1. a.

Fill in the following table with the carbon and energy source and an example (the correct scientific name) of each type of organism. Autotrophs fix CO2 and assemble into organic molecules (mainly sugars). Heterotrophs use preformed organic molecules. Phototrophs obtain energy from chemical reactions triggered by light. Chemotrophs obtain energy from oxidation-reduction reactions. Lithotrophs use inorganic molecules as a source of electrons. Organotrophs use organic molecules. Type of organism Photoautotroph (obtain energy from chemical reactions triggered by light.) Photoheterotroph Chemoautotroph Chemoheterotroph Carbon source Carbon dioxide Energy source sun

Organic compounds from enviroment Organic Form own organic compounds

light Organic Lipids, carbs, proteins

b. In which class would you place an organism that uses? i. glucose as the sole carbon and energy source? Chemoheterotroph ii. elemental sulphur as the energy source and CO2 as its sole carbon source? Lithotroph 2. What do the terms anabolism and catabolism mean? Explain the connection between them. Anabolism synthetic reactions require energy. It synthesises complex molecules. Catabolism breakdown of reactions that release energy and its conserved as ATP. One product may serve as a substrate for another reaction. The three main pathways are: fermentation, respiration and photoheterotrophy. Amphibolic both anabolic and catabolic. 3. In your own words define phototroph, chemoorganotroph and chemolithotroph. Use your textbook to find a bacterial example of each. Phototroph by using light as energy it obtains energy from chemical reactions of respiration and biosynthesis. E.g Chemoorganotroph - oxidize the chemical bonds in organic compounds as their energy source. E.g. Chemolithotrophs - an organism that obtains its energy from the oxidation of inorganic compounds

4. Name the three most significant electron carriers in eukaryotic and prokaryotic cells. What is the oxidised and reduced form of each? Eukaryotic NADH (donor) -> NAD+, NADPH, FADH (FADH2 reduced form, FAD oxidised form) Prokaryotic -

5. What are the 3 main mechanisms of ATP synthesis? Which of these mechanisms is the primary mechanism of ATP synthesis in: ATP contains a base, a sugar and 3 phosphates. Hydrolysis releasing phosphate (Pi) [hydrolysis of ATP at the terminal phosphate consumes H2O to produces ADP and pi. This release energy from ATP which can then be transferred to a coupled reaction of biosynthesis e.g. building amino acids. The 2 reactions use an enzyme with binding sites specific for ATP. Hydrolysis releasing pyrophosphate (PPi) [ATP can hydroloyze at te middle of phosphate realeasing pyrophosphate (ppi). The pyrophosphate usually hydrolyzes shortly after making 2p, meaning more energy is spent from ATP. But this in turn drives the reaction forward because twice as much energy will be needed to reverse the reaction. Phosphorylation of an organic molecule [ATP can transfer its phosphate to the hydroxyl group of a molecule such as glucose to activate the substrate for a rapid reaction. No water is consumed.

6. Explain the major differences between: a. aerobic and anaerobic respiration aerobic respiration uses oxygen in reactions in the
Aerobic: 1. occurs in presence of oxygen. 2. CO2 and water is produced 3. lot of energy is liberated (38 ATP) 4.occurs in plants' and animals' cells 5. C6H12O6 --> CO2 + H2O + ATP ( Energy) Anaerobic: 1. occurs in absence of oxygen 2. Lactic Acid or Alchol is produced 3. relatively small energy is liberated (2ATP) 4. occurs in many anaerobic bacteria and human muscle cells. 5. in muscles, lactic acid is produced while bacteria as Yeast produces Alcohol (Ethanol) 6. C6H12O6 --> Lactic acid / C2H5OH + ATP 1. Aerobic respiration take place in the cristae of mitochondria, while anaerobic respiration occurs in the cytoplasm.

b. respiration and fermentation What are the differences between respiration and fermentation? Respiration: the physical and chemical processes by which an organism supplies its cells and tissues with the oxygen needed for metabolism and relieves them of the carbon dioxide formed in energy-producing reactions.

Fermentation: an enzymatically controlled anaerobic breakdown of an energy-rich compound (as a carbohydrate to carbon dioxide and alcohol or to an organic acid). Doesnt need oxygen

c. substrate-level phosphorylation and oxidative phosphorylation

Substrate-level phosphorylation occurs during Glycolysis and the Kreb's Cycle and involves the physical addition of a free phosphate to ADP to form ATP. Oxidative phosphorylation, on the other hand, takes place along the electron transport chain, where ATP is synthesized indirectly from the creation of a proton gradient and the movement of these protons back accross the membrane through the protein channel, ATP synthase. As the protons pass through, ATP is created.

Substrate-level: production of ATP molecules via transfer of a phosphate group from an intermediate high-energy substrate directly to ADP. Oxidative: production of ATP molecules from the redox reactions of an electron transport chain Gylcolysis and Krebs cycle use substrate-level phosphorylation. Electron transport chain uses oxidative phosphorylation.

d. active transport & group translocation as mechanisms of nutrient uptake

Suggestion: Think about other terms you could be asked to compare. 7. a. Explain the difference between an oxidation reaction and a reduction reaction.
In an oxidation reaction one of the reactants is releasing, or giving up, electrons, making its oxidation number to increase. In a reduction reaction just the opposite happens, one of the reactants is gaining electrons and reducing its oxidation number.

b. Nitrate is reduced by some bacteria during anaerobic respiration. What is the formula for nitrate? What are the possible end-products of nitrate reduction? What is the environmental significance of this process? NO3- is the formula End products - ammonia, nitrite, or molecular N2 It involces the most oxidation steps, and cannot survive without prokaryotes. C. some bacteria use ammonia as an energy source and convert it to nitrate. Is the ammonia oxidised or reduced in this reaction? What are the names of the two key enzymes responsible for this conversion? What are their roles? Ammonia oxidation. Its called nitrification, a form of lithotrophy. Ammonia monooxygenase which oxidizes ammonia to hydroxylamine, and nitrite oxidoreductase, which oxidizes nitrite to nitrate. 8. There are 3 major pathways for the conversion of molecules such as glucose to pyruvate in prokaryotes. a. Name the pathways and identify which, if any, are unique to prokaryotes. Fermentation Glycolysis Cellular respiration b. Draw a table to compare the pathways in terms of ATP produced per molecule of glucose, reduced electron carriers and intermediates for biosynthesis.

Fermentation 2ATP per glucose (in the embden-meyerhof-parnas pathway, glucose is activated through 2 substrate phosphorylations by ATP. The breakdown f 2 molecules of glucose to pyruvate is coupled to a net production of 2 ATP and 2 NADH. Aerobic respiration releases 38 ATP Glycolysis 2 expended, 4 produced

c. Name the process by which ATP is produced in these pathways. Fermentation substrate-level phosphorylation Aerobic respiration Glycolysis d. What is the role of NAD+ in these pathways? Fermentation reproduces NAD+ e. Many bacteria utilise more than one of these pathways. Can you suggest why?

9. What are the substrates and products of the citric acid (or TCA) cycle? State the number of each of the products for each molecule of glucose.
acetyl CoA, ADP, Pi, NAD+, FADH+

10. Where do the enzymes for the citric acid (or TCA) cycle occur in a) eukaryotes and b) prokaryotes? in the mitochondria of eukaryotes and in the cytosol of prokaryotes 11. Explain the connection between the TCA cycle and the electron transport chain.
the Krebs cycle and electron transport chain are both involved in celluar respiration. They also both produce ATP

12. Most organisms, even if they do not carry out aerobic respiration, have at least some of the enzymes of the TCA cycle? Explain why. It uses the same process as their bacterial ancestors, who were all once anerobic. Its an amphibolic pathway.

13. What is the proton motive force? How is it generated how is it linked to electron transport? How is it linked to production of ATP? Where does this occur in prokaryotic cells? Why is it important for bacteria to generate a proton motive force (PMF)? The transfer of H through a proton pump that generate an electrochemical gradient of protons. It drives the conversion of ADP to ATP via ATP synthase. It is bound on the membrane. It is important for the movement of flagella, uptake of nutrients and flushing out toxic materials. When protons are pumped across the membrane, energy is stored in two different forms: The electrical potential (Dy) arises from the separation of charge between the cytoplasm and solution outside the cell membrane. The pH difference (DpH) is the log ratio of external to internal chemical concentration of H+. 14. A bacterium normally grows on 6 carbon sugars using glycolysis and aerobic respiration to conserve energy. Can these pathways still be used if the bacterium is grown on a medium with 5 carbon sugars as the only carbon source? Explain. By using the pentose phosphate pathway. It generates one ATP with no NADH, but 2 NADPH for biosynthesis. 15. Draw a diagram showing the major steps in the metabolism of glucose by E. coli when it is growing: a. aerobically b. anaerobically in a medium containing nitrate c. anaerobically in a medium without nitrate. ii. Compare the amount of ATP that would be produced in each of these conditions. 16. Use the following choices to answer i.-iv. a. E. coli growing in glucose broth at 35oC with O2 for 5 days b. E. coli growing in glucose broth at 35oC without O2 for 5 days c. Both a and b d. Neither a nor b. i. Which culture produces the most lactic acid? ii. Which culture produces the most ATP? iii. Which culture uses NAD+? iv. Which culture uses the most glucose? 17. To a culture of E. coli growing fermentatively you add 1g/l of NaNO3. Would you expect the growth rate of the culture to increase or decrease? The growth yield? Why? There would be no effect. OR e.coli will use the nitrate as a source. 18. In E. coli the synthesis of nitrate reductase (the enzyme that transfers electrons to nitrate) is repressed by oxygen. Why might this repression have evolved? Because oxygen is used as an electron acceptor. 19. Using the diagram on the following page to explain: a. why more ATP is produced when NADH is the electron donor and oxygen rather than nitrate is the final electron acceptor

b. why more ATP is produced when NADH donates the electrons rather than FADH2 c. why less ATP is produced by a bacterium growing aerobically with nitrite as an energy source (a nitrifying bacterium) compared to a bacterium growing aerobically on glucose.

Reference: Prescott, L.M., Harley, J.P. & Klein, D.A. (2005) Microbiology, Boston: McGraw Hill, Figure 8.7)

20. Why does the growth rate of a bacterium slow down when it is switched from aerobic to anaerobic growth conditions? Because it has to find different sources of energy and use a new pathway. The things needed for it to grow may only be small. 21. a. Define fermentation. What are the similarities and differences between fermentation and aerobic respiration? If oxygen is absent, many cells are still able to use glycolysis to produce ATP. electrons and hydrogen ions from the NADH produced by glycolysis are donated to another organic molecule.
They are both methods employed by a cell to derive energy. fermentation is anaerobic so that means O2 is not present in the process. Aerobic is more efficient because you get more ATP with the same amount of nutrients in comparison to anaerobic respiration.

b. Explain the role of NAD+/NADH in respiration and fermentation. How is redox balance maintained? (i.e. how is the number of oxidation and reduction reactions balanced?).
NADH (Reduced B-nicotinamide adenine dinucleotide) is a molecule formed in everycell in the body from niacin (vitamin B3). NADH functions as a major co-factor in energy production. NADH increases the production of ATP, the cells major "energy currency." This increase in ATP in turn increases cellular energy production. n electron carrier. It's not an enzyme but a coenzyme (which is an organic molecule that attaches allosterically/non competitively or competitively on active site, but in this case attaches in active site). It doesn't provide energy, yet.... because NAD+ is in it's oxidized form. When it acts as an oxidizing agent for glucose, it will become reduced (gain 2 electrons.. and one proton) to NADH (which is what releases the energy step-by-step in the ETC). Both store energy. Fermentation - It accepts electrons during one of the reactions of glycolysis and is reduced to NADH. It then donates those electrons back in one of the reactions of the specific fermentation.

So, for example, in a yeast carrying out ethanol fermentation, the pyruvate that is formed by glycolysis undergoes 2 more reactions. In the first, CO2 is removed from pyruvate, and the 2-carbon molecule gains the electrons from NADH to become ethanol. The NAD+ is then available to gain electrons again in glycolysis Because it is an Aerotolerant Lactofermentive Anaerobe, Which Only Uses Glycolysis for Energy

23. Explain the meaning of the terms nitrification and denitrification. What is the environmental significance of these two processes? Nitrification the oxidation of reduced nitrogen to nitrate or nitrite. Used in fertilizer to breakdown nitrogen into ammonia. Dentrification nitrateNO3 is reduced to nitriteNO2, diatomic nitrogenN2 and ammoniaNH3. It is important in the nitrogen cycle for soil, where it uses it as an electron acceptor in the absnese of oxygen.

24. Give examples of molecules that serve as the final electron acceptor in anaerobic respiration. To what are these molecules converted when they accept electrons (i.e. when they are reduced)? Nitrate Pyruvate - lactate NADH - NAD 25. What is lignocellulosic waste? What are its major components? Which of these components is fermentable? Fuel that is made up of plant structures, cellulose, lignin, hemicelluloce the fermentation of
lignocellulosic biomass to ethanol
[1]

is an attractive route to fuels that supplements the fossil fuels. .

Lignocellulosic biofuel production involves collection of biomass, deconstruction of cell wall polymers into component sugars (pretreatment and saccharification), and conversion of the sugars to biofuels (fermentation)

26. Discuss examples of microbial fermentations important in: Cheese -

Involves a standard series of steps: - Milk is filtered, centrifuged, and subjected to flash pasteurization. - Fermenting microbes are added as a starter culture. - The solid curd is then cut, or cheddared. - Curd is then heat-treated. - The pressed curd is shaped into a mold.
- The cheese is the ripened (or aged). Saccharomyces cerevisiae: Bakers yeast

Pyruvate Ethanol + CO2 - CO2 causes bread to rise. - Easier to chew and digest . ethanol removed by baking.

- More gluten more rise using fermentation systems. Batch fermentation:

one of the methods used in the industrial production of microorganisms, where the sterile growth medium is inoculated with the microorganisms and no additional growth medium is added. Continuous fermentation: A process in which cells or micro-organisms are maintained in culture in the exponential growth phase by the continuous addition of fresh medium that is exactly balanced by the removal of cell suspension from the bioreactor.{industrial fermentation is the use of biotechnology.}

in the gut of some animals. 27. Name and compare two microorganisms that are utilised in production of bioethanol. S. cerevisiae is good at converting glucose to ethanol Saccharomyces and Zymomonas 28. Bioprospecting and genetic modification are both used to source microorganisms for industrial applications. Briefly explain how. Bio-prospecting is the search for organisms with potential commercial applications. An important aspect is mining the genome. The cloned genes are transferred into an industrial strain, which must have the following attributes: - Genetic stability and manipulation - Inexpensive growth requirements - Safety - High level of protein expression - Ready harvesting of product Genetic modification - Baculovirus - Can be used to clone genes in insect genomes - Desired gene (e.g., antibody) is spliced into a bacterial transfer vector. - Insect culture cells are co-transfected with recombinant vector and a fluorescent marker protein. - The two DNAs recombine within the cell, and a recombinant baculovirus is produced. - Can be used to infect caterpillars (in their food) 29. By understanding metabolic pathways in microorganisms, scientists can devise novel ways of harnessing their reactions for industrial applications. One such application is the microbial fuel cell. , explain the principle of a microbial fuel cell.

30. Draw a diagram to summarise how chemolithotrophs such as nitrifying bacteria:

a. generate ATP b. generate reducing power for biosynthesis c. acquire carbon for synthesis of organic molecules

On your diagram show the flow of electrons. What is the source of electrons (electron donors) and what molecules act as electron acceptors? (Hint: Figure 5.23 in Madigan & Martinko textbook may provide a starting point) Sample essay questions 1. Microorganisms have evolved diverse strategies for energy conservation. Discuss the similarities and differences in the ways in which the ATP and reducing power needed for biosynthetic reactions is generated by chemoheterotrophs such as E. coli under aerobic and anaerobic conditions.

2. During the formation of the end products of fermentation pathways NADH is oxidised to NAD+. Why is this essential for the cell? Explain and compare how NAD+ is oxidised and reduced in aerobic and anaerobic chemoheterotrophs and chemolithotrophs.

3. Explain the central role of pyruvate in the metabolism of chemoheterotrophic bacteria. Pyruvate can be converted to carbohydrates or fats which can be used by chemoheterotrophs as a source of carbon.

SECTION 2: GENETIC DIVERSITY OF MICROORGANISMS 1. Name three methods by which microbial cells regulate enzyme synthesis and three methods by which they regulate enzyme activity. Synthesis Activity Amount (regulate level of transcription, translation. A slower process) Activity (temp. inactivation of the protein through either covalent/non-covalent changes in the enz. Structure. (post-translational regulation, rapid process)

2. Explain the mechanisms of induction and feedback regulation based on the operon model. Feedback inhibition mechanism for turning off the reactions in a biosynthetic pathway. (the end product binds to the first enzyme making it allosteric)

Operon consists of a promoter, operator and a series of genes. All of the genes are transcribed in a single mRNA molecule which are then translated into protiens. Inducible (presence of substrate) for catabolic need an inducer. repressible for anabolic. (presence of product) 3. The lac operon is a well-studied operon that is under both positive and negative control. are the genes in this operon? What are the products of these genes?

In the presence of lactose lacI binds as a tetramer to the operon region. And it represses the lac operon by preventing the formation of RNA polymerase, preventing transcription. Presence of lactore - b-galactosidase, if at low conc. cleaves/rearranges lactose to make

the inducer allolactose. It binds to LacI, reducing its affinity to the operator and this allowing induction of the operon.

CAP cyclic AMP receptor protein. It coordinates regulation of catabolite operons. Exists as active(cAMP bound) or inactive. It must bind to the site before RNA polymerase can bind to the promoter. Negative control of transcription binding of regulatory protein (repressor) at DNA regulatory site (operator) inhibits transcription.

Positive promotes transcription. Protein activated by inducer.

4. Catabolite repression is an example of global control mechanism. Explain what it means. What is meant by diauxic growth?

Diauxic a biphasic cruve of a culture growing on 2 carbon sources.

5. The best studied example of gene regulation by quorum sensing is bioluminescence in Vibrio fischeri. Explain what is meant by the term quorum sensing, and briefly describe the major components of the quorum sensing system in V. fischeri. Quorum sensing the process where bacterial cells work together at high density. Discovered in V.F; which is a bioluminescent bacteria that colonizes the light organ of the Hawaiian squid. So when there are large numbers in the squid there are enough cells to make visual impact. By using QS gene system it requires the molecule called an autoinducer, which binds to regulator. So the more cells the more autoinducers. 6. Transcription and translation of genes and regulation of these processes involves four different types of RNA. Name the four types and briefly describe their function. (See Section 10.5 of the text for type 4). mRNA messenger RNA ( tRNA transfer RNA rRNA ribosomal RNA sRNA Small RNA (doesnt function as the other 3. Can inhibit/enhance translation. Regulate gene transcription/expression. Antisense means that they can bind to mRNA and stabilize it or make it a target for degradation. (plasmid replication). It does not require protein synthesis. 7. Genetic recombination and mutation introduce the genetic diversity into a microbial population. Explain the difference between the two. Genetic recombination the breaking and re-joining of DNA strands to form new molecules of DNA encoding a novel set of genetic information. Mutation Mutation occurs when there are changes of genes, generating new genes and new phenotypes in follow. While genetic recombination occurs in Prophase I when non-sister chromatids from each

homologous chromosome touch each other called crossing over that leads to an exchange of genes from each non-sister chromatid, generating various forms of genotypes but no change of genes. However, both mutation and genetic recombination can lead to evolution because they contribute to genetic variation in populations that finally contribute to difference in ability to survive in certain environments.

8. Define the terms mutation, genotype, phenotype and wild-type strain. Mutation a perminant, heritable alteration in a DNA sequence (harmful/beneficial/ Neutral) Genotype The genetic constitution of an individual organism (genetic) Phenotype physical characteristics by the environment and genotype Wild type strain A strain, gene, or characteristic that prevails among individuals in natural conditions, as distinct from an atypical mutant type

9. Differentiate between: point and frame-shift mutations Point change in a single nucleotide Frame-shift changes open reading frame missense, nonsense and silent mutations Missense changes amino acid sequence to another Nonsense changes amino acid sequences to a stop codon Silent doesnt change the amino acid sequence. transition and transversion mutations Transition changing a purinepurine or pyrimidinepyrimidine Transversion swapping a purinepyrimidine spontaneous and induced mutations Spontaneous- arise without exposure to external agents, can result in DNA error (insertion/deletion) Induced a mutation that is produced by treatment with a physical or chemical agent that affects the
deoxyribonucleic acid molecules of a living organism.

10. Identify the different types of mutagens and outline their modes of action.

11. Describe a method for the detection and isolation of an auxotrophic mutant (eg hismutant). The Ames test relies on a mutant bacterial strain that is defective in hisG. - Cannot grow on a medium lacking histidine Identifying Mutagens - A mutagen-containing disk is placed on an agar plate with the mutant. - Mutagen causes reversion mutations, and colonies start to appear around the disk. 12. Discuss the significance of site-directed mutagenesis as a tool in microbial genetics.
a mutation is created at a defined site in a DNA molecule. "Playing god" - When and why use site directed mutagenesis? Constructing deletions Constructing point mutations at random sites in a particular region of DNA Constructing point mutations at a particular base pair Reversion of site-directed mutations

13. Briefly describe the major mechanisms of repair of DNA damage (including mutations). - Error-proof repair pathways, which prevent mutations Methyl mismatch repair - is based on recognition of the methylation pattern in DNA bases. - Uses methylation of the parental strand to discriminate from newly replicated DNA Nucleotide Excision Repair - An endonuclease removes a patch of single-stranded DNA containing certain types of damaged bases, including dimers Base Excision Repair - Specialized enzymes can recognize specific damaged bases and remove them without breaking the phosphodiester bonds. Recombinational Repair A single-stranded segment of the undamaged daughter strand can be used to replace a gap in the damaged daughter strand. by RecA

Error-prone repair pathways, which risk introducing mutations - Operate only when damage is so severe that the cell has no other choice but to die SOS (Save Our Ship) repair- Induced by extensive DNA damage - RecA coprotease activity stimulates autodigestion of the LexA repressor.- Expression of many DNA repair enzymes- Among them, 2 sloppy DNA polymerases that lack proofreading activity- However, the cell has no other option but to mutate or die.

14. Name three methods by which DNA is transferred from donor to recipient bacterial cells. Horizontal gene transfer donor cell contributes part of genome to recipient cell Three types Transformation Transduction Bacterial conjugation Recombination Two different DNA molecules in a cell can recombine by one of several mechanisms (generalized and site specific)

Mobile genetic elements - Transposable elements move from one DNA molecule to another, 15. Explain what is meant by transduction. Differentiate between generalised and specialised transduction. Transduction - is the process in which bacteriophages carry host DNA from one cell to another. - Generalized transduction: Can transfer any gene from a donor to a recipient cell (during viral assembly) - Specialized transduction: Can transfer only a few closely linked genes between cells 13. Compare and contrast transformation and conjugation as methods of genetic transfer in bacteria. Transformation - is the process of importing free DNA into bacterial cells. The cell needs to be competent or needs an artificial manipulation (disturbing the membrane by chemicals CaCI2 or electroporation). Gram negative bact. Dont need competent factors (Because they already are). They dont use transformers. The protein system allows DNA to move across G positive/neg cell walls.

Conjugation the transfer of DNA from one bacterium to another, following cell-to-cell contact. (imitated by pilus protruding from donor cell) J. Lederberg and E. Tatum demonstrated that the transfer of genes between bacteria depends on:direct cell to cell contact mediated by the F pilus unidirectional DNA transfer from donor to recipient

Bacterial plasmids - small, autonomously replicating DNA molecules that can exist independently or, as episomes, integrate reversibly into the host chromosome conjugative plasmids such as the F plasmid can transfer copies of themselves to other bacteria during conjugation Conjugation also contains the fertility factor. (Q 14) 14. Distinguish between F+, Hfr and F- strains of E. coli with respect to their physical nature and role in conjugation. F factors contain the information for formation of sex pilus attach F+ cell to F- cell for DNA transfer during bacterial conjugationF factors have insertion sequences (IS)assists in plasmid integration F+ - donor cell F - - recipient cell. a copy of the F factor is transferred to the recipient and does not integrate into the host chromosome donor genes usually not transferred F factor codes for sex pilus Type IV secretion system that makes contact between cells that DNA moves across plasmid is replicated by rolling circle method

HFr - The F-factor plasmid can integrate into the chromosome. - The cell is now designated Hfr, or high-frequency recombination strain. An Hfr cell is capable of transferring chromosome parts into a recipient cell. - Genes are transferred in order. - Entire chromosome takes about 100 minutes to transfer. - The process can be used to map genes.

F Conjugation result when the F factor incorrectly leaves the host chromosome some of the F factor is left behind in the host chromosome some host genes have been removed along with some of the F factor

these genes can be transferred to a second host cell by conjugation 15. Outline the role of transposons and plasmids in creating genetic diversity in bacteria. Transposons elements that from one DNA molecule to another. They exist in virtually all life forms and can move within and between chromosomes. Insertion sequences are smile trans. Elements that contain a transposase gene, flanked by a short inverted repeat sequence (targets for the transposase enzyme) segments of DNA that move about the genome in a process called transposition can be integrated into different sites in the chromosome are sometimes called jumping genes the simplest transposable elements are insertion sequences transposable elements which contain genes other than those used for transposition are called composite transposons segments of DNA that move about the genome in a process called transposition can be integrated into different sites in the chromosome

Simplest transposons are insertion sequences, which have no more than two inverted repeats and a gene for transposase Complex transposons contain one or more genes not connected with transposition (e.g., antibiotic resistance) 16. What are the roles of a cloning vector, restriction enzymes and DNA ligase in molecular cloning? They are nucleic acid molecules that deliver a gene into a cell. They are small enough to manipulate, survive n cells, contain recognizable genetic marker and ensure genetic expression.

Restriction enzymes - Bacterial enzymes that cut DNA molecules only at specific locations (restriction sites) there are two types ones that cut with sticky or blunt ends.

DNA ligase a ligase that catalyzes the linkage between two free ends of double-stranded DNA
chains by forming a phosphodiester bond between them, as in the repair of damaged DNA.

17. Outline the main stages involved in gene cloning and the techniques associated with these.

18. Why do bacteria produce restriction enzymes?

It is believed that restriction enzymes evolved as a defense mechanism againstviruses (phages). The enzymes do not cut the host bacterial DNA, as it is modified at the recognition site of the enzyme by methylation.

19. You are attempting to insert a gene for enzyme that degrades phenol into a bacterial cell using a plasmid as the cloning vector. In addition to the desired gene, you add a gene for ampicillin resistance to the plasmid. What is the purpose of the antibiotic resistance gene?
The ampicillin resistance gene (AmpR) is called a selectable marker gene and is incorporated into several plasmids that are commonly used in a wide range of genetic engineering and molecular biology experiments. The function of a selectable marker gene is to provide the host containing the plasmid a certain property that is not inherently present in the host. For example, the AmpR gene codes for a protein that makes cells containing this gene resistant to the antibiotic ampicillin. Therefore, when plasmids are successfully transfected into bacterilal cultures, one can assess which colonies have taken up the plasmid by looking at which ones survive on ampicillin-containing media. Those that do not survive do not have the plasmid. The surviving colonies can then be picked up and carried on to the next stage of experimental research.

20. Explain how insertional inactivation is used in the selection of a recombinant vector, giving specific examples.
a technique used in recombinant DNA engineering where a plasmid (such as pBR322) [3] disable expression of a gene.
[2]

is used to

The inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence. Any future products from the inactivated gene will not work because of the extra codes added to it. An example is the use of pBR322, which has genes that respectively encode polypeptides that confer resistance to ampicillin and tetracyclin antibiotics. Hence, when a genetic region is interrupted by integration of pBR322, the gene function is lost but new gene function (resistance to specific antibiotics) is gained.

21. Outline the polymerase chain reaction (PCR) highlighting the key factors considered important for its success. Large number of identical molecules of DNA produced in vitro Repetitive process consisting of three steps Denaturation Priming Extension Can be automated using a thermocycler Critical to amplify DNA in variety of situations Epidemiologists use to amplify genome of unknown

reaction mix contains primers target DNA thermostable DNA polymerase such as Taq polymerase each of the four deoxyribonucleotide triphosphates Multiple PCR - Pairs of primers that amplify species-specific genes can be combined in a single reaction Real time PCR a fluorescent assay able to monitor the progress of PCR as it occurs. 22. What are the essential characteristics of a cloning vector? What characteristics of plasmids make them especially useful as vectors for molecular cloning?

Origin of replication (ORI) -This process marks autonomous replication in vector. ORI is a specific sequence of nucleotide in DNA from where replication starts. Selectable Marker - This gene permits the selection of host cells which bear recombinant DNA (called transformants) from those which do not bear rDNA (non-transformants). Restriction sites- It should have restriction sites, to allow cleavage of specific sequence by specific Restriction Endonuclease. Restriction sites in E.coli cloning vector pBR322 include HindIII , EcoRI , BamHI , SalI, PvuI, PstI, ClaI etc.

23. Describe two examples of successful applications of recombinant DNA technology in medicine, industry or agriculture. Medicine - Genetically engineered viruses are being tested as gene therapy vehicles to deliver DNA to correct inherited diseases in humans. - e.g.: Adenovirus and adenoassociated viruses- Proteins from pathogenic bacteria and viruses cloned into fruits and vegetables may provide a cost-effective way to vaccinate large populations. Industry Agriculture Genes encoding insecticidal proteins from Bacillus thuringiensis have been engineered into plant genomes to protect crops from insect devastations. Sample essay questions 1. Microorganisms can respond rapidly to changes in their environment by regulating the amount or the activity of particular enzymes. Select and discuss three such mechanisms. In prokaryotes, regulatory proteins are often controlled by nutrient availability. This allows organisms such as bacteria to rapidly adjust their transcription patterns in response to environmental conditions. In addition, regulatory sites on prokaryotic DNA are typically located close to transcription promoter sites and this plays an important part in gene expression. Activator proteins bind to regulatory sites on DNA nearby to promoter regions that act as on/off switches. This binding facilitates RNA polymerase activity and transcription of nearby genes. At the same time, however, other amino acids would bind to negative regulatory proteins called repressors, which in turn bind to regulatory sites in the DNA that effectively block RNA polymerase binding As previously mentioned, enhancer sequences are DNA sequences that are bound by an activator protein, and they can be located thousands of base pairs away from a promoter, either upstream or downstream from a gene. Activator protein binding is thought to cause DNA to loop out, bringing the activator protein into physical proximity with RNA polymerase and the other proteins in the complex that promote the initiation of transcription. Different cell types express characteristic sets of transcriptional regulators. In fact, as multicellular organisms develop, different sets of cells within these organisms turn specific combinations of regulators on and off. Such developmental patterns are responsible for the variety of cell types present in the mature organism 2. Compare and contrast conjugation, transformation and transduction as mechanisms of genetic exchange in bacteria.
Transformation: a bacterium acquires DNA from the environment; Transduction: a virus infects a bacterium and some bacterial DNA is entrapped in the viral capside; this virus infects another bacterium and transfers the DNA of the previous bacterium; Conjugation: a bacterium transfers DNA directly to another bacterium. Transformation is when an organism can take up naked DNA from its environment usually from a dead host. Transduction is when Bacteriophages infect different bacteria, and while doing this incorporate some of the hosts DNA into their own. Then when the Bacteriophages offspring are released they also contain this new host DNA to be carried to otherBacteria Conjugation is when two living cells swap and share their plasmids with each other.

SECTION 3: MICROBIAL EVOLUTION AND SYSTEMATICS 1. What is the endosymbiotic hypothesis? How does it explain the evolution of eukaryotic cells? What evidence is there to support it? Occurs when one partner grows within the body of another. e.g.: The alga Chlorella growing in Paramecium - Alga provides nutrients. - Protozoa shelter alga. Lynn Margulis et al. have shown that assimilation of endosymbionts as mitochondria and chloroplasts played a central role in eukaryotic evolution. - Mitochondria evolved from an alphaproteobacterium, as or shortly after the eukaryotes diverged from the Archaea. - Chloroplasts evolved from cyanobacteria at some point before the divergence of red and green algae. 2. In which sequence did aerobes, anaerobes, chemolithotrophs and cyanobacteria evolve? What evidence is there to support your answer?

3. What is the definition of a species? Is it the same for bacteria and animals? Explain. Species a single, specific type of organism, designated by a genus and species name. Bacteria also have sub-classes and ranks due to differences in genetic diversity. Phenotypic categories, ecological categories, disease categories. 4. What is meant by phylogenetic classification? Development of phylogenetic classification schemes for prokaryotes has taken much longer to develop than schemes for animals and plants. Can you explain why? A phylogenetic tree or evolutionary tree is a branching diagram or "tree" showing the inferred evolutionary relationships among various biological species or other entities based upon similarities and differences in their physical and/or genetic characteristics. Because of mutations

5. What is a phylogenetic tree? How are phylogenetic trees developed? What is meant by evolutionary distance? The frequency of differences between homologous sequences can be used to generate a phylogenetic tree. - Estimates the relative amounts of evolutionary divergence between the sequences - Divergence data can be used to infer the length of time since two species shared a common ancestor. - Assuming mutation rate is same All phylogenetic trees depend on complex mathematical analysis to determine the most probable tree. Two common approaches are: - Maximum parsimony (The best fit tree is defined as the one requiring the fewest mutations to fit the data.) - Maximum likelihood (The probability that such a tree would have produced the observed DNA sequences) Phylogenetic trees can be drawn in different ways: rooted and unrooted.- Rooted trees indicate position of the common ancestor; unrooted trees do not. Evolutionary distance - The number of base substitutions per homologous site that have occurred
since the divergence of two deoxyribonucleic acid sequences.

6. Draw a phylogenetic tree that shows the linkages between the three domains into which living organisms are classified.

7. Why is rRNA chosen for studying evolutionary relationships between living organisms? Because rRNA is still shown in living organism and show common ancestory, even during evolution. 8. Explain the differences between: a. archaeal and bacterial cell membranes archaeal - differ from Bacteria and Eukarya in having branched chain hydrocarbons attached to glycerol by ether linkages polar phospholipids, sulfolipids, glycolipids, and unique lipids are also found in archaeal membranes bacterial - fatty acids attached to glycerol by ester linkages 9. What are plasmids? Explain their significance in classification schemes based on phenotypic characteristics.

10. Use diagrams to explain the principle of DNA hybridisation. What information does this technique give us about the relatedness of microorganisms?

11. a. Explain how a nucleic acid probe could be used to detect the presence of the bacterium Salmonella in a sample of powdered milk. basically, you will extract all DNA from some milk a do some qPCR or chip, when will you detect DNA specific for your patogen. If is it present, you know, it was infected, if it's not, it was under your detection limit (including absence)

b. Compare this process to the detection of Salmonella using conventional microbiology techniques (i.e. culturing and biochemical tests). Consider the need for a pure culture, the specificity of the tests and the sensitivity of the tests (i.e. how many cells can be detected?). it's definitelly simplier, faster, more specific... 12. Antigen-antibody reactions are the basis of immunological tests used in bacterial identification. a. Explain how these tests distinguish between different microorganisms. b. Name three methods by which Ag-Ab reactions can be detected. c. What are the advantages of immunological tests compared to culture-based methods of identifying microorganisms? 13. When tracing the source of an outbreak of a communicable disease, it is necessary to distinguish between strains of the same species. a. Distinguish between the terms strain and species. Species are reproductively isolated. Strains
can mate.

b. Name 4 test methods that can distinguish between strains within a species.

14. If two organisms have similar rRNA sequences, you can conclude all the following except a. they are related b. they evolved from a common ancestor c. they will have similar G-C ratios d. they will both ferment lactose e. none of the above 15. Biochemical tests are used in bacterial identification to determine a. staining characteristics b. amino acid sequences c. base composition of nucleic acid d. enzymatic activities e. all of the above 16. Amino acid sequencing provides direct information about: a. enzymatic activities b. nucleotide bases making up a gene c. identification of an organism d. antigenic composition e. morphology 17. Which of the following best defines a strain? a. a population of cells with similar characteristics b. a group of organisms with a limited geographical distribution c. a pure culture d. a group of cells all derived from a single parent e. the same as a species. 18. Serological testing is based on the fact that: a. all bacteria have the same antigens b. antibodies react specifically with an antigen c. the human body makes antibodies against bacteria d. antibodies cause the formation of antigens e. bacteria clump together when mixed with any antibodies. 19. Which of the following indicates that two organisms are closely related? a. both are cocci b. both ferment lactose c. their DNA can hybridise d. both normally occur in chains e. all of the above. 20. The phylogenetic classification of bacteria is based on: a. cell morphology b. Gram reaction c. rRNA sequences d. Habitat e. all of the above 21. Phage typing is based on the fact that: a. bacteria are destroyed by viruses b. viruses cause disease c. bacterial viruses attack specific cells

d. Staphylococcus causes infections

e. Phage and bacteria are related. 22. Some Archaea live in extreme environments. What mechanisms do the following groups of organisms have to enable them to survive in their natural habitats? i. Hyperthermophiles - The cell membrane contains high levels of saturated fatty acids which are usually arranged in a C40 monolayer to retain its shape at high temperatures. ii. Extreme halophiles Sample essay questions 1. Based on studies of ribosomal RNA living organisms are currently classified into three domains. Identify and compare the three domains and discuss how rRNA studies have contributed to our understanding of the phylogenetic relationships between them. Bacteria prokaryotic,consists of prokaryotic cells possessing primarily diacyl glycerol diaster lipidsin their membranes and bacterial rRNA, no nuclear membrane, traditionally classified as bacteria, contain most known pathogenic prokaryotic organisms (see [3] for exceptions), studied far more extensively than Archaea Kingdom Eubacteria Examples: Cyanobacteria photosynthesizing bacteria Spirochaete Gram-negative bacteria that include those causing syphilis and Lyme disease Firmicutes Gram-positive bacteria including Bifidobacterium animalis which is present in the human large intestine Achaea prokaryotic, no nuclear membrane, distinct biochemistry and RNA markers from eubacteria, possess unique ancient evolutionary history for which they are considered some of the oldest species of organisms on Earth; traditionally classified as archaebacteria; often characterized by living in extreme environments Kingdom Archaebacteria Examples: Methanogens metabolize hydrogen and carbon dioxide into methane Halophiles thrive in salt Thermoacidophiles thrive in acid and high temperatures (up to 110 degrees Celsius) Eukarya eukaryotes, nuclear membrane Kingdom Fungi or fungi Examples: Saccharomycotina includes true yeasts Basidiomycota includes shiitake mushrooms Kingdom Plantae or plants Examples: Bryophyta mosses Magnoliophyta flowering plants Kingdom Animalia or animals The ribosomal RNA is distinct between the three domains and can be used to trace evolutionary lineages. The molecular make up for the ribosomal RNA is identical between the three domains proving the theory of a common ancestor. The ribosomal RNA distinctions are easier to represent on paper than phylogenetic relationships. The genetic make-up of the three domains is so similar that it

makes it difficult to make genetic distinctions between species. Because rRNA molecules throughout nature carry out the same function, their structure changes very little over time. Therefore similarities and dissimilarities in rRNA nucleotide sequences are a good indication of how related or unrelated different cells and organisms are.

2. In classical microbiology microorganisms are classified and identified by studying a range of phenotypic characteristics. Compare this approach to the identification of bacteria based on molecular and immunological methods Because phenotypic techniques only allow for a very basic classification, and further studies are needed to classify, study, compare and distinguish different bacteria.