Bacterial Genetics: Mechanisms of Genetic Variation

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Chapter 13

• Bacterial Genetics Mechanisms of Genetic
  Variation
• 122209

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Horizontal (Lateral) Gene Transfer (HGT) in
bacteria

• Haploid geneome
• Exogenote (Donor)
• Endogenote (Recipient)
• Merozygote
• Transconjugants
• Transformants
• Transductants

Episomal form
Figure 13.16
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Homologous recombination

• Reciprocal recombination
• Double-Strand Break Model
• results from DNA strand breakage and reunion,
  leading to crossing-over

Figure 13.17
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Nonreciprocal Homologous Recombination

• incorporation of single strand of DNA into
  chromosome, forming a stretch of heteroduplex DNA
• proposed to occur during bacterial transformation

Figure 13.18
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Site-specific recombination

• insertion of viral genome into host chromosomes
• insertion of nonhomologous DNA
• Only a small region of homology is required
• Transposition carried out by transposable
  elements
• Simple transposition
• Replicative transposition

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Transposable Elements

• transposition
• the movement of pieces of DNA around the genome
• transposable elements (transposons)
• segments of DNA that carry genes for
  transposition
• widespread in bacteria, eukaryotes and archaea

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Types of transposable elements

• IS element
• 2 IR transposase gene
• composite transposons
• 2 IS elements
• additional genes
• Replicative transposons
• Resolvase gene

Figure 13.19
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Simple Transposition- cut and paste
- Generation of direct repeats in host DNA
flanking a transposon
Figure 13.20
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Replicative Transposition

• - insertion generates direct repeats of flanking
  host DNA
• usually transposon replicated, remaining in
  original site, while duplicate inserts at another
  site
• Tn3

Figure 13.21
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Effects of transposition

• mutation in coding region
• disruption of coding sequences
• arrest of translation or transcription
• interruption of promoter regions
• interruption of ribosomal binding sites
• activation of genes
• provide strong promoter sequences
• generation of new plasmids
• Multi-drug resistance plasmids

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R Plasmid results from Tn3 Transposition
R plasmid
Figure 13.22
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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
• R plasmids
• have genes for resistance to antibiotics
• some are conjugative
• usually do not integrate into chromosome

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Other Types of Plasmids

• Col plasmids
• encode colicin to kill E. coli
• virulence plasmids
• carry virulence genes
• confer resistance to host defense mechanisms
• encode toxins (e.g., anthrax toxin)
• metabolic plasmids
• carry genes for metabolic processes
• encoding degradative enzymes for pesticides
• Encoding proteins for nitrogen fixation

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Bacterial conjugation

• The transfer of genes between bacteria that
  depends on direct cell to cell contact
• mediated by the sex pilus encoded by F plasmid

Figure 13.24
Figure 13.23
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F plasmid integration
Homologous recombination mediated by IS sequence
Figure 13.25
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Evidence for bacterial conjugation

• transfer of DNA between cells
• - 1946, discovered by Lederberg and Tatum

Figure 13.26
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The U-tube experiment
demonstrated that direct cell to cell contact was
necessary
Figure 13.27
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F x F- Mating

• F donor
• F- recipient
• replicated by rolling-circle and the duplicate is
  transferred
• recipients usually become F
• donor remains F
• donor genes usually not transferred

Figure 13.28(a)- a polar gene transfer
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HFr Conjugation

• donor Hfr cell has F factor integrated into its
  chromosome
• donor genes are transferred to recipient cell
• a complete copy of the F factor is usually not
  transferred

Figure 13.28 (b) (c)
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F conjugation
Fig. 13.29

• formed by incorrect excision from 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

F x F- Mating
Figure 13.30
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DNA transformation

• uptake of naked DNA molecule from the environment
  and incorporation into recipient in a heritable
  form
• competent cell
• capable of taking up DNA
• may be important route of genetic exchange in
  nature

Figure 13.31
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Bacterial transformation in
Streptococcus pneumoniae
Smooth- and rough-form S. pneumoniae S? mouse
dead R? mouse alive S R ? mouse dead Heated S
R? dead
Figure 13.32
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DNA Uptake System
Natural competence G(-) Neisseria sp.
G() Bacillus sp.
Figure 13.33
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Transduction- transfer of genes by phages
Figure 13.34
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Generalized transduction

• any part of bacterial genome can be transferred
• occurs during lytic cycle
• during viral assembly, fragments of host DNA
  mistakenly packaged into phage head

Figure 13.35
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Specialized Transduction

• carried out only by temperate phages that have
  established lysogeny
• only specific portion of bacterial genome is
  transferred
• occurs when prophage is incorrectly excised

Figure 13.36
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Chapter 34

• Antimicrobial Chemotherapy
• 122209
• 122309

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Chemotherapeutic agents

• Chemical agents used to treat disease
• destroy pathogenic microbes or inhibit their
  growth within host
• most are antibiotics
• microbial products or their derivatives that kill
  susceptible microbes or inhibit their growth

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The Development of Chemotherapy

• Paul Ehrlich (1904)
• selective toxicity magic bullet
• dyes ? African sleeping sickness
• Sahachiro Hato (1910)
• arsenic compounds ? syphilis
• Gerhard Domagk (1927)
• Prontosil red? pathogenic streptococci and
  staphylocccci
• Jacques and Therese Trefouel (1935)
• 1939, Domagk received Nobel prize for discovery
  of sulfonamides and sulfa drugs

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Penicillin
Staphylococcus

• 1896, first discovered by Ernest Duchesne
• 1928, Alexander Fleming
• observed penicillin activity on contaminated
  plate
• 1939, Florey, Chain, and Heatley ? effectiveness
• 1945, Fleming, Florey and Chain received Nobel
  Prize
• Without Fleming, no Chain or Florey without
  Florey, no Heatley without Heatley, no
  penicillin.

Penicillum notatum
Figure 34.1
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Later discoveries

• 1944, Waksman screened 10,000 soil bacteria?
  Streptomycin (produced by Streptomyces griseus)?
  tuberculosis?1952, Nobel Prize
• 1953, chloramphenicol, terramycin, neomycin, and
  tetracycline isolated
• Box 34.The use of antibiotics in research
• chlporamphenicol? bacterial protein synthesis
• rifampin ? bacterial RNA synthesis
• cycloheximide? eukaryote protein synthesis
• mitomycin D? eukaryote DNA synthesis

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General Characteristics

• selective toxicity
• therapeutic index
• ratio of toxic dose to therapeutic dose
• toxic dose
• drug level at which drug becomes too toxic for
  patient (i.e., produces side effects)
• therapeutic dose
• drug level required for clinical treatment

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Properties

• Bacteriacidal kill
• Bacteriastatic inhibit growth
• broad-spectrum
• attack many different pathogens
• narrow-spectrum
• attack only a few different pathogens

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Antibiotic Production

• bacteria and fungi are natural producers
• penicillin G and penicillin V
• synthetic chemotherapeutic agents
• Semisynthetic antibiotics
• chemically modified ? less susceptible to
  pathogen inactivation
• Ampicillin, amoxycillin

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Level of antimicrobial activity

• Effectiveness
• minimal inhibitory concentration (MIC)
• lowest concentration of drug that inhibits growth
  of pathogen
• minimal lethal concentration (MLC)
• lowest concentration of drug that kills pathogen
• Antimicrobial tests
• Dilution Susceptibility Tests
• inoculating media containing different
  concentrations of drug
• Disk Diffusion Tests
• disks impregnated with specific drugs? observe
  clear zones (no growth) around disks

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Kirby-Bauer method - Disc diffusion test

• sensitivity and resistance determined using
  tables that relate zone diameter to degree of
  microbial resistance
• table values plotted and used to determine if
  concentration of drug reached in body will be
  effective

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The Etest- strip test
The MIC concentration is read from the scale at
the point it intersects the zone of inhibition
Each strip contains a gradient of antibiotic
AB Biodisk, Solna, Sweden
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Action of Antimicrobial Agents

• Targeting some function necessary for its
  reproduction or survival
• Targeted function is very specific to pathogen ?
  higher therapeutic index
• inhibitors of cell wall synthesis
• Penicillins, cephalosporins, vancomycin
• protein synthesis inhibitors
• metabolic antagonists
• nucleic acid synthesis inhibition

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Penicillins- PG synthesis inhibitors
inhibit the last step of
transpeptidation

• b-lactam ring
• resistant organisms? b-lactamase (penicillinase)

Fig. 34.5
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Cephalosporins

• structurally and functionally similar to
  penicillins

Figure 34.6
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Cell wall synthesis inhibitors

• Semisynthetic penicillins
• have a broader spectrum
• resistance continues to be a problem
• 1-5 of adults in US are allergic to penicillin
• allergy ?a violent allergic response and death
• broad-spectrum antibiotics (used to treat
  penicillin-allergic patients)
• Vancomycin and Teicoplanin
• glycopeptide antibiotics
• vancomycin ? treatment of antibiotic resistant
  staphylococcal and enterococcal infections
• drug of last resort

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Protein synthesis inhibitors

• many specifically to the prokaryotic ribosome
• Aminoglycoside antibiotics
• contain a cyclohexane ring and amino sugars
• bind to 30S ribosomal subunit
• others inhibit a step in protein synthesis
• aminoacyl-tRNA binding, peptide bond formation,
  mRNA reading, translocation

Figure 34.7
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Tetracyclines

• broad spectrum and bacteriostatic
• inhibits bind of aminoacyl-tRNA molecules to the
  A site of the ribosome
• sometimes used to treat acne

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Macrolide antibiotics
Figure 34.9

• 12 22-C lactone rings linked to sugar(s)
• erythromycin
• broad spectrum and usually bacteriostatic
• binds to 23S rRNA (inhibits peptide chain
  elongation)
• used for patients allergic to penicillin

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Chloramphenicol

• chemically synthesized
• binds to 23s rRNA to inhibit peptidyl transferase
  
• toxic with numerous side effects
• only used in life-threatening situations

Figure 34.10
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Metabolic Antagonists

• Antimetabolites
• antagonize or block functioning of metabolic
  pathways by competitively inhibiting the use of
  metabolites by key enzymes
• are structural analogs
• molecules that are structurally similar to, and
  compete with, naturally occurring metabolic
  intermediates? block normal cellular metabolism

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Sulfonamides or sulfa drugs

• Structurally related to sulfanilamide, a
  p-aminobenzoic acid (PABA) analog used for the
  synthesis of folic acid

precursor for synthesis of purines and pyrimidines
Fig.34.11.12
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Synergistic Interaction
Trimethoprim - synthetic antibiotic that also
interferes with folic acid production
Figure 34.14
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Nucleic acid synthesis inhibition

• Block DNA replication
• inhibition of DNAP or helicase
• Block transcription
• inhibition of RNAP
• Not as selectively toxic as other antibiotics
• Quinolones
• Broad spectrum
• Synthetic drugs
• inhibit DNA-gyrase complex

first synthesized 1962
used to treat anthrax in 911 (2001)
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Factors influencing antimicrobial drugs

• ability of drug to reach site of infection
• susceptibility of pathogen to drug
• ability of drug to reach concentrations in body
  that exceed MIC of pathogen
• Modes of administration
• Oral- some drugs destroyed by stomach acid
• topical
• parenteral routes
• Non-oral routes of administration
• drug can be excluded by blood clots or necrotic
  tissue

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Drug Concentrations in the Blood

• must be gt MIC at infection site to be effective
• Factors influencing the concentrations
• amount administered
• route of administration
• speed of uptake
• rate of clearance (elimination) from body

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Drug resistance

• Antibiotic misuse (Box 34.2)
• Can be transmitted
• Arise spontaneously
• Superbug
• 2002, an MSRA that developed resistance to
  vancomycin
• this VRSA (from VRE) also resisted to most other
  antibiotics

Fig 34.17
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Mechanisms of drug resistance

• Natural resistance- prevent entrance
• G(-) resist to Penicillin G
• Mycolic acid layer of Mycobacterium
• (1) alteration of target
• use of alternative pathways or increased
  production of target
• (2), (3) inactivation of drug
• chemical modification of drug
• (4) pump drug out

Figure 34.18
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Origin and spread of resistance
genes
Figure 34.19

• chromosomal genes
• spontaneous mutations in the drug target
• R plasmids
• can be transferred by conjugation, transduction
  and transformation
• can carry multiple resistance genes
• transposons
• Integrons may be gt 100 kb
• Gene (cassettes) capture

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Preventing drug resistance

• give drug in high concentrations
• give two or more drugs at same time
• use drugs only when necessary
• possible future solutions
• continued development of new drugs
• use of bacteriophages

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Antibiotic misuse and drug resistance

• Overuse and misuse of antibiotics
• Over 90 of colds and upper respiratory
  infections are caused by viruses
• Many patients always do not complete their
  course of medication
• The use of antibiotics in animal feeds
• As much as 70 of the antibiotics are added to
  livestock feed in USA
• Increase the number of drug resistant bacteria
  in animal intestinal tracts

Box 34.2
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Superinfection

• development and spread of drug-resistant
  pathogens
• caused by drug treatment, which destroys drug
  sensitive strains
• pseudomembranous enterocolitis
• caused when treatment with certain antibiotics
  kills intestinal flora, leaving Clostridium
  difficile to flourish and produce a toxin

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Antifungal drugs
superficial mycoses treatment
polyene antibiotic from Streptomyces

• fewer effective agents because of similarity of
  fungal cells and human cells

Disrupt membrane permeability and inhibit sterol
synthesis
Disrupts mitotic spindle may inhibit protein and
DNA synthesis
Figure 34.20
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Treating systemic infections
binds sterols in membranes
5FC
disrupts RNA function
Figure 34.20
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Antiviral Drugs-

• relatively few because difficult to specifically
  target viral replication
• Anti-flu
• Amantadine
• blocks uncoating of influenza virus
• Tamiflu ???
• a neuraminidase (NA) inhibitor
• not a cure for influenza (to shorten course of
  illness)
• Emerging resistant strain

Figure 34.21
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inhibits herpes virus enzymes involved in DNA and
RNA synthesis and function
Figure 34.21
inhibits herpes virus and cytomegalovirus DNA
polymerase
inhibits herpes virus DNA polymerase
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Broad-spectrum anti-DNA virus drugs
inhibits viral DNA polymerase
papovaviruses, adenoviruses, herpesviruses iridovi
ruses, and poxviruses
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Anti-HIV drugs

• Target to reverse transcriptase
• Target to protease