (2010). Textbook of Microbiology. I. K. International Pvt Ltd, 2010. ISBN: 9380026447, 9789380026442. 23. PELCZAR (2010). MICROBIOLOGY:APPLICATION BASED APPROACH. Tata McGraw-Hill Education. ISBN: 1259081753, 9781259081750. 24. "Potato Dextrose Broth". Merck KGaA. Archived from the original on 2006-05-16. Retrieved 2005-05-29. 25. Pulvirenti A., Solieri L., Gullo M., De Vero L., Giudici P. (2004). Occurrence and dominance of yeast species in sourdough. Lett Appl Microbiol. 2004;38(2):113-7.

217

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

26. Prescott Harley, Klein. Microbiology (7th ed.). London: McGraw–Hill. p. 1040. ISBN 978-0-07-110231-5. 27. R. Saravanamuthu (2010). Industrial Exploitation of Microorganisms. I. K. International Pvt Ltd. ISBN: 9380026536, 9789380026534. 28. "Red yeast rice (Monascus purpureus)" (2010). Mayo Clinic. 2009-09-01. http://www.mayoclinic.com/health/red-yeast-rice/NS_patient-redyeast. Retrieved 2010-02-01. 29. Rex Bookstore, Inc. (2007).Foundations in Microbiology' (sixth Edition). ISBN: 0071262326, 9780071262323. 30. Sansing G.A., Lillehoj E.B., Detroy R.W. (1976). Synergistic toxic effect of citrinin, ochratoxin A and penicillic acid in miceToxicon14213220 31. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages. 32. Simona Ivana, Nicolae Starciuc, Delia Costache, Cristea Costache (2017). Penicillin and Sir Alexander Fleming: Historical Perspectives. Bio-Innovations in Translational Immunology and Applied Microbiology, Vol. 1 (1), pp. 11-13, December, 2017 Issue.Available online http://indigoroyal.com. 33. Shore R. (15 February 2011). "Eureka! Vancouver scientists take the headache out of red wine". The Vancouver Sun. Archived from the original on 17 February 2011. 34. S. K. Soni (2007). Microbes: A Source of Energy for 21st Century. New India Publishing. ISBN: 8189422146, 9788189422141. 35. "The Art of Mould" (2015). (http://discardstudies.com/2012/01/02/the-art-of- mould/), (Discard Studies), retrieved May 11, 2015. 36. Young Linda, Cauvain Stanley P. (2007). Technology of Breadmaking. Berlin: Springer. p. 79. ISBN 0-387-38563-0. The scientific name for baker's yeast is Saccharomyces cerevisiae.

218

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

CHAPTER 15 The microbiome (optional course)

15.1. General characteristics 15.2. Beneficial microbes 15.3. Definition of a healthy microbiome 15.4. Enterotypes of human gut microbiome

Campylobacter jejuni is a species of bacteria commonly found in animal feces. It is curved, helical-shaped, non- spore forming, Gram-negative, and microaerophilic. C. jejuni is one of the most common causes of human gastroenteritis in the world. C. jejuni is commonly associated with poultry, and it naturally colonises the digestive tract of many bird species. It is normally a harmless commensal of the gastrointestinal tract in cattle.

It can cause campylobacteriosis Campylobacter jejuni in calves. nickname ”Seahorse”

Learning objective

Metatranscriptomics, metaproteomics and metabonomics will be useful to explore the functional aspects of the gut microbiome

Key points

The gut microbiome is very important in maintaining both gastrointestinal and immune function

219

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

15.1. The microbiome

Microbial communities carry out the majority of the biochemical activity on the planet, and they play integral roles in processes including metabolism and immune homeostasis. The Nobel Prize laureate Joshua Lederberg called this ecosystem a microbiome [23].

Feature 15.1. Joshua Lederberg

[13] Joshua Lederberg, (May 23, 1925 – February 2, 2008) was an Americanmolecular biologist known for his work in microbial genetics, artificial intelligence, and the United States space program. He was just 33 years old when he won the 1958 Nobel Prize in Physiology or Medicine for discovering that bacteria can mate and exchange genes [27]. He shared the prize with Edward L. Tatum and George Beadle who won for their work with genetics. A microbiota is "the ecological community of commensal, symbiotic and pathogenic

microorganisms that literally share our body space" [15].

Joshua Lederberg coined the term, emphasizing the importance of microorganisms [22; 27] inhabiting the human body in health and disease .

The human body is inhabited by trillions of bacteria and other microbes. The human microbiome plays a key role in human health and is associated with numerous diseases. The human body is home to numerous microbial species and several complex microbial ecosystems. Nowadays, advances in sequencing technologies and metagenomics allow researchers to characterize the composition of species that inhabit the human body and the variation these communities exhibit in health and disease. Recent studies of the microbiome have found tremendous variation among healthy individual [11] and demonstrated clear association between species composition and several host phenotypes including obesity [25; 16], inflammatory bowel disease (IBD) [19] and diabetes [19], as well as with external factors such as diet. Diet has been demonstrated to be a strong predictor of intestinal microbiota composition and may, accordingly, be the primary link between host macroecological state and community composition [8; 30].

220

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Fig. 15.1. Human microbiome

Obesity, metabolic syndrome and type 2 diabetes are major public health challenges, affecting approximately 26 million children and adults in the US [5]. Recently, interest has surged in the possible role of the intestinal microbiome as a potential contributor to the rapidly increased prevalence of obesity [24]. Microbes are also implicated in depression. Borrelia burgdorferi (Lyme diseases) causes depression in up to 2/3 of all cases. Gut microbes are also implicated in anxiety disorders. The bacteria Campylobacter jejuni has been shown to cause anxious behavior in mice. Autistic populations have a unique microbiome consisting of more clostridial species. Half of all autistic children with gastrointestinal dysfunction were found to have the bacteria Sutterella [10; 28]. Microbiome is regarded as a ”newly discovered organ” and is understood to have potentially over whelming impact on human health. Modern dietary and nondietary intake may serve to unbalance the beneficially balanced population of our non-human intimate colleagues and partners (our microbiome ”zoo”) [23]. Metatranscriptomics, metaproteomics and metabonomics will be useful to explore the functional aspects of the gut microbiome from top down. Real-time analysis of the intestinal microbiome is a useful tool in the development of personalized approaches to targeted therapies [24]. Metabonomics can be described as the study of metabolic responses to chemicals, the environment and diseases and involves the computational analysis of spectral metabolic data that provide information on temporal changes to specific metabolites.Metabonomics provides global

221

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

metabolic profiling of an individual in real time. The combination of metabolic profiling and metagenomic studies of gut microbiota permits the study of host and microbial metabolism in great detail [24]. The gut microbial community includes approximately 1014 bacteria that normally reside in the gastrointestinal tract, reaching a microbial cell number that greatly exceeds the number of human cells of the body [24]. The collective genome of those microorganisms (the microbiome) contains millions of genes (a rapidly expanding number, compared to roughly 20,000-25,000 genes in the human genome). This microbial ”factory” contributes to a broad range of biochemical and metabolic functions that the human body could not otherwise perform [24; 17].

Fig. 15.2. The microbiome

skin

oral

vaginal

gut

ral

222

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Recent evidence suggests that the gut microbiota affect nutrient acquisition, energy harvest, and a myriad of host metabolic pathways. The gut microbiota has an important role in regulating weight and may be partly responsible for the development of obesity [24]. Initial evidence of the relationship between obesity and gut microbial composition was reported 3 decades ago, when surgically included weight loss by gastric bypass and weight gain through lesions of the ventromedial hypothalamic nucleus were found to be associated with changes in gut microbial ecology [4; 6; 24]. The gut microbiome is very important in maintaining both gastrointestinal and immune function it is also crucial for the digestion of nutrients, and this fact has been confirmed by studies of germ-free mice [14]. Important metabolic functions of the gut microbiome include the catabolism of dietary toxins and carcinogens, synthesis of micronutrients, fermentation of indigestible food substances and assistance in the absorption of electrolytes and minerals [24]. The production of short-chain fatty acids (ScFAs) by the gut microbiome affects growth and differentiation of enterocytes and colonocytes. The gut microbiome can affect whole-body metabolism and alter physiological parameters in multiple body compartments. Intestinal bacterial taxa differ with respect to their abilities to utilize dietary and host-derived carbohydrates (e.g. mucus components) [24]. Strains of the genus Bifidobacterium contain genes encoding glycan-foraging enzymes that enable these gut bacteria to acquire nutrients from host-derived glycans [7]. Beside their capacity to hydrolyze starch, gut microbes have developed the ability to degrade numerous plant and host-derived glycoconjugates (glycans) and glycosaminoglycans including cellulose, chondroitin sulfate, hyaluronic acid, mucins and heparin [24]. Microbial catabolic enzymes such as endoglycosidases may act on dietary substrates to release complex N-glicans from human milk and other dairy sources. Fluctuations in diet may have functional consequences for bacteria and the host, so that the ”cannibalization” of indigenous mammalian carbohydrates may result in augmentation of beneficial features, prevention of diseases, or predisposition to different disease state. For example, bifidobacteria grown on human milk oligosaccharides stabilize tight junction in the epithelium and promote the secretion of the anti-inflammatory cytokine, interleukin-10 [24]. The biogeography of the microbiome may be relevant because specific genes/pathways such as simple carbohydrate transport phosphotransferase systems are more prominent in the small intestine than in the colon. Intestinal bacteria including probiotics produce a diverse array of fatty acids that may have health-promoting effects. Intestinal bifidobacteria produce conjugated linoleic acid (CLA) which modulate the fatty acid composition in the liver and adipose tissue in murine models [18]. Roux-Y Gastric Bypass (RYGB) surgery is a major bariatric intervention to treat morbid obesity. Before surgery, increased quantities of Bacteroides were observed, but reductions in Bacteroides and enhanced quantities of Proteobacteria were detected following surgery. These microbial population shifts are likely to change the metabolite profiles and the relative preponderance of different fatty acids (SCFAs) [24].

15.2. BENEFICIAL MICROBES

Beneficial microbes such as bifidobacteria and lactobacilli produce biologically active compounds derived from amino acids, including a variety of biogenic amines. Aminoacids derived from dietary protein sources may serve as substrates for luminal bioconversion by the gut microbiome [24].

223

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Nutrients consumed by the host may be converted by intestinal microbes into several bioactive compounds that could affect the health and disease states of the host and the intestinal microbiota [21]. SCFAs are short-chain fatty acids. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases. Such anti-inflammatory aminoacid metabolites may ameliorate pathologic processes in obesity and diabetes [24]. The human body is actually a super-organism that is composed of 10 times more microbial cells than our body cells. The bacterial diversity analysis showed that about 1000 bacterial species are living in our gut and a majority of them belong to the divisions of Firmicutes and Bacteroides. In addition, most people share a core microbiota that comprises 50-100 bacterial species when the frequency of abundance at phylotype level is not considered, and a core microbiome harboring more than 6000 functional gene groups is present in the majority of human gut surveyed till now [33; 2]. The human colon harbors a highly complex microbial ecosystem, at concentrations of 1012 microorganismsper gram of gut content. The gut microbiota composition of each individual is unique and is influenced through a legacy acquired at birth, genotype and physiological status of the host, diet, and lifestyle [32]. Colonic bioconversion of polyphenols is most well described for flavonoids and is highly variable because of three main reasons. First. Large interindividual differences have been noted in the bioconversion of specific flavonoids. This variability can be attributed to the individual colonic microbiota composition and has led to the recognition of low to high flavonoid converters [12]. Second. Small differences in substitution pattern of flavonoids can lead to major changes in colonic bioconversion. Third. A third factor is the dietary context of the ingested polyphenols that can modulate polyphenolmicrobiota interaction. Besides bioconversion of active dietary compounds into less active metabolites, a number of bacterial transformations are also known to produce metabolites with increased biological activity. A good example are the phytoestrogens such as say isoflavones, prenylflavonoids from hops, and lignans, for which the pseudoestrogenic activity is determined by intestinal bacterial activation followed by absorption of the microbial metabolites. The holistic view provided by functional genomics can be overwhelming, thus we have adopted a strategic approach.

224

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Figure 15.1. Biological system complexity

Human intervention trials

Humanized mice models

System In-vivo colonic models

Biological

Complexity

Upper digestive

tract

Organs

Colon Phase and I II

Polyphenols

HostMetabolome

Dietary Context

Food Food Metabolome

Microbiome

Metabolomics data Nitrikinetics

Biological Biological Microbiomics Integration signatures

Microbiome – Metabolome interactions Bioavailability Bloactivity relations

RESEARCH NEWS

Gut microbiome could be regulated through electrical signaling

The UC San Diego biologists discovered in their laboratory work, that a biofilm composed of a single species of Bacillus subtilis bacteria was able to recruit bacteria of a different species (in this case: Pseudomonas aeruginosa), through electrical signaling. Using microfluidic growth chambers, the biologists documented the process by which potassium ion electrical signaling generated by Bacillus subtilis biofilms attracted

distant cells within the chambers to

the edge of electrically oscillating biofilms. Bacteria (Suel et. all) living in biofilm communities communicate with one another electronically through proteins called ”ion channels”, an electrical signaling

method similar to that used by neurons in the human brain. The composition of mixed species bacterial communities, such as our gut microbiome could be regulated through electrical signaling.

225

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

15.3. DEFINITION OF A HEALTH MICROBIOME

Experimental strategy for assessment of the metabolic fate of polyphenols in the human super-organism. Biological complexity can be addressed by combining In Vitro Colonic Models, Humanized Mice Models, and Human Intervention Trials [12]. The resident colonic microbiota can be regarded as a separate organ within the human host, which can perform many functions of which the human host is incapable. These strong and symbiotic microbiota-host interactions have led/to the recognition of humans as superorganisms, in which the colon operates as a bioreactor with a virtually unlimited metabolic potential.

Figure 15.2. Biological complexity of a Healthy Microbiome

Resistant starch scFAs

fermentation hydrolysis

Amines ligans phenols

aromatic Metabolic functions of the

fission indigenous gut Proteins microbiota

phytoestrogens HEALTHY HEALTHY

MICROBIOME deconjugation

Multiple Xenobiotics metabolites (drugs, toxins)

The gut microbiota can carry out a suite of biochemical activities that can convert luminal compounds to secondary metabolites. These conversion reactions can alternately detoxify ingested toxins but in other cases can result in the production of compounds that can be deleterious. The specific composition of the gut microbiota can thus determine the balance between beneficial and harmful chemical conversion in the gut lumen. The gut microbiota, can signal to the host immune system and gut epithelium to set the tone of mucosal immunity. In response to the gut microbiota, the host will secrete a variety of cytokines and host defense effector molecules that can in turn shape the indigenous microbiota community and prime host responses to environmental stimuli. Certain microbial distributions may make a person more susceptible to infection or disease. For example: alteration of the indigenous gut microbiota by antibiotics can put an individual at risk for developing infections from an opportunistic pathogen, such as Clostridium difficile [9].

226

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

The presence of microbes that convert luminal compounds into potential carcinogens also puts one at increased risk for cancer and can lead to adverse responses to chemotherapeutic agents [26]. A recently proposed definition of human health suggested that health can be considered ”a dynamic state of wellbeing characterized by a physical, mental and social potential, which satisfies the demands of a life commensurate with age, culture and personal responsibility” [3]. The core microbiome (based on clusters of orthologous groups) may differ across continents, ethnicities, diets or other factors. The colonizing microbiota are established early in life but can shift with changes in age, diet, geographical location, intake of food supplements and drugs, and likely other causes as well [9]. Recently, the concept that all humans can be divided into one of three discrete gut enterotypes based on the composition of the microbiota was proposed [1]. Enterotypes appear to be independent of gender and nationality. Arecent study suggested that long-term food preferences may contribute to formation of different enterotypes [30]. 1. Bacteroidaceae; Bacteroides; 2. Prevotellaceae; Prevotella; 3. Firmicutes. It can be demonstrated that specific members of a bacterial community can play an important functional role in the realm of resistance to infection. The ability of specific resident microbes to occupy host niches and mount an effective resistance to pathogens may depend on a unique functional activity. For example: transfer of fecal microbes (species of Prevotella) conferred resistance to Citrobacter rodentium infection in a mouse model of infections colitis [29]. ”Nondigestible” components include plant cell wall polysaccharides (including cellulose, xylan, and pectin) and certain storage polysaccharides such as inulin and oligosaccharides that contain bonds that are resistant to mammalian hydrolytic enzymes. Even dietary starch includes an important component (”resistant starch”), that is not fully digested in the small intestine, for example, because of retrogradation or starch granule structure. This nondigested residue provides the major source of diet-derived energy for the growth of microorganisms in the large intestine, and therefore has the potential to profoundly influence microbial ecology and competition between species within the colonic microbial community.

A surprising discovery was the key role of some bacteria emerged as our great friends and symbiotic: 1. Bacteroides thetaiotaomicron: - it is a champion of complex carbohydrates; - it is able to break down the complex metabolism sugars presentin many vegetables and plants into glucose and other simple and digestible sugars; - our genome lacks completely these enzymes since these bacteria inhabits our intestine; - they are the interface that allow us to extract nutrients from oranges, apples, potatoes or wheat germs; - the bacteria fermentspolysaccharides. 2. Bacteroides fragilis: - Polysaccharide A is critical fro the health of the intestine: - produce a complex sugar: polysaccharide A (PSA) to inhibit intestinal inflammation (cholitis); - mice in which Bacteroides does not produce PSA develop chronic inflammation in the gut;

227

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

- the presence of PSA stimulates the development of regulatory T cells and switch off the inflammatory T cells, restoring health. 3. Helicobacter pylori is the etiologic agent of Peptic ulcer: - the study of microbiome has rehabilitee part of the bad reputation of Helicobacter; - in 1980 Barry Marshall and Robin Warren discovered its key role as the causative agent of peptic ulcer; - it is one of the few bacteria that thrive happily in the acidic environment of the stomach that is lethal to most other living forms; - the rate of Helicobacter induced peptic ulcers has dropped by more than 50%; - Martin Blaser, a scientist which has been studying Helicobacter for the past 25 years sees the microbe from a totally different perspective as a commensal; - gastric acid production – cag A: in most people it regulates gastric acidity; - if there is too much acid, it express a gene called cag A and start producing a protein that signal the stomach to stop producing acid. Appetite regulation – Ghrelin: The new fascinating discovery is that another function of Helicobacter is regulating appetite. We know that ghrelin tells the brain that the body needs to eat and leptin signal the brain that the stomach is full and no more food is needed. - children repeatedly exposed to antibiotics have major changes of their interface ecology with wide effects on nutrition, immune response and inflammatory response set point; - this ecological change of intestinal microbiome contribute to today epidemic of obesity; - the microbiome works as a sensor and based upon its ecology determine cell differentiation into fat, muscle or bone; - the abuse of antibiotics interferes with physiological sensing and signaling and causes and overproduction; - the microbial flora regulate the differentiation of T helpers into: Treg, Th17, Th2. 4. Probiotics are live microorganisms that are thought to be beneficial to the host organism. According to the currently adopted definition by FAO/WHO, probiotics are: ”Live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Lactic acid bacteria (LAB) and bifidobacteria are the most common types of microbes used as probiotics. Probiotic are commonly consumed as part of fermented foods with specially added active live cultures; such as yogurt, soy yogurt or as dietary supplements. Probiotics are also delivered in fecal transplants, in which stool from a healthy donor is delivered like a suppository to an infected patient. 5. Prebiotics are non-digestible food ingredients that stimulate the growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial to health. They first identified and named by Marcel Roberfroid in 1995. As a functional food component, prebiotics are conceptually intermediate between foods and drugs.

15.4. ENTEROTYPES OF HUMAN GUT MICROBIOME

An enterotype is a classification of living organisms based on its bacteriological ecosystem in the gut microbiome. There are three human enterotypes: Type 1 is characterized by high levels of Bacteroides; Type 2 has few Bacteroides but Prevotella are common;

228

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Type 3 has high levels of Ruminococcus . Those who eat plenty of protein and animal fats typical of Western diet have predominantly Bacteroides bacteria, while for those who consume more carbohydrates, especially fiber, the Prevotella species dominate [31]. Prevotellas specialize in digesting sugar-covered proteins in mucin, the mixture of viscous proteins in the gut-an ability shared by people with a ruminococcus ecosystem. The only difference identified was in the vitamins produced. Bacteroides had a higher proportion of bacteria that make high amounts of vitamins C, B2, B5 and H, and prevotellan guts had more bacteria that make vitamin B1 and folic acid.

Type 1 Type 2 Veillonella Akkermansia Helicobacter Alkaliphilus Catenibacterium Ruminococcaceae Geobacter Staphylococcus Leuconostoc Prevotella

Parabacteroides Eggerthella Peptostreptococcaceae Bacteroides Clostridiales

Lactobacillus Desulfovibrio Methanobrevibacter Holdemania Escherichia/Shigella Slackia Rhodospirillum

Positive correlation (˂ 0,4) - positive correlation (˂ 0,4) main contributors genero co- occurring with main contributors

Type 3

Sphingobacterium Akkermansia Gordonibacter

Ruminococcus Staphylococcus Ruminococcaceae

Dialister Marvinbryantia Symbiobacterium

229

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

Blood groups, finger prints, iris sc ans and DNA bar codes are several ways to distinguish or identify individual humans. According to Arumugam and colleagues (2011) we can now also be distinguished by the microbial communities in our faeces, our ”enterotypes”.

230

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

References

1. Arumugam M., Raes J., Pelletier E., Le Paslier D., Yamada T., Mende D. R., Fernandes G. R., Tap J., Bruls T., Batto J. M., et al. (2011). Enterotypes of the human gut microbiome, Meta HIT Consortium, Nature 473, 174-180. 2. Baoli Zhu, Xin Wang, Lanjuan Li (2010). Human gut microbiome: the second genome of human body, Protein Cell 2010, 1(8): 718–725 DOI 10.1007/s13238-010-0093-z. 3. Bircher J. (2005). Towords a dynamic definition of health and disease. Med. Health Care Philos. 8, 335-341. 4. Calvani R., Miccheli A., Capuani G., Tomassini Miccheli A., Puccetti C., Delfini M., et al. (2010).Gut microbiome-derived metabolites characterize a peculiar obese urinary metabotype. Int J Obes. 2010;34:1095–8. 5. Cowie C.C., Rust K.F., Ford E.S., Eberhardt M.S., Byrd-Holt D.D., Li C., et al. (2009). Full accounting of diabetes and pre-diabetes in the U.S. population in 1988-1994 and 2005-2006. Diabetes Care. 2009;32:287–94. 6. Clement K. (2011). Bariatric surgery, adipose tissue and gut microbiota. Int J Obes. 2011;35(Suppl 3):S7–15. 7. Dethlefsen L, Relman DA (2010). Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation. Proc Natl Acad Sci U S A. 2010; 108(Suppl 1):4554–61. [PMC free article] [PubMed]. 8. Faith J.J., McNulty N.P., Rey F.E., Gordon J.I. (2011). Predicting a human gut microbiota’s response to diet in gnotobiotic mice. Science. 2011;333(6038):101–104. 9. Fredrik Bäckhed, Claire M. Fraser, Yehuda Ringel, Mary Ellen, Sanders R., Balfour Sartor, Philip M. Sherman, James Versalovic, Vincent Young B., Brett Finlay (2012). Defining a Healthy Human Gut Microbiome: Current Concepts, Future Directions, and Clinical Applications, Volume 12, Issue 5, p. 611–622, 15 November 2012, Cell Host and Microbe, DOI: http://dx.doi.org/10.1016/j.chom.2012.10.012. 10. Finegold SM, Molitoris D, Song Y, et al. (2002). "Gastrointestinal Microflora Studies in Late‐Onset Autism". Clinical Infectious Diseases 35 (Suppl 1): S6–S16. doi:10.1086/341914. 11. Huttenhower C., et al. (2012). Human Microbiome Project Consortium (2012) Structure, function and diversity of the healthy human microbiome. Nature 486(7402):207–214. 12. John van Duynhovena, Elaine E. Vaughana, Doris M. Jacobsa, Robèr A. Kempermana, Ewoud J. J. van Velzena, Gabriele Grossa, Laure C. Rogera, Sam Possemiersd, Age K. Smildec, Joël Doréb, Johan A. Westerhuisc, Tom Van de Wieled (2010). Metabolic fate of polyphenols in the human superorganism. vol. 108 no. Supplement 1, 4531-4538. DOI10.1073/pnas.1000098107. PubMed ID20615997. 13. Joshua Lederberg, Rockefeller University's fifth president, dies at 82. 14. June L. Round, Sarkis K. Mazmanian (2009). The gut microbiome shapes intestinal immune responses during health and disease. Nat Rev Immunol. Author manuscript; available in PMC 2014 Jul 14.Nat Rev Immunol. 2009 May; 9(5): 313–323. doi: 10.1038/nri2515. 15. Lederberg J., McCray A.T. (2001). ’Ome sweet ’omics—a genealogical treasury of words. Scientist. 2001;15:8. 16. Ley R.E. (2010). Obesity and the human microbiome. Curr Opin Gastroenterol 26(1):5–11. 17. Neish, A.S. (2009). Microbes in gastrointestinal health and disease. Gastroenterology, 136, 65-80. 18. O'Shea E.F., Cotter P.D., Stanton C., Ross R.P., Hill C. (2012). Production of bioactive substances by intestinal bacteria as a basis for explaining probiotic mechanisms:

231

GENERAL MICROBIOLOGY SIMONA -plasticine collection - IVANA

bacteriocins and conjugated linoleic acid. Int J Food Microbiol. 2012 Jan 16;152(3):189-205. doi: 10.1016/j.ijfoodmicro.2011.05.025. Epub 2011 Jun 14. 19. Qin J., et al. (2010). MetaHIT Consortium (2010). A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464(7285):59–65. 20. Qin J, et al. (2012). A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490(7418):55–60. 21. Peera Hemarajata, James Versalovic (2013).Effects of probiotics on gut microbiota: mechanisms of intestinal immunomodulation and neuromodulation. Therap Adv Gastroenterol. 2013 Jan; 6(1): 39–51. doi: 10.1177/1756283X12459294. 22. Simona Ivana (2016). Manual of General Microbiology, New Edition, Plasticine Collection. Printech Publishing House. ISBN: 978-606-23-0640-3, 194 pages. 23. Simona Ivana, Nicolae Starciuc, Delia Costache, Cristea Costache(2017). The biodiversity of microbiome and human health. BITIAM December 2017 Vol: 1(1): 1-10. http://indigoroyal.com/. 24. Sridevi Devaraj, Peera Hemarajata, James Versalovic (2013). The Human Gut Microbiome and Body Metabolism: Implications for Obesity and Diabetes. Clin Chem. Author manuscript; available in PMC 2014 Apr 3.Clin Chem. 2013 Apr; 59(4): 617–628. Published online 2013 Feb 11. doi: 10.1373/clinchem.2012.187617. 25. Turnbaugh P.J., et al. (2009). A core gut microbiome in obese and lean twins. Nature 457(7228):480–484. 26. Wallace B. D., Wang H., Lane K. T., Scott J. E., Orans J., Koo J. S., Venkatesh M., Jobin C., et al. (2010). Alleviating cancer drug toxicity by inhibiting a bacterial enzyme, Science 330, 831-835. 27. Warwick K. (2001). "The Joshua Lederberg Papers: Profiles in Science, National Library of Medicine", Biography, Volume 24, Number 4, Fall 2001, pp. 978-982. 28. Williams BL, Hornig M, Parekh T, Lipkin WI. (2012). "Application of Novel PCR- Based Methods for Detection, Quantitation, and Phylogenetic Characterization of Sutterella Species in Intestinal Biopsy Samples from Children with Autism and Gastrointestinal Disturbances". MBio 3(1):e00261–e00211. doi:10.1128/mBio.00261-11. 29. Willing B. P., Russell S. L., Finlay B. B. (2011). Shifting the balance: antibiotic effects on host-microbiota mutualism. Nat. Rev. Microbiol. 9, 233-243. 30. Wu G.D., et al. (2011). Linking long-term dietary patterns with gut microbial enterotypes. Science. 2011;334(6052):105–108. 31. Zimmer Carl (2011). "Bacteria Divide People Into 3 Types, Scientists Say". The New York Times. Retrieved 21 April 2011. a group of scientists now report just three distinct ecosystems in the guts of people they have studied. 32. Zoetendal, E.G., Cheng, B., Koike, S., and Mackie, R.I. (2004). Molecular microbial ecology of the gastrointestinal tract: from phylogeny to function. Curr Issues Intest Microbiol 5: 31–48. 33. Zhu B., Wang X, Li L. (2010). Human gut microbiome: the second genome of human body. Protein Cell. 2010 Aug;1(8):718-25. doi: 10.1007/s13238-010-0093-z. Epub 2010 Aug 28.

232