Microbiology: Bacterial Structure and Physiology II

Microbiology: Bacterial Structure and Physiology II

Kimberly Watkins pg. 11

Slide 27

The things that make a gram-positive are thick cell wall, teichoic acids and lipoteichoic acids. Those are the big features that would distinguish it from a gram-negative. This is a peptidoglycan backbone with the GlcNac and MurNac and on that is tightly linked a teichoic acid structure. Onto these teichoic acids may be proteins that are bound (which are not shown) to the teichoic acids. In addition, many bacteria, including gram-positive, have what are called capsules, so polysaccharides on their outer surface. Those are probably linked to the peptidoglycan through this GlcNac residue. If you simply isolate for peptidoglycan, you actually end up getting a multitude of structures coming with it because of other covalently linkages to that peptidoglycan backbone. So this forms the outer surface of the gram-positive cell.

Slide 28

In the gram-negative cell it is going to be different in several respects. First of all, we have the typical lipid bilayer that is the cytoplasmic membrane. Out from there is also a cell wall; it is going to be thinner than what we observed in gram-positive organisms. There is another membrane layer beyond the peptidoglycan backbone, called the outer membrane and is composed of several things. It is not the typical lipid bilayer, unlike the cytoplasmic membrane which is a typical lipid bilayer. Here the outer structure of this bilayer is a structure called lipopolysaccharide.

Slide 29

For the cell wall the peptidoglycan is thin and is only about 2 nm is size as opposed to the 10 to 100 nm in size of the gram-positive. If you take it all apart, it is effectively only 1 layer. If you look at the way I have it drawn on this figure (Slide 28) what you see is that it is not a single layer going all the way around you still have the cross-links in forming a typical cell wall. You have those cross-links that allow it to form but if you take it all apart and put it back together it is not much more than a full layer of peptidoglycan once around. However, because you divide it up and make cross-links that 1-1½ layers worth is enough to give you a rigid cell wall. In that cell wall, unlike the gram-positive there is no teichoic acids—no wall teichoic acid located in the peptidoglycan and no lipoteichoic acid located in the membrane. So, teichoic acids are exclusive to gram-positives.

The area between the inner membrane or the cytoplasmic membrane and the outer membrane where the cell wall is located is called the periplasmic space or the periplasma. In that area, there are a lot of functions going on. There are digestive and protective enzymes and also transport systems that have to mediate things getting in and out of the cell that have to transit through the periplasma.

Outside the periplasma is the outer membrane. The outer membrane alone is going to block the entry of large molecules—the things larger than 800 Daltons in size. The outer membrane is not the typical lipid bilayer; it is formed by the LPS (lipopolysaccharide). In addition to having that form, the outer membrane is going to be responsible for getting things into the cell so it has to have transport.

Slide 30

What does the lipopolysaccharide (LPS) look like? LPS is also called endotoxin .Endotoxin is exclusive to gram-negative. Gram-positives do not have endotoxin but they can have toxins. Endotoxin is responsible for toxic shock that occurs in gram-negative infections. The LPS is on the surface. If we start from the outer membrane, we have the inner lipid membrane, on the next layer we have Lipid A, then a core polysaccharide, then O Ag (O antigen) which is the furthest point on the outside. The Lipid A part itself is responsible for the toxic properties (points out Lipid A on the bottom left) and this is where it is inserted into the lipid membrane. The core polysaccharide varies with the species of bacteria and is formed of several sugars (bottom right). Outside of that is O Ag that is a polysaccharide. The polysaccharide structure has 3-4 sugars per repeating unit and there can be 25 or more repeating units to form the outer layer of the O Ag. That is going to be quite variable within species. For example, E. coli O 157, the O 157 refers to the O Ag. If you take all the strains of E. coli and do serology of all the O antigens, you find that you can make different antiserum to different O antigens. You test a new strain with all the O antigens to find out which one it reacts with.

The distinguishing features of gram-negatives are thin cell wall, the presence of an outer membrane, and the lack of teichoic acids.

Slide 31

Those are the things that are necessary for any gram-positive or gram-negative that you consider a bacterium; they have to have those elements. There are optional features that will be important not necessarily in the structure or the viability of the bacterium under favorable conditions. However, under non-favorable conditions, such as surviving in a host or being able to cause infection these may be critical to their survival. Both gram-positives and gram-negatives can have these.

Capsules will surround the outer surface of the cell. In gram-positives, this would be beyond the layer of the cell wall (outside of cell wall). In a gram-negative, the capsule will be located on the outside of the outer membrane. It will be on the furthest surface regardless of which cell it is. Capsules are polysaccharides most always. There are some examples of proteins. These occur in Bacillus, such as Bacillus anthracis, which causes anthrax. Regardless of what it is, capsules perform the action of being antiphagocytic. They are antiphagocytic in that they either block complement deposition onto the bacterial cell or they permit the recognition of complement that has been deposited on the cell so it winds up being underneath the capsule and not recognizable. They can also play roles in attachment. In many oral bacteria (which adhere to the tooth surface) it is actually a polysaccharide capsule that allows for adherence.

There may be surface proteins that can be anchored in the teichoic acids, cell membrane or the outer membrane. They perform (like capsules) functions in antiphagocytosis or attachment. In the upper left picture, it shows a capsule growing on a cell on an agar plate. In the lab you grow bacteria on solid medium and some of them will look more like these small colonies growing on a cell. This is a non-encapsulated isolate of this strain which is making a capsule and most of what you see is actually the capsule, not just the bacteria. The lower left picture shows the capsule under the microscope (points out the bacterial cells in the center and the capsule around the outside).

Flagella are made of proteins and they allow the bacteria to rotate and move. Flagella also allow them to move toward things they are attracted to, such as food sources. They move away from things they are repelled by such as toxic substances. They actually form a structure in the cell that comes from the cytoplasmic membrane to the outer membrane and forms a hook that basically spins around allowing propulsion (lower middle). Flagella can be found on just one end of the cell, such as unipolar (lower right) or they may be found all over the cell (upper right). So they are important in motility, getting to things it needs and/or virulence. Attachment is one way that bacteria can cause virulence.

Slide 32

Pili are similar to flagella but they have a different structure. They do not rotate or cause any locomotion. They are shorter and narrower structurally so when you see them you can usually distinguish them. There are several kinds that are important in attachment (peritrichous are the ones all over the cell) and others that are important in gene transfer. The pili allow the bacteria to interact with each other and ultimately exchange DNA.

There may be many toxins that are present; these are excreted and act on the host cells. Some of these are actually encoded by phage or viruses that are in the bacterial cell not by bacterial genes. Only bacterial isolates that have that phage are going to cause disease because that is where the toxin is coming from. This is true of the botulinum toxin; it is a phage encoded toxin.

Other enzymes that may be present include hylauronidase, proteases, DNases. Whenever the bacterium finds itself in a new situation with the host, it may need these to degrade and find a niche in the host where it can attach. It may also use these to breakdown host products, such as glycoproteins, and take into the host and grow.

A few bacteria form endospores, which are effectively dehydrated cells. Examples include Clostridium and Bacillus. They form spores which can survive under the most extreme conditions. Once conditions become more favorable, they are able to rehydrate and sporulate and reform the bacterial cell. There is interest in Bacillus, in anthrax, with spores being spread and understanding the mechanism. Once you inhale the spores, they are in a favorable environment where they can germinate, sporulate and have the full fledge bacterium to cause disease.

Slide 33

What the bacteria requires to grow in the host and what it requires in the lab may be different. Most of what bacteria need is WATER because water comprises the majority of the cell. It is also going to require CARBON AND ENERGY SOURCE and often times these can be the same. Most of the bacteria (all of the pathogens you are going to encounter) are chemoheterotrophs, which means they use organic molecules for both their carbon and energy sources. Examples of what they can use are: monosaccharides, disaccharides, organic acids, amino acids, alcohols, and fatty acids. These are the ones that are frequently used by pathogenic bacteria. These two (monosaccharides and disaccharides, I think) are used as a way to distinguish bacteria. For example, some bacteria may be able to use sucrose while another cannot. So, you can distinguish bacteria based on growth requirements.

Slide 34

All bacteria are going to require NITROGEN to grow. Nitrogen can come from an inorganic source, often times that is (1)ammonia and it will be taken into the cell where it is ultimately converted into glutamate and glutamine. Some bacteria are capable of (2)nitrogen fixation; they fix elemental nitrogen, convert that to ammonia, which goes into glutamate and glutamine. There is also (3)nitrate reduction which ultimately you get ammonia. (4)Denitrifiers actually release nitrogen from the cell. In addition to using an inorganic source, some bacteria may preferentially use an organic source and they may be able to use both depending on their environment. Amino acids are frequently the source with glutamate and glutamine being commonly used. Ultimately what you see is that you are basically trying to get to these amino acids whether you take them in directly or you start with ammonia and work your way to these amino acids.

Slide 35

The growth of bacteria and their AERATION requirements are critical for not only growing them in lab but also to isolate them from a patient’s sample. Some bacteria are going to be highly sensitive to oxygen (aerated environments). If you are trying to get it from a patient sample to the lab and have it survive you need to be aware of bacteria that might be killed by oxygen (you have to isolate and prepare in the right way). Some bacteria are strict aerobes, which will always require oxygen and the reason being is that they are not capable of undergoing fermentation. Fermentation is the transfer of electrons and protons directly to an organic receptor. They always have to transfer to oxygen so it always has to be present.

On the other extreme are strict anaerobes, which are going to be killed by oxygen (they have to be grown its absence). These bacteria will lack the enzymes that are necessary to degrade toxic oxygen byproducts and will consequently ferment. Oxygen itself is not toxic; it is the byproducts of oxygen metabolism that are. In the first reaction, the toxic byproduct is hydrogen peroxide (H2O2). Bacteria have to be able to get rid of H2O2 and catalase is the enzyme that does that. Other toxic byproducts are superoxide radicals. Those are gotten rid of by superoxide dismutase, which in the process generate H2O2 and it can be converted by catalase back into the harmless water and oxygen which continues to go through the cycle. Bacteria that are strict anaerobes lack these two enzymes and therefore will be killed in the presence of oxygen by the toxic byproducts.

Slide 36

Some where in between these two extremes are all the others. We have facultative organisms that are able to grow with or without oxygen. In the presence of oxygen they are going to respire and in the absence they will ferment. Whatever situation they are in they will adapt. Aerotolerant anaerobes are anaerobe organisms that will always ferment (regardless of the situation) but they are not killed by oxygen. They have the enzymes needed to survive in that environment but they just do not make use of that oxygen. Microaerophilic organisms are the ones best grown under low conditions and can usually grow without. These may have lower levels of those enzymes that allow them to survive in the anaerobic conditions.

Slide 37

Other growth requirements are TEMPERATURE and pH. Bacteria can grow at all extremes. There are thermophiles, even among the eubacteria, that can grow at high temperature and also psychrophiles than can grow at low temperatures. Most bacteria, particularly the pathogens that you are focusing on, are called mesophiles and they grow somewhere in between. 20° to 40° is optimum temperature conditions. They grow mostly at a neutral pH. However, there are conditions where they have to grow at extremes. If you think about one that is a GI pathogen (one that you eat), it has to be able to survive at the pH of the stomach before it is going to be able to get into the GI tract and cause disease. Many bacteria get phagocytized and at phagosome-lysosome fusion it dumps them into a very acidic environment. Bacteria that can survive go on to cause disease. They have to be able to adapt and have multiple conditions in which they can survive. The pathogens that we know now have survived the defenses of the host; the host has killed off those they could not survive.