03/04: bacterial cytology

Transcribed by Amit Amin July 03rd, 2014

[Microbiology] [3] – [Cytoplasmic components] by [Dr. Boylan]

[20] – [Example of a monomer of peptidoglycan2 amino sugars and a tetra peptide T][Dr. Boylan] – Some of you talked after the first class and asked about how to do the attendance. So last night I sent an email (11:40) and I tried to clarify and so the attendance is not a required as I said yesterday. It’s totally up to you but I recommend it. You come to lecture and study the materials and you do well. The system here is that randomly I will take attendance. The way to do it was used to be with a clicker (sometimes works and sometimes doesn’t). I thought I’d just distribute the scantrons. Pick it up, write down your name, and then your E numbers. If you want to write down your seat numbers that’s fine. The E numbers is key here. I will collect the scantron after the lecture. I won’t collect in the middle if there are 2 hours. After the lecture I will leave two boxes (one in the front and 1 in the back and drop it there). Then we will do this counting. Again it is totally randomly. This is to help students who really work hard and want to get extra points in the course. If you have any questions feel free to email me or talk to your curriculum representative. Good afternoon, so how do we get that down. I like that. Dim the lights. Started off talking about microbiology and 4 different microbes are included among that group. The work of Pasteur and then we started to talk about bacteria and we discussed the cell membrane and the functions that are carried on the membrane especially since they don’t have a lot of the organelles that they eukaryotes don’t. Bacteria are prokaryotes and are simple cells. They lack a nuclear membrane. They have a nucleoid DNA. Other names of DNA bacteria are chromosome genome. It’s replican b/c it replicates. WE talked about its lengths and how its super coiled. Let’s get back to what I consider the most important feature (the cell wall) and here’s a slide I showed 2 days ago. The building block of peptidoglycan (made up of peptides and glycans). We see here the two sugars. This one here is N-acetyl glucosamine. It’s a glucose molecule w/ a N-carboxyl group there molecule there. It’s N-acetyl. It’s an amino sugar. This is the other amino sugar and is the same as this one except it has a lactic acid group attached to it. That makes it a sugar called N-acetylmuramic acid. These are two amino sugars that form the sugar backbone of peptidoglycan. Then you have the peptide portion. The common one is L-alanine and D-glutamic acid really, Lysine, and then D-alanine which is only found in bacterial cell wall. Lysine as you recall is the amino acid that’s (some word that I can’t understand what he says b/c he mumbled. I tried to slow it down and it didn’t help) by DAP which looks like Lysine but it has a one arm difference so go back and review that. If you look at the backbone here, the glucosamine, muramic acid, etc. these paired sugars combine over and over again to form a lattice around the whole cell. The dark circle represents the Lysine or DAP. This would be the L-alanine as the #1 sugar, glutamic acid #2, lysine #3, and D-alanine as #4.

[21] – [ Cross-linking in peptidoglycan ] [Dr. Boylan] – Just to show it here again. Here we have the peptide bond (cross-linking) formed. 1,2,3, here formed w/ the lysine in this peptide and the D-alanine of

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an adjacent peptide. That’s the peptide bond that keeps the peptidoglycan together and forms this structural component. Preventing it from getting so large that it would burst. Without the peptide bond shown there, there probably wouldn’t be any bacteria. That’s how important the bond is. Without peptidoglycan all cells would lyse. In gram-positive bacteria you have layer after layer of peptidoglycan. This goes around the whole cell. But cells have openings and pores so things can get outside the bacterial cell (foods, metabolites, and all kinds of good things pass through it to get into the interior of the cell where growth occurs). Don’t just think of it as a dense component where nothing can pass through.

[22] – [ Close-up of a type of bridge ] [Dr. Boylan] – Here’s that linkage again. Here’s DAP. D-ala is the 3 so amino acid 3 and 4 on adjacent strands.

[23] – [ Cross-linking in peptidoglycan ] [Dr. Boylan] – Here it is again. Anybody notice one thing different about the peptidoglycan has shown in the lower part (B) that what we have seen in the previous slides. Look at here. Ahhh which one. Here and here and here. So usually the linkages are 4 amino acids (tetra peptides) but really when they are first synthesized as peptidoglycans are being produced they are formed as pentapeptide w/ two D-ala at the ends. Ala as #1, glutamic acid, DAP, D-ala, D-ala. The last D-ala is cleaved and lost and the peptidoglycan is synthesized. We’ll show you why it’s an important role that this is D-ala, D-ala peptide, well what the purpose of the cleavage is later one. Initially it’s a pentapeptide and then it becomes a tetra peptide. The last one is cleaved and lost for a very important function.

[24] – [Example of another type of bridge – in Staphylococcus aureus (left)] [Dr. Boylan] – And one other type of linkage b/w peptidoglycan strands here we’re talking about we see this type of already, (1,2,3,4) is that the one found in the bacterium on S. aureus has a slightly unique linkage b/w it’s strands of peptidoglycan in that it has 5 residues of glycine so glycine 1,2,3,4,5 complicating things a bit. Here’s the 1 strand, here’s the lysine, here’s the adjacent strand. But between these two strands you have pentaglyciene (5 glycine) which is found in S. aureus which will help the strands separate and aren’t as close together when they have this gly bridge b/w them. There are all kinds of variation in bacteria but this one is an important one.

[25] – [Peptidoglycan - additional notes][Dr. Boylan] – Peptidoglycan protects it from bursting and gives it the shape of the cell. It’s also the spot for the action of an enzyme lysozyme. Let’s go back a couple slides and we see that we mentioned it here. Here’s the sugar backbone (2 amino sugars) and see this linkage hydrolyzed by lysozyme. The sugar backbone of peptidoglycan can be broken apart and destroyed by this enzyme known as lysozyme. I don’t know what that T up there means so forget about that. Lysozyme what it does (it’s an enzyme and we have it in all our secretions in our body). This

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enzyme is one of our defenses against bacterial infections (host defenses against bacterial infections). If we happen to pick up bacteria that is pathogenic and touch our eye. Teardrops will have lysozyme in them, which will hopefully destroy the bacteria by cleaving the sugars (the bond b/w the two amino sugars). I want to point out that that’s how that works. The linkage of the formation of the peptide bonds b/w adjacent strands is carried out by transpeptidase called transpeptidation and penicillin is the anti-biotic that inhibits the peptide bond b/w the strands of peptide glycan. Penicillin is an antibiotic and is one of the best of all time. Bacteria are becoming resistant to it. The wonder drug that saved so many thousand lives in WWII, we’ve overused it. You hear problems about antibiotic resistant and they are resistant to penicillin and other antibiotics. It’s a wonder drug that works against that particular bond of peptidoglycan.

[26] – [No title][Dr. Boylan] – There is much more peptidoglycan in cell walls of what we call gram-positive bacteria than gram negative.

[27] – [Teichoic acids][Dr. Boylan] – Let’s take at each of these in turn. We will first look at gram-positive bacteria and then gram negative shortly. Positive’s cell wall is about 80% peptidoglycan and negative is 15%. The cell wall of gram negative is only 15% roughly peptidoglycan so a lot less. The only other major component in positive is teichoic acid. Teichoic means wall in I think Greek. You remember in building blocks and biochem that glycerol (3 carbon compound). Well there are two types of teichoic acid. One of them is made up of glycerol linked to other glycerols over and over again by a phosphorous. Glycerol phosphate, etc. Here’s another large macromolecule (t. acid) and you put these monomers together to put chain upon chain of glycerol residues. In addition to that you have ribitol phosphate, which is connected to other ones by ribitol phosphate. T. Acids are found in the wall and are made up on glycerol or ribitol and they stick to the peptidoglycan for about 20% of the wall. They have a strong negative charge, which is why the surface of the bacteria has a strong negative charge. We’ll see the Importance of it later. The wall t. acid as I just mentioned. Another type is the lipoteichoic acid we see here. These stick right on up through the peptidoglycan. These strands originate in the cell membrane. They are called lipoteichoic acids. This little part of them that is stuck here is a lipid. Here’s the lipid and here’s the lipoteichoic acid going up. It’s always made up on glycerol phosphate never ribitol. So t. acid, wall t. acids, ribitols, or glycerol phosphates residues and chains and that are found in the cell wall in the peptidoglycan. The specific ones are lipoteichoic acids in the cell membranes and they stick on through the peptidoglycan. Both of these t. acids are exterior. They kind of poke up on top of the peptidoglycan. The rolls of t. acids are antigenic and can be involved to adherence and they give a negative charge. Antigenic we will talk about later. These t. acids do stimulate an immune response in us and they are involved in adherence. Once again our friend staph. They have a lot of t. acids in them to help them adhere and stick to our epithelial cells. These things are repeated over and over again. S. aureus (we all have it on us) and they really like to live on

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our skin or in the anterior part of the nose where it’s a bit cooler. These S. aureus use the t. acids to stick to the epithelial tissues in the front part of the nose.

[28] – [Gram-negative wall components][Dr. Boylan] – Here’s a gram-negative cell wall. You can see it’s a bit more complicated than a gram positive. Let’s start off in the bottom here and work our way up. This is cell envelope meaning membrane and wall together. Here we have the cell membrane, the cytoplasmic membrane we’ve already discussed, phospholipid bilayer, and proteins in it. Then we go up here and we see another what we call outer membrane. So here’s the layer of phospholipids but it’s only one layer of phospholipids. Not a bilayer. In the outer part of that bilayer we have a structure called LPS. Lipopolysaccharide. That’s the stuff that’s right here which goes through here and up like this. They make up the bulk of the outer leaflet. Here’s a double leaflet and another bilayer and only the inner leaflet is a bilayer. LPS also known as endotoxin. Very important molecule b/c many times we have an infection caused by gram negative bacteria, the LPS is responsible for the fever we get in infections and it can also induce shock. The LPS can shut down organ function leading to death. It’s a very important biological molecule. Of course we have gram-negative bacteria in our intestinal tract but that’s the ok LPS that doesn’t cause us any harm. The one that causes infections can lead up to death b/c of the function of the role of LPS. The next slide will show more detail about LPS. We have porins in the outer membrane. Here we have the trimers (3 peptides that open and close and once again regulate what passes through from the outside). Bacteria that grow have to get food from the outside. They sometimes can’t get large polysaccharides to pass through into the interior so the porins can regulate the size of what passes through from the exterior and works its way down into the cell interior. They restrict passage of proteins through roughly 1,000 Daltons. Any molecules larger than that will be restricted and prevented from passing through. Anything smaller than that (sugar/ amino acids) can go through but anything larger, the cell needs to break them down into smaller components. The space b/w the cell membrane and the outer membrane are called the periplasmic space. It’s this white here which is filled w/ fluid. Space b/w the cell membrane and the outer membrane and this is where you find peptidoglycan in gram-negative bacteria. Peptidoglycan shown here. You can only see a monolayer in gram-negative bacteria in contrast to the multilayer in gram-positive bacteria. It is needed and is found in the periplasm in gram negatives. Then you have nutrient binding proteins. Here we have binding proteins shown here. Some of these proteins are called PBPs for penicillin binding proteins and are found in the periplasm of gram-negative bacteria. Lipoproteins are what bind the peptidoglycan to the outer membrane preventing it from floating away. This is essential the cell wall of a gram-negative bacterium.

[29] – [LPS close-up][Dr. Boylan] – Here are the LPS that we just saw before. It’s called the endotoxin as well. Once again it’s toxic which causes fever. The part that causes fever is the lipid portion (lipid A). That’s all we want to know about that. There may be hundreds and thousands of copies of LPS. The lipid portion is responsible for fever, hypertension,

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and shock in negative infections. There are 3 parts to LPS (#1- lipid A found in the outer membrane, #2 core polysaccharide- 4,5 sugars (almost the same in all negative bacteria), #3-o antigen shown here). Once again we have that familiar story of repeating monomers. Look at this, 4 sugars that is synthesized by a gram negative bacterium and then another unit is created and added on to the top of that over and over and over again up to 40 unites for this sugar. Different bacterium makes different combinations of sugars (thousands of types of o antigens). They have one basic until of sugars though. In a way, the composition of sugars in o antigen helps us identify in the lab different bacteria b/c the differences in the composition in the o antigen. You can pretty much tell what genus/ species you’re working with.

[30] – [ Cell wall-defective cells ] [Dr. Boylan] – Cell wall defective cells. So far we have talked about the composition of the positive/ negative walls but protoplasts are positive bacteria that have completely lost their cell wall. If they lose their cell wall the cells will die and burst. The will pop or burst. We have protoplast that are positive and have no cell wall at all. How do they exist? Ordinarily there are some other factors that help prevent them from bursting. If we have an infection from gram-positive bacteria and we treat it with penicillin it’s going to destroy the peptidoglycan but one of these bacteria is hiding in our tissues and they are stabilized in the environment of our lung tissue. W/o a cell wall they might survive for a while and maybe even grow. Protoplasts are gram positive w/o the wall and they survive. What’s the consequence of that? You’ve heard of infections that are recurrent and you’re taking antibiotics and you feel better but a few weeks later the infection comes back. Ya the infection was treated successfully but you have a few bacteria that hid in tissue in your body and when the penicillin was taken away they came out and they synthesis all over again. You have a recurrent infection. Spehroplasts are negative that have lost their peptidoglycan but still retain the outer membrane. Protoplasts are positive and spheroplasts are negative both of which have lost peptidoglycan. W/ spheroplasts they survive even longer w/o the peptidoglycan. L-forms refer to protoplast that continue to grow and divide. Once again if they are protected from bursting they can grow for a while and L forms are the names of cells that do grow w/o a cell wall. They have the capacity to grow a cell wall and eventually they will and blossom and cause recurrent infections. Mycoplasmas are a genus of bacteria that never has a cell wall. How does it survive w/o a peptidoglycan? It has sterols in its cell membrane. It’s not a hearty bacterium at all. It doesn’t last long outside the body but it can survive since it has sterols that make that membrane a bit tougher than the other bacteria we know about it nature.

[31] – [ Capsule ] [Dr. Boylan] – Now we are going to talk about something further exterior. The capsule. The capsule is a nice name for the slime layer. Extracellular and not a part of the cell envelope. A lot of bacteria but not all for what is called a capsule. Another name for it is glycocalyx. Some microbiologists differentiate b/w the two but essentially they are the same. They are made up of a polysaccharide sugar, which is why it’s called a slime layer. The capsule forms around the bacteria. They can

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survive w/o it but when they do have it, they have many advantages that other bacteria don’t have. The composition is polysaccharide sugars and among the types that have capsules there is different compositions. The exception to being polysaccharide is bacillus anthraces. This is the one that causes anthrax. For some reason during the evolution of it the capsule is not going to be polysaccharide but a capsule of single glutamic acid (polymer of it). It’s different from all other capsules. The others are polysaccharide. The functions of the capsule. Never underestimate the power of the capsule. Bacteria that have a capsule can be extremely pathogenic causing many serious infections. Among them, most of the bacteria that cause pneumonia have a capsule. Most of the meninges of the brain have a capsule as well. A capsule helps bacteria become sever pathogens. Why does it help them? It prevents these bacteria from being eaten or destroyed by our white blood cells. Our white blood cells known as phagocytes. These white blood cells are constantly in our blood looking for invaders. When they have this slimy capsule around them, they can slip away. It is harder for these phagocytes to engulf. They are anti-phagocytic (what capsules are). Prevent phagocytosis or destruction by white blood cells or macrophages. That’s what helps them be so difficult to eradicate/ get rid of when they start to cause an infection and produce these serious diseases. Another goal of the capsule is that there’s a polysaccharide layer that surrounds the bacteria (you can see this large capsule around them) is dehydration prevention. Bacteria want to survive and how do they keep going on? If someone has a severe case of pneumonia and they cough up these bacteria. They lodge all of the place. While most bacteria would die w/in minutes/ hours, but when they have capsules, this mucoid polysaccharide slime layer doesn’t dry up as fast. They aren’t desiccated as rapidly. They can survive on surfaces for hours or days b/c they don’t dry up due to the capsule. Other bacteria w/o a capsule would not survive as long in the environment. It helps them to keep going on and become transmitted from person to person. The longer they can survive the more likely someone will touch the contaminated object and pick up the bacteria. Glycocalyx w/ strep mutans is the bacterium responsible for carries. It’s found in the plaque on our teeth. These bacteria in plaque produce all kinds of polysaccharides and capsules and other things to stick to our teeth and each other as well. S. mutans is the culprit in caries and it uses a capsule made up of dextrans and glucose which helps bond tenaciously to our enamel. It’s tough to brush them away b/c of the dextrin capsule it produces. Other bacteria are there as well but that’s the one that sticks to our teeth and does the damage.

[32] – [ Cytoplasmic components ] [Dr. Boylan] – No we can go through some of these things since we have talked about some already. Nucleoid is single and a coil that’s a double helix. Made up of DNA. Ribosomes, polysomes are a bunch linked to each other. Inclusions also bacteria have that we will talk about as well.

[33] – [ Bacterial chromosome ] [Dr. Boylan] – Chromosome. Haploid, circular, linked about a thousand times longer than the cell. Base pairs can be up to 5 million. The way I remember is the number of genes. How many genes can a bacterial cell have? E. Coli has about 4,500 genes in its

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chromosome. Some have more or less. How many genes do a human have? 23. We use to think that we more but we only have 23,000 genes all together. Now we can modify our genes a lot through genetics but if you look at genes itself 4,500 to 23,000 genes in humans. So here’s bacterium w/ a nucleoid. Here’s what happens when you burst the cell releasing the stuff and you can see how much longer it is than the cell (1000x more) [34] – [ CHROMOSOME REPLICATION ] [Dr. Boylan] – I have a quick question before I discuss this problem. We have haploid chromosome that means one chromosome but it’s cut in a double helix (coiled) and the origin. Here’s the chromosome and this chromosome has started to replicate. One chromosome to two. Mother cell will grow and divide into daughter cells. Each needs an intact chromosome. This darker area represents the initial strand so double strand. The blue represents the newly synthesized strand, which started at the origin, and it begins to replicate the DNA into complementary strand. Semi-conservative replication occurs. DNA is going to have one strand from the mother and one new strand. It then goes around and once it gets to this point here the whole chromosome will be replicated and that’s the terminus. It begins at the origin and ends at the terminus. Semi-conservative replication. This process of replication in bacteria takes about 40 minutes to replicate its chromosome. A around of replication takes 40 minutes. Not only that, but look at the way it’s going on. It goes from 12 o’clock and goes clockwise and counterclockwise. It goes in both directions. That is caused bi-directional replication. We don’t have that. Ours don’t’ divide that way. Speeds up the replication but it still takes 40 minutes. Here’s the question I want to pose. I mentioned before that some can divide in 20 minutes. Here’s a cell called a mother cell and in 20 minutes there are 2 daughter cells but they have chromosomes. Wait you said it takes to replicate the chromosomes but these cells divide in 20 minutes. How is that possible? That was asked for many years. Some of you may want to think about that and we can talk about it the next time we meet. There is a way this happens.

[35] – [Plasmids - properties][Dr. Boylan] – The plasmids closed loops and circular. It’s made up of only DNA. That means it has genes. There is a bacterial cell that has a chromosome stuck to a protein. Here we have the extra chromosomal DNA (plasmid). These are extra genes in the plasmid. This is and advantage. The properties these bacteria can get when they are a host to a plasmid, they can have a profound effect on the life of a bacterial cell and what happens to us during the course of an infection. Plasmid can allow benefit for the bacterium and that means they are more dangerous to us. Extra chromosomal and are autonomous meaning they replicate independently. When the chromosome replicates the plasmid does but does it only it’s own. The size is 3-4% of the chromosome and has 10-15 genes compared to the 4500 genes in the chromosome. Copy number refers to the number of plasmids in the cell (up to 20). There can be different types of plasmids in a cell. A bacterial cell may have 2-3 plasmids. Autonomous means they replicate on themselves. Dispensable means it’s not needed for survival but just provide advantage. They are transmissible meaning

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they can be transmitted from one cell to another. They are contagious and is why one type of gene (destruction of antibiotics) and at the same time they are being transmitted among different bacterial populations. More are picking up plasmids and more are being resistant to these antibiotics. R plasmids stand for resistance to antibiotics. These genes here. Some are called R plasmids and these have genes that make the cells resistant to antibiotics. Resistant that destroys penicillin or tetracycline. There are other ways to become resistant but this is a major one. Plasmids are also involved in recombinant DNA work. They play an important role in the life of a bacterial cell. Recombinant DNA is used to produce drugs in a lab setting.

[36] – [ Ribosomes ] [Dr. Boylan] – Ribosomes are the sites of protein synthesis. In our cells they are 80S. Here’s eukaryote and has 2 subunits which are 60S and 40S. Depending on how far they move in a centrifuge w/ a solution in it you see how far down it goes. The bigger they are the farther they go down into the centrifuge. When separated the subunits have different shapes. The larger is 60S and the smaller is 40S. Bacterial cells have 70Sà 50S, 30S. You’ll see this again dealing w/ antibiotics. Bacterial cells and our cells have ribosomal cells for protein synthesis. There is a difference in size and composition. Some drugs can inhibit effect in bacteria b/c of the differences but have little effect on our cells. Protein synthesis is an essential function in all living cells but if you stop synthesis you will stop it from growing that’s great for us. Our healthy cells can keep functioning. 50S has 5S and 23S. The 30S has 16SRNA. I mention that b/c if you want to identify it on a molecular level, 16S RNA is unique to bacteria. Every bacterium has it’s own unique 16SRNA. The part of DNA that codes for it is unique. You can identify it most specifically looking for 16S RNA or the gene for it and you’ll know exactly what kind of bacterium it is. Polysomes are ribosomes linked together and are sites of protein synthesis. I’ve already mentioned the importance of ribosomal sizes and antibiotics since they will only bind to bacterial cells and stop protein synthesis by stopping them from growing or killing them.

[37] – [ Inclusions ] [Dr. Boylan] – We talked about the ribosome and of course they are in the cytoplasm inside synthesizing proteins. There may be 10,000 or more ribosomes when they are growing rapidly. Thinking of a cell that grows and replicates every 20 minutes. Just think about how quickly it has to synthesize the organelles. Another type of body found inside bacterial cells are inclusions. For the most part they are storage or granules where bacteria can store materials that they would like to hang on to and store them for a while. They don’t need them right away but maybe in the next day or hour they will need to call upon the reserves of nutrients of growth and division. We know we will have 3 meals a day so therefor we can know how much to eat. We don’t have to stop ourselves thinking this may be our last meal. bacteria don’t know that. They will store nutrients and not eat them right away but maybe the next day they won’t be in the same environment so they can store stuff. They are storing organic and inorganic materials. Lipids they store are polymers of beta- hydroxybutyric acid. They can store sugars in granulose. They need the sugar for

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growth. Inorganic inclusion is called volutin or metachromatic granules. Those are made up of phosphate for their nucleic acid synthesis. We always need to mention that b/c we use to have labs and every time we had labs and do this stain using methlylblue but when we added this stain to cells w/ volutin they would become red. So metachromatic (stain would change to red when it would bind to phosphate). V

[38] – [Bacterial appendages Flagella][Dr. Boylan] – There are two appendages. Flagella and pilli. Flagella are for motility. here is a cell w/ one flagellum. One of its ends or its poles. Along thing, wavy, for motility. They are so thin you cannot see them using the light microscope under normal staining conditions. They are too thin (than a bacterial cell). How do you see them? Well, what we do in the lab if you want to look to see if it has a flagellum or not, we coat the bacterium w/ a silver nitrate or a salt. We keep dumping salts on the bacterial cell (on a microscope side). We keep dumping the salt on and it keeps sticking to the flagella and eventually it looks bigger and is visible in even in a light microscope. It’s made up of flagellin monomer. Linked together to form a long filamentous flagellum. As you can see here is a flagellum but what happens is that it goes around within these bodies called basal bodies in green here. It rotates around that. You can see it goes in a counter clockwise formation. They are found in the cell membrane and wall of the membrane. It rotates through these bodies. We have flagella in the sperm cells but they act in a wave like motion to move cells along but the flagella act like a boat propeller. You see here the flagellum moves around in these basal bodies. It’s moving in a counterclockwise. We know that b/c people have studied it. We stick the tips through glass slides to keep the bacteria in place and then see, which way the bacteria rotate. We know that when bacteria are moving they are going in a counterclockwise direction. Not in a whip like fashion as in eukaryotes.

[39] – [Role of flagella][Dr. Boylan] – Bacteria exhibit chemotaxis. Taxis means movement in a chemical gradient. A gradient of chemicals. Chemicals that are high conc. to low conc. They like to move one-way to another and it’s the gradient of chemicals. What does that mean? We’ll show you soon. Let’s look at these lower figures. We have a beaker w/ some liquid in it. Some kind that we stick a capillary tube in. What’s going through to come through this hollow tube from here to here? Well it says no attractant so nothing that the bacteria will benefit/ is attracted to. It’s not an attractant for bacteria. So what’s going to happen then when the bacterium over here is going and moving around and is moving back and forth over and over again. It has an erratic movement. It winds up over here. What was coming through the tube did not attract the bacterium to come newer it so it moved away. You notice here that what happens is, it moves over from this spot to this spot. It’s thinking to itself if I’m in a better spot or worse spot than where I was. Then it stops and we move here, stop, sense the environment and then it continues to move. When it stops you see the flagella stick out. The point I’m trying to make is that the flagella are moving in a

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counterclockwise direction and then they stop and they sense their environment. Their flagella start to move in a clockwise direction. They are sensing their environment and thinking is it better or worse. They are sensing on the receptors of the surface. When they move, it’s called running. When the bacteria move they run. They’re moving along. When they stop to sense their environment they tumble. Let’s try this way let’s try that way. On the left, the bacterium moves in an unusual pattern. What happens now, and you put an attractant. What’s an attractant? Glucose or another sugar. Something that it’s really attracted to. Something that it can use to grow and wants to survive. Let’s see the difference now in the way the bacteria initially moves. It moves along stops here, and tumbles. Sensing this environment and sees what the conc. of the attractant says. I’m moving along here and the conc. at this site is more than before so I move in that direction stopping and sensing the environment until it moves in that direction. It doesn’t move in a straight fashion but back and forth. It eventually gets there. That helps the bacteria in the body. The ones that want to cause infection want to borough into the tissue and find sites where there are nutrients for us to grow. What will happen if there were not an attractant but a repellant? Maybe using acetic acid. They would do the same thing but they would be as far away from the repellant than normally. Either way it’s a chemical gradient. You know here, the gradient is much more attracted in this area as it is introduced in the beaker than it is here. It moves in a chemical gradient from low to high and then it grows a lot better as a result. The same thing happens in the body since it helps them borough in the tissues. Even flagella can be appendages that help bacteria cause infection.

[40 [ ARRANGEMENT OF FLAGELLA (-trichous)][Dr. Boylan] – Arrangement of flagella can be used to classify. Trichous means flagella. Mono- means one flagella. Lopho- tuff of flagella. Amph-/ Peri- are all around the cell. These are ways to characterize all of bacteria. Some of them have one and some have tufts at both poles. This is another way to identify bacteria by the arrangement of the flagella.

[41] – [ Flagella in spirochetes ] [Dr. Boylan] – An unusual flagella is found in spirochetes that we talked about in the first lecture. The flagella in them stays inside the periplasm. They stay within the space in the cell membrane and outer membrane. It leads to a twisting rotating motion (corkscrew). This helps the bacteria borough into the tissue. It’s called endoflagulation since they are inside the cells. It helps them rotate in that spiral fashion. Here we have the flagellum inside. Here’s the outer membrane and inner membrane and it’s not sticking out.

[42] – [Bacterial AppendagesPilli][Dr. Boylan] – Another this is pilli. We can see they are here. They are much shorter and straighter than flagella. They are made up of proteins and the protein here is pilin. Flagella are made up of flagelin. Their main function is to help adhere. In the

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infections disease they have to stick to something. The bacteria that have pilli are the main source of adhesion. We talked before the capsule, which can help the bacteria adhere, but the pilli that’s their main function. Host colonization is when one bacteria will stick to epithelial cells and say I like it here. Now I can grow and double to form colonies over time. They form little colonies inside of us. Not just one cell but they colonize. They are also there to help w/ biofilm formation where you have mixtures of bacteria (100s of species) like dental plaque. Living film of bacteria (30-40 species). They work together and grow and they use their pilli to adhere. Biofilms are also formed in other parts of the body for infections. Importance for the pilli is demonstrated here. This question is often in the boards is this. The importance of the pilli is always on the gonococcus. It has pilli or fimbriae plus or negative. If it has pilli it’s called Film+ and if it doesn’t its called Flm-. If you get rid of the pilli from gonococcus they will not be infectious at all. You can inject someone with millions of it but if they don’t have pilli they won’t cause an infection. That points out the importance of pilli. If they can’t stick they are not going to cause and infection. W/ gonorrhea they can’t stick at all. There is a special pilus called the sex pilli and it’s not shown here but it’s also protein pilus. It’s a little bit larger than the ones shown here we’ll see what happens. The special pilus is formed as a sex pilus, which helps bacteria stick to each other and mate. We will talk about this next week. The combine and unite and having sex meaning they exchange their DNA. They do this by the sex pilus. It helps the bacteria conjugate for the exchange of DNA.

[43] – [ Spores ] [Dr. Boylan] – And then we have bacterial spores. Spores are bacterial cells. They are also called endospores. They are called endospores because they are found inside the bacterial cells. Just like endotoxin, we’ll talk later about exotoxin. In bacteria they are called spores or endospores. Later we will talk about spores or fungi that are formed inside the cell. Only two types of genus produce spores, Bacillus and clostridium. Positive bacteria, bacillus is a stripped aerobe and clostridium is a gram positive as well and is anaerobic. Here we have two gram positive rod bacterium which look the same. Both are gram positive and both form spores. One can grow in the presence of oxygen but they can form spores. They are growing anywhere in the body/ test tube and they grow for a while. All of a sudden they sense they are running out of nutrients. They are going to starve. Most bacteria will starve and will die. But not these two genera. Once they sense they are running out of food they sense if there is a lot of carbon in the environment, nitrogen and as they deplete their food they start to run out of the nutrients which they detect as a reduction of carbon and nitrogen sources. They thus form spores so that they will survive even when they run out of food. Spores in contrast in fungi are produced for reproduction. Spores in fungi are for reproduction while in bacteria they are used for survival. They can survive for hundreds of years. They are living bacterial cells but they are completely inert. They don’t carry out metabolism or grow. They are viable and are alive but they don’t carry out any kind of reactions inside. They are metabolically inert. Properties of spores, they are dormant. They have resistance to heat and chemicals. This is why they are important. They are resistant to heat and chemicals. They are formed to survive and it’s possible later on that this inert form

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may begin to grow again. They are resistant to heat and chemicals. Here’s a spore. The interior portion is called the core. Spore, inert, no metabolism, but inside this core is DNA, RN, enzymes, lipids, sugar, everything else it will need if it wants to function as a normal bacterium again. It’s resistant to heat and chemicals because there is no water in the core. It is dehydrated. Think about why are our cells destroyed when we boil them in water. Without any water in the core of these spores even boiling water won’t effect them. The other reason is so resistant to heat b/c of the dipicolinic acid (just remember the name), which is often associated with calcium. It forms up to 15% of the core. It also helps the spore becomes resistant to heat. Lack of water, this acid, and calcium helps build resistance to heat. This is why we have to special conditions to kill them. Lets not go through the other layers except for the coat. Remember the core, acid, and protein coat. The coat is shown here and it surrounds the core is a thick protein. It’s a keratin like protein meaning it is very thick. This coat protects the spore from the passage of chemicals from outside to inside. This coat is a thick keratin like protein that makes the spore chemical resistant. These are important traits that help spores survive. What’s the problem if they are inert? They can come back and form normal active cells.

[44] – [Spore components][Dr. Boylan] – So we went through that already. Sporulation. Here’s a bacterial cell we’ll see in the slide sensing it’s running out of food (triggering events). No carbon or nitrogen and thus form spores to survive. It’s a differentiation process meaning that these bacteria always had the genes in their chromosomes to form spores. They always had the genes but those genes as you know can be turned off or repressed. I like it/ this is good for me so I don’t need to form a spore. When it starts to run out of food, I will form a spore and I will survive. It turns off a gene and turns on other genes. A whole bunch of genes in this bacteria are always there but then they are repressed. Turning off that they were using before and turn on the genes for repression.

[45] – [Sporulation and germination][Dr. Boylan] – Here we have the growth and preparation just to show that spores are formed. Here we can see the spore is formed. This is just normal growth. It begins to divide. Notice that the septum is closer to one end and not in the middle as normal division. The spore is forming inside the cell (endospore). DNA, RNA, proteins, enzymes. Lose water and form the coat and others and finally the intact spore is formed inside the cell. The cell lyses releasing a free spore. This is the living inert dormant cell resistance to heat chemicals. As said before it can survive for maybe 100s of years. Sometimes it comes back to life. It’s kind of like a chick coming out of an egg cell. Under the right circumstances, sensing its environment. Somebody drop these spores in a nice rich carbon broth environment. Let’s break out of this shell and emerge. They can germinate and grow once again as normal bacterial cells. One cell that comes out can multiply into thousands and millions of cells from a spore. 1 cellà 1 spore à 1 cellà millions of other cells. One more term I could use. You talk about these other cells called vegetative cells. All bacteria are

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bacteria cells in contrast to spores that are unique. A vegetative cell pops out and grows out over and over and over again.

[46] – [Review][Dr. Boylan] – Review. Some properties of positive and negative cells. Can be seen b/c of the composition of their cell wall. The gram stain is an important distinction for these cells. Outer membrane only in negative. Cell wall and thicker proteoglycan in positive. 80% here and 15% here. LPS only in negative. Endotoxin only in negative (just like LPS). T. acid never found in the negative bacteria. If you had a test looking for a t. acid you’d know it was positive if the test was positive. If you had something that identified LPS you know it’s negative since only negative have it. Sporulation only see in in positive. Not all bacteria have a capsule in causing infection. Lysozyme. Sensitive I wouldn’t completely agree w/ this statement. It’s an enzyme not an antibiotic. It cleaves the sugar bonds. Gram negative bacteria have peptidoglycan but less so they are only partially sensitive to lysozyme. They won’t have a serious effect on negatives as they do on gram positives. The cell will then lyse. The same regions penicillin acts on positive more than negative. We will see a lot of infections that are treated w/ penicillin. It doesn’t mean the negative bacteria are completely resistant to penicillin.

[47] – [Review 2][Dr. Boylan] – If you just want to go through and see the differences we discussed. I think we went over all of these things. Porins, capsule.

[48] – [Comparison of viruses, bacteria and fungi][Dr. Boylan] – We will talk next week about viruses too. Looking at the three major microbes let’s just give you a preview of their differences. Let’s just look only at bacteria. Living cells, they can grow as living cells. Size is 1-5 um. Both types of nucleic acid. No mitochondria, rigid cell wall. They divide by binary fission. Viruses: cells? No. They are not alive. They cannot reproduce on their own. They are not cells. Smaller than bacteria. They do not have both types of nucleic acids. They either have DNA or RNA. No nucleus, mitochondria, or ribosomes. They are not motile or divide by binary fission. Fungi are neat cells. Larger than bacteria. Eukaryotes. Why are they eukaryotes and not prokaryotes? They have a nuclear membrane. Large ribosomes, rigid cell wall, and they divide by budding and mitosis. Just a preview of the difference we be discussing.

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03/04: bacterial cytology

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