Micro 2124 - EXAM I - Summer 1991

Part 1 - Multiple Choice (worth 3 points each). Please read each statement carefully and choose the best answer by writing the appropriate letter in the left margin.

1. The carrier used in the biosynthesis of fatty acids is:
a. tRNA
b. acyl carrier protein
c. flavoprotein
d. nucleoside diphosphate.

2. When E. coli are moving away from an agent which is chemotactic for them they tend to:
a. swim faster
b. swim slower
c. undergo catabolite repressi
on
d. tumble frequently.

3. What type of bacteria would tend to reduce highly oxidized inorganic compounds?
a. those that undergo anaerobic respiration
b. fermenters
c. Gram positives
d. phototrophs.

4. Chemotrophy and phototrophy refer to:
a. temperature requirements for growth
b. the structure of bacterial spores
c. differences in carbon sources
d. differences in energy sources.

5. The peptidoglycan of a bacterium:
a. is located primarily in the spore
b. is formed in the capsule
c. contains short peptides
d. is found only in Gram positive bacteria.

6. A governing principle of anaerobic respiration is that:
a. ATP is not produced
b. oxygen is not used as the terminal electron acceptor
c. only fats are used as substrates
d. alcohol is produced.

7. Procaryotes differ from eucaryotes because they:
a. do not have transfer RNA
b. do not have cytoskeletal elements
c. do not have unit membrane
d. do not have ribosomes.

8. Electron transport leads to:
a. substrate level phosphorylation
b. the creation of high energy bonds
c. increased gyrase activity
d. the extracellular transport of protons.

9. Most of the energy derived from the TCA cycle comes from:
a. substrate level phosphorylation
b. fermentation
c. GTP
d. electron transport.

10. The photosynthetic apparatus usually involves a reaction center, electron transport and:
a. the antenna
b. microtubules
c. photosynthetic vacuoles
d. the electron sink.

11. Enzymes which convert protonmotive force into ATP are called:
a. phosphatases
b. proteases
c. ATPases
d. catalases

12. Which of the following is not true about the outer membrane of Gram negative bacteria?
a. it is a lipid bilayer
b. it contains teichoic acids
c. it contains lipopolysaccaride
d. it forms the outer layer of the periplasm.

13. Chlorosomes are internal structures of bacteria which:
a. control elevation in liquids
b. are involved in photosynthesis
c. are the site of C02 fixation
d. orient bacteria in a magnetic field.

14. (A) substance(s) found in procaryotes but not in eucaryotes include(s):
a. polyhydroxybutyrate
b. tRNA
c. steroids
d. cytochromes.

15. Photosystem II is required in addition to photosystem I for photosynthesis if:
a. water serves as the source of electrons
b. the organism has no thylakoid membranes
c. light in the UV range is absorbed
d. carotenoids are used instead of chlorophylls.

16. The Calvin-Benson cycle is used by autotrophs to:
a. utilize reduced inorganics as an energy source
b. convert NAD to NADH
c. harvest light for transfer to reaction centers
d. utilize C02 as a carbon source.

17. The pentose phosphate cycle is important because it leads to the production of 5 carbon sugars and:
a. ATP
b. amino acids
c. NADPH
d. phospholipids.

18. The cell membranes of most procaryotes contain:
a. permeases
b. biosynthetic enzymes
c. esterified fatty acids
d. all of the above.

19. The primary difference between organelles in eucaryotes and organelles in procaryotes is the presence or absence of:
a. DNA
b.peptidoglycan
c. unit membrane
d. glycoproteins.

20. The tertiary structure of a protein is determined by:
a. binding to other proteins
b. interactions between amino acids in close proximity
c. interactions between widely separated amino acids
d. tautomeric shifts.

21. Introns are portions of:
a. DNA which do not code for protein
b. proteins which have no apparent function
c. receptors found in cell membranes
d. microtubules which are bound to the nuclear membrane.

22. Eucaryotes include:
a. fungi and bacteria
b. plants and blue-green algae
c. bacteria, protozoa and fungi
d. fungi, protozoa, plants and animals.

23. Another name for glycolysis is:
a. the Entner-Doudoroff pathway
b. the Embden-Myerhoff pathway
c. the citric acid cycle
d. gluconeogenesis.

24. Specialized proteins which bind to DNA and regulate its conformation are called:
a. histones
b. nucleons
c. regulons
d. helicons.

25. A nucleotide consists of:
a. a purine or pyrimidine base only
b. a base plus ribose or deoxyribose
c. a base plus ribose or deoxyribose plus a phosphate ion
d. none of the above.

Part II - Short Essay (worth 5 points each). Please answer the following questions completely but concisely.
1. Compare eucaryotic and procaryotic chromosomes (structure, number, histones and replication).

2. Define the following: chemoautotroph, chemoorganotroph, fermentation, substrate level phosphorylation and electron transport.

3. Compare the cell wall structure (everything outside of the cell membrane) of Gram positive and Gram negative bacteria.

4. List and describe each of the three main steps which occur during translation.

5. Draw a simple diagram showing how energy from sunlight can ultimately be converted into ATP in a chemoorganotroph. (Hint: Include at least one other group of procaryotes in the diagram.)

Epidemiology and Public Health Microbiology

Epidemiology is the study of the occurrence, distribution, and control of disease in populations.
Epidemiologists are interested in the life history of pathogens. What it does to the host, the spread of diseases, where did it start and what is its mode of transmission.

Terms used by epidemiologists
Prevalence is the percentage of individuals infected with the disease. The incidence of a disease is the number of diseased individuals in a population.

Epidemic occurs when a disease occurs in high numbers in a community at the same time. Pandemic is a widely distributed epidemic. Endemic is a disease constantly present in a population usually in low incidence.

Mortality expresses the incidence of death in a population. Morbidity refers to the incidence of disease in a population and includes both fatal and nonfatal.

Progression of disease
A. Infection
B. Incubation period - time between infection and symptoms. Can be short or a long period of time. Depends on inoculum size, virulence of pathogen, and resistance of the host.
C. Acute period - disease at its height.
D. Decline period - disease symptom are subsiding.
E. convalescent period - regain strength and health

Disease reservoirs (see table 22.2)
origins of the infectious agent. Can be inanimate or animate.

Zoonosis - a disease that is primarily in animals but may go to humans. Maintenance of the pathogen in nature requires animal to animal transfer.
Two examples include bovine tuberculosis and brucella. Eliminating infected animals was central in their control. Also pasteurization of the milk controlled tuberculosis since this was a primary mode of transmission.

Carriers - a carrier is someone without clinical signs but carriers of the disease agent. May be individuals in the incubation period - referred to as acute carriers. Respiratory infections are common among these since we are non clinical for a while before coming down the symptoms. Chronic carriers may be recovered patients or subclinical infections. These individuals may appear to be healthy but they harbor and transmit the agent - typhoid mary for example.

Transmission of pathogens
Host to host - respiratory route (for example the common cold or flu) or direct contact are common. Pathogens that are transferred by direct contact frequently cannot survive outside the host for significant periods of time. Examples include sexually transmitted diseases such as gonorrhea or syphilis. They are both extremely sensitive to drying effects and do not last on exposed surfaces for even the briefest period of time. Direct contact required for many skin pathogens such as Staphylococci or fungi.

Indirect host to host transmission - vectors such as arthropods (such as fleas, ticks, or mites) or vertebrates (such as dogs or rodents). Many of the arthropods require a blood meal where they acquire the pathogen from an infected individual. In the arthropod, the pathogen population builds up and now becomes sizable.

Inanimate objects such as toys, books, bedding are transmit disease. These objects are called fomites.

Epidemics (See figure 22.5) can be divided into common-source epidemics and host-host epidemics. In common source epidemics a large number of individuals become infected through a common source such as food or water. Pathogens are usually intestinal and are due to fecal contamination of food or water sources. Characterized by a rapid rise to a peak number of individuals infected and a slower still rapid decline.

Host-host epidemics shows a relatively slow, progressive rise in infected individuals.

Host community - Excellent example of coevolution of the host and pathogen. Originally the host population is sensitive to the pathogen and epidemics are common. But with each epidemic, the number of resistant hosts increases and finally there is a balance between the host and the pathogen. The host usually develops resistance to the pathogen and the pathogen is trying to overcome the resistance. Example of this is a virus and rabbits in Australia.
Rabbits were rampant during the 1950s after their introduction in the 1800s. Myxoma virus, which is pathogenic to the European rabbit at the time but non pathogenic to its host the south American rabbit, was introduced into Australia. Transmitted by mosquitoes. In a brief time (6 years) both the virus and host rabbits had changed. During the first year about 95% of the infected rabbits died. in 6 years the mortality rate dropped to 84% and the virus was less virulent and changes in the host resistance were noted. There were genetic changes in both the host and virus.

Hospital infections - Nosocomial infections occur in about 5% of all patients up to 10% in intensive care units. In all there are about 2 million nosocomial infections each year leading directly or indirectly to about 80,ooo deaths!!! Due to disease sources and selection for certain agents by the hospital environment. Most nosocomial agents are endemic not epidemic. E. coli, P. aeruginosa and Enterococcus are ommon urinary tract pathogens.

Staphylococcus aureus is common in septicemia, surgical wounds, and lower respiratory infections. Other species of Staphylococcus are also found in septic patients and wound infections. The source is generally a healthy carrier such as hospital personel. Staphylococci are resistant to drying and can survive long periods of time on dust and other fomites..

Pseudomonas aeruginosa is important in lower respiratory and urinary tract infections and burn victims. These organisms can show high resistance to antibiotics due to plasmids they carry often multiple resistance. Staphylococcus is generally less resistant and Escherichia are most susceptible.

How do we control epidemics? A number of factors have reduced the incidence of certain diseases. Generally there has been an increase in the general well-being of the population. Better nutrition, less crowding, and lighter work loads have contributed to this increase in well being. There have been successful public health measures that have reduced the incidence of specific diseases such as typhoid fever, diphtheria, polio and burcellosis.
Control diseased domesticated animals by eradicating diseased animals and immunization procedures. It is difficult to take the same measures against wild animals that are reservoirs for things like rabies.

Insect reservoirs have been controlled by pesticide sprays. The malaria spreading mosquito has been controlled in North america by originally spraying DDT which is now banned from use. Sometimes the measures we use to control vectors/reservoirs have been unhealthy themselves.
Control the transmission of pathogens. In japan, many people wear face masks to prevent the transmission of respiratory infections. The spread of water-borne pathogens is controlled by water purification procedures implemented.
Immunization schedules have gone a long way in controlling smallpox, diphtheria, tetanus, polio, and pertussis (whooping cough). We shouldn't become laxed about these either, we have not erradicated the viruses or bacteria responsible for these diseases!

Quarantine is another measure to prevent the spread of disease. Simply restrict the movement of an infected individual about the population. There are six diseases that are quarantinable by international law - smallpox, cholera, plaque, yellow fever, typhoid fever, and relapsing fever.

Emerging and resurgent infectious diseases
Emerging diseases are those that suddenly become prevalent and include not only "new" diseases but old ones that have become less well controlled. Syphilis, the plague, influenza, legionellosis, AIDS and lyme disease are examples. Why have these emerged? Outline some factors:

Human demographics
Populations are more dense than 1800's therefore disease spreads more easily.

Human behavior
Sexual promiscuity, intravenous drugs use

Technology and industry advances
Includes advances in medicine and hospitals where we have explosive increase in nosocomial infections. Increase in antibiotic resistance and multiple resistances. Transportation, bulk processing and central distributions have led to increases in some diseases such as E. coli strain O157:H7 in meat.

Economic development
Awan High Dam in Egypt has increased Rift Valley fever incidence since there is an increase in mosquito breeding grounds.

Land use
Lyme disease has become more common. Reforestation practices have helped increase the deer population and the reservoir of lyme disease. More ticks are carrying the disease. Also more people like to live and recreate in forests so there is more contact with these ticks.

International travel and commerce
Travel to and from exotic places could lead to the movement of endemic disease agents.

Microbial adaptation
Evolution of new strains contributes to their emergence. RNA viruses constantly mutate since they use reverse transcriptase to make the replicative intermediate. Reverse transcriptase has no editing function so when errors are introduced they become incorporated into the new virus. Examples include the HIV virus and influenza virus.
Bacteria also evolve using mechanisms such as transposons, bacteriophages and plasmids to facilitate their evolution.

Breakdown of public health measures
This happens occassionally. The cholerae epidemic of Peru was due to a breakdown in water sanitation. Their municpal water supply was contaminated. Similarly in Milwaukee, WI a chlorine resistant protozoa, Chryptosporidium, caused 370,000 cases of intestinal disease and 4000 required hospitalization.
Vaccination programs are neglected. Measles and whooping cough have made a resurgence due to lack of proper immunization programs.

Abnormal natural occurrence
Changes in the weather patterns for example. Hantavirus infects rodents and is a human pathogen. In SW US recently there were some lethal cases reported. Due to abundant mice since there was adequate rains and a long growing season coupled with a mild winter. More mice were around, leaving their droppings that people came in contact with which resulted in more people coming down with the sickness.

Viruses
General properties of viruses
Viruses occur either extracellularly as virus particles or virions or intracellularly where the virus replicates in the infected host cell. Viruses may be either DNA or RNA viruses, that is their genome is either type of nucleic acid. Viruses don't function without a host cell since the virus is metabolically inert. There are plant and animal viruses.
Virions are very small - between 10's to 100's of nanometers in size. Their genomes are correspondingly small upwards to 190 kilobase pairs compared to 1000's of kilobase pairs for bacteria.
Virions composed of nucleic acid, a coat protein called the capsid, which may be composed of subunits that self-assemble assisted by molecular chaperones. The complex of nucleic acid and capsid is called the nucleocapsid. Some viruses are enveloped such the nucleocapsid is enveloped in a lipid bilayer membrane which is derived from the host cell. The membrane may have glycoproteins embedded in it - these are coded by the virus.
The virions are symetrical in shapes of rods or helical symetries or spheres such as the icosahedron with 20 faces. Some bacteriophages have a protein helical tail.
Few viruses have their own enzymes, e.g., nucleic acid polymerase. For example, retroviruses are RNA viruses that have reverse transcriptase that generates DNA from the RNA nucleic acid during replication. Other enzymes include neuraminadases to break down glycoproteins, glycolipids of animal cells to liberate the virus. Some Bacteriophages have lysozyme to make small holes in the cell so that the viral nucleic acid can enter the cell.

Quantifying viruses
Virus infection unit is the smallest unit that causes an effect on the host cell. How do virologists measure virus infection units?
Plaque assay is a way to measure virus infection units. A monolayer of host cells are plated out on appropriate medium and where a viron initiates an infection there will be a clear zone of dead due to lysis or inhibited cells called a plaque. Usually one mixes the host cells with a suspension of viruses and spreads this mix out on the plate. The plate is incubated for the host and plaques are detected after the host grows.
Efficiency of plating is usually much less than 100%. This means that not all virions cause a plaque but for some unknown reason, less than all of the virions cause a plaque. Bacteriophage plating efficiencies are usually greater than 50% while many animal viruses are less than 1%. So virologists often report the numer of plaque forming units of a virus suspension which is not the absolute number of viruses in the suspension.

Virus life cycle
The problem is to infect a cell, replicate and be released to reinfect another host cell.
One-step growth curve of viruses
During the latent period the virus undergoes an eclipse when the nucleic acid is separated from the virus coat protein and becomes less infective if it where released. Next comes the maturation period when the virus' nucleic acid is packaged in the capsid. During maturation period the virus titer per cell rises dramatically. The number of viruses per cell may be between 10 to 1000s of viruses.

Steps: Attachment - receptor on the host cell. Could be one of many things like a flagella, a transport protein, polysaccharides, lipo-polysaccharides, or other usual cell structures.
Penetration - Complicated steps that depend on the virus and host. Well studied T-4 virus attaches and injects its DNA into the host bacterium cell. Animal viruses are transported by endocytosis like phagocytosis.
Early steps of replication -
Replication
Synthesis of coat proteins
Assembly
Release

Restriction and modification
Bacteria and other organisms have evolved methods to restrict virus infection. One is to cut up the virus' nucleic acid before it is replicated this is called restriction. Host cell produces restriction endonucleases that digest or cleave foreign DNA at specific sequences that are usually palindromes of 4 or 6 nucleotides long. The host protects its own DNA by modifying nucleotides in it's palindromes so the restriction endonucleases will not cleave at these sites. Common form of modification is methylation of one or more of the bases. Some viruses have devised their own modification systems to overcome the host cell's restriction system.
Some host restriction systems recognize modified palindromes so that when a virus infects on bacterium, replicates, becomes modified, is released and infects the second cell, this second cell cleaves the DNA. Obviously the second cell does not have the modification system.

production of viral nucleic acid and proteins
Single and double stranded RNA viruses and single stranded DNA viruses present unique problems for their replication and protein synthesis. Virologists talk about positive and negative strand DNA or RNA viruses (See figure 8.12).
Positive strand RNA viruses can translate the RNA into proteins directly. These viruses code for their own RNA polymerase which makes the negative strand RNA which will be used as a template to make more positive strand RNA.
Some positive strand RNA viruses must go through a ds strand DNA intermediate, e.g., HIV, via reverse transcriptase an enzyme that makes DNA complement to RNA.
Negative strand RNA viruses must synthesize the positive strand RNA before proteins can be made. But there is a dilemma here, how will these viruses make positive strand RNA since the cell doesn't have an RNA-dependent RNA polymerase? They inject their own RNA-dependent RNA polymerase into the cell.
Viral proteins - two broad catagories of proteins:
1. Early proteins - made soon after infection and are generally required for replication of virus nucleic acid
2. Late proteins - synthesized later and include the coat proteins.

Viruses
RNA bacteriophage - many single stranded RNA viruses. All are icosahedral. Viruses of the enteric bacteria infect only the male cells since these viruses infect by attaching to the pili coded for by the F-plasmid. All are very small, 26 nm, in size and have a small genome, 3569 nucleotides long.
See Figure 8.15
MS2 is a positive strand RNA enteric virus. Walk through Figure 8.15.
Note: this virus has overlapping genes for coat and replicase gene with the lysis protein gene overlapping.

Single stranded DNA icosahedral virus
Circular DNA viruses that use the host cell DNA replication machinery for virus DNA replication.
Best studied example is [phi][chi]174 like other small viruses it has been completely sequenced to the nucleotide level. There are overlapping genes, genes within genes, and genes with translation start sites at different positions.
Replicative form is the double stranded intermediate form made soon after infection. A process similar to the lagging strand synthesis of the chromosomal DNA is used to make the replicative form (See Figure 8.16). Notice that there are no virus proteins used in making the replicative form, the cell provides a primase, DNA polymerase, ligase and gyrase to make the RF DNA which is double-stranded, circular molecule with extensive supercoiling. The RF ds DNA is replicated using conventional DNA replication procedures we have talked about. The formation of viral ss DNA is carried out by the rolling circle mechanism (see Figure 8.17). There is a nick in the plus strand, the 3-prime end of the nick is a primer for DNA polymerase which uses the unnicked strand as a template. During synthesis the growing point displaces the original plus strand and synthesizes a copy of the plus strand to replace it. This continues over many rounds of synthesis.

Single-stranded filamentous DNA bacteriophages - have a helical symmetry rather than icosahedral. M13 is a well studied ss DNA virus that infects only male Escherichia coli cells after attachment of the male-specific pilus. Unlike the previously described viruses which lyse the cell to be released, M13 virus are released from the cell without killing the cell but they slow down the cell's growth. Therefore plaques are not clear but instead are areas of reduced growth. These viruses are used extensively in molecular biology since they make exellent cloning vectors and may be sequenced directly.

Double-stranded DNA bacteriophages - a group of linear ds DNA viruses have been well studied including T4 and T7. These infect E. coli and are small virions with icosahedral heads. They economize on their genome by using internal translational initiation sites, internal frameshifts and gene overlap strategies.
T 7 virus - Injection of DNA into the host cell begins with the left end where there early protein genes are found (see Figure 8.19). These early genes including T7 RNA polymerase are transcribed by the host's RNA polymerase which is soon shut down and T7's RNA polymerase takes over and transcribes only from phage promoters.
DNA replication is unusual since it the DNA is linear. It begins nearer to one end and is bidirectional (See Figure 8.20). Upon completion, there is a 3-prime overhang since there is no primer available for the DNA polymerase. To fill this overhang, completed strands hybridize by forming complementary bonds between the terminal repeats found at the ends of the DNAs and DNA polymerase and ligase fills in the gaps. .

The above viruses have been lytic viruses - that is as soon as they are mature, the cell is lysed to release the virus for further infections. But not all viruses lyse their host - are virulent. Some viruses are temperate and enter into lysogeny as prophage where they become part of the hosts genome and are replicated with the host's chromosome. Prophage may be integrated in the host chromosome or synchronously replicated with the host chromosome. The bacteria are called lysogens, and under appropriate conditions will be induced to produce virions of the temperate virus.

Lambda virus - a bacteriophage that has been the subject of intensive studies. Genome is a linear chromosome that has 5-prime termini extensions (cohesive ends) which are complementary. The purpose of these extensions is to make a circular chromosome when it is injected into the host cell.

Lytic cycle - we begin with this since this is, after all, the fate of these viruses. Refer to figure 8.26 or Click on the Biology Learning Center (this is a hot link that you may want to review while looking through the following notes)

Infection of host cell - First round of transcription begins with the host DNA-dependent RNA polymerase transcribing the genes cro from the right hand promoter and N from the left hand promoter. N and cro are regulatory proteins - N is an antiterminator of transcription and cro is a transcription regulatory protein.
N allows the transcription ofcIII from P_L and cII, O, P, and a little Q from P_R. Q, an antiterminator too, allows transcription of the late genes for the lytic cycle.
Cro blocks transcription from P_L and P_R by binding to O_R and O_L - Cro is a repressor protein! No cII and cIII are made either and these are needed for the cell to enter lysogeny! You guessed it this cell is headed towards lysis.

DNA replication - rolling circle replication. (see Figure 8.28) There is a nick in one strand, the 3-prime end of the nick is a primer for DNA polymerase which uses the unnicked strand as a template. During synthesis the growing point displaces the original nicked strand and synthesizes a copy of this strand to replace it. This continues over many rounds of synthesis. This leads to a single stranded DNA molecule, so concomittant with the synthesis of the nicked strand, the unrolled DNA is primed and the complement is made to synthesize a double stranded DNA molecule.

Lysogeny - lysogeny requires I) the shut down of the late genes and ii) the integration of lambda DNA into the host's chromosome.
Shutting down the late genes - simply (ha ha nothing is simple) need the protein from cI to repress the synthesis of all of lambda's genes! cI gene is transcribed from P_E in the opposite direction of P_R. P_E is for Promoter establishment. This promoter must be activated by some other element. What is that element? cII
cII is the activator of cI promoter. This gene product remember was made early in infection, but it is extremely unstable and is degraded by a host's protease. cIII, a protein made early on, stablizes cII so it can activate P_E to transcribe cI gene.
cI is lambda repressor and binds to O_R and O_L, the same operators that cro binds to, but in a different fashion so as to shut down cro synthesis quickly. Also, lambda repressor has a greater affinity for these operators than cro so it can outcompete it for these operators.
Problem here - if lambda repressor binds to O_R and O_L it blocks its own synthesis from P_E by the activation by cII. There is another promoter, P_M, which is activated by lambda repressor binding to O_R. So when lambda repressor binds to O_R the repressor is acting as an activator of P_M and a repressor of P_R.

Integration of DNA into host chromosome - lambda integrates at a specific site in the chromsome. Once integrated, only the lambda repressor is made to repress lambda's genes. Also, lambda repressor is responsible for preventing a second virus from infecting the cell since the repressor will effectively shut down all of the second virus' genes too.

lysogeny or lytic cycle? This is a race in essence. A race as to whether cro or lambda repressor will be synthesized in higher amounts. Though cro is made first, it must be in higher amounts to repress the synthesis of lambda repressor since cro has a weak affinity towards the operators.
Another control is the physiological state of the cell. The protease that degrades cII is subjected to catabolite repression (remember what that is?). Therefore the protease genes are not transcribed when a cell with lots of glucose and high intracellular ATP and lysogeny occurs. If the cell is nutrient poor, cAMP levels are high, the protease gene is activated and the lytic cycle occurs.
Other signals for lysogen to undergo lytic cycle. DNA damaging agents such as ultraviolet light, x-rays, or chemicals. These agents induce the SOS response and RecA protein which as a protease activity. This protease can degrade the lambda repressor and derepress lambda lytic genes resulting in phage production.