Microbiology Study Guide

Chapter 5 – Short answer

a) Phosphorylation - the addition of phosphate to a chemical compound

b) What are the 3 mechanisms of phosphorylation used by organisms? • Substrate level phosphorylation – ATP is generated when a high-energy phosphate is directly transferred from a phosphorylated compound (substrate) to ADP • Oxidative phosphorylation – electrons are transferred from a group of organic compounds to a group of electron carriers (NAD+ and FAD); occurs in inner mitochondrial of eukaryotes and in plasma membrane of prokaryotes; the sequence of electron carriers is called electron transport chain; the transfer of electrons from one electron carrier to the next releases energy, used to generate ATP from ADP in a process called chemiosmosis • photophosphorylation – occurs in photosynthetic cells, which contain light-trapping pigments (chlorophyll); light cause chlorophyll to give up electrons. Energy released from the transfer of electrons (oxidation) of chlorophyll thru a system of carrier molecules is used to generate ATP.

c) Oxidation – the removal of electrons

d) Chemiosmosis – process whereby energy is released when protons moves along a gradient and is used to synthesize ATP; responsible for most of the ATP generated; process – electrons from NADH pass down the ETC. Carriers in the chain actively transport protons across membranes thru proton pumps. One-directional pumping established a protein gradient; excess H+ on one side of membrane makes that side positively charged compared with the other side, creating a proton motive force…protons on the side of the membrane with higher proton diffuse thru special proton channels that contain ATP synthase; when this flow occurs, E is released and used by the enzyme to synthesize ATP from ADP and Pi

e) Holoenzyme – apoenzyme and cofactor that make a whole, active enzyme

f) Reduction – the gain of electrons

g) fermentation – releases energy from oxidation of organic molecules; doesn’t require oxygen or use the Krebs cycle or ETC; uses an organic molecule as the final electron acceptor

h) respiration – ATP generating process in which molecules are oxidized and the final electron acceptor is an inorganic molecule. 2 types: aerobic/anaerobic; aerobic – for organisms that use oxygen – final electron acceptor is O2; anaerobic – final electron acceptor is inorganic molecule other than O2, or rarely, and organic molecule

i) Coenzyme – cofactor that is a non-protein organic molecule; eg coenzyme A, flavin coenzymes (FAD, FMN), NAD+ and NADP+

3) State what happens during the following steps of carbohydrate metabolism:
a) Glycolysis – oxidation of glucose (6-carbon sugar) to two 3-carbon sugars and then to 2 molecules of pyruvic acid with the production of some ATP and energy-containing NADH

b) TCA/Kreb cycle – series of biochemical reactions in which the large amount of potential chemical energy stores in acetyl CoA is released step by step. In this cycle, a series of oxidations and reductions transfer that potential energy, in the form of electrons, to electron carrier coenzymes, chiefly NAD+. The pyruvic acid derivatives are oxidized and the coenzymes are reduced; for every 2 molecules of acetyl coA that enter the cycle, 4 molecules of CO2 are liberated by decarboxylation, 6 molecules of NADH and 2 of FADH2 are produced by oxidation-reduction reactions, and 2 molecules of ATP are generated by substrate-level phosphorylation. Summary - oxidation of acetyl CoA (derivative of pyruvic acid) to carbon dioxide, with the production of some ATP and energy containing NADH, and another reduced electron carrier FADH2 (reduced form of flavin adenine dinucleotide)

c) Electron transport chain – consists of a sequence of carrier molecules that are capable of oxidation and reduction. As electrons are passed thru the chain, there occurs a stepwise release of energy, which is used to drive the chemiosmotic generation of ATP. In eukaryotic cells – ETC is in inner mitochondria membrane; plasma membrane in prokaryotic. 3 classes of carrier molecules: flavoproteins, cytochromes and ubiquinones. Summary: NADH and FADH2 are oxidized, contributing to the electron they have carried from the substrates to a cascade of oxidation-reduction reactions involving a series of additional electron carriers. Energy from these reactions is used to generate a considerable amount of ATP.

4) Describe factors that influence enzymatic activity • Temperature – optimum: 34-40 degrees C; can be denatured by really high or low temps • PH (acidity) – optimum: 4 - 6; can be denatured by really high or low pH • Substrate concentration – activity will increase as substrate concentration increases • Competitive inhibition – competitive inhibitor binds on enzyme’s active site, taking the space of the substrate ex: sulfanilamide competes w/ PABA and takes its place; as a result, bacteria are unable to synthesize folic acid and can’t grow since they need PABA to synthesize it • Non-competitive inhibition (allosteric inhibition) – inhibitor binds to allosteric site instead of active site, which alters the structure of the active site, inhibiting the substrate from binding there

5) You look in the refrigerator and find some orange drink you had forgotten was there. The drink now has an off taste and bubbles. What is the most likely explanation for the changes in the drink?
When metabolize glucose will get ATP, Acid and CO2. The acid breaks down glucose – causes off taste; bubbles because of the gas from CO2

6) Describe mechanisms that can be used to generate ATP within the cell
Glycolysis, respiration – krebs cycle/ETC, fermentation, chemiosmosis – see above for descriptions

Chapter 6 – Short answer

a) culture medium – nutrients prepared for microbial growth in the lab

b) complex medium – extracts and digests of yeasts, meat or plants; each chemical composition varies slightly from batch to batch; ex: nutrient broth, nutrient agar

c) generation time – time required for a cell to divide or a population to double

d) selective medium – suppress unwanted microbes and encourage desired microbes; growth of unwanted organisms is prevented by salts, dyes or other chemicals

e) streak plate method – isolation method most commonly used to get pure cultures; sterile inoculating loop is dipped into a mixed culture that contains more than 1 type of microbe and is streaked in a pattern over the surface of the nutrient medium; the last cells to be rubbed off the loop are far enough apart to grow into isolated colonies, that can be picked up and transferred to a test tube to form 1 type of bacteria

f) pour plate method – empty plate is inoculated, melted nutrient agar is added to plate, swirl to mix, colonies grow on and in solidified medium

g) differential medium – make it easy to distinguish colonies of different microbes; type of selective media

2. Draw a bacterial growth curve and describe the different phases of growth • Lag phase – cells adjusting to media • Log phase – bacteria multiply at the fastest rate (exponentially, cells active metabolically • Stationary phase – equilibrium between cell division and death • Death phase – number of deaths exceed the number of new cells formed

3) List any 3 methods used to:

a) directly measure microbial growth: • plate counts – perform serial dilutions of a sample, most frequently used method to measure populations, measures # of viable cells, takes about a day for colonies to form • filtration – when quantity is small, at least 100ml of water is are passed thru a thin membrane filter whose pores are too small to allow bacteria to pass • most probable number MPN test – statistical estimating technique based on the fact that the greater the # of bacteria in a sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to grow in the tubes in a dilution serious • direct microscopic count – measured volume of a bacterial suspension is placed w/in a defined area on a microscopic slide eg: count # of bacteria in milk

b) Indirectly measure microbial growth: • turbidity – using a spectrophotometer to monitor bacterial growth in a liquid medium – as bacteria multiplies, the medium becomes turbid or cloudy with cells; amount of light striking the light-sensitive detector on the spectrophotometer is inversely proportional to the # of bacteria - the les light transmitted, the more bacteria in the sample • metabolic activity – assumes that the amount of a certain metabolic product is in direct proportion to the # of bacteria present eg: microbiological assay test • dry weight – microbe is removed from growth medium, filtered to remove extraneous material, and dried in desiccator. It is then weighed

Chapter 7 - Essay Questions

Terms • Sterilization – removal of all microbial life including endospores • Disinfection – process of reducing or inhibiting microbial growth on non-living tissue • Antisepsis – removal of pathogens from living tissue; sepsis – microbial contamination • Degerming – removal of microbes from a limited area • Sanitization – treatment intended to lower microbial counts on eating/drinking utensils to safe public health levels

1. Describe 5 physical methods Microbial growth:
-Heat and Moist heat ex autoclave (steam under pressure) - denatures proteins, Pasteurization - reduces spoilage organisms and pathogens; Dry heat sterilization – flaming, incineration, hot-air sterilization
-Filtration – removes microbes; ex hepa filters, membrane filters
Low temp – reduce metabolic rate, eg: refrigeration, deep freeze, lyophilization
High pressure – denatures proteins; alteration of molecular structure of proteins and carbs
Desiccation – prevents metabolism; eg: lyophilization – freeze drying to preserve microbes
Osmotic pressure – causes plasmolysis
Radiation (ionizing – X-rays, gamma rays, electron beams/non-ionizing - UV)– damages DNA

2. State 4 factors that influence the effectiveness of antimicrobial agents • Concentration of disinfectant (# of microbes) – the more microbes there are to begin with, the longer it takes to eliminate the entire population • Time – endospores require extended exposure; in heat treatment, longer exposure can compensate for lower temps • Environmental influences – presence of organic matter often inhibits action of chemical antimicrobials; in hospitals, the presence of organic matter eg vomit or blood, influences the choice of disinfectant • Microbial characteristics – influence choice of physical and chemical control methods selected

3. How do salty and sugars preserve food? Based on effects of osmotic pressure – high concentrations create hypertonic environments that cause water to leave cell; deny cell moisture needed for growth; eg: concentrated salt solutions to cure meat and thick sugar solutions to preserve fruit

4) The results below were obtained from a use-dilution test of two disinfectants. Cultures were inoculated into tubes with varying concentrations of disinfectants and incubated for 24 hr at 20°C, then subcultured in nutrient media without disinfectants. (+ = growth; - = no growth)

Disinfectant 1 Disinfectant 2
Concentration Initial Subculture Initial Subculture
1:10 - + - -
1:90 + + - -
1:900 + + - -
1:90,000 + + - -
1:900,000 + + - +
1:9,000,000 + + - +

a. In Table 7.4, what is the minimal bacteriostatic concentration of each disinfectant? 1:10

b. Which compound is bactericidal? At what concentration? Disinfectant 2 at 1:90,000

Chapter 8 – Short answer and Essay Questions

a) DNA replication: semiconservative process; DNA molecule contains one original (conserved) and one new strand; accurate process (proof-reading done by DNA polymerase); replication occurs in replication fork point; free nucleotides present in the cytoplasm are matched up to the exposed bases of the single-stranded DNA (T-A; G-C); copied by DNA polymerase; in the 5’ – 3’ direction; leading strand synthesized continuously and lagging strand discontinuously

Define the following
a) Plasmid – self-replicating, gene containing circular pieces of DNA about 1-5% the size of bacterial chromosomes
1. conjugative – carries genes for sex pili and for the transfer of the plasmid to another cell
2. dissimilation – encode enzymes for catabolism of unusual sugars and hydrocarbons
3. r (resistance) factors – encode antibiotic resistance
4. col plasmids - carry genes for the synthesis of bacteriocins, toxic proteins that kill other bacteria
5. Virulence plasmids - turn the bacterium into a pathogen

b) Transformation – “naked” DNA; genes are transferred from one bacterium to another as naked DNA in a solution – study of this phenomenon led to the conclusion that DNA is the genetic material; Griffith experiment

c) general Transduction – bacterial DNA is transferred from a donor cell to a recipient cell inside a virus that infects bacteria, called a bacteriophage or phage

d) Translation – process by which info contained in a molecule of mRNA is used to synthesize a protein; mRNA is translated in codons (3 nucleotides); begins at start codon AUG and stops at UAA, UAG or UGA

e) Transcription – synthesis of complementary strand of RNA from DNA template

f) conjugation – requires contact between cells; mediated by one kind of plasmid (fertility of F factor); cells must be of opposite mating types; donor cells must carry the plasmid and the recipient cells usu dont

g) codon – groups of 3 nucleotides; language of mRNA; sequence determines sequence of amino acids

h) specialized transduction – only certain bacterial genes can be transferred this way; steps: 1. prophage exists in galactose-using host (containing gal gene) 2. Phage genome excises, carrying with it the adjacent gal gene from host 3. Phage matures and cell lyses, releasing phage carrying gal gene 4. Phage infects a cell that cant utilize galactose (lacking gal gene) 5. Along w/ the prophage, the bacterial gal gene becomes integrated into the new host’s DNA 6. Lysogenic cell can now metabolize galactose

mutagen – agent that causes mutations eg chemicals (nitrous acid, nucleoside analog) and radiation (x rays, gamma rays, UV light)

Describe the lac operon.

The I-gene is the regulatory gene, which codes for a repressor protein; the promoter is a region of DNA where RNA polymerase initiates transcription; the operator acts like a go or stop signal for transcription of structural genes; the operon is a set of operator and promoter sites, and the structural genes they control are what define an operon.

If no lactose in environment, means that bacteria doesn’t have to make enzymes – repressor protein will bind to operator region. Therefore, no transcription, no RNA, no proteins or enzymes formed. If lactose is present, bacteria will need to make enzymes to break down lactose. Lactose (allolactose) will act like inducer and bind to the repressor protein. Repressor protein can no longer bind to the operator bc of change in structure – it is removed. RNA polymerase comes and binds to promoter region and starts transcription resulting in production of enzymes used to metabolize lactose.

Define a mutation. Describe any 4 types of point mutations.
Change in the base sequence of DNA; can be neutral, beneficial or harmful 1. Missense mutation – change in one base resulting in change in amino acid eg sickle cell disease 2. Nonsense – change in a codon for an amino acid into a termination codon 3. Frameshift - result from insertion of extra bases or the deletion of existing bases from the DNA sequence of a gene 4. Silent – change in DNA base sequence causes no change in activity of product encoded by gene

List and state the functions of 5 enzymes involved in DNA replication, expression and repair • DNA polymerase – synthesizes, proofreads and repairs DNA • RNA polymerase – copies RNA from a DNA template • DNA ligase – makes covalent bonds to join DNA strands; joins Okazaki fragments and new segments in excision repair • Photolases – use visible light energy to separate UV-induced pyridimine dimers • Primase – makes RNA primers from a DNA template • Helicase – unwinds double-stranded DNA • Ribozyme – RNA enzyme that removes introns and splices exons together

Chapter 13 – Short answer
1) Name any DNA or RNA viruses that could cause cancer? HPV (human papillomavirus) can cause cervical cancer – usu specific strains such as HPV-16

2) You are growing Bacillus subtilis in nine 16,000-liter fermenters to produce enzymes for industrial use. The Bacillus cultures had been growing for 2 days when the cells in one of the fermenters lysed. Explain what happened in this fermenter. The cells were lysed by a bacteriophage.

a) virion – complete, fully developed, infectious viral particle composed of nucleic acid and surrounded by a protein coat that protects it from environment and is a vehicle of transmission from one host to another
b) provirus – a virus genome that is integrated into the DNA of a host cell; never comes out of chromosome and protects host’s immune system and antiviral drugs
c) prophage – inserted phage DNA; a phage (viral) genome inserted and integrated into the circular bacterial DNA chromosome. A prophage is any virus in the lysogenic cycle; it is integrated into the host chromosome or exists as an extrachromosomal plasmid. Technically, a virus may be called a prophage only while the viral DNA remains incorporated in the host DNA. This is a latent form of a bacteriophage, in which the viral genes are incorporated into the bacterial chromosome without causing disruption of the bacterial cell.
d) eclipse period – period during viral multiplication when complete, infective virions are not yet present
e) oncogenes – parts of genome affected by cancer-causing alterations to DNA; can be activated to abnormal functioning by a variety of agents, inc mutagenic chemicals, high energy radiation, and viruses

4) List 4 characteristics that define a virus – contain either DNA or RNA (never both); contain a protein coat (capsid), sometimes enclosed by an envelope composed of lipids, proteins and carbs; obligate intracellular parasites – replicate in host; have few or no enzymes of their own for metabolism – lack enzymes for protein synthesis and ATP generation

5. Describe the 5 distinct stages of the Lytic cycle of a T-even bacteriophage
1. attachment – phage attachs by tail fibers to host cell
2. penetration – phage lysozyme opens cell wall, tail sheath contracts to force tail core and DNA into cell (use phage lysosome to break bacterial cell wall)
3. biosynthesis – production of phage DNA and proteins (phage takes over and uses host’s ribosomes, enzymes, aa etc)
4. maturation – assembly of phage particles
5. release – phage lysozyme breaks cell wall

6. What are the 3 important results of lysogeny?
1. phage conversion – host cell may exhibit new properties
2. lysogenic cells become immunr to re-infection with the same phage
3. specialized transduction – bacteria cells can be picked from a bacterial coat and transferred to another cell

7. How do prions differ from viruses?

Prions are different because they do not infect and replicate in the same way that viruses do. Viruses have RNA or DNA components and use body cells to replicate in. Prions are infectious proteins and they cause a structural change to other proteins that they interact with and that is how they cause damage. Probably the most famous prion disease is Bovine Spongiform Encephalitis (mad cow) where the structural change that the prion causes to the proteins in the brain causes holes to appear in the tissue, making it look like a sponge.

8. Describe how prions can be infectious

1. PrPC (cellular prion protein) produced by cells is secreted to the cell surface
2. PrPsc (scrapie protein) may be acquired or produced by an altered PrPC gene
3. PrPsc reacts with PrPC on the cell surface
4. PrPsc converts the PrPC to PrPsc
5. The new PrPsc converts more PrPC
6. The new PrPsc is taken in by endocytosis
7. PrPsc accumulates in endosomes
8. PrPsc continues to accumulate as the endosome contents are transferred to lysosomes – result is cell death

Essay Questions

1) Describe the viral:

a) lysogenic cycle
In the lysogenic phase there is no pathology. Under certain conditions the lysogenic lifestyle can switch to a lytic lifestyle. A virus is found at this stage under harsh conditions and is a prophage at this stage.
-The virus binds to bacteria (host)
- The virus inserts its DNA into the bacteria
- The viral DNA gets incorporated into the cell's chromosome
-Viral DNA is replicated along with chromosomal material

In the lysogenic cycle, the viral DNA or RNA enters the cell and integrates into the host DNA as a new set of genes called prophage. That is, the viral DNA becomes part of the cell's genetic material. No progeny particles, like in the lytic phase, are produced. Each time the host cell DNA chromosome replicates during cell division, the passive and non-virulent prophage replicates too. This may alter the cell's characteristics, but it does not destroy it.

There are no viral symptoms in the lysogenic cycle; it occurs after the viral infection is over. But the viral DNA or RNA remains in the cell and it may remain there permanently. However, if the prophage undergoes any stress or mutation or is exposed to UV radiation, the viral lysogenic cycle can change into the viral lytic cycle. In which case, there will be symptoms of a new viral infection.

Some viruses first replicate by the lysogenic cycle and then switch to the lytic cycle.

b) Lytic cycle
In the lytic stage, many viral particles are made and copies are sent back into the environment. A virus is found in this phase when conditions are favorable, i.e. when bacteria is "growing like crazy"
- The virus attaches to bacteria (host)
- The virus inserts its DNA into the bacteria
- The virus takes over the cell's machinery
- The virus reproduces itself and self-assembles. The host cell is destroyed

In the lytic cycle, which is considered the main cycle in viral replication, once the viral DNA enters the cell it transcribes itself into the host cell's messenger RNAs and uses them to direct the ribosomes.

The host cell's DNA is destroyed and the virus takes over the cell's metabolic activities.

The virus begins using the cell energy for its own propagation. The virus produces progeny phages. These replicate fast, and soon the cell is filled with 100-200 new viruses and liquid. As the cell starts getting overcrowded, the original virus releases enzymes to break the cell wall. The cell wall bursts – this process is called lysing - and the new viruses are released.

Differences Between Lytic and Lysogenic Cycles
In the Lytic Cycle:

•Viral DNA destroys Cell DNA, takes over cell functions and destroys the cell.
•The Virus replicates and produces progeny phages.
•There are symptoms of viral infection.
•Virtulant viral infection takes place.
In the Lysogenic Cycle:

•Viral DNA merges with Cell DNA and does not destroy the cell.
•The Virus does not produce progeny.
•There are no symptoms of viral infection.
•Temperate viral replication takes place.