Chapter 4

A Survey of Prokaryotic Cells and Microorganisms Characteristics of cells and life

All living things (single and multicellular) are made of cells that share some common characteristics:

– Basic shape – spherical, cubical, cylindrical

– Internal content – cytoplasm, surrounded by a membrane

– DNA chromosome(s) , ribosomes, metabolic capabilities

Two basic cell types: eukaryotic and prokaryotic

Characteristics of cells Eukaryotic cells: animals, plants, fungi, and protists – Contain membrane-bound organelles that compartmentalize the cytoplasm and perform specific functions – Contain double-membrane bound nucleus with DNA chromosomes

Prokaryotic cells: and archaea – No nucleus or other membrane-bound organelles Characteristics of life

• Reproduction and heredity – genome composed of DNA packed in chromosomes; produce offspring sexually or asexually

• Growth and development

• Metabolism – chemical and physical life processes

• Movement and/or irritability – respond to internal/external stimuli; self-propulsion of many organisms

• Cell support, protection, and storage mechanisms – cell walls, vacuoles, granules and inclusions

• Transport of nutrients and waste

Whittaker’s 5 kingdom classification Bacterial shapes, arrangements, and sizes • Vary in shape, size, and arrangement but typically described by one of three basic shapes:

– Coccus – spherical

– Bacillus – rod

• Coccobacillus – very short and plump

• Vibrio – gently curved

– Spirillum – helical, comma, twisted rod,

• Spirochete – spring-like Common bacterial shapes Pleomorphism Variation in cell shape and size within a single species

Some species are noted for their pleomorphism: Corynebacterium diphteriae what are metachromatic granules? Volutin granules Inorganic polyphosphate granules

Appear red when stained with methylene blue

Bacterial arrangements Arrangement of cells is dependent on pattern of division and how cells remain attached after division:

– Cocci: • Singles • Diplococci – in pairs • Tetrads – groups of four • Irregular clusters • Chains • Cubical packets (sarcina)

– Bacilli: • Diplobacilli • Chains • Palisades Classification systems for Prokaryotes

1. Microscopic morphology

2. Macroscopic morphology – colony appearance

3. Bacterial physiology

4. Serological analysis

5. Genetic and molecular analysis Bacterial based on Bergey’s Manual

Bergey’s Manual of Determinative Bacteriology – five volume resource covering all known prokaryotes

– Classification based on genetic information –phylogenetic

– Two domains: Archaea and Bacteria

– Five major subgroups with 25 different phyla

– Table 4.3 Structural plan of a prokaryote Structure of a bacterial cell Model of a bacterial cell

Cytoplasm = consists of a gel-like network

Cell membrane = encloses the cytoplasm

Cell wall = covers the cell membrane

Nucleoid = area of the cytoplasm that contains the chromosome in the form of looped coils

Flagellum = external helical filament whose rotary motor propels the cell External structures Appendages

Motility Attachment Channels Fimbriae Flagella Pili

Axial filaments (periplasmic flagella)

Surface coating- Glycocalyx Flagella

Prokaryotes that are motile generally swim by means of flagella

• Rotates 360o

• Functions in motility of cell through environment Flagella Filament – long, thin, helical structure composed of protein flagellin

The filament is rotated by a motor driven by the proton motive force

Hook – curved sheath

Basal body – stack of rings firmly anchored in cell wall

http://www.rowland.harvard.edu/labs/bacteria/movies/misc.php Flagellar arrangements Monotrichous – single Amphitrichous – flagella at both flagellum at one end ends of cell Vibiro cholerae

Aquaspirillum sp.

Lophotrichous – small bunches Peritrichous – flagella dispersed emerging from the same site all over surface of cell Proteus vulgaris

Spirillum serpens www.microbeworld.org Flagellar responses Guide bacteria in a direction in response to external stimulus: Chemical stimuli – chemotaxis; positive and negative Light stimuli – phototaxis Signal sets flagella into motion clockwise or counterclockwise: Counterclockwise – results in smooth linear direction – run Clockwise – tumbles Periplasmic flagella

Internal flagella, enclosed in the space between the outer sheath and the cell wall peptidoglycan

Produce cellular motility by contracting and imparting twisting or flexing motion

Examples: Spirochetes like Borrelia burgdorferi Fimbriae

Fine, proteinaceous, hairlike bristles emerging from the cell surface

Function in adhesion to other cells and surfaces Pili Rigid tubular structure made of pilin protein

Mostly found in Gram-negative bacteria

Function to join bacterial cells for partial DNA transfer called conjugation

Pili involved in conjugation are called sex pilus or F-pilus Glycocalyx Coating of molecules external to the cell wall, made of sugars and/or proteins Two types: 1. Slime layer - loosely organized and attached 2. Capsule - highly organized, tightly attached

Functions of the Glycocalyx Protect cells from dehydration and nutrient loss

Inhibit killing by white blood cells by phagocytosis, contributing to pathogenicity

Attachment - formation of biofilms How biofilms form Biofilm on a catheter Inside the bacterial cell

Cell cytoplasm:

– Dense gelatinous solution of sugars, amino acids, and salts

– 70-80% water

• Serves as solvent for materials used in all cell functions

Nucleoid • Chromosome – Single, circular, double-stranded DNA molecule that contains all the genetic information required by a cell

• Plasmids

– Free small circular, double-stranded DNA

– Not essential to bacterial growth and metabolism

– Used in genetic engineering - readily manipulated and transferred from cell to cell

Bacterial ribosome Ribosomes

– Made of 60% ribosomal RNA and 40% protein

– Consist of two subunits: large and small

– Prokaryotic differ from eukaryotic ribosomes in size and number of proteins

– Site of protein synthesis

– Found in all cells A portion of an E. coli chromosome being transcribed (left to right) and being simultaneously translated. The arrow points to the putative site where RNA polymerase is first bound to the DNA. The chains of dark bodies are polysomes, that is, several ribosomes on the same mRNA molecule.

Miller OL Jr, Hamkalo BA, Thomas CA Jr. Visualization of bacterial genes in action. Science. 1970 Jul 24;169 (3943) :392-5

http://schaechter.asmblog.org/schaechter/ Bacterial internal structures Inclusions and granules Intracellular storage bodies

Vary in size, number, and content

Bacterial cell can use them when environmental sources are depleted

Magnetosomes Aquaspirillum sp. poly β hydroxyl butyrate (PHB) granules

Ralstonia eutropha

Wahl et al. BMC Microbiology 2012, 12:262 Bacterial internal structures Gas vesicles = to increase buoyancy

Filaments (chains of cells) of the Gram-negative phototroph Halochromatium roseum. Gas vesicles (seen as white spots) provide buoyancy, enabling the phototroph to remain at the surface of the water, exposed to light. Source: P. Anil Kumar et al. 2007. Int. J. Syst. Evol. Microbiol. 57:2110–2113. Bacterial internal structures Cytoskeleton – Many bacteria possess an internal network of protein polymers that is closely associated with the cell wall Bacterial internal structures Endospores – Inert, resting, cells produced by some G+ genera: Clostridium, Bacillus, and Sporosarcina • Have a 2-phase life cycle: – Vegetative cell – metabolically active and growing – Endospore – when exposed to adverse environmental conditions; capable of high resistance and very long-term survival

– Sporulation - formation of endospores • Hardiest of all life forms • Withstands extremes in heat, drying, freezing, radiation, and chemicals • Not a means of reproduction

– Germination - return to vegetative growth

Sporulation cycle Endospores

• Dehydrated, metabolically inactive

• Thick coat

• Longevity verges on immortality, 250 million years

• Resistant to ordinary cleaning methods and boiling

• Pressurized steam at 120oC for 20-30 minutes will destroy them The cell envelope External covering outside the cytoplasm

Composed of two basic layers: – Cell wall and cell membrane

Maintains cell integrity

Two different groups of bacteria demonstrated by Gram stain:

– Gram-positive bacteria: thick cell wall composed primarily of peptidoglycan and cell membrane

– Gram-negative bacteria: outer cell membrane, thin peptidoglycan layer, and cell membrane

Structure of cell walls Determines cell shape, prevents lysis due to changing osmotic pressures

Peptidoglycan is the primary component:

– Unique macromolecule composed of a repeating framework of long glycan chains cross-linked by short peptide fragments

Structure of cell walls Determines cell shape, prevents lysis due to changing osmotic pressures

The bacterial cell wall (sacculus) consists of a single interlinked molecule.

Peptidoglycan (murein) is the primary component:

Unique macromolecule composed of a repeating framework of long glycan chains cross-linked by short peptide fragments

http://schaechter.asmblog.org/schaechter/2008/12/the-secrets-of-the-sacculus.html Peptidoglycan (Murein)

Peptidoglycan is unique to bacteria

Thus, the enzymes responsible for its biosynthesis make excellent targets for antibiotics

Penicillin inhibits the transpeptidase that cross-links the peptides

Vancomycin prevents cross-bridge formation by binding to the terminal D-Ala-D-Ala dipeptide

Unfortunately, the widespread use of such antibiotics selects for evolution of resistant strains Gram-positive cell wall

– 20-80 nm thick peptidoglycan

– Includes teichoic acid and lipoteichoic acid: function in cell wall maintenance and enlargement during cell division; move cations across the cell envelope; stimulate a specific immune response

– Some cells have a periplasmic space, between the cell membrane and cell wall. Gram-negative cell wall – Inner and outer membranes and periplasmic space between them contains a thin peptidoglycan layer

– Outer membrane contains lipopolysaccharides (LPS) • Lipid portion (endotoxin) may become toxic when released during infections • May function as receptors and blocking immune response • Contain porin proteins in upper layer – regulate molecules entering and leaving cell

Periplasmic space

High magnification image of the envelope showing the plasma membrane (PM) enclosed by a low-density inner wall zone (IWZ) which is bound by a high-density outer wall zone (OWZ). Matias, V. R. F. and Beveridge, T. J. (2005), Cryo-electron microscopy reveals native polymeric cell wall structure in Bacillus subtilis 168 and the existence of a periplasmic space. Molecular Microbiology, 56: 240–251. Structures of Gram-positive and Gram-negative bacterial cell walls Structures of Gram-positive and Gram-negative bacterial cell walls Comparison of Gram-positive and Gram-negative cell walls Nontypical cell walls

Some bacterial groups lack typical cell wall structure, i.e., Mycobacterium and Nocardia

– Gram-positive cell wall structure with lipid mycolic acid (cord factor)

• Pathogenicity and high degree of resistance to certain chemicals and dyes

• Basis for acid-fast stain used for diagnosis of infections caused by these microorganisms

Some have no cell wall, e.g. Mycoplasma sp. – Cell wall is stabilized by sterols – Pleomorphic Cell membrane structure • Phospholipid bilayer with embedded proteins – fluid mosaic model • Functions in:

– Providing site for energy reactions, nutrient processing, and synthesis – Passage of nutrients into the cell and discharge of wastes – Cell membrane is selectively permeable Major prokaryote taxonomic groups

• Domain Archaea – primitive, adapted to extreme habitats and modes of nutrition

• Domain Bacteria:

Phylum – Gram-negative cell walls

Phylum Firmicutes – mainly Gram-positive with low G + C content

Phylum Actinobacteria – Gram-positive with high G + C content Diagnostic scheme for medical use Uses phenotypic qualities in identification

Restricted to bacterial disease agents

Divides bacteria based on

(i) cell wall structure,

(ii) shape,

(iii) arrangement, and

(iv) physiological traits Medically important bacteria Species and Subspecies

Species – a collection of bacterial cells which share an overall similar pattern of traits in contrast to other bacteria whose pattern differs significantly

Strain or variety – a culture derived from a single parent that differs in structure or metabolism from other cultures of that species (biovars, morphovars)

Type – a subspecies that can show differences in antigenic makeup (serotype or serovar), susceptibility to bacterial viruses (phage type) and in pathogenicity (pathotype)

Prokaryotes with unusual characteristics

Free-living nonpathogenic bacteria

Photosynthetic bacteria – use photosynthesis, can synthesize required nutrients from inorganic compounds

Cyanobacteria (blue-green algae)

Green and

Gliding, fruiting bacteria

Cyanobacteria (blue-green algae) Gram-negative cell walls

Extensive thylakoids with photosynthetic chlorophyll pigments and gas inclusions

Green and Purple Sulfur bacteria

Photosynthetic

Contain photosynthetic pigment bacteriochlorophyll

Do not give off oxygen as a product of photosynthesis

Unlike plants, algae, and cyanobacteria, do not use water as their reducing agent, so no oxygen is produced

Use hydrogen sulfide, which is oxidized to produce granules of elemental sulfur.

This sulfur may be oxidized to form sulfuric acid. Gliding and Fruiting bacteria Gram-negative E.g.: Myxobacteria sp. Glide over moist surfaces

L- forms L- forms arise from mutations of cell wall forming genes

Can be induced in lab by treating bacteria with lysozyme or penicillin

Treated Gram positive cells lose cell walls completely forming protoplast – fragile cell bound by only membrane

Treated Gram negative cells lose peptidoglycan but retain outer membrane forming spheroplast – less fragile than protoplast

http://schaechter.asmblog.org/schaechter/2010/05/mysteries- of-the-bacterial-lform-can-some-of-them-be-unveiled.html Unusual forms of medically significant bacteria

Mycoplasmas

Lack cell wall

Cell membrane has sterols- prevents lysis of cells

Pleomorphic

Mycoplasma pneumoniae: primary atypical pneumonia

Colony shows “fried egg” morphology

G.A. Meloni et al. J. Gen Microbiol. 1980 Unusual forms of medically significant bacteria Rickettsias

• Obligate intracellular parasites Very tiny, Gram-negative bacteria

Most are pathogens

Cannot survive or multiply outside of a host cell

Rickettsia rickettisii – Rocky Mountain spotted fever

Transmitted by ticks and fleas http://wwwnc.cdc.gov/travel/yellowbook/2014/chapter-3-infectious-diseases-related-to- travel/rickettsial-spotted-and-typhus-fevers-and-related-infections-anaplasmosis-and-ehrlichiosis Unusual forms of medically significant bacteria

Chlamydias

Tiny, Obligate intracellular parasites, Gram-negative bacteria

Not transmitted by arthropods

Chlamydia trachomatis – severe eye infection and one of the most common sexually transmitted diseases

Chlamydia pneumoniae – lung infections

Chlamydia psittaci - psittacosis (parrot fever) is a zoonotic infectious disease contracted from parrots

http://www.cdc.gov/std/chlamydia/ HeLa cells infected with Clamydia trachomatis

Suchland R J et al. Infect. Immun. 2000;68:360-367 Comparing mycoplasma, rickettsias, chalmydia, viruses

Mycoplasma Rickettsia Chlamydia Viruses Size 0.1-0.3 µm 0.25-0.4 µm 0.3 µm nm Cell wall No Yes Yes No Gram stain N/A Gram negative Gram negative N/A

Obligate intracellular No Yes Yes Yes parasites Growth in cell free Yes No No No media

1 µm = 1000 nm Archaea: The other prokaryotes More closely related to Eukarya than to Bacteria

Have unique membrane lipids and cell walls

Archaea Live in the most extreme habitats in nature, extremophiles

Adapted to heat, salt, acid pH, pressure, and atmosphere

Includes: methane producers, hyperthermophiles, extreme halophiles, sulfur reducers