I.

General Structures
A. Qualities of Prokaryotes i. No Nucleus ii. DNA is loose, not wound around histones, and posses a single chromosome. iii. Cell wall mostly made up of peptidoglycan iv. No intermembrane structures or organelle, but they do posses a ribosome. v. Smaller than Eukaryotes vi. Similarities between all prokaryotes a) Single chromosomes b) External cell membrane c) Ribosomes d) Cytoplasm

B. Parts of a bacteria a) Flagellum: Appendage that rotates that gives the bacteria mobility for traveling towards a food source, fleeing from danger, and away from toxins. Made of three major parts The filament is a rigid helical structure which extends outside the cell. It is hollow and filled with a hook around which are protein rings enabling motion. The hook which is inside the filament and allows for movement.

A series of rings which are embedded inside different parts of the membrane. Basal body proteins are those that make up the flagellum, and a proton gradient moving through them powers the flagellum. The type III secretion system is responsible for assembling the flagellum If basal body proteins are injected through the flagellum, it forms more of the flagellum leading to its construction. It is also thought that the type III secretion system was responsible for the evolution of the flagellum. This structure is in both gram positive and gram negative with only a minor difference. Bacteria can have more than one flagellum. Monotrichious: one flagellum peritrichious: more than one flagellum from the same area lophoitrichious: one flagellum on opposite sides of the cell. Amphitrichious: lots of flagellum from lots of areas

b) Pili: There are two types of pili Type one: Used for adhesion. Examples: Sticking to the urethra in a urinary tract infection, respiratory infection, etc... Type two: Sex pili are used for the transference of DNA, in the form of plasmids, from one cell (does not have to be the same species) to the other by opening a hole in the cytoplasms of the cells to enable the exchange. Example: if one

c)

d) e) f) g) h)

bacteria was resistant to an antibiotic it might transfer that gene to another bacteria. They are very fragile so they are often replaced. Capsule: Is a polysaccharide layer outside of the cell wall. Used for protection to the cell and also helps bacteria stick. The capsule helps protect from phagocytosis which is the eating of bacteria by phagocytes (white blood cells) by making it harder for the phagocyte to recognize the bacteria and therefore harder for it to eat it. It is very slippery and hard to stick to It creates difficulties doing a gram stain because it covers the peptidoglycan and prevents the dye from getting into there. It is a virulence factor, which means that it increases the chances of spreading disease because it can escape from phagocytes. Cell wall: A semi permeable layer in the cell that only lets certain things in and out of the cell. Cytoplasm: Water based membrane that contains ribosomes, DNA, etc... Plasma Membrane: A membrane that gives the cell more flexibility and some more control of what goes in and out of the cell. Ribosomes: An organelle responsible for protein synthesis. Phospholipid bilayer: A semipermeable membrane that filters what goes in and out. The phospholipids posses a polar head (on the outside of the membrane) and a nonpolar tail (on the inside of the membrane) leading to the formation of a micelleesque structure in an aqueous environment. In eukaryotes the phospholipid bilayer helps with endocytosis and exocytosis, the absorption and removal of materials in and out of the cell. Small, non-charged molecules enter the cell via passive transport. Larger molecules enter the cell via active transport. The main jobs of proteins embedded in the phospholipid bilayer are either to enable the transportation of materials in and out of the cell and to act as receptor proteins. There are two types of proteins, integral proteins and peripheral proteins. Only the integral proteins are bound to the fatty inside of the phospholipid bilayer. Integral proteins allow for the transfer of signals from the outside of a cell to the inside while the peripheral proteins aid in either cell recognition or the start of a relay inside the cell. The polar tail of a phospholipid is composed of one unsaturated (bent) leg and one saturated leg (straight). The interactions between the unsaturated and saturated legs is not as strong as that between two saturated ones and thusly this dual leg nature gives the membrane flexibility by allowing proteins/particles to pass through the areas between saturated and unsaturated legs.

C. The three shapes of bacteria i. Sphere shaped: cocci/coccus ii. Rod: bacilli/bacillus iii. Vibrio (curved like a banana) iv. What is the difference between the use of the term bascillus and bacillus? a) Bascillus is a shape, but bacillus is a genus. b) Not all bacillus species are bacillus in shape. D. Gram Positive Bacteria: i. To stain, bacteria are exposed to the dye crystal violet and then then treated with iodine. If the bacteria is purple after this procedure, it is gram positive, and if it is pinkish, it is gram negative. ii. The gram positive bacteria: http://www.cehs.siu.edu/fix/medmicro/pix/walls.gif iii. Gram positive cell walls contain a thick layer of peptidoglycan. The thicker the layer the more defense the cells have against phagocytes and also helps prevent osmotic lysis. This layer is linked to a layer of polysaccharrides. a) It is held together like this http://pathmicro.med.sc.edu/fox/ec-pep.jpg. The peptidoglycan is held together by chains of amino acids that cross link (which adds strength) sugars that form the main body.

iv. Only gram positive bacterias possess LTAs (lipotheicoic acids) that are negatively charged chemicals that serve as anchors. E. Gram Negative i. 1. What is the function of the inner membrane? a) Another name for the inner membrane is plasma membrane. The plasma membrane holds the cytoplasm, which is very useful in cells without cell walls, and it separates the cell from the surroundings. The plasma membrane allows ions and molecules to pass through its permeable membrane either into or out of the cell, while preventing the movement of other molecules and ions. The membrane holds a variety of crucial metabolic processes including respiration, photosynthesis, the synthesis of lipids and cell wall constituents. Finally, the membrane contains special receptor molecules that help bacteria detect and respond to chemicals in their surroundings. ii. 2. What does gram negative mean? a) Gram-negative of bacteria being of or relating to a bacterium that does not retain the violet stain used in Gram's Method. iii. 3. Describe the peptidoglycan layer in Gram negative cells. Does it serve the same function as it does in the Gram positive cell? a) Peptidoglycan (pep''ti-do-gly'-kan), also known as murein, is the single most important component of the bacterial cell wall. It is a polymer so large that it can be thought of as one immense, covalently linked molecule. It forms a chain around a bacterium that resembles multiple layers of chain-link fence. b) http://www.mansfield.ohiostate.edu/~sabedon/biol1080.htm#inner_membrane c) There is a difference between the gram positive cells peptidoglycan and the Gram negative cells. The peptidoglycan in the gram-positive cell wall prevents osmotic lysis which is when the cell bursts because of osmotic pressure from a hypotonic environment. d) http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/gncw.html e) The peptidoglycan in Gram Negative cells function as enzymes. They also serve as adhesions which help the bacteria attach to other cells. Sometimes they function as invasions which are proteins that allow bacteria to penetrate hostcells. http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/gncw.html f) Gram positive cells can keep the initial crystal violet dye during the Gram stain process and holds a purple color, however, gram negative cells decolorize during the Gram stain procedure and appear pink. g) http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/cw.html h) The thick layer of peptidoglycan help determine the organisms form ranging from rod, spherical, or helical shaped in Gram positive cells. Gram Negative cells have a thin layer of peptodoglycan outside their cell membrane. http://www.daviddarling.info/encyclopedia/P/peptidoglycan.html iv. 4. Describe LPS. What is its function? a) The lipopolysaccharides, consist of a lipid portion called lipid A embedded in the membrane and a polysaccharide portion extending outward from the bacterial surface. http://freespace.virgin.net/r.barclay/edtxsch1.htm b) The LPS from the outer membrane of the gram-negative cell wall is thought to add strength to the outer membrane, in a manner similar to the glycopeptides and teichoic acids of the gram-positive cell wall. The LPS portion of the outer membrane is also an endotoxin in that it is recognized by the immune systems of the animals in which the bacteria is located.

c) Endotoxin recognition: http://tiny.cc/4mash d) Once LPS binds to TLR4/MD-2, the mechanism whereby the receptor becomes activated is unclear. http://tiny.cc/s5gsz e) 5. What is a lipoprotein? Lipoproteins connect the outer membrane to the peptidoglycan and are also responsible for the transport of small molecules through the outer membrane f) 6. Describe the role of porins. Porins allow for the transport of small molecules in and out of the cell. They can be thought of as small holes through which things like sugars, amino acids, and ions can pass through. v. 7 . What is the periplasmic space and how does it function? a) The periplasm, or periplasmic space is the material between the outer membrane, the peptidoglycan, and the cytoplasmic membrane, and the periplasm contains enzymes and binding proteins that allow for nutrient breakdown and transfer through the cytoplasmic membrane. vi. 8 . What characteristics does the outer membrane confer on Gram negative bacteria? a) The outer membrane contains lipoproteins phospholipids, proteins, and LPS. The outer membrane acts as a filter that does not allow toxic substances such as penicillin G to enter. Vesicles form on the outer membrane so that the cell can fuse with another gram negative cell to communicate. This can be used in some cases to connect with a host cell to transfer toxins to the host. b) Gram Stain/Comparison What structure causes some bacteria to stain and not others? Crystal violet is positively charged, so therefore it sticks to the negatively charged peptidoglycan. The large peptidoglycan in the gram positive cell means that the dye sticks really well to it and not to the small peptidoglycan layer in gram negative cells.

F. Endospores i. A tough, dormant protective mechanism that bacteria use to protect themselves from environmental stress likes chemicals, nuclear waste, UV rays, etc... ii. Layers of protection: a) Endosporian and Spore coat: made of proteins and prevent things from coming into the cell

b) Cortex: peptidoglycan and DPA + calcium which prevent things from coming in c) Core: contains the cytoplasm, DNA, cell structures, and ribosomes. It is dehydrated and contains only 20-30% of a normal cells water allowing it to withstand high temperatures. SASP, found in the core, is a small, acid soluble protein that protects the DNA.

d) Process of becoming an endospore During the vegetative state the cell has a normal metabolism, etc... and is in a good environment and living normally. When the bacteria realizes it is under stress, it begins the endospore-forming process which lasts about 8 hours. The DNA starts to replicate, and a wall, called the septum, forms which creates a region where a double membrane starts to form around the DNA. The spore coat forms around the DNA and the rest of the endospore is made. The rest of the cell, known as the mother cell, then dissipates away.

e) When the endospore is in a favorable environment, the spore germinates, becomes metabolically active, and reforms a vegetative cell. f) Most endospore formers are gram negative, and only certain genuses of bacteria can become endospores. Most endospore formers are not disease causing. G. Qualities of Eukaryotes i. Posses a Nucleus ii. DNA wound around histones bound into chromosomes iii. Cell wall not made up of peptidoglycan iv. Contain many membrane bound organelles such as the golgi body, ribosomes, etc... that

prokaryotes do not posses. H. Gram Stain: A gram stain is a technique by which bacteria are identified by the color they turn when stained with crystal violet.

ii. How exactly does gram staining work? Charges, peptidoglycan, etc... iii. Simple Stain: Coloring a sample for visualization. Shape differences are visible, but not color. iv. Differential stain: Coloring a sample for differentiating bacteria by color and shape. a) Gram Stain Dyes: crystal violet, iodine, water (rinsing), etoh, water (rinse), safranin Procedure Put a small drop of water on the slide unless the sample is aqueous. Take an inoculating loop and move a small amount of bacteria into the petri dish and spread them around the in drop of water. Let the water dry Heat fix the bacteria to the slide by running the slide over the bunsen burner 4-5 times with the bacteria side up. Add a few drops of crystal violet to stain the bacteria Rise off the dye with water Add a few drops of gram's iodine (to help affix the stain to gram positive bacteria) and let sit for 20 seconds.

Tilt the slide and add drops of alcohol (to totally remove the dye from gram negative bacteria), drop by drop, until the drops running off the side of the slide are colorless. The alcohol dehydrates the peptidoglycan which causes the peptidoglycan to contract and retain the crystal violet. Since the peptidoglycan is so much bigger in gram positive bacteria, the dye is retained much better in gram positive than gram negative. Counterstain: Add a few drops of safranin O and let sit for 20 seconds, rinse off the safranin, blot the slide dry. The counterstain is done so that we can see the gram-negative bacteria that did not get stained by crystal violet. The gram positive bacteria will not show this stain because of its light color, allowing us to differentiate between the two types of bacteria easily.

II.

Metabolism

A. Anaerobic Metabolism

i. Glycolysis

a) Glycolysis is the process of glucose metabolism mediated by enzyme-coupled reactions, which combine spontaneous and non-spontaneous reactions into one reaction with a negative delta G, allowing for further steps to be taken towards the end product of pyruvate and a net gain of two ATP. All non glucose sugars are converted to glucose, except for fructose which enters in a later step of the process. 2 NADH molecules are also gained (4 electrons) for use in the electron transport chain. b) 2 ATP are required for glycolysis (for the formation of required phosphorylated sugars), and 4 ATP are gained in later steps. The net result is +2ATP. Energy is gained from ATP by breaking down the chemical bonds holding the phosphates together. The phosphates have 4 negative charges in close proximity, making ATP an unstable molecule, so the loss of the first phosphate (which has 2 negative charges) is spontaneous, requiring no energy to be removed. Therefore, Gibbs free energy is negative. The reverse reaction is not spontaneous, so it requires energy to put the phosphate on the now ADP, making ATP a form of energy storage. In step 4 of glycolysis, the 6 carbon sugar is changed into two 3 carbon sugars, both of which yield 2 ATP for a combined gain of 4. When taken into consideration with the 2 ATP cost of glycolysis, the net result is +2ATP. c) The steps in glycolysis are all mediated by various enzymes which change one molecule to another. The steps that require ATP input are mediated via an enzyme coupled reaction.

d) In the above picture, glyceraldehyde 3-phosphate donates electrons to NAD+, and in doing so becomes 1,3 bisphosphoglycerate which is a positively charged molecule. The NAD+ gains a negative charge through this reaction and then has an overall neutral charge. This is a redox reaction. The NAD+, now an NADH, is an electron carrier.

THE PICTURE ABOVE IS WRONG (NADH+ + H+, it should be NAD+ NADH) ii. Krebs Cycle (Citric Acid Cycle) a) In the Krebs cycle, pyruvate is oxidized, leading to the reduction of NAD+. A continuous cycle of redox reactions leads to the complete oxidation of glucose. One molecule of glucose yields 24 electrons (including glycolysis) and a net gain of 2ATP. The electrons and then carried to the electron transport chain by NADH. Pyruvate turns into Acetyl CoA by being oxidized by NAD+ which itself becomes an NADH. A CO2 is also produced in this step. In further steps, 2 more CO2 molecules are produced, completely breaking down the pyruvate. At each one of these steps, the sugar molecule is oxidized by NAD+ which becomes NADH. There are a further two steps to produce FADH and another NADH, making the krebs cycle an overall gain of 4 NADH and 1 FADH, or 10 electrons. Also, one molecule of GTP is produced for each pyruvate going through the krebs cycle, so 2 in total for each molecule of glucose. Because two pyruvates are produced in glycolysis, there is a net gain of 8 NADH and 2 FADH, or 20 electrons, through 10 redox reactions. Also, 6 CO2 molecules are released. With the krebs cycle and glycolysis combined (counting both molecules of pyruvate produced), there is a gain of 24 electrons in the form of 10 NADH and 2FADH.

iii. Electron Transport System:

a) The electron transport system acts as if it were one big enzyme because it takes place through enzyme-coupled reactions via proteins embedded in the cell membrane. b) NADH is oxidized and its electrons are removed (spontaneous reaction). These electrons are combined with hydrogens (H) and Oxygens (O2) and combine with them to form water. c) While this is happening, H+ moves from inside the cell (low concentration) to the outside of the cell (high concentration) which is a non-spontaneous reaction. The work required to do this is produced from the removal of electrons from NADH and the subsequent formation of water. d) In the last step, ATP synthase creates ATP from ADP and a P (non spontaneous). The work required for this reaction is produced by the influx of protons moving into the cell from an area of high concentration (spontaneous) that was established by moving H+ into the environment previously. iv. Total reaction from glycolysis, krebs cycle, and the electron transport chain: a) 6H2O +C6H12O6 6CO2 + 12H2O v. Types of Bacterial metabolism a) Anerobic: Use inorganic elements (nitrogen, sulfur, etc...) as their final electron acceptor. These organisms die in the presence of oxygen because oxygen is a more favorable electron acceptor than what they use, and because they can't effectively

utilize oxygen as an electron acceptor, they die. b) Aerobic: Oxygen as a final electron acceptor The vast majority of ATP produced inside the cell is produced through the electron transport system which uses Oxygen as its final electron acceptor. When oxygen is not present the cell is unable to synthesize the majority of its ATP. When Oxygen is removed the cell can't turn the ever growing NADH produced through glycolysis to NAD+. In glycolysis, there is a step that requires NAD+, so when it runs out and can't be replenished the cell gets stuck and can't produce anymore ATP, thus dying. c) Facultative: Can use oxygen as a final electron acceptor, but don't have to. Unlike the aerobic cell which can't replenish NAD+ without oxygen present, facultative cells have another method to produce NAD+ known as fermentation

In fermentation the cell proceeds with glycolysis, but when pyruvate is formed it is reduced into lactate and by NADH which is converted into NAD+. This prevents the same stall (no production of NAD+) that aerobic organisms experience when no oxygen is available, allowing for the survival of the facultative cell.

vi. Transport Processes

a) Types of Ports: Uniport: Moving in one direction either way Symport: Two moving in the same direction at once either way Antiport: One moving out, one moving in.

b) Diffusion: Molecules moving through the membrane due to concentration differences. Passive and requires no input energy to happen.

c) Facilitated Diffusion: The passive movement of larger molecules through the membrane via integral proteins in the membrane. These molecules move relatively fast because of the integral proteins. Only specific molecules can move through any given integral protein.

d) Active Transport: Requires the use of ATP to provide the energy for the diffusion of the molecule through an integral protein. Because active transport moves molecules from low to high concentration, this process requires energy in the form of a coupled reaction breaking down ATP to ADP. e) Secondary Active Transport: The movement of molecule X from high concentration to low concentration is used to drive the movement of molecule S from low concentration to high to move both inside the cell. The build up of the concentration gradient of molecule X requires energy which is garnered from the hydrolysis of ATP into ADP. The two compounds, when moving inward, can either bind weakly or the force of the movement of X brings S in with it.

III.

Virulence

A. Antibiotic: A product, usually of a fungus, competing bacteria, or synthetic origin, that inhibits or kills susceptible bacteria. i. Compound types: a) Compounds that inhibit bacterial growth are known as biostatic compounds. b) Compounds that kill bacteria are known as bacteriocidal compounds. c) Selectively toxic compounds only kill certain bacteria. ii. Methods of activity a) Inhibition of cell wall synthesis. Can be selective in the sense that there is a difference between the cell walls of gram positive and gram negative bacteria, as well as that of the walls between prokaryotic cells and eukaryotic cells. Biocidal Selectively toxic b) Inhibition of the use of metabolites (stopping enzymes, preventing uptake of food, etc...). Biostatic Selectively toxic c) Inhibition of protein synthesis via targeting of ribosomes. Very selectively toxic in

that ribosomal size differs between eukaryotes and prokaryotes and thus only certain bacteria will be targeted. Selectively toxic Biostatic d) Inhibition of reproductive machinery (DNA polymerase, helicase, etc...). Biostatic Not selectively toxic B. Drug immunity a) In order for a bacteria to be resistant to an antibiotic, it must acquire a gene that codes for something that will grant resistance. Mutations for antibiotic resistance are favorable in an antibiotic rich environment, and by the process of natural selection it will be the bacteria to reproduce the most. An acquisition of an rPlasmid, a plasmid that contains a gene for an antibiotic resistance, allows for a bacteria to gain a resistance to a particular antibiotic. A plasmid is defined as a small, circular, extrachromosomal, self-replicating, and not critical for the cell's survival (but could be adventageous in certain situations). If the cell is in a bad situation in which resources are limited, a bacteria will expel the plasmid to save replication resources.

b) Plasmid transferral can occur in three ways:

Conjugation is the process of bacterial sex (via sex pili) by which genetic material is transferred. The pilus of the donor comes into contact with the recipient and both connects and brings the cells together. At this point, the plasmid is copied and transferred, via the pilus, to the recipient.

Bacterial Transformation is the transfer of naked genetic material from a dead cell to a recipient cell. The naked genetic material, once taken up becomes a single stranded piece of DNA. At this point, it can either become integrated into the host cell's chromosome, or not, and become degraded.

Transduction is the transfer of a plasmid via viral delivery. A bacteriophage (virus) that has injected its DNA into a bacteria can copy its DNA and make more bacteriophages, and in the process accidentally incorporate a bacteria's DNA into their own genome. Then, if enough bacteriophages are made to bleb out of the cell, and not kill it, then these transducing phages, ones with the DNA gained from the cell, can infect another host and transfer that DNA to the other cell which might or might not incorporate it into its own genome. The bacteriophages degrade the host DNA which is why this kind of transfer can take place because this degraded DNA, if somehow reformed, is much more likely to incorporate something new into the genome.

c) Conjugation is most efficient at DNA transfer, transduction next, and transformation least. d) One can tell whether or not a gene was added to a host cell through transformation or transduction based on whether or not neighboring sequences to the added DNA are viral in nature or not. e) Types of resistance. Preventing access to the cell (alterations of the cell wall, etc...) Antibiotic degradation

Antibiotic alteration The development of an efflux pump which rapidly expels the antibiotic out of the cell via a use of active transport.

C. Toxins a) Exotoxins: Toxins that are released by the cell when it is still living and are byproducts secreted by the cell during normal growth. Super Antigens A/B Toxins Consist of two polypedtides that work together to attack the cell. The A is the enzyme, or active, polypeptide. The B is the binding component. The A/B complex enters the cell through endocytosis, via the binding component. The A part kills the cell once the complex is inside, and the B part leaves the cell. In this picture, the A part causes ADP-Ribosylation which disrupts protein synthesis. However, there are many different A/B toxins that act through various means.

Membrane disrupting toxins: Type 1: Proteins that bind to the host cell, insert themselves into it, and form a channel or pore in the cell causing cell lysis (cytoplasmic contents rush out, water rushes in). Type 2: Toxins that release phospholipase which cleaves the hydrophilic heads of the victim cell's membrane, making it unstable, causing cell lysis.

Examples of Exotoxins: Neurotoxic: Prevents signal transduction between neurons (pesticides, snake venom, botulism toxin). Enterotoxin: A toxin that forms pores in the cell membrane, usually in epithelial cells in the intestinal wall. After a while, toxins lose their toxicity and become toxoids, which are inactive toxins that retaint heir antigentic properties. As a result, white blood cells can use these toxoids to gain an immunity to its active form. Vaccines utilize this principle by using dead cells with toxoids in them to give the vaccinated person an immunity to the toxin. Environmental Signals Bacteria excrete toxins when they grow, so the better they grow, the more toxins they excrete. How well bacteria grow are based on the environment, of which factors include iron concentration, CO2 levels, temperature, pH. Because of the fact that toxin production takes energy, when the cell is in an unfavorable environment then it won't produce much toxin. b) Endotoxins: Toxins that are part of the structure of the cell and are released when it dies/lyses. D. Phase Variation a) Phase variation is a random change in a specific DNA sequence, caused by an enzyme, that affects the phenotype of a cell's outer structure. By changing outer structure, the cell can change an outer structure so that antibodies and phagocytes do not recognize the cell. The immune system produces antibodies much slower than the cell has phase variations.

Many of these outer structures are PAMPS (pathogen associated molecular patterns) that let phagocytes know when a cell is virulent. These changes are reversible and happen very frequently, more so than mutations. They are also inheritable. b) Inversion: When an enzyme flips the promoter/sequence of a gene so that it reads it in reverse/a different order Only certain bacteria can phase vary because they posses the enzymes necessary. Enzymes are specific for certain genes, so one enzyme can not invert everything but only one certain sequence.

Types of inversion In A, the blue and yellow coding sequences are switched. If there was no termination sequence between the two, then it would read yellow first and then blue, but after the inversion it would read blue and then yellow, changing the way that the mRNA would be translated. In B, there is a termination sequence between the yellow and the blue genes. Normally only yellow is made because the promoter changes is pointing that way, but when it is flipped only blue is made. c) Slip Strand Mispairing

Slip Strand Mispairing occurs when an enzyme removes or injects a bases into a gene, changing the protein that it codes for. In the above picture, an enzyme has removed a CTCTT string of bases, making the stop codon come sooner, changing the protein coded for.

d) Under certain conditions, such as different temperature and pH, phase variation is more or less likely to occur. When the cell is under stress, it will produce more of the enzyme to phase vary a gene that it could help it. If, for example, there were high amounts of antibiotics, the cell could produce more enzymes to phase vary the genes that code for the cell wall to make itself more resistant to the antibiotics. E. Biofilm: An aggregate of micro-organism when cells stick to each other or a surface.

a) In the picture above, the surface is known as the substratum, and the peach colored fluid is known as the EPS (extracellular polymeric substance). At first planktonic cells, the floating cells that can move around in the media via a flagellum, form the first layer of the biofilm and change their phenotype via phase variation in such a way as to lose their flagellum at that point. The EPS, or extracellular polymeric substance, is the medium secreted by the cells that form the biofilm and is made up of complex polysacharrides, extracted DNA, and proteins. This medium is very sticky, and is tough for things like antibiotics to penetrate, giving the aggregate of cells the ability to stick to things and get protection. b) For the biofilm to begin forming, the concentration of potential biofilm forming cells needs to reach a quorum, or a critical amount of cells that signals the start of biofilm formation.

For the cells to known when this happens, quorum sensing signaling molecules are sent out. Quorum sensing in itself is a method of communication between bacteria that allows the coordination of group behavior based on population. Autoinducer molecules are secreted by the bacteria, so when there are a lot of bacteria there are a lot of autoinducer molecules. When the concentration of these autoinducer molecules is high enough, the molecules bind to an enzyme with which it complexes. This complex will then go to the biofilm relevant gene sequence(s) and start the transcription of the sequence. c) Biofilm Formation

For a biofilm to form, the environment needs both nutrients and moisture. Firstly, the cells, which have reached a concentration high enough (a quorum) to begin forming a biofilm, adhere to the substratum to form a monolayer off of which more layers can be made. As it grows, the EPS begins to form. The aggregate then grows (steps 2-5) until it becomes a mature biofilm, at which point it can only change in shape and size. At this point it can send out planktonic cells to go and form more biofilms, or even detach a small part of the aggregate to go to another place and mature. While in the biofilm, gene transfer via conjugation can take place. Biofilms can form on things like teeth, on contacts, in sewage pipes, showers, etc... Many, many places. d) Microbes, when they switch to a biofilm mode of growth, undergo a phenotypical change so that they can group together with other bacteria. This change in mode is due to environmental stress and is done via phase variation. e) When drugs are made to combat the production of biofilms, the drugs are made to target the autoinducer molecules. F. Influenza a) A virus, like influenza, is a microorganism that contains genetic material and can not grow or reproduce apart from host cell. b) Influenza A, unlike B and C, causes disease.

The virulence of influenza is determined through the organization of two surface proteins. The HA helps the virus to enter the cell, and NA helps it to exit. There are 16 kinds of HA, and 9 types of NA. c) The method by which the virus changes is through either the processes of antigenic drift and antigenic shift. Antigenic drift is point mutations that occur when the virus is replicating itself inside the host cell.

Antigenic shift occurs when two different strains of a virus infect the same host, and combine to form a new strain that combines the surface antigens (HA and VA) of the two viruses. When the lipid envelope surrounding the genetic material of the virus opens up inside the cell to allow for replication to occur, the genetic material of it and another strain could mix to produce a new strain or serotype.

G. Rous Sarcoma Virus i. ********************************

IV.

Immunology

A. Hallmarks of immunology i. Rubor Redness a) Leaky blood vessels due to histamines so phagocytes can get to the site ii. Calor Heat a) The heat slows down cell division iii. Dolor Pain a) So you stop moving and don't hurt yourself more iv. Tumor Swelling (edema) a) The cell infiltration into the affected area as well as things being secreted there make the area swell. B. White Blood Cells i. White blood cells are most encountered in the bloodstream

ii. Types of white bloodcells a) Phagocytes Eat PAMPS Found in bloodstream b) Monocytes: Macrophages Eats pathogens Uses lysosomes near the cell membrane to break down bacteria Normally found in the bloodstream, but can move to infection location Is nonspecific and has no memory While digesting cells, it releases cytokines to call other cells to the area Function 2: Acts as an antigen presenting cell (the dendridic cell also does this). After it chews up the cell, it shows the adaptive immune system (B cells) what it needs to look for by pushing specific parts (antigen) of the chewed up cell to its surface c) Neutrophil: phagocyte It only phagocytes cells and releases cytokines Found in the tissue and in the blood. Has internal digestive enzymes to break down bacteria d) Mast Cell Found all over the body in places like the blood, skin, nerves, etc... The mast cell has something inside of it called granules that are tightly packed, pre-formed cytokines and histamines (histamines make holes in blood vessels) When a trauma or infection occurs, the mast cell releases the granules which disperse and trigger an immune response Most of the mast cell's cytokines are pro inflammatory e) T Cell (lymphocyte) Made in the thymus and end up in the lymphnode Cytotoxic T cell (CD8+) kills virally infected cells Helper (CD4) controls immune response f) B Cell (lymphocyte) Made in bone marrow and end up in lymphnodes Recognizes a large variety of antigens Produce antibodies (plasma cell variety) and have a memory (memory cell variety) of past infections and antibodies. iii. Communication a) Immune cells communicate with chemokines, chemicals that immune cells produce and receive Cytokines serve the same role as the chemokine, to allow immune cell communication. These two types of chemicals can be pro immune response, and negative (to stop the immune response once the infection is gone) Interlukins are a type of chemical that signal the cell to do certain things (abbreviated: IL) In order for phagocytes to receive the mast cell's signal, vasodialators are required to open up the tightly packed blood vessel walls to allow for the cytokines to go out and phagocytes to come into the infected tissue.

Chemicals in the mast cell's granules signal for the production of proteins on the walls of the blood vessels that act as a velcro for phagocytes that allow them to stick more effectively to the walls until they encounter a hole that they can go through to reach the affected site. This process is called extravasation. When we are sick, we take things like benedril, which is an antihistamine, that is a vasodialator and helps spread the signal and bring in phagocytes by expanding the blood vessel walls and adding more space to it.

C. Host defense i. Physical Defense (1st line of defense, nonspecific)

a) Physical obstructions Tears Mucus Saliva Earwax Cilia Skin b) Chemical barriers Saliva (digestive fluids and enzymes) Stuff in earwax and mucus ii. Innate immunity (2nd , nonspecific) a) Phagocytosis Phagocytes eat PAMPS Monocytes eat PAMPS b) Inflamation

c) Chemokines iii. Adaptive immunity a) Is specific, has memory, and only activated when needed b) Order of events in adaptive immune response: An antigen presenting cell, such as a macrophage or a dendritic cell, goes to the lymphnode and finds a helper t cell specific for the antigen being presented and presents it to it via the MHC Class II complex, and the antigen binds to the T cell receptor. The helper T-cell activates, cytokines are released by the APC, and TH1 and TH2

helper cells are produced/activate. TH2 activates the b cell specific for the antigen, and TH1 activates cytotoxic t cells (CD8), all via cytokines. The activated CD8 cells, told by the cytokines to go to the infected area, go to there and find infected cells. The infected cells have MHC Class I that they use to show antigens, and the CD8 that is specific for the antigen being presented (many CD8 cells go the infected site, but only the antigen specific one becomes fully activated once the specific antigen is encountered) will kill the infected cell. The TH2 cells activate B cells via cytokines, and the B cells go to the activated T cell, and the one out of many that binds to its T cell receptor via its MHC Class II (it is an antigen presenting cell that can recognize specific antigens via its B cell receptor) and becomes further activated. It then proliferates into plasma cells which make a lot of antibodies specific to the antigen, and also memory b cells. B cells have IgM antibodies on their surfaces that can bind to their specific antigens, allowing the B cell to ingest them, and present it to a CD8 cell via its MHCII complex. Though, this order of events doesn't happen too much. c) Humoral (Antibody production) B cell's are responsible for a lot of antibody production Antibody Function Increase, by a lot, phagocytosis, in a process called opsonization in which antibodies are used to coat pathogens making them more easy to find and eat. Neutralize viruses and toxins by binding to the B part of an A/B toxin so it can't enter the cell via a receptor. Cause pathogens to agglutinate, or stick together, making them easier for phagocytes to take care of. Activate the complement cascade. The proteins involved are found in the blood, and are inactive. The complement cascade is generally ineffective against viruses.

 Alternative Pathway Trauma and Inflammation cause C3 to become cleaved and break into C3A and C3B C3A is an anaphylatoxin, a compound that causes mast cells to release histamine and recruit more white blood cells. The more histamine produced, the more the blood vessels open, and the more complement cascade proteins come out of the blood. Some C3B molecules act as an opsonin (coats antigens to make them easier for phagocytes to find and eat). Other C3B molecules bind to C5, resulting in the cleaving of the compound to produce C5a and C5b. C5a is an anaphylatoxin like C3A. C5b combines with other proteins to make the membrane attack complex (MAC). The MAC makes pores in the bacterial membrane causing cell lysis to occur. Classical Pathway Activated by antibodies that bind to the surface of a pathogen. When the antibodies bind, a series of complement proteins bind to the antibodies and become cleaved.

 Many proteins get cleaved and activated, eventually leading to a step where the classical pathway merges with the alternative. C3 can also bind to PAMPS, and when enough of it is bound to the bacteria, the bound C3 will cleave and activate itself, leading to the activation of the alternative pathway, leading to the death of the bacteria. The pathways are not as effective against encapsulated bacteria and, to a lesser extent, bacteria with a thicker cell wall because encapsulated bacteria have an extra layer of protection that helps them prevent the binding of C3 to their PAMPS. As for the thicker cell wall, it is just a bit harder to make a pore in its cell wall because of the increased thickness.

All antibodies share the same general structure (shown above) made up of two heavy chains (dark green) which are identical to each other, and two light chains (light green) which are identical to each other. Holding the parts together is the hinge region. The structure of the antibody gives it a dual purpose. The constant region, the first part of the two heavy chains which are not attatched to the light chains, tells effector cells (macrophages, etc...) to eat. Based on one of 5 constant regions, we can classify antibodies as certain types IgA is found in secretions (such as in mucus membranes) where it makes things clump together. If they clump together they can be say, swallowed into the stomach. IgD is involved in B cell development IgE is involved in allergic reactions IgM is the first antibody made in response to a pathogen. IgG is made in huge amounts during an active infection. To have a healthy immune system, we need to be able to recognize about 10^11 antigens, but we have about 25,000 genes only.

The above picture is a picture of B cell germ line (germ line DNA is the same in all B cells) DNA whose change results in B cell differentiation. The V, D, and J genes in the germ line can be snipped out in many different ways, and since these genes are responsible for the structure of the antigen binding region, many different VDJ combinations can be produced. This VDJ recombination is done for each cell, and the final VDJ combination varies.

The variable, VDJ region is upstream of genes that code for IgM, IgD, etc... Because the IgM is closest to the variable region, it is always made first. When other kinds of antibodies are needed, genes can be cut out in such a way as to allow for the production of say, IgA, or IgE, etc... The variable region (antigen binding site) of the antibody binds to the antigen. The variable region is determined by the specificity of the cell that is creating them, and this is related to the specificity they gain through VDJ recombination. This means that antibodies, the B cell receptor, and the t cell receptor, if specific for the same antigen, will have regions of same protein sequences. d) Cell mediated (Cells causes pathogen death) Presentation Topic: Rickettsia rickettsii