EXAM II Study Guide (Microbiology)

8/10/2019 EXAM II Study Guide (Microbiology)

1/25

EXAM II Study Guide Virus/Phage + DNA + Immunology + Pathogenicity

VIRUS/PHAGE

Define viruses and phages. Differentiate Viroid, Prion, GTA

VirusGenetic element containing either RNA/DNA surrounded by a protein capsid and that replicates

only inside host cells

Phage Virus that infects only bacterial cells

Viroid Naked molecules of RNA important infectious disease agents in plants

PrionAn infectious agent composed only of protein that is responsible for causing a variety of

spongiform encephalopathies (e.g., scrapie).

GTA* Phage-like element produced by several bacteria that mediates horizontal gene transfer

*Gene Transfer Agent

Which of these is not UV sensitive?

Estimate the abundance of viruses in aquatic systems and postulate the significance of these pathogens in

terms of controlling primary productivity and horizontal gene transfer.

A. 106- 10

9ml

-1in seawater

B. Control 1 productivity: Each infection has the potential to introduce new genetic information into an

organism or progeny virus, thereby driving the evolution of both host and viral assemblages

a. Viruses kill ~20% of biomass/day

Contrast viruses with bacteria in terms of genome structure, metabolic activity, life cycle and size (in nm).

Bacteria Virus

Cell wall Peptidoglycan/LPS No cell wall; protein coat

Reproduction Fission; asexual Lysogenic; lyticCellular organization Complex Simple

Ribosome + -

Enzymes + Some

DNA/RNA Both DNA/RNA DNA ORRNA

Life cycle Some obligate IC parasites Obligate intracellular parasites

Size 1-5 m 10-400 nm

UV sensitivity: Bacteriophage > Bacterium > Gene > Prions


2/25

Viral Genome

Structureo Genetic information may be coded as DNA or RNA and in double-stranded or single-stranded form.

o The viral genome may contain exotic bases.

Replication

o Takes place only after successful infection of an appropriate host.

o Proceeds when the injected viral genome subverts normal replicative processes of the host,

producing new virus particles.

Bacteriophage types

o May be discerned by their mode of propagation.

o Lytic phages quickly produce many copies of themselves as they kill the host.

o Temperate phages can lie seemingly dormant in the host (prophage state), timing replication of

prophage genetic material to replication of the host cell. Various activation signals trigger the

prophage to enter a lytic cycle, resulting in host death and the release of new phages.

Sketch these viral morphologies:


3/25

Virus Morphology

TMV (Tobacco Mosaic Virus) Helical

T4 Phage Complex

HIV Enveloped

HSV Enveloped

Polyomavirus Icosahedral

Explain the role of reverse transcriptase, RNA replicase, holin and endolysin in the life cycle of viruses and

phages.

Enzyme Role Type of Phage

Reverse transcriptase Generation of cDNA from RNA; integration of retrovirus into host Retrovirus

RNA Replicase Catalyzes replication of phage RNA in host Lysogenesis

Holin Host lysis; produces lesions in cell membrane T4 phage protein

Endolysin Attacks peptidoglycan T4 phage protein

Contrast the biology of common phages:

Virus Genome Transmission

Cycle

Mode of Transmission Morbidity Mortality

Polio

+ ssRNA

Non-human

host

Fecal - oral

Endemic Pakistan

< 1%

WNV

+ ssRNA

Vector borne

zoonotic

Arthropod vector

Differential

4%

Ebola

- ssRNA

Zoonotic

Contact

Endemic (Africa)

High (5090%)

influenza

- ssRNA

sgmtd

Zoonotic

Aerosols

Humidity

50,000/year; seasonal

Adults: 5-10%

Kids: 20-30%

HIV

retro

Human

STD

---

100% w/o therapy

HSV

dsDNA

Human

STD; mother fetus

5060 million US

Low

Membrane = Peplomeres (spikes)


4/25

smallpox

dsDNA

Zoonotic

Contact

eradicated

High

Discuss how the above examples maintain virulence and evade specific immunity through antigenic drift

and shift.

A. Antigenic drift: natural mutagenesis of viruses as viruses replicate results in altered antigenic profile

B. Antigenic shift: segmented genome of select viruses (influenza) allows reassortment of different

strains; occurs through infection of same host cells and new-intermixed viral genome

Sketch lytic and lysogenic cycles using lamba phage as an example.

Lytic: phage life cycle that culminates with host cell bursting, releasing virions

Virulent phages: phages that lyse their host during the reproductive cycle i.e. T4Phage

(1) Early mRNA synthesis (2) Synthesis of proteins that (3) Enable T4 to take over host cell (4) Phage

DNA replication (5) Late mRNA synthesis (6) encode capsid proteins and other proteins needed for

phage assembly

Lysogenic: Nonlytic relationship b/t phage-host; integration of phage genome into host DNA

Defined terms:

a. Prophageintegrated phage genome (lysogeny)

b. Temperate phagephages able to establish lysogeny

c. InductionAn event in the life cycle of some viruses (e.g., temperate bacteriophage) that results in

the provirus initiating synthesis of mature virions and entering the lytic cycle.

Explain why stress triggers induction events

Triggered by drop in levels of lambda repressor caused by exposure to UV light and chemicals that

cause DNA damage

a.

Catalyzed by excisionase: binds integrase which enables integrase to reverse integration process

DNA/RNA

Describe these key experiments that elucidated the central dogma of molecular biology in terms of

objective, methods, results & conclusions:

a. Gliffith (1928), Avery (1944)

i. Griffith: Transformation; Dead S (infectious) + Live R strain injected Mouse dies (live S strain

isolated); conclusion: hereditary element can be transformed from dead to live cells


5/25

ii. Avery: Protein not transforming agent; R & S culture PRO/lipid removed transforming

element had DNA chemistry (destroyed by DNAse)

b. Heshey & Chase (1952)

i. DNA injected by viruses; bacteriophage infected cells had radioisotope labeled DNA (phosphorus) +

protein (sulfur) agitation/centrifugation revealed only P isotope still present in cells

c. Fraenkel-Conrat (1957)

i.

NA (RNA), not protein coat is infective agent; Tobacco infected with hybrid: TMV protein coat andHRV RNA observed lesions characteristic of HRV

d. Watson & Crick and Meselson & Stahl (1958)

i. Double helix 2 strands (parental & complementary)

ii. Meselson & Stahl radioisotope labeled DNA revealed semi-conservative replication in DNA

Identify the sugar in nucleic acids and features associated with I ', 2', 3' and 5' positions

Explain difference In strength of G-C pairing versus A-T.

A. GC capable of 3 H bonding

Contrast the structure(s) and function(s) of DNA & RNA including mRNA, tRNA & rRNA

Describe how these enzymes: gyrase, DNA polymerase, helicase and ligase, contribute to DNA replication

A. Gyrase: Remove supercoil

B.

DNA Polymerase: polymerizes dNTP in 5 3C. Helicase: Unwinds double-helix

D. Ligase: Fuses okazaki fragments

Describe polypetide synthesis using the terms transcription and translation.

RNA polymerase (a large multi-subunit enzyme) is responsible for the synthesis of RNA

The core enzyme (2, , ' subunits) catalyzes RNA synthesis

The sigma subunit () helps the core enzyme bind DNA at the appropriate site


6/25

sequences centered at -35 and -10 BP before the transcription starting point are important in directing

RNA polymerase to the promoter

Prokaryotic mRNA can code for one polypeptide (monogenic) or many polypeptides (polygenic)

Leader sequences consist of 25 to 150 bases at the 5 end of the mRNA, and precede the initiation

codon

Spacer regions separate the segments that code for individual polypeptides in polygenic mRNAs

Trailer regions are found at the 3 end of the mRNA after the last termination codon

Identify where sigma factors bind to activate transcription and explain their role stress response and

antibiotic resistance.

A. Binds to RNA polymerase @ specific promoter sequence; each factor binds to unique sequence

Sketch a biphasic growth curve using the example of diauxic growth observed when E. coli is grown on

glucose and lactose as sole carbon sources.


7/25

Discuss transcription regulation in this lac operon example using these terms: cAMP, promoter,

repressor, polygenic and inducer.

Discuss how eukaryotes and bacteria generate genotype and phenotype diversity.

Explain degeneracy and wobble in the genetic code and how point mutations and frame-shift mutations in

DNA alter polypeptide sequences or, in the case of silent mutations, don't.

Define wild type, prototrophs and auxotrophs to explain how the Ames test works and how horizontal gene

transfer (MGT) was quantified Lederberg and Zinder (1951).

a. Auxotrophrequires an organic growth factor

b.

Prototrophcan synthesize all necessary growth factors

Using the terms pili, cell-to-cell contact, prophage, lytic phase and phage- like particles, contrast

conjugation, transformation, specialized transduction, generalized transduction and gene transfer agents.


8/25

Conjugation

Define plasmids, episomes and conjugative plasmids.

PlasmidSmall replicons, double-stranded, usually circular DNA molecules; have their own origin of

replication; can exist as single copies or as multiple copies

Episome Plasmids that can exist either with or without integrating into chromosome

onjugationThe form of gene transfer and recombination in procaryotes that requires direct cell-to-cell

contact

Conjugative

Plasmids

Have genes for pili (ex F factor in E. coli); can transfer copies of themselves to other bacteria during

conjugation


9/25

CuringElimination of plasmid; spontaneous or induced by treatments that inhibit plasmid replication but no

host cell reproduction

F FactorF plasmid; the fertility factor; a plasmid that carries genes for bacterial conjugation and makes its E.

coli host the gene donor during conjugation

eneralized

ransduction

The transfer of any part of a prokaryotic genome when the DNA fragment is packaged within a virus

capsid by mistake

HGT The process by which genes are transferred from one mature, independent organism to another

Prototroph A microorganism that requires the same nutrients as most of the members of its species

Specialized

ransduction

A transduction process in which only a specific set of bacterial or archaeal genes is carried to a

recipient cell by a temperate virus

ransduction The transfer of genes between bacterial or archaeal cells by viruses.

ansformationMode of gene transfer in prokaryotes in which a piece of free DNA is taken up by a cell and stably

maintained

ranspositionThe movement of a piece of DNA around a cell's genome. transposon A mobile genetic element that

carries the genes required for transposition

Explain relationship between methylation of cytosine in viral DNA replication and restriction enzymes.

Contains hydroxymethyl-cytosine (HMC) instead of cytosine

o Synthesized by two phage encoded enzymes

Protects phage DNA from host restriction endonucleases

Restriction defends against viral infection

Contrast restriction enzymes and CRISPR as bacterial defenses against phages. Insertion Sequences

a. Short DNA sequences (6003,000 bp)

b. Carry transposease

Transposons

a. Known for carrying antibiotic resistance

b. Include conjugative

CRISPR (clustered regularly interspaced shortpalindromic repeats)

a. Bacteria andArchaea not only produce restriction endonucleases (Sections 8.6 and 11.1) that

function to destroy incoming foreign DNA, they also have an RNA-based defense program to

destroy invading DNA from viral infection and sometimes conjugation. This type of prokaryotic

immune system helps preservegenome stability and is called the CRISPR system, which stands

for clustered regularly interspaced shortpalindromic repeats

b. DNA repeats spacing between DNA complementary to foreign DNA

c. Processed to generate crRNAs


10/25

The CRISPR region is transcribed as a whole into a long RNA molecule that is then cleaved in

the middle of each of the repeated sequences by the nuclease activity of Cas proteins. This

converts the long RNA molecule into spacer segments of small RNAs called CRISPR RNAs

(crRNAs)

2 Methods of Microbial Genetic Diversity

Mutationalteration in existing DNA sequence

DNA transfer (horizontal gene transfer)acquisition of DNA from another source

Discuss, citing Corvaglia et al. (2010), the importance of type Ill-like restriction enzymes in the virulence of S.

aureus through acquisition of multiple antibiotic resistance.


11/25

Explain how the roles of RecA and LexA, uvrD in the SOS response.

LexA:repressor; in absence of DNA damage LexA binds to operator

RecA: inducer; DNA damage activates; cleave LexA

UvrD: helicase; removes thymine dimer

Compare structure and organization of chromosomes and genes in archaea, bacteria and eukaryotes usingthe terms intron, start amino acids, size (bp), circular/linear, haploid/diploid.


12/25

Explain the function of 5' cap and poly A tail in eukaryotic primary transcripts.

Protect from exonuclease activity in Eukaryotes

Capping is the addition of a methylated guanine nucleotide at the 5-phosphate end of the mRNAa. The cap nucleotide is added in reverse orientation relative to the rest of the mRNA molecule and is

needed to initiate translation.

Poly A tail

a. The tail recognition sequence, AAUAAA, is located close to the 3 end of the primary transcript.

b. The poly(A) tail stabilizes mRNA and must be removed before the mRNA can be degraded.

Discuss how alternative splicing, insertion sequences, methylation and RNA interference contribute to

complexity of organisms.


13/25

Alternative splicing: # of introns; increases w/ complexity of organism

Insertion sequences: Carry transposease (transposons); most bacteria have few; increase variability of

organisms (antibiotic resistance)

RNA interference

Explain why obligate mutualist bacteria generally have small genomes and few insertion sequences.

No selective pressure to undergo evolutionary beneficial adaptations

Describe in general terms nonspecific responses to infection.

Innate Immune System (PAMPs)

a. Cells: Macrophages + Neutrophils + NK cells

b. Proteins: Complement (opsonization; c3b) + Interferons

c. Systemic: Inflammatory + Temperature

i.

Cytokines: Proinflammatory (IL-1 + TNF- + IL-6)

Discuss why these PAMPs illicit a general immune response by binding PRRs and identify the class of

microbes associated with them: LPS, teichoic acid, peptidoglycan, chitosan, CpG DNA (methylationpatterns), SS DNA and DS DNA.

PAMPs:

Class of

MicrobesPAMP

PRR (Patern

Recognition

Receptor)

Result

Gram - LPS TLR-2/4 Phagocyte activation + inflam

Gram + Teichoic Acid CD14TLR-4 Phagocyte + inflam

Gram -/+ Peptidoglycan TLR-2; NOD1/2

Phagocyte activation + inflam; Antimicrobial

peptide production + proinflamm cytokinesFungal cell

wallChitosan TLR-6 Phagocyte activation + inflam

Bacteria

CpG DNA

(methylation

patterns

TLR-9 Phagocyte activation + inflam

Virus ss DNA TLR-7 Phagocyte activation + inflam

Virus ds DNA TLR-3 Phagocyte activation + inflam


14/25

Describe how these PAMPs stimulate systemic and local responses by stimulating the release of

endogenous pyrogens, particularly IL-I and TNF-a, or, for viral associated PAMPs, interferon.


15/25

Discuss evidence that interferon production interferes with viral replication.


16/25

Contrast the three pathways of complement activation and explain the central role of C3.

Explain these effector roles of complement:

Inflammation (C3a & C5a)

Lysis (membrane attack complex

Opsonization.


17/25


18/25

Memory B and T cells generated, which confer long-term immunity

Describe the role of macrophages, neutrophils, dendritic cells and in the immunes system.

Macrophage: phagocytosis

Neutophils: phagocytosis

Dendritic: antigen presentation

Describe two mechanisms of activating natural killer cells.

Nonspecific activation signal on target cell

Absence of MHC I (endogenous) marker on target cell for NK cells

Cell-mediated CD16 binds to Fc region of Ig (+) NK cells

Differentiate MHC class I and Il in terms of endogenous'exogenous peptides and CDS & CD4 positive cells.

Rule of 8

MHC I = CD 8+ = endogenous = T killer

MHC II = CD4+ = exogenous = T-helper

Differentiate the two arms of acquired immunity, humoral and cellular, in tenns of cells (B and helper and

cytotoxic T) involved.


19/25

Define antigens, epitopes and haptens. Sketch the basic structure of antibodies including variable and

conserved regions and light and heavy chains.

Identify the structure and function of the five isotypes of antibodies: lgG, IgA, IgM, IgD and lgE.


20/25

Identify those found on mature B cells, where IgM functions as the B cell receptor, and produced following

activation.

IgD

Differentiate active and passive imtiation of specific Immune response, with natural and artificial examples.

Identify the isotypes (IgG and lgA) involved in natural, passive immunity acquis'tion by the fetus and

newborns. Describe how HIV evades the immune system.

List Koch's Postulates.

Pathogen presentin every case of disease

Isolatepathogen and growin pure culture

Isolated pathogen causes diseasewhen inoculatedinto lab animal

Pathogen isolated from inoculatedanimal identical to original organism

Differentiate the terms disease and pathogen.

Contrast mutualism, commensualism and parasitism.

Macro


21/25

Define virulence or pathogen and lethal dose 50

Differentiate opportunistic and nosocomial infections.

Differentiate vectors and fomites.

Vectorsliving organism transmits pathogen

Fomitesinanimate object that transmits pathogen

Explain the relationship between virulence, mode of transmission and reservoirs.

Virulenceextent of pathogenicity

Discuss role of probiotics in prevention of disease including the role of attachment and competition forspace in establishment of infection.

Define these pathogens in these genera as either facultative intracellular, obligate intracellular, or

extracellular: Yersinia, Chlamydia, Vibrio and Pseudomonas.

YersiniaFacultative IC

ChlamydiaObligate IC

Vibrio - EC

Psuedomonas - EC


22/25

Compare exotoxins and endotoxins in terms of chemical nature, host responses, gram positive/negative,

heat stability and toxicity.

Contrast these exotoxin types: superantigens, type AB and cytotoxins.

Superantigens

Cause T cells to release cytokines

e.g., toxic shock syndrome

AB

diptheria

neurotoxins

target nerve tissue

e.g., botulinum toxin

enterotoxins

target intestinal mucosa

e.g., cholera toxin

cytotoxins


23/25

target general tissues

Include hemolysin and membrane disruptors

Identify MHC and TCR bound by superantigens.

Contrast A and B proteins in AB-types.

A = active; effector

B = mediates attachment

Explain how pore-forming and phospholipases cause cytotoxicity.

Pore-forming

a. bind cholesterol

b. e.g., hemolysins, leukocidins

Phospholipases

a. destroy membrane integrity

b. e.g., gangrene toxin

Explain how siderophores and lgA proteases are virulence factors and help bacteria evade the immune

system.

Collagenase

a. Hydrolyzes collagen


24/25

IgA protease

a. Destroys secreted antibodies

sidephores

a. Take essential nutrient, iron, from host enzymes

Capsules

a. not antigenic

b. e.g., Streptococcus pyogenes

N. gonorrheaapproaches

a. genetic variation of surface antigens

b. production of IgA proteases

Interfere with antibody-mediated opsonization

a. produce proteins that bind Fc portions of immunoglobins

b. Can bind macrophages as well as normal antibody response

c.

e.g., Streptococcus pyogenes

Describe how type Ill secretion systems are acquired as pathogenicity islands and contribute to virulence.

Present evidence that capsules and fimbriae are virulence factors for S. pneumonia and E. coli respectively.


25/25

Documents

EXAM II Study Guide (Microbiology)