MB 351 Test 2

Microbial growth is defined as:

An increase in the number of cells.

The final electron acceptor in the ethanol fermentation pathway is:

An organic compound

Recognize the enormous impact fermentation has had, and continues to have, on industrial products.

,,,

Be able to describe the methods used to measure growth of bacterial cultures (and their limitations).

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The best way to obtain a pure culture would be to use the streak plate method (also called T streak or isolation streak). In this method, you would start with a sterile loop. You would streak out the sample in one quadrant of the plate. After streaking, you would flame the loop again. Once cool, you would place the loop in the edge of the quadrant previously inoculated and streak into the next quadrant. This same procedure is repeated for section of the plate. If done properly, you will have well isolated colonies. Ideally, you would pick an isolated colony from this plate and repeat the entire procedure to be absolutely sure that you have a pure culture for further study.

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The size and shape of the microbes are only the starting point towards establishing any relationship between the microbes of interest and Earth microbes. Phylogenetic relationships are based on evolutionary relatedness. The best way to determine if such relationships exist between the microbes of interest and Earth microbes would be to isolate the nucleic acid from both and compare ribosomal RNA genes, or analysis by some other genomic method.

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Compare the phases of microbial growth. Be able to describe what happens to microbial populations in each phase and why these changes occur.

1. Lag Phase: (length of time is highly variable) • dependent on "history" of inoculum • Change in cell composiMon but no cell division • AdaptaMon to new medium condiMons • Resynthesis of damaged cell constituents 2. Log (Exponential) Phase: (often short) • # of cells doubles in fixed Mme period • Cells are most active (good for experiments) • Balanced growth phase - cell component synthesis is occurring at a constant rate relative to each other 3. Staitonary Phase: (most bacteria) • Growth limited by nutrient availability, toxic metabolites, and limiitng space. • Dying cells = dividing cells (viable count doesn't change) • Important time for 2o metabolite production, endospore formation 4. Death Phase • Essentially a reverse of exponential growth phase • Exponential loss of cell viability

Compare and contrast aerobic and anaerobic respiration.

Aerobic Respiration: Final electron acceptor-molecular oxygen, Substrate-level and oxidative phosphorylation, 36 ATP produced by eukaryotes and 38 by prokaryotes Anaerobic Respiration: Final electron acceptor-Usually an inorganic substance (such as NO3-, SO42-, or CO32-) but not molecular oxygen (O2), Substrate-level and oxidative phosphorylation, Variable ATP produce (fewer than 38 but more than 2)

Explain the roles and sources of macroelements, microelements and growth factors.

All cells consist mainly of what I call "CHNOPS" which stands for carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur. These elements are what we call macronutrients, meaning they are required in large amounts by the cell. The key macronutrients are C and N. All cells need C. About half the dry weight of a typical bacterial cell is made of C. It is the major element in all macromolecules. Oxygen and Hydrogen is required, it is usually attached to C. N is ~13% by dry weight of the cell. N is a key element in proteins, nucleic acids, and other cell parts. P (occurs as organic or inorganic phosphates) is needed for synthesis of nucleic acids and phospholipids. It is also found in the high energy bonds of ATP. S is needed in the amino acids cysteine and methionine, and also in vitamins like thiamine, biotin. S originates from inorganic sources in nature (sulfate or sulfide). Oxygen is an important element in organic compounds and macromolecules. Molecular oxygen (O2) requirements vary between different species of microbes. Other important elements are all metals used as cofactors in enzymes, to stabilize membranes and cell structure, etc. Mineral ions are necessary in most microbes, elements such as iron, potassium and sodium, play important roles in enzymes and macromolecules. So where do microbes get their macronutrient Carbon? Well, the majority of bacteria are "heterotrophs" rely on other organisms to form the organic compounds (such as glucose) that they use as carbon sources. Heterotrophs get most of their carbon from the source of their energy- organic materials such as proteins, carbohydrates, and lipids. This usually supplies their H and O requirements. The Autotrophs assimilate inorganic CO2 as a carbon source, reducing it (adding hydrogen atoms) to make complex cell constituents made of C, H, and O. These organic compounds can then be used by heterotrophs. Autotrophs are classified as Photoautotrophs which use light for photosynthesis, whereas Chemoautotrophs (aka chemolithotrophs, lithotrophs) gain energy by oxidizing inorganic substances such as iron or ammonia. Both these types of autotrophs carry out the autotrophic process of CO2 fixation. N is approximately 13% of dry weight. Needed for protein synthesis. Used primarily to form the amino group of amino acids of proteins. DNA and RNA synthesis needs N, think nitrogenous bases. Most microbes get it from decomposing protein containing material (organic cellular material) and re-incorporating the amino acids into new proteins. Some use NH4+ (ammonium ions) , some get nitrogen from nitrates (NO3-)in solution, while some photosynthetic cyanobacteria use N2 gas through nitrogen fixation. Sulfur is used in amino acids (cysteine and methionine), and in several vitamins (thiamine, biotin, and lipoic acid). Sulfur is obtained from either S containing-amino acids (organic sources) or from sulfate (oxidized) or sulfide (reduced), which are inorganic sources. Phosphorus is needed for nucleic acids, and phospholipids of cell membranes, in ATP. Phosphorus is usually supplied to a cell as inorganic phosphate (PO42- ). Microbes require several metals for growth, typically in very small amounts and are referred to as trace elements. Eg. Fe, Cu, Mo, Zn, B, Co, Mn. Since they are required in such small amounts (microgram/ug quantities), we call them "micronutrients". Chief among them is iron, which plays a key role in cellular respiration by serving as the redox center or function as cofactors in enzymes involved in electron transport reactions. Although sometimes these elements are added to the lab medium, they can be naturally present in tap water or other media components. Unlike trace elements, growth factors are typically organic, however, like trace metals, they are required in only small amounts (a micronutrient) and only by certain organisms. Many microorganisms can satisfy their own requirements for growth factors through biosynthetic processes (making their own), but some require one or more pre-formed from the environment and must then be supplied in the media culture in the lab. Three main types: Amino acids for proteins, Purines/pyrimidines for Nucleic acids, and Vitamins: mainly B-type vitamins, most vitamins function as coenzymes, which are nonprotein components of enzymes. These growth factors may be needed in varying concentrations, ranging from none to mg quantities per liter.

List the characteristics of the Bacteria, Archaea, and Eukarya domains.

Archaea- prokaryotic, varies in composition; contains no peptidoglycan, composed of branched carbon chains attached by glycerol by ether linkage, methionine, not antibiotic sensitive, lacking rrna loop, lacking trna Bacteria- prokaryotic, contains peptidoglycan, composed of straight carbon chains attached by glycerol by ether linkage, Formylmethionine, antibiotic sensitive, rrna loop, trna Eukarya- eukaryotic, aries in composition; contains carbohydrates, composed of straight carbon chains attached by glycerol by ether linkage, methionine, not antibiotic sensitive, lacking rrna loop, trna

Biosafety Levels (BSL)

BSL1: If you work in a lab that is designated a BSL-1, the microbes there are not known to consistently cause disease in healthy adults and present minimal potential hazard to laboratorians and the environment. An example of a microbe that is typically worked with at a BSL-1 is a nonpathogenic strain of E. coli. Work can be done on an open lab bench, requires a sink. Requires personal protective equipment (PPE) such as lab coats, gloves, eye protection. BSL 2: BSL-2 builds upon BSL-1. If you work in a lab that is designated a BSL-2, the microbes there pose moderate hazards to laboratorians and the environment. The microbes are typically indigenous and associated with diseases of varying severity. An example of a microbe that is typically worked with at a BSL-2 laboratory is Staphylococcus aureus. It includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosols in a lab setting, such as Clostridium difficile, most Chlamydiae, hepatitis A, B, and C, influenza A viruses, Salmonella. BSL-2 differs from BSL-1 in that: laboratory personnel have specific training in handling pathogenic agents and are directed by scientists with advanced training; access to the laboratory is limited when work is being conducted; extreme precautions are taken with contaminated sharp items; and certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment. BSL-3 is required for work involving indigenous or exotic agents, and they can cause serious or potentially lethal disease that are transmitted through the air (via aerosols). Respiratory transmission is the inhalation route of exposure. Laboratory personnel must receive specific training in handling pathogenic and potentially lethal agents, and must be supervised by scientists competent in handling infectious agents and associated procedures. Lab personnel are under medical surveillance and might receive immunizations for microbes they work with. All procedures involving the manipulation of infectious materials must be conducted within Biosafety Cabinets (BSCs) (shown at the top image), or other physical containment devices, or by personnel wearing appropriate personal protective equipment (eg respirators). A BSL-3 laboratory has special engineering and design features that prevent the release of microorganisms to the environment. Facilities have hands free sink, exhaust air cannot be recirculated, entrance is through two sets of self closing and clocking doors. Microbes that are worked on in BSL3 facilities are Mycobacterium tuberculosis which cause tuberculosis, etc. BSL-4 labs builds on the containment requirements of BSL-3 and is the highest level of biological safety. BSL-4 labs is required for work with dangerous and exotic agents that pose a high individual risk of life- threatening disease, aerosol transmission or unknown risk of transmission. The microbes in BSL4 labs cause infections that are frequently fatal and generally there are no vaccines or treatments for these infections. There are only a small number of such labs in the U.S (<10) and the world. Laboratory staff must have specific and thorough training in handling extremely hazardous infectious agents. Access to the laboratory is controlled by the laboratory supervisor. All handling of agents must be performed in a gas tight Class III Biosafety Cabinet or by personnel wearing a positive pressure protective suit. BSL-4 Laboratories have special engineering and design features to prevent microorganisms from being released into the environment. The lab is in a separate building or isolated restricted zone of the building, and has a dedicated supply and exhaust air, as well as vacuum lines and decontamination systems. Personnel mush change clothing before entering and shower upon exiting. Most of the pathogens worked on are viruses: Crimean-Congo hemorrhagic fever caused by Ebola, Junin, Lassa, Machupo, Marburg viruses , and tick-borne encephalitis virus complex (including Absettarov)

Identify the roles played by early pioneers in discovering chemical control of microbial growth (Lister, Semmelweis, etc.)

Back in the 17th C. in many European cities, hospitals for childbirth became common. These "lying- in" hospitals were established at a time when there was no knowledge of antisepsis (destruction of pathogens on living tissue) or epidemiology (the study of the distribution and determinants of health-related states). Patients were subjected to crowding, frequent vaginal examinations, and the use of contaminated instruments, dressings, and bedding. It was common for a doctor to do an autopsy and then go into the obsteric ward to deliver babies. The doctor would deliver one baby after another, without washing his hands or changing clothes in between. This resulted in high numbers of childbed fever/puerperal fever. The most common infection causing puerperal fever is genital tract sepsis (bacterial contamination) caused by contaminated medical equipment or unhygienic medical staff who contaminate the mother's genital tract during the delivery. Other types of infection that can lead to sepsis after childbirth include urinary tract infection, breast infection (mastitis) and respiratory tract infections. America consistently reported death rates between 20% to 25% of all women giving birth prior to the late 1800s. And in Europe, The death rates for mothers could range from 5-30%. Giving birth was dangerous business, and mothers knew it, in fact, they had a better chance of survival by staying away from these birthing hospitals. Along came, Ignaz Philipp Semmelweis, a Hungarian physician who is now called the "saviour of mothers". He discovered, by 1847, that the incidence of puerperal fever, could be drastically cut by use of hand washing standards in obstetrical clinics. He introduced hand washing with chlorinated lime solutions for interns who had performed autopsies. This immediately reduced the incidence of fatal puerperal fever from about 10 percent (range 5-30 percent) to about 1-2 percent. At the time, diseases were attributed to many different and unrelated causes-the whole "bad air/ miasmas, evil spirits, " etc. argument. Remember, this was before the work of Robert Koch and Louis Pasteur. Along came, Ignaz Philipp Semmelweis, a Hungarian physician who is now called the "saviour of mothers". He discovered, by 1847, that the incidence of puerperal fever, could be drastically cut by use of hand washing standards in obstetrical clinics. He introduced hand washing with chlorinated lime solutions for interns who had performed autopsies. This immediately reduced the incidence of fatal puerperal fever from about 10 percent (range 5-30 percent) to about 1-2 percent. At the time, diseases were attributed to many different and unrelated causes-the whole "bad air/ miasmas, evil spirits, " etc. argument. Remember, this was before the work of Robert Koch and Louis Pasteur. Along came, Ignaz Philipp Semmelweis, a Hungarian physician who is now called the "saviour of mothers". He discovered, by 1847, that the incidence of puerperal fever, could be drastically cut by use of hand washing standards in obstetrical clinics. He introduced hand washing with chlorinated lime solutions for interns who had performed autopsies. This immediately reduced the incidence of fatal puerperal fever from about 10 percent (range 5-30 percent) to about 1-2 percent. At the time, diseases were attributed to many different and unrelated causes-the whole "bad air/ miasmas, evil spirits, " etc. argument. Remember, this was before the work of Robert Koch and Louis Pasteur. Another pioneer in the control of microbial growth was Lord Joseph Lister (1867), a contemporary of Louis Pasteur and Robert Koch, he was a British surgeon. Joseph Lister became aware of Semmelweis's work and together with Pasteur's work, he realized the true nature of infectious disease. He then recognized that he could use this idea to help his surgery patients. At this time, major injuries, broken bones or surgery would often result in infection of the damaged area, sometimes leading to amputation or death. Lister found he could greatly reduce the number of microorganisms on wounds and incisions by using bandages treated with phenol (aka carbolic acid), an aromatic organic compound that killed microorganisms by denaturing their proteins and disrupting the cell membrane. During surgery he began the practice of spraying the wound with a fine mist of phenol using this spray bottle to kill microbes. Then he started washing everything in his operating room-hands, scalpels, wiping tables, etc. This image at the top right shows a operating theatre with someone using one of those carbolic acid/ phenol sprays. These practices greatly reduced the rate of infection and mortality of surgery patients, lending further credence to the germ theory of disease. Lister is credited for developing the use of antiseptics (compounds used on living tissue that kill microbes) and aseptic surgery (techniques to minimize microbial contamination). Lister earned a reputation as the world's safest surgeon.

Define microbial growth in bacteria, and describe process of binary fission.

Bacterial growth refers to an increase in bacterial numbers, not an increase in the size of the individual cells. In most bacteria, growth first involves increase in cell mass and number of ribosomes, then duplication of the bacterial chromosome, synthesis of new cell wall and plasma membrane, partitioning of the two chromosomes, septum formation, and cell division. This asexual process of reproduction is called binary fission and results in two daughter cells that are genetically identical. 1. cells elongate to approximately twice their original length and 2. then form a partition that constricts the cell into two daughter cells. This partition is called the septum and results from the inward growth of the cytoplasmic membrane and cell wall from opposing directions; 3. septum formation continues until the two daughter cells are 4. pinched off. By definition, when one cell divides to form two, one generation has occurred, and the time required for this process to occur is called generation time.

Discuss the various physical and chemical requirements for growth to occur and how they affect bacterial growth.

Carbon, Hydrogen, Nitrogen, sulfur phosphorus, etc. all key elements. We'll talk about them in a bit. Various micronutrients : trace elements and growth factors are needed in smaller amounts. Molecular oxygen (O2), a certain concentration may or may not be required. Various organic growth factors, usually special vitamins or other factors like amino acids. Plenty of water as most microbes obtain almost all their nutrients in solution from the surrounding aqueous environment. What about "physical requirements"? temperature, pH, and osmotic pressure, we will cover this in the next lecture.

Using examples, describe the differences between defined, complex, enrichment, selective and differential media.

Chemically Defined Media: are prepared by adding precise amounts of pure inorganic or organic chemicals to distilled water. Therefore the exact composition of a defined medium (in both a qualitative and quantitative sense) is known. For example, in Table 6.2, for a chemoheterotroph like E. coli, the chemically defined medium must contain organic growth factors , such as glucose, that serves as a source of carbon and energy. Defined media is used extensively for well characterized isolates in the lab, but may not be as useful when the nutritional requirements are unknown for a particular microbe. In this case, you might resort of a complex medium. Complex media are made from digests of microbial, plant, or animal products, such as casein (milk protein), beef (beef extract), soybeans (trypic soy broth), yeast cells (yeast extract), or any other number of highly nutritious substances. The disadvantage is that is nutritional composition is not known precisely. Complex media are often referred to as "Rich" media, which means that it contains the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Complex media usually provide the full range of growth factors that may be required by an organism so they may be more handily used to cultivate unknown bacteria or bacteria whose nutritional requirement are complex (i.e., organisms that require a lot of growth factors, known or unknown). Selective media are designed to suppress/inhibit the growth of unwanted bacteria and encourage the growth of desired microbes. Eg. Brilliant Green Agar is a highly selective medium used for the isolation of Salmonella species other than typhoid bacilli from feces and other materials. Brilliant green dye is used in this medium to inhibit gram-positive bacteria and most gram- negative bacilli for the selective isolation of gram negative Salmonella species from feces and other clinical specimens. Enrichment media: it is usually a complex medium base to which additional nutrients such as serum or whole blood are added. These added nutrients better mimic conditions in the host and are required for successful lab culture of some human pathogens like Streptococcus pyogenes (strep throat) and Neisseria gonorrhoeae (gonorrhea). For example: Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients. Or Chocolate agar, this does not contain chocolate! It is enriched with heat- treated blood (40- 45°C), which turns brown and gives the medium the color for which it is named. Another time when you will need enrichment cultures: Since microbes may be present in small numbers can be missed, especially if other microbes are present in much larger numbers, like for soil or fecal samples. The medium for an enrichment culture provides nutrients and environmental conditions that favor the growth of a particular microbe but not others. In that sense it is also a selective medium, but it is designed to increase very small nos. of the desired type of organism to detectable levels. Eg. Want a phenol metabolizing microbe, take soil sample and put in enrichment medium where phenol is the only source of Carbon and energy, microbes unable to metabolize phenol don't grow. The medium is allowed to incubate for a few days, then a small amount is transferred to another flask of the same medium. After a series of such transfers, the surviving populations will be made of microbes capable of metabolizing phenol. Differential media make it easier to distinguish colonies of the desired organism from other colonies growing on the same plate. It is a media that supports growth of many different organisms, but differentiates between them. This type of media will allow identification of microbes based on their growth, color, and appearance on the medium. Eg. Blood agar contains 5% sheep's blood.

In the Elvis case study (CC 3), according to your boss, Elvis was treated with ethanol, triclosan, and the plastics were impregnated with chlrohexidine. This treatment of Elvis was:

Disinfection

List some products of fermentation

Ethanol: Beer, Wine, Fuel Lactic Acid: Cheese, Yogurt, Rye bread, Sauerkraut, Summer Sausage

An intact cell membrane is not used for energy generation by respiration.

False

Fermentation is more efficient than respiration in generating energy.

False

Respiration requires an initial electron donor but not a final electron acceptor.

False

Per molecule of glucose, what are the net products of glycolysis? (2 pts.) Per molecule of glucose, what are the net products of Krebs, including the bridge/link step? (2 pts.)

Glycolysis = 2 pyruvic acid, 2 ATP, 2 NADH (reduced electron carriers) Bridge step + Kreb's cycle = 2 ATP, 8 NADH, 2 FADH2

Describe the differences between substrate level phosphorylation and oxidative phosphorylation.

In substrate level phosphorylation, ATP is made when a high energy phosphate is directly transferred from a phosphorylated compound (a substrate) to ADP. Generally, the phosphate has acquired energy during an earlier rxn in which the substrate itself was oxidized. In this figure, the substrate is a 3 carbon compound called PEP (phosphoenol pyruvate, one of the breakdown products of glycolysis) which has one of these high energy phosphates, when this bond gets broken, the energy released is used to put the phosphate onto ADP to create ATP. This reaction is happens to be catalyzed by an enzyme (that's why you see this reaction within the "folds" of this purple blob representing an enzyme.

Which of the following statements about anaerobic respiration is FALSE?

It involves glycolysis only.

The 16S rRNA gene is used for phylogenetic studies of Bacteria because:

It is found in all bacteria and contains variable and constant regions

What is the fate of pyruvic acid in an organism that uses aerobic respiration?

It is oxidized in the Krebs cycle

Describe the chemical reactions of fermentation (lactic and ethanol fermentation)

Lactic acid bacteria are Gram positive nonsporulating bacteria that produce LA as a major or sole fermentation product from the fermentation of sugars. The two important genera of LA bacteria are the Streptococcus and Lactobacillus. LA fermentation can result in food spoilage, however, the process can also produce yogurt from milk, sauerkraut from fresh cabbage, and pickles from cucumbers. In Alcohol fermentation, carbon dioxide is released from pyruvic acid to form the intermediate acetaldehyde, which is quickly reduced to ethyl alcohol by electrons from NADH. Alcoholic fermentation, although rare in bacteria is common in yeasts, and is used in making bread and wine.

Define metabolism, and describe the fundamental differences between anabolism and catabolism

Metabolism is the term that refers to the sum of all chemical reactions within a living organism. It has two counteracting processes: Catabolic rxns and Anabolic rxns. When a microorganism consumes/takes in large complex molecules for energy, such as carbohydrates (complex sugars, starches), proteins, or nucleic acids even, they need to break it down to release energy. We call those reactions catabolic rxns, which generates smaller molecules (carbons get broken down to simple molecules like glucose units,proteins to individual amino acids,etc). These reaction share generally "oxidative", meaning that there is a removal of electrons from an atom or molecule, a reaction that often produces energy. The oxidation of these complex molecules results in energy conserved and stored as ATP. Since it's not a 100% efficient process, some heat is released during these reactions.

Be able to classify the different types of microbes according to their oxygen requirement (aerobes, facultative, anaerobes, etc.)

Obligate aerobes require O2 for growth; and can grow at full oxygen tensions (air is 21% Ox); these microbes use O2 as a final electron acceptor in aerobic respiration. Aerotolerant anaerobes do not use oxygen, but can tolerate it, so they grow throughout the tube. Obligate anaerobes (occasionally called aerophobes) do not need or use O2 as a nutrient. In fact, O2 is a toxic substance, which either kills or inhibits their growth. Anoxic (O2 free) environments are common in nature (mud, marshes, deep subsurface of earth). Obligate anaerobic prokaryotes may live by fermentation, anaerobic respiration, bacterial photosynthesis, or the novel process of methanogenesis. Facultative anaerobes (or facultative aerobes or just "facultative") are organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions (no O2) they grow by fermentation or anaerobic respiration, but in the presence of O2 they switch to aerobic respiration. A microaerophile requires oxygen to survive, but requires environments containing lower levels of oxygen than are present in the atmosphere. Therefore they need oxygen levels that are <20% concentration(which is atmospheric oxygen concentration). Many microphiles are also capnophiles, as they require an elevated concentration of carbon dioxide too.

Which of following is NOT necessary for cellular respiration?

Oxygen

Discuss the genotypic and phenotypic tools used in taxonomy

Phenotype: Morphology: cell and colony shape, spores, flagella, cell wall pattern Gram Reaction: positive, negative, acid fast Biochemical: lipid analysis, enzyme protein analysis, serotype Nutritional: phototrophic, autotrophic etc. Physiological: aerobic, anaerobic, fermentative etc.. Others: storage compounds, temperature optima, pH range, symbioses, antibiotic sensitivity, gaseous needs, phage sensitivity Genotypic: most precise method for classification G:C content, DNA hybridization, nucleic acid sequence analysis (whole genome sequencing, 16S rRNA analysis). Plasmid analysis, analysis of chromosomal DNA sequences.

Explain how microbial control techniques work (physical and chemical) and their range of uses and limitations. Be able to give examples of each.

Physical: Heat: There is "Dry" heat: provided by flame or incineration: like a Bunsen burner, or by infrared heat using a bacticinerator. Or use of hot air sterilization, which is forced ventilation of heated air: like that provided by a drying oven. Dry heat kills the organisms by destructive oxidation of essential cell constituents. Killing of the most resistant endospores by hot air sterilization requires a temperature of 170 °C for 2 hours. Dry heat is most often employed for glassware; syringes, metal instruments and paper wrapped goods, which are not spoiled by high temperatures. You will often see these in doctor's offices. It is also used for anhydrous fats, oils and powders that are impermeable to moisture. There is "moist" heat obviously this type of heat requires a liquid. Moist heat kills microbes by coagulation of proteins (denaturation), which is caused by breakage of Hydrogen bonds that hold the proteins in their 3D structure. There are variations in moist heat: pasteurization is heat treating a food or liquid to eliminate pathogens, however Pasteurization will not achieve sterilization. You can also boil the liquid, which will kill most pathogens, but not necessarily all endospores, therefore boiling is not always a reliable sterilization procedure. For reliable sterilization with moist heat,this requires temperatures above that of boiling water. Radiation that kills microbes (sterilizing radiation) is of two types: ionizing and nonionizing. Ionizing radiation includes Xrays, gamma rays, or hi energy electron beams. These types of radiation have high penetration and can ionize molecules; particularly ionizing H20 = forming highly reactive hydroxyl free radicals which results in DNA damage, killing the microbe. Used for sterilizing pharmaceuticals and medical, dental, supplies. Non-ionizing radiation has a wavelength longer than that of ionizing radiation (>1nm). Ultraviolet light (UV) is a type of nonionizing radiation, it causes thymine dimers which inhibit correct DNA replication. UV is very effective but doesn't get through glass or plastic very well, but we do use it in closed environments, like biosafety cabinets (an enclosed, ventilated lab workspace for safely working with materials contaminated with pathogens. Filtration, this physically removes microbes by passage through a screen-like material with pores small enough to retain microbes. You can readily purchase disposable filters of various sizes- 0.22um or 0.45um, even some 0.01um, and these should capture most microbes, given that the average E. coli is ~1 um. In addition, know that there are a bunch of other ways that we won't discuss: such as Cold , High atmospheric pressure , Desiccation Osmotic pressure, etc. We will instead focus on the use of moist heat treatments since this is the most common method. Chemical: • Phenolic compounds (derivatives of phenol): Remember Lister and phenols/carbolic acid? they denature proteins and membranes...but are irritants and toxic to humans.. • Alcohols: denature proteins and membranes, dissolves lipids. • Halogenated compounds: oxidizing, damages protein synthesis • Gaseous chemosterilizers: sterilizes in a closed chamber, such as Ethylene Oxide: extremely toxic.. Alkylates proteins, pretty much kills everything. • Aldehydes: extremely toxic, inactivates proteins by forming covalent cross links with several organic functional groups in proteins (-NH2, -OH, -COOH) Unfortunately, formaldehyde is a human carcinogen, limiting its use....unless you're dead. Glutaraldehyde and formaldehyde are used by morticians for embalming. I know, how morbid. • Quaternary ammonium compounds (Quats): these cationic detergents are the most widely used surface active agents around. Mostly likely destroy cell permeability by interfering with cell membranes. • Finally, Biguanides: injures the plasma membrane by blocking an enzyme needed for lipid synthesis. We will talk a bit more about the more popular ones used.

Recent research supports the theory that organelles such as the mitochondria and chloroplasts, and indeed all cells, and major pathways evolved from_____________.

Prokaryotes

Explain how bacteria can support respiration using electron acceptors other than O2.

Some microbes are capable of using nitrate as their terminal electron acceptor. The ETC used is somewhat similar to aerobic respiration, but the terminal electron transport protein (nitrate reductase) donates its electrons to nitrate NO3- instead of oxygen. Nitrate reduction in some species (the best studied being E. coli) is a two electron transfer where nitrate is reduced to nitrite NO2-. Starting at the beginning of the ETC, NADH donates its two electrons which flow through the cytochrome b/c1 complex (the purple protein) and the quinone pool (Q, QH2) , then to nitrate reductase (red protein) resulting in the transport of protons across the membrane as discussed earlier for aerobic respiration. These accumulated protons generate a PMF, which will drive the production of ATP via ATP synthase. The reaction for nitrate reduction is shown here on top. N03-, nitrate; N02-, nitrite. This reaction is not particularly efficient. Nitrate does not as willingly accept electrons when compared to oxygen and the potential energy gain from reducing nitrate is less. If microbes have a choice, they will use oxygen instead of nitrate, but in environments where oxygen is limiting and nitrate is plentiful, nitrate reduction takes place. The product of this reaction, nitrite NO2- is either excreted or could be further reduced to ammonia NH3- if the microorganism contains a "nitrite reductase" to perform this conversion, however, it doesn't conserve any energy.

Describe how the process of denitrification can occur in a model organism like Paracoccus.

Some organisms, like E. coli, are capable of only carrying out Nitrate respiration/ reduction. They are able to use nitrate as the final electron acceptor and reduce it to nitrite using the enzyme nitrate reductase. This metabolic capability is dictated by the genetic of this organism. However, some microbes are able to serially reduce nitrate completely to dinitrogen/nitrogen gas N2. These bacteria are called "Denitrifying bacteria" and this process is called Denitrification. Eg. Pseudomonas stutzeri and Paracoccus species are capable of denitrification because they have the genes that encode for all the enzymes (nitrate, nitrite, nitric oxide, nitrous oxide reductases) that perform this pathway. The process is carefully regulated by the microbe since some of the products of reduction of nitrate to nitrogen gas are toxic to metabolism. The advantage of a cell for carrying out this complete reduction of nitrate is: the ETC serves as a place to oxidize NADH back to NAD+ and free it up for accepting electrons again, and movement of electrons through this respiratory chain provides an opportunity to pump protons across the membrane to create a PMF to make ATP.

Define taxonomy, phylogeny.

Taxonomy: • The science of identifying, naming, and organizing living organisms into systems of classification. • Provides a reference for identifying organisms • Provides universal names (nomenclature) for organisms. • Example: Salmonella typhii (Genus, species) • Organizes organism into categories (taxa)- predictive • Microbial taxonomy combines: • Phenotypic • Genotypic Phylogeny: • The study of the evolutionary history of organisms • Each species retains some characteristics of its ancestor • Grouping organisms according to common properties implies that a group of organisms evolved from a common ancestor • Anatomy • Fossils • Analyzing DNA sequences

Explain how temperature, pH, osmotic pressure, affects growth.

Temperature affects microbes in two opposing ways, as temperatures rise, the rate of enzymatic reactions increases and growth becomes faster. However, above a certain temperature, proteins or other cell parts may be denatured or irreversibly damaged. For every microbe, there is a minimum temp below which growth is not possible, and optimum temperature at which growth is most rapid, and a maximum temperature above which growth is not possible. These 3 temperatures are called the cardinal temperatures, and are characteristic for any given microbe and can vary dramatically between species. For example, some microbes can grow optimally at 0degC, while others this can be higher than 100degC. The optimum temperature reflects a state in which all or most cellular components are functioning at their maximum rate and is usually closer to the temperature maximum than to the minimum. Each species has its own characteristic and particular range of values in which it grows and reproduces best. These boundary values define the maximum and minimum temperature at which life can exist (and grow). Each species of microbe has its own, unique upper and lower limit, which is a defining characteristic for that species. In analogy to a temperature range, every microbe has a pH range, typically about 2- 3pH units, within which growth is possible. Also, each microbe shows a well defined pH optimum, where growth occurs best. Most natural environments have a pH between 3-9. Terms used to describe organisms that grow best at a neutral range (pH >5.5 and <8) are called neutrophils. The approximate pH for optimum growth is 7. Acidophiles grow at pH<5.5, there are different classes of acidophiles, some grow best at moderately acidic pH (pH5) and others at very low pH (pH 1). Some acidophiles are obligate (require) acidic conditions: sulfolobus, thermoplamsa, ferroplasma. If the pH is raised to 7, the cytoplasmic membrane of acidophiles are destroyed and cells lyse. These microbes are not just acid tolerant but require high concentrations of H+ for membrane stability. Alkaliphiles prefer pH of 8 or above. Again, the pH optima for different microbes may vary from pH8 to 10. Many of these alkaliphiles are found in highly alkaline habitats such as soda lakes and high carbonate soils. Osmotic pressure is the pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane (like the plasma membrane). It is also defined as the minimum pressure needed to nullify osmosis. High osmotic pressures have the effect of removing necessary water from a cell. When a microbe is in a solution whose concentration of solutes is higher than in the cell (the environment is hypertonic to the cell), the cellular water passes out through the plasma membrane to the high solute concentration. This osmotic loss of water causes plasmolysis, or shrinkage of the cell's cytoplasm. This will inhibit cell growth. Thus adding salts to a solution results in the increase of osmotic pressure can be used to preserve foods. If you've ever had salted fish or meat, this is how it works-adding the salt literally draws the water out of any microbial cells that are present and thus prevents growth.

Discuss the limitations of a five-kingdom classification system.

That was the most accepted classification system at the time. In the 5 kingdom system, prokaryotes were placed in kingdom monera, and eukaryotes were in the other 4. This was based on macroscopic and microscopic observations. But DNA-based phylogenetic analysis (the study of the evolutionary history or organisms) revealed that the 5 kingdoms do not represent 5 primary evolutionary lines. Instead, cellular life on earth evolved along three primary lineages called Domains. Bacteria and archaea are exclusively microbial and are made only of cells that lack a membrane enclosed nucleus (prokaryotes). The third lineage, is eukaryotes and is primarily microbial (unicellular) and includes all the other original five kingdoms except bacteria.

Describe how the ETC works and how a proton gradient is generated.

The ETC starts with NADH oxidation and regenerates NAD+, the electrons have to go somewhere... they are transferred through a series of membrane bound carrier molecules and ultimately to a terminal electron acceptor. Energy from the electrons is used to actively transport (pump) protons (H+) across the membrane (aka proton translocation), thereby establishing a proton gradient that generates ATP via a process called chemiosmosis.

A key feature of cellular respiration is the removal of electrons from fuel molecules (oxidation) and the ultimate acceptance of these electrons by a low-energy electron acceptor. The process involves the use of electron carriers, NAD+ and FAD, which play crucial roles in multiple steps of the metabolic pathways. The overall equation for cell respiration is shown below. C6H12O6+6O2+38ADP+38Pi→6CO2+6H2O+38ATP Why do NAD+ and FAD NOT appear in the overall equation?

The NAD+ and FAD are initially reduced then oxidized to their original state, so they do not appear in the net equation.

Define Cellular respiration and Fermentation. Name the stages associated with each.

The cellular respiration of glucose occurs in 3 principal stages: glycolysis, krebs, and ETC. 1. Glycolysis (aka Embden Myerhof pathway) is the oxidation of glucose to pyruvic acid with production of ATP and energy containing NADH 2. Krebs cycle (aka TCA or citric acid cycle): oxidation of acetyl CoA (derivative of pyruvic acid) to CO2 with production of some ATP, NADH and another reduced e- carrier, FADH2. 3. ETC: NADH and FADH2 are oxidized, contributing e-s they have carried from substrates to a cascade of oxid-red rnxs involving a series of additional e- carriers. Energy from these rxns is used to make a lot of ATP. In respiration, most ATP is made in the third step. In the process of Fermentation, the first step is also glycolysis, but once it occurs, the pyruvic acid is converted into one or more different products via various fermentative pathways, depending on the type of cell. Products include alcohols and various acids. Unlike cellular respiration, there is no Krebs cycle or ETC here, and significantly less ATP is made.

Summarize endosymbiotic theory.

The endosymbiotic relationship of mitochondria with their host cells was popularized by Lynn Margulis. The endosymbiotic hypothesis suggests that mitochondria descended from bacteria that somehow survived endocytosis by another cell, and became incorporated into the cytoplasm. The ability of these bacteria to conduct respiration in host cells that had relied on glycolysis and fermentation would have provided a considerable evolutionary advantage. In a similar manner, host cells with symbiotic bacteria capable of photosynthesis would have had an advantage. The incorporation of symbionts would have increased the number of environments in which the cells could survive. This symbiotic relationship probably developed 1.5-2 billion years ago.

Pasteurella multocida is a facultative anaerobe and can switch to anaerobic metabolism. Predict which of the following is most likely to occur as a result of the switch from aerobic to anaerobic metabolism.

The organisms will grow more slowly because they will produce less ATP compared to aerobic metabolism.

Discuss the advantages of the three-domain system.

The three-domain system emphasizes the similarities among eukaryotes and the differences among eukaryotes, bacteria, and archaea. These organisms are classified by cell type in the three domain system. Classification is based on differences in ribosomal RNA (rRNA), the 3 domains also differ in membrane lipid structure, transfer RNA, and sensitivity to antibiotics. In this widely accepted scheme, animals, plants, and fungi are kingdoms in the Domain Eukarya. The Domain Bacteria includes all of the pathogenic prokaryotes, as well as many of nonpathogenic ones found in the soil and water. Photoautotrophic prokaryotes are also in this domain. The domain Archaea includes prokaryotes that do not have peptidoglycan in their cell walls. They often live in extreme environments and carry out unusual metabolic processes. Recent research has indicated that perhaps the mitochondia was originally of bacterial origin. This was proposed in the theory of endosymbiosis, suggesting that mitochondria and chloroplasts descended from bacteria that somehow survived endocytosis by another cell, and became incorporated into the cytoplasm.

Explain what is meant by generation time and be able to calculate it.

The time required for a cell to divide (and its population to double) is called the generation time. To calculate the number of genera2ons a culture has under gone, cell numbers must be converted to logarithms. Standard logarithm values are based on 10. The log of 2 (0.301) is used because one cell divides into two. Number of generations = Log # of cells (end) - Log # of cells (beginning)/0.301

Compare and contrast batch vs. continuous cultures.

There are two types of broths, batch cultures and continuous or chemostat cultures. The more common is the batch culture where microbes are cultured in an enclosed vessel (tube, flask) with a broth medium that supplies its nutritional requirements. Microbes are inoculated into this medium and placed in an appropriate environmental condition. Microbes will adjust and grow in this medium until nutrients run out, and/or toxic waste products are accumulated, and there are too many cells-running out of space (physical limitation), and growth ceases. In order to overcome these limitations of a batch culture, chemostats might be set up. In a chemostat, the growth chamber is connected to a reservoir of sterile medium. Once growth is initiated, fresh medium is continuously supplied from the reservoir. The volume of fluid in the growth chamber is maintained at a constant. Fresh medium is allowed to enter into the growth chamber at a rate that limits the growth of the bacteria. The bacteria grow (cells are formed) at the same rate that bacterial cells (and spent medium) are removed by the overflow. The rate of addition of the fresh medium determines the rate of growth because the fresh medium always contains a limiting amount of an essential nutrient. Thus, the chemostat relieves the insufficiency of nutrients, the accumulation of toxic substances, and the accumulation of excess cells in the culture, which are the parameters that initiate the stationary phase (where cell growth is equal to cell death) of the growth cycle. The bacterial culture can be grown and maintained at relatively constant conditions, depending on the flow rate of the nutrients. A practical advantage to the chemostat is that a cell population can be maintained in the exponential growth phase (where cells are rapidly dividing) for long periods, days or even weeks. There are wide range of uses for chemostats in experimental studies: microbial ecology, for enrichment and isolation of bacteria for nature by selecting for a stable population under nutrient and dilution rate conditions chosen. For microbial physiology: to mimic low substrate concentrations often found in nature and probe which microbes in mixed

Discuss how the disk diffusion method works.

Use the disk diffusion method where a disk of filter paper is soaked with a chemical and placed on an agar plate that has been previously inoculated and then incubated. The size and appearance of the "zone of inhibition", which is a clear zone representing inhibition of growth, will indicate that an organism is sensitive to that agent. Disks with antibiotics are commercially available and used to determine microbial susceptibility to antibiotics. In the plates shown here, we have 3 different bacteria exposed to four different chemical agents, which microbe is most sensitive to all chemical agents? S. aureus, it has a zone of inhibition around all the chemicals. Which is the most resistant? The Pseudomonas aeruginosa, it appears to only be sensitive to chlorine.

Define microbial death rate and describe its significance in microbial control. Be able to estimate decimal reduction time.

We use the "decimal reduction time (D)" to refer to the length of time it takes for an antimicrobial treatment to decrease the population by 1 log.

Define the following key terms related to microbial control: aseptic technique, sterilization, disinfection, antiseptic, bacteriostatic, bacteriocidal, bacteriolytic.

aseptic techniques: these are techniques to prevent microbial contamination of instruments, lab benches and workstations, patients, surgical rooms. These techniques are also applied in work at research labs, the food industry, and pharmaceutical companies that produce medicines and other health products. Sterilization: is a term that is often used and misused. In microbiology, this term has a very specific meaning, which is the complete destruction or removal of ALL viable (living) microorganisms. It is frequently achieved by chemical or physical treatments, with heating as the most common method used for killing microbes. Vegetative cells are cells that are actively growing or dividing, and endospores are highly resistant dormant forms of microbes. Sterilization refers to the destruction of both vegetative and endospores. Disinfection: The use of physical or chemical agents to inhibit or destroy microbes. Most often, one is reducing numbers of pathogens to point where they no longer cause disease. This is usually achieved using chemicals. "Antiseptics" refer to those chemical agent applied to living tissues to kill microbes, eg. Many topical antiseptics such as Alcohol, hydrogen peroxide, betadine solutions. "Disinfectants" refer to those chemicals applied to inanimate objects in order to achieve disinfection. These disinfectants may be too toxic to use on humans or animals Eg. Pine oils, bleach, alcohols. Some chemical agents are both, such as alcohols can be applied to human tissue, and also to instruments for disinfection. Bacteriostatic: are those chemical agents that do not kill but only inhibit growth. These are typically inhibitors of some important biochemical process, such as protein synthesis, and bind relatively weekly; if the agent is removed, the cells resume growing. Some antibiotics fall in this category. Bacteriocidal refers to those chemical agents that kill bacteria. While fungicidals kills fungi and viricidals kills viruses. The bacteriocidal agents will bind tightly to their cellular targets and by definition, kill the cell. However, dead cells are not usually lysed, and total cell numbers reflected in the turbidity of the culture remain constant Bacteriolytic agents kill cells by lysing them and releasing their cytoplasmic contents.

Microbial growth rates are controlled in a chemostat by

controlling the amount of a limiting nutrient.

Match the following cellular process with the correct cellular location in the prokaryotic cell.

fermentation → cytoplasm, glycolysis → cytoplasm, Krebs → cytoplasm, electron transport chain → cytoplasmic membrane

Match the following description with the correct biosafety level.

for pathogens not easily transmitted by aerosol → BSL2, requires full-body, air-supplied suit → BSL4, requires closed toe shoes → BSL1, requires non-circulating air flow → BSL3

Which of the following molecules is broken down in cellular respiration, providing fuel for the bacterial cell?

glucose

Discuss the importance of biofilm communities, how they form, and their potential for causing infection.

n nature, microbe seldom live in isolated single-species colonies that we see on lab plates. They more typically live in communities we call biofilms. Biofilms usually consist of a mixed bacterial population, but they may also consist of a single bacterial species. Some biofilms contain bacteria, viruses, and even eukaryotic cell types (most often yeast). These biofilm communities consist of sessile organisms, meaning that they grow attached to a surface. Biofilms on surfaces have a characteristic structure consisting of microcolonies enclosed in a hydrated matrix of microbially produced : proteins, nucleic acids, and polysaccharides, often called a slime or hydrogel. In this complex biofilm network, the cells act less as individual entities and more as a collective living system, often with channels to deliver water and nutrients to the cells at the inner portion of the biofilm. Biofilm organisms are significantly more resistant to environmental stresses or microbially deleterious substances (such as antibiotics and biocides) than planktonic (floating or free-living) cells. Biofilm cells present on infected tissues or medical devices are less susceptible to host immune responses than planktonic cells. The development of a biofilm in vitro involves the following 5 stages:Stage 1: reversible attachment of bacterial cells to a surface, Stage 2: irreversible attachment mediated by the formation of exopolymeric (polysaccharides, etc) material, Stage 3: formation of microcolonies and the beginning of biofilm maturation, Stage 4: formation of a mature biofilm with a 3-dimensional structure containing cells packed in clusters with channels between the clusters that allow transport of water and nutrients and waste removal, and Stage 5: detachment and dispersion of cells from the biofilm and initiation of new biofilm formation; dispersed cells are more similar to planktonic (that is, nonadherent) cells than to mature biofilm cells. The bacteria that become part of a biofilm engage in quorum sensing, a type of decision-making process in which behavior is coordinated through a "chemical vocabulary." Although the mechanisms behind quorum sensing are not fully understood, the communication process allows, for example, a single-celled bacterium to perceive how many other bacteria are in close proximity. If a bacterium can sense that it is surrounded by a dense population of other pathogens, it is more inclined to join them and contribute to the formation of a biofilm. There are a few advantages to the growth pattern of biofilms: First, bacteria are protected from the inhibitory effects of antimicrobial compounds, biocides, chemical stresses (such as pH and oxygen), and physical stresses (like pressure, heat, and freezing). Second, the polymeric matrix increases the binding of water and leads to a decreased chance of dehydration of the bacterial cells—a stress that planktonic cells are subject to. And third, close proximity of the microorganisms in biofilms allows nutrients, metabolites, and genetic material to be readily exchanged. Microbes in a biofilm can work cooperatively to carry out complex tasks. Eg. The digestive systems of ruminant animals such as cattle require many different species to break down cellulose. Most of the microbes are in a biofilm lining its digestive tract. This probably works the same way in your digestive tract! Often times though, biofilms are in important factor in health, for example, microbes in biofilms were found to be 1000 times more resistant to antibiotics! According to a recent public statement from the National Institutes of Health, more than 70% of all microbial infections are caused by biofilms.... UTI, periodontitis, ear infections, chronic wounds, cystic fibrosis, chronic sinusitis - One study found that biofilms are present on the removed tissue of two-thirds of patients undergoing surgery for chronic inflammation of the sinuses. The development of a biofilm allows for the cells inside to become more resistant to the body's natural antimicrobials as well as the antibiotics (antibiotic resistance) administered in a standard fashion. Biofilms can also be a problem in industry: they can be a problem in pipes and tubing where their accumulations impede circulation, such as in indwelling medical devices, including mechanical heart valves and catheters. Biofilms can do long term damage to water distribution facilities and other public utilities, causing fouling of equipment and contamination of products. The study of biofilms is thus an important aspect of medical and industrial microbiology.

Organisms that can grow with oxygen present are

obligate aerobes and facultative anaerobes

Discuss why ATP yields might vary, even within a single organism.

ot all bacteria make the same number of ATPs using cellular respiration, it will depend on the several factors such as the environmental conditions, and the metabolic (and thus genetic) capability of that organism. For example, an organism like Escherichia coli, is highly versatile, it is a facultative aerobe, so it can use oxygen when it is available as a final electron acceptor, but if it is not available, it can switch to an anaerobic mode of metabolism and use alternative final electron acceptors, such as nitrate, sulfate, etc., depending on what's available in its environment. It has this great metabolic potential and versatility because it contains all the genes capable of carrying out these metabolisms. The cell is sensitive to different environmental conditions and will sense, for example, varying oxygen concentrations in its environment, and will adjust its expression of genes for specific respiratory chains (carrier molecules in ETC) to respond to these changes. In this figure, under 1. High and intermediate Oxygen concentrations (~20%, which is normal atmospheric oxygen), E. coli will express the following respiratory chain that contains "DH1" or dehydrogenase complex 1, that is able to oxidize NADH, taking its electrons and pumping out 4 H+. The DH1 gets reduced. The electrons in DH1 then get shuttled along the ETC to Q, then to "QO1" quinone oxidase 1 complex, QO1 gets reduced when it gets the electrons (DH1 gets oxidized because it loss its electrons to QO1). QO1 is capable of pumping 4H+. This is now the end of the chain, the electrons join with protons and oxygen (O2) in the matrix fluid to form water(H2O). So for each NADH, you get 8H+ pumped in this first respiratory chain, the more H+ you have, the greater the proton gradient and will result in more ATP being via the ATP synthase. As you can see in scenarios 2, 3, and 4, you get different (generally lower) Oxygen concentrations (intermediate, low and intermediate), which causes E. coli to respond by expressing different respiratory chain electron carriers like DH2 (that doesn't pump H+), or QO2 (that pumps only 2 H+). Just by varying the oxygen concentrations, you can see E.coli is capable of four different electron transport systems and thus you will get different ATP yields depending on which one is expressed.

Describe the start and end products of glycolysis in terms of carbon compounds, ATP (net and total) and NADH. Identify where in the glycolytic process do you generate ATP and NADH and how this happens.

the oxidation of glucose to pyruvic acid producing ATP and NADH • 2 ATP are used • Glucose is split to form 2 glucose-3- phosphate • Two glyceraldehyde-3- phosphate oxidized to two pyruvic acid • 4 ATP produced • 2 NADH produced Net ATP from glycolysis: 2

Which of the following is an direct method for estimating the number of living microbes in a sample?

viable plate counts

Identify the products of the Krebs cycle in terms of ATP, FADH2, NADH and CO2.and how they are generated.

• OxidaIon of Acetyl-CoA produces some ATP and the reduced coenzymes: NADH and FADH2 (which contain most of the energy originally stored in glucose). • During next phase of respiraIon, a series of reducIons indirectly transfers energy from NADH and FADH2 to ATP during electron transport chain. AXer two cycles for 2 pyruvate: • Two ATP generated by Substrate level phosphorylaIon. • 8 NADH (remember the bridge step!) • 2 FADH2 1 NADH = 3 ATPs 1 FADH2 = 2 ATPs

Explain why the 16S rRNA gene is used for phylogenetic studies.

• Present in all cells and always has the same function • Enough sequence information in ~1500 residues • Contains both rapidly (variable) & slowly evolving (constant) regions - the fast regions are useful for determining closely-related species, and slow regions are useful for determining distant relationships. • Horizontal transfer of rRNA genes does not occur • Ribosomal database project (RDP): database has about 2 million aligned 16S rRNA sequences available

Describe the Chemiosmotic theory for ATP generaIon.

• The two processes ETC and OXPHOS are linked together by the ChemiosmoIc Theory. • This theory proposes that energy generaIon in biological systems is driven by proton gradients that are established across membranes: A proton gradient is established by two important processes: 1) The transfer of electrons between electron carriers in the membrane 2) Reactions that consume protons on one side of the membrane