03: Genetic Basis of Variation of Bacteria

Question 1

What did Griffiths discover regarding the genetic basis of variation in streptococcus pneumoniae?

Answer 1

Rough (R) strain was benign (lacked protective capsule –> recognized & destroyed by host’s immune system).

Smooth (S) strain was virulent (polysaccharide capsule prevented detection by host immune system).

Different morphologies led to different pathologies.

Question 2

What did Griffiths discover regarding the conversion of bacteria?

Answer 2

Heat-killed virulent bacteria, when added to live non-encapsulated nonvirulent bacteria, are lethal to mice.

Somehow, the live bacteria were able to convert from non-pathogenic to pathogenic.

Question 3

What did Avery et al. discover about the genetic basis of variation?

Answer 3

When RNA extracted from S strain bacteria was added to R strain bacteria and plated, no S transformants arised.

However, when DNA extracted from S strain bacteria was added to R strain bacteria and plated, S transformants formed.

Revealed that DNA is the transforming principle.

Question 4

Describe how genetic material of bacteria is organized within chromosomes.

Answer 4

• Most bacteria contain a single chromosome, along with extrachromosomal elements.
• Some bacteria also have 2-3 replicons, considered to be either megaplasmids or minichromosomes.
  
  • Ex: Rhodobacter sphaeroides has 3.0Mb and 0.9 Mb replicons
• Some bacteria harbour large replicons essential for survival in a specific ecological niche
• Few bacterial genera contain more than 1 chromosome.
  
  • Ex: Brucella has 2.1Mb and 1.2 Mb chromosomes
• Genes are very tightly packed; very little intergenetic sequences

Question 5

Describe how genetic material of bacteria is organized into plasmids.

Answer 5

• Extrachromosomal segments of DNA which are either circular or linear and range from 2-100+ kb in size.
• Non-essential
• May carry supplemental genetic information or may be cryptic.
• Employ host functions for most of DNA metabolism; use of cellular machinery for replication and maintenance encoded by plasmid (“parasitic elements”).
• May bring about features beneficial or detrimental to the host (e.g., tetracycline resistance, organism death).

Question 6

Describe the types of point mutations.

Answer 6

• Transitions (4 types): Purine-purine (A-G) or pyrimidine-pyrimidine (C-T)
• Transversions (8 types): Purine-pyrimidine

Question 7

Describe how DNA rearrangements contribute to genetic variation.

Answer 7

• Insertion sequence (IS) elements may disrupt a gene and change an organism from non-pathological to pathological.
• IS elements are the simplest type of transposable element found in bacterial chromosomes and plasmids.
  
  • Transposable elements can change their position within the genome, sometimes creating or reversing mutations and altering the cell’s genome size
• Encode only genes for mobilization and insertion; transposase enzyme allows their insertion.
• Range in size from 768bp to 5kb.
• The ends of all known IS elements show inverted terminal repeats (ITRs).
• Ex.1: Interruption of toxin repressor by IS element results in the toxin being expressed.
• Ex.2: IS element insertion results in strong promotor, strong expression of beta-lactamase gene, protecting organisms against ampicillin.

Question 8

Rank from low to high frequency the sources of genetic variation.

Answer 8

Lowest (10^-8)

1. Point mutations (but highest diversity)
2. Transposition
3. Plasmid transfer
4. Homologous recombination (but lowest diversity)

Highest (10^-1)

Question 9

What is the pathogenesis of Shigella?

Answer 9

• Derived from E. coli ancestor
• Transposable elements (SHI-1 & 2) incorporated into chromosome
• Virulence plasmid encoding Shigella toxin introduced
• Two important regulators of bacteriophysiology (ompT & cadA) lost

Question 10

What did the Luria-Delbruck test reveal about transmission of genetic information?

Answer 10

Hypothesized that resistance to mutation is either a physiological response or arises randomly in time.

Plating study resulted in unequal surviving colonies (“jackpots”), fitting with expectations that resistance by mutation arises randomly in time.

This leads to linear transmission (daughter cells acquire mutation; becomes fixed in population) and horizontal transmission (transfer of information to genetically-independent lineage) of genetic variation.

Question 11

Describe the process of transformation.

Answer 11

• Gene transfer in which donor cell is lysed and DNA uptaken from environment by host bacterium’s chromosome.
• Although this is the simplest form of genetic exchange (no physical contact requirement), it is not an efficient system (extracellular enzymes may destroy DNA).
• Factors affecting transmission:
  
  • DNA size and state
  • Competence of the recipient (state at which becaterium is accepting DNA)
• Double-stranded DNA (a⁺) brought into cell by pilus (one strand enters while the other is degraded); formation of triple-strand with host chromosome (a/a/a⁺); recombination by double crossover; displaced strand degraded by nucleases ==> chromosome with segment of a+/a heteroduplex DNA; half of replicated DNA is transformant, half is nontransformant.

Question 12

Describe the phases of transduction.

Answer 12

• Infection phase: DNA injected by phage into the host.
• Lysogenic phase: DNA uptaken by host chromosome but does not promote the formation of phages; host replicates stably (introduced by temperate bacteriophage).
• Induction phase: DNA uptaken by host chromosome spontaneously induced to produce phages
• Lytic phase: Alternative route in which injected DNA does not become incorporated into chromosome, but rather independently takes over the cell and within an hour, makes many phages and lyses out of cell (introduced by lytic bacteriophage).

Question 13

What did the Ledereberg & Zinder experiment reveal?

Answer 13

• Demonstrated the transmission of genetic variation via transduction.
• Phe/Trp-deficient strains placed in U-tube that allowed medium to pass across a filter (but not cells); on other side of filter, Phe/Trp-positive strains. When deficient strains plated on minimal medium and incubated, they grew (were Phe/Trp-positive).
• Able to prove that phenotype was the result of transduction due to the facts that:
  
  • DNAse presence ruled out transformation.
  • Filter prevented contact (no conjugation).
  • Reducing filter pore size below size of phage inhibited transduction.

Question 14

How large is the average bacteriophage DNA sequence?

Answer 14

20-100 kb

Question 15

What are the two types of transduction?

Answer 15

• Generalized transduction: DNA fragment transferred from one bacterium to another by lytic bacteriophage carrying donor bacterial DNA due to error in maturation during lytic life cycle.
• Specialized transduction: DNA fragment transferred from one bacterium to another by temperate bacteriophage carrying donor bacterial DNA due to error in spontaneous induction during lysogenic life cycle.

Question 16

What is the central mechanism of transduction?

Answer 16

Transduction arises from the accidental packaging of bacterial host DNA into a phage particle, which is then transferred to a new bacterium.

Question 17

Describe the steps of generalized transduction.

Answer 17

1. Lytic bacteriophage adsorbs to susceptible bacterium.
2. Bacteriophage genome enters bacterium and directs bacterium’s metabolic machinery to manufacture phage components & enzymes (chops up DNA of host cell).
3. Occassionally, phage head/capsid assembles around fragment of donor bacterium’s nucleoid/plasmid instead of phage genome by mistake.
4. Bacterophages are released from host.
5. Bacteriophage carrying the donor bacterium’s DNA adsorbs to a recipient bacterium.
6. Bacteriophage inserts donor bacterium’s DNA into recipient.
7. Donor bacterium’s DNA exchanged for some of recipient’s DNA (this recombination occurs only if there is high homology between DNA sequences; rare (1 in 10 million), but since infections common, happens relatively frequently).

Question 18

Describe the steps of specialized transduction.

Answer 18

1. Temperate bacteriophage adsorbs to susceptible bacterium and injects its genome.
2. Bacteriophage genome inserted into bacteriums nucleoid to become a prophage.
3. During spontaneous induction, small piece of donor bacterium’s DNA picked up as part of phage’s genome in place of some of the phage DNA which remains in the bacterium’s nucleoid.
4. As the bacteriophage replicates, the segment of bacterial DNA replicates as part of the phage’s genome; every phage now carries that segment of bacterial DNA.

Question 19

What is the central mechanism of conjugation?

Answer 19

Genetic recombination in which DNA transferred as plasmid from living donor bacterium to recipient bacterium via sex pilus.

Conjugative plasmids found in approximately 30 genera of bacteria, mostly gram-negative (antibiotic-resistance plasmids RP4 & R68.45 can propogate and promote conjugation in virtually any gram-negative bacterium).

Some gram-positives conjugate, such as Streptococci, Staphylococcus, Streptomyces, Clostridium and Bacillus.

Question 20

What is F+ conjugation?

Answer 20

• Genetic recombination in which there is a transfer of a large (95kb) F+ plasmid (“fertility factor;” coding only for a sex pilus), but not chromosomal DNA, from a male donor to a female recipient bacterium; recipient F- cell becomes F+.
• Other plasmids present in the cytoplasm of the bacterium (such as those coding for antibiotic resistance) may also be transferred.
• F can be transferred from E. coli to Salmonella, Shigella and Proteus.

Question 21

What is R-plasmid conjugation?

Answer 21

R-factors are drug resistance plasmids.

1. Bacterium with R-plasmid is multiple antibiotic resistant and can produce a sex pilus (serve as genetic donor).
2. Sex pilus adheres to an F- female (recipient); one strand of the R-plasmid breaks.
3. Sex pilus retracts and a bridge between the two bacteria is created. One strand of the R-plasmid enters the recipient bacterium.
4. Both bacteria make a complementary strand of the R-plasmid, and both are now mutliple antibiotic resistant and capable of producing a sex pilus.

Question 22

What is conjugative transposition?

Answer 22

• Process through which plasmid inserts itself into the host chromosome as a composite transposon and is stably maintained.
  
  • Carry genes (e.g., for antibiotic resistance) flanked on both sides by IS elements (which supply transposase and ITR recognition signals)
• Can then excise self from host chromosome, transfer to new cell and integrate self into new chromosome.

Question 23

CTX phage

1. Means of transmission
2. Bacterium
3. Gene product
4. Phenotype

Answer 23

1. Transduction
2. Vibrio cholerae
3. Cholerae toxin
4. Cholera

Question 24

Lambda phage

1. Means of transmission
2. Bacterium
3. Gene product
4. Phenotype

Answer 24

1. Transduction
2. Escherichia coli
3. Shigalike toxin
4. Hemorrhagic diarrhea

Question 25

Clostridial phages

1. Means of transmission
2. Bacterium
3. Gene product
4. Phenotype

Answer 25

1. Transduction
2. Clostridium botulinum
3. Botulinum toxin
4. Botulism (food poisoning)

Question 26

Corynephage beta (phage)

1. Means of transmission
2. Bacterium
3. Gene product
4. Phenotype

Answer 26

1. Transduction
2. Corynebacterium diphtheriae
3. Diphtheria toxin
4. Diphtheria

Question 27

Meningococcal prophage

1. Means of transmission
2. Bacterium
3. Gene product
4. Phenotype

Answer 27

1. Transduction
2. Neisseria meningitidis
3. Neisseria toxin
4. Cerebrospinal meningitis

Question 28

Bacillus spp. (anthracis, cereus & thuringiensis)

1. Means of transmission
2. Plasmids involved
3. Plasmids encoding
4. Phenotype

Answer 28

1. Conjugation
2. pXO1 & pXO2
3. Anthrax toxin & capsule
4. Anthrax

Note that there is extensive homology between the three Bacillus species, although only anthracis has the insertion of anthrax virulent genes; can be conjugated into other two species resulting in similar phenotypes.

Question 29

What bacteria are competent?

Answer 29

1. Bacillus
2. Haemophilus
3. Neisseria
4. Streptococcus

Question 30

Which bacteria participate in conjugation?

Answer 30

• Mostly GNs
• GPs: Streptococc, staph, streptomyces, clostridum, bacillus