FUNDAMENTALS 2 Scribe: LOUISA WARREN

10-18-2010 Proof: CHRISTINE SIRNA

YOTHER BACTERIAL PHYSIOLOGY Page 9 of 10

I. GRAM-NEGATIVES [S30]:

a. Cytoplasmic membrane similar to that of gram positive

b. Cell wall following that

c. Then it starts to get different- outside of the cell wall, there is another membrane

i. This is called the outer membrane (OM)

ii. It is not like the cytoplasmic membrane (inner membrane)

iii. OM is lipopolysaccharide (LPS), and the other structures on here are various proteins

II. GRAM-NEGATIVES [S31]

a. Gram negative differs from gram positive in that it has a very thin peptidoglycan (PG) layer- about 2 nm in structure

i. If you were to take all of this PG and add it up, it is about enough to go around the cell one time

ii. But it still has cross-links to give it integrity

1. Like the cross-links in the gram positive cell wall

2. Gives it a double layer with thickness of 2 nm

3. Still thin compared to gram positive cell wall

b. No Teichoic acids in gram negative- they are unique to gram positive

c. In between Inner and outer membrane is the periplasmic space

i. Site of many digestive and protective enzymes

ii. Some mechanisms that allow for antibiotic resistance contained here

iii. Where transport occurs from the outside of the outermembrane to the inside of the cell

iv. Where the PG is located

d. Outer membrane is found outside the PG

i. Primary function of OM: blocks entry of very large molecules (ones greater than 800 daltons)

ii. PG is attached to the OM by a lipoprotein

iii. The OM is not a typical lipid bilayer- it is made of an LPS, which is the outer leaflet of the OM

iv. If you look back (S27), the inner leaflet of the OM has typical lipids (the black structure) and outside of that is LPS (green)

1. The outer leaflet is not typical in the bilayer

v. Transport is important for the OM- things have to get through there to get to the cell, and there are specific mechanisms for that

III. LIPOPOLYSACCHARIDE (LPS) [S32]

a. LPS is endotoxin- responsible for toxic shock- fever, hypotension, all of the properties of toxic shock

b. Looking at the membrane you see the inner leaflet, then the outer leaflet of the OM which is made of Lipid A, followed by the core polysaccharide further outside, then outside is the O Antigen (O Ag)

c. Lipid A

i. Anchoring component that allows LPS to be the outer part of the OM

ii. Has the toxic properties of LPS (endotoxic shock)

d. Core polysaccharide is short, made of 7 or so sugars

i. Structure varies with different bacterial species

ii. When you talk about the species, back to the Borrelia example

1. Borrelia was the genus

2. Burgdorferi was the species (the more specific part of the bacteria)

3. So the core polysaccharide will vary with the different Borrelia species, or the different Salmonella species, for example

e. O Antigen

i. Made of polysaccharide, repeating units that are 3-5 sugars in length to make a very long chain

ii. Outermost surface of the cell is the O Ag polysaccharide

iii. Can vary with particular strain

1. For example, within Borrelia Burgdorferi, there can be strain A, B, C, or D- different isolates

2. So the O Ag can vary with different isolates of the same bacterial species

3. Becomes important in serotyping

a. For example, E. Coli has hundreds of O Ag

b. Can be serotyped using antiserum specific for individual O Ag

c. E. Coli O157- O refers to the O Antigen, and that is the serum type

IV. OPTIONAL FEATURES (GRAM +/-) [S33]

a. Remember:

i. Unique to gram positive- teichoic acids, lipoteichoic acids, thick cell walls

ii. Gram negative- thin cell walls, OM, LPS

b. These features can occur on either gram positive or gram negative cells

c. These features may not be essential for viability, but may make it possible for the cells to be pathogenic organisms that cause disease or survive in multiple different environments

d. Capsules

i. Almost always made of polysaccharides

1. A few exceptions

2. Bacteria that causes anthrax has capsule made of protein

ii. Can work in a couple of different ways to block the complement or antiphagocytosis

1. The cell wall, particularly in gram positive, activates complement with C3b binding of the complement

2. Phagocytic receptors normally recognize this and allow it to be phagocytized

3. Capsule can come along and block the phagocytic receptors from being able to recognize that deposited complement

4. Alternatively, capsule can be there to block activation of complement

5. So it can either block activation or block the recognition if activation has occurred and C3b has deposited

6. In the gram negative cell, the O Ag would be the outside part, and what activates the complement is the LPS

a. The blocking can occur in the same way- either preventing it from being recognized or from ever taking place

e. Surface Proteins are anchored

i. Can be anchored in the teichoic acid, PG, OM

ii. Also serve functions of attachment and antiphagocytosis (both capsules and surface proteins can serve attachment functions)

iii. One important polysaccharide that serves an attachment function is an oral bacteria, streptococcus mutans

1. Has an outer polysaccharide glucan polymer that is essential for being able to attach to the surface of the tooth

2. In the absence of that, it is not able to anchor and cause dental caries

iv. One of the bacteria that causes disease- streptococcus pneumoniae- causes pneumonia in the elderly, recurrent ear infections in children, also causes many eye infections

1. The capsule is an absolutely essential component for it to cause systemic disease

2. Non-encapsulated organisms are what cause eye infections

3. So it is a unique part of the environment, and eye infections are the only instance where capsule is not essential for streptococcus pneumoniae

f. Flagella are native proteins

i. Structure shown in color

ii. Have a basal hook/body that anchors them to membranes

iii. Flagella spins around- this allows for motion to propel the cell

iv. For motility, chemotaxis, getting bacteria to things that are good and away from things that are bad, and important in virulence

v. Many different kinds

1. Peritrichous- all around the cell

2. Unipolar- on one end of the cell

3. Bipolar- two

g. Capsules in pictures on left show when bacteria is grown on agar plates

i. Strain on the right is the same as what is on the left

ii. Right group is unable to produce capsule- so what you see in the colony on a plate (left) is the large amount of capsule the bacteria is able to make

iii. Under the microscope, the chains with black dots are streptococci growing in the cell

1. This is a capsule that has been reacted with an antibody to emphasize the capsule, and you can see large amounts of capsule around the cell

h. Bacteria that make capsules have a huge amount on the outer surface of the cell

V. OPTIONAL FEATURES (GRAM +/-) [S34]

a. Pili are proteins, shorter and smaller than flagella

i. Many involved in attachment

ii. Others involved in gene transfer (in later lecture)

b. Toxins

i. Excreted from the cell to act on host cells

ii. Not the same as endotoxin

1. Endotoxin is LPS, which is uniquely gram negative

2. Endotoxin is not excreted from the cell; it remains attached with the cell

iii. Toxins can be made by gram positive or gram negative bacteria, and they act on host cells

c. There are many enzymes that bacteria make

i. Hyaluronidases, proteases, DNAses

ii. Need to be able to degrade things in their environment not only to survive by utilizing them as nutrition but also to invade through tissues

d. Endospores are dehydrated cells

i. Survive long-term in dry conditions and extreme environments- why they are useful in terrorist activities

ii. Can rehydrate to form normal bacterial cell, which can cause disease

1. Anthrax spores can be inhaled and germinate inside the body/ lungs

iii. Mainly formed by 2 bacteria

1. Clostridium- causes food poisoning/ botulism- spores are important in passing the disease along

2. Bacillus

VI. BACTERIAL GROWTH AND METABOLISM [S35]

VII. GROWTH REQUIREMENTS [S36]

a. Bacterial cell is mostly made of water- about 70% is water, and the rest is what we talked about in the beginning of lecture

b. Carbon and energy sources are required for growth for all bacteria

i. These may be the same thing

ii. Most bacteria and all of the ones that are pathogens are chemoheterotrophs- use an organic molecule for both their carbon and energy source

1. Some of the things they can use frequently are glucose, galactose, all kinds of monosaccharides, disaccharides, organic acids, amino acids, alcohols, ribitols, fatty acids

2. There is probably some bacterium somewhere that can grow on anything

c. A lot of these are utilized in the laboratory not only to grow bacteria but to help identify bacteria based on what conditions they will grow under

VIII. GROWTH REQUIREMENTS- NITROGEN [S37]

a. Nitrogen is a requirement and can come from 2 ways: inorganic source or organic

b. Inorganic

i. Many bacteria can take ammonia and ultimately convert that to Glu or Gln

ii. Others can fix nitrogen to ammonia and then to Glu/Gln

iii. Some are able to do nitrate reduction to get ammonia

iv. Others are denitrifiers- a different thing/ utilization

v. All ultimately get this into pathways as Glu or Gln

c. Organic source

i. Some bacteria are able to take up Glu and Gln directly and utilize them in pathways

IX. GROWTH REQUIREMENTS- OXYGEN [S38]

a. Important for bacterial cell and whether it is able to grow in oxygen for its own survival, but also important clinically (as are all growth requirements) because it is important to process samples in an appropriate way so that they are able to make it to the laboratory where they can be identified

i. Oxygen is important because some cannot survive in aerated environments, and the reason goes back to their basic metabolism

b. There are 5 ways to divide bacteria regarding their oxygen requirements. These are the 2 extremes:

i. Strict Aerobe- requires oxygen under all circumstances to be able grow

1. Cannot ferment (ferment- transfer electrons and protons directly to an organic receptor)

2. Always have to respire (transfer to oxygen) to grow

ii. Strict Anaerobes- Killed in O2

1. The ones you have to worry about transporting, they may not survive for too long in oxygen

2. Always ferment/ transfer to an organic receptor

3. Lack the enzymes necessary to degrade toxic oxygen metabolites

iii. Oxygen itself is not toxic- biproducts of oxygen metabolism are toxic

1. Oxygen can be converted to hydrogen peroxide

a. Toxic to many bacteria

b. Avoid that toxicity through an enzyme called catalase, which breaks this back down to water and oxygen

2. Oxygen can also be converted to a superoxide radical

a. Toxic

b. The enzyme superoxide dismutase breaks that down to oxygen and hydrogen peroxide, which can then be converted by catalase to oxygen and water

3. Anaerobes are strict anaerobes because they lack catalase and superoxide dismutase

c. There are bacteria between aerobes and anaerobes

i. Some lack these enzymes, some only have the machinery for respiration or fermentation

X. GROWTH REQUIREMENTS- OXYGEN [S39]

a. Facultative organisms are able to grow in the presence or absence of oxygen- they do whatever the environment dictates

i. In oxygen, they respire

ii. In the absence of oxygen, they ferment

b. Aerotolerant anaerobes are able to grow with or without oxygen

i. Don’t have machinery to respire

ii. Always do fermentation

iii. Tolerate oxygen because they have the enzymes to degrade the toxic biproducts, but they don’t utilize oxygen in their growth

c. Microaerophiles grow best with low oxygen conditions

i. Able to grow without oxygen but prefer to have oxygen

XI. GROWTH REQUIREMENTS [S40]

a. Temperature

i. Most pathogenic organisms are mesophiles- optimum growth temperature at 20-40 degrees C

ii. There are bacteria that can grow in extremes

1. High temperatures above 50

2. Low temperatures down to 4 and up to 20 degrees

iii. Most pathogens prefer the mid-range- even though some of them can survive at lower temperatures, they will grow best at the higher temperatures

b. pH

i. Grow best at 6 to 8/mostly neutral range, but bacteria will have to survive extremes of pH

1. Bacteria that cause gastrointestinal diseases have to go through the stomach or intestines and be able to survive in the acid of the stomach if they are going to cause disease

c. Many bacteria that cause disease are able to do so because they can survive in a given environment

i. Bacteria that causes pneumonia doesn’t cause GI disease because it can’t survive in the GI tract

ii. They have been selected for by the human host to cause the diseases that they cause- you are causing selective pressure on these with the different environments (pH, aeration, etc)

d. Other things required for growth are usually considered trace elements, but they have to be there

i. Sulfur, phosphorus, various minerals

ii. Growth factors, etc.

iii. The chromosome size is where this “other” can be very small or very large, depending on whether the bacterium is able to synthesize these things on its own

1. Many can synthesize all the amino acids they need, so you don’t have to add them

2. With others like the mycosplasma, you have to add serum and other things to get them to grow in culture

XII. NUTRIENT UPTAKE [S41]

a. Bacteria have to be able to take up the nutrients they are going to survive on, and one of the ways of doing that is by hydrolyzing those using various proteases, nucleases, lipases

b. Have to be able to break down nutrients into something they can take in