Genetic Variation, Gene Transfer and the Evolution of Virulence

Question 1

Describe the mechanisms that generate genetic diversity within a bacterial species and how these contribute to the evolution of virulence.

Answer 1

1. Spontaneous Mutation. Single base changes, deletions and insertions occur spontaneously within a population.
2. Recombination. Either site-specific or homologous recombination within a particular organism (e.g. Gonorrhoeae switching pili), or genetic exchange and recombination between closely related organisms can contribute to the emergence of strains with new properties.
3. Acquisition of new DNA segments by lateral transfer from other bacteria, even from unrelated species and possibly eukaryotic organisms.

• –Transposable elements (transposons, IS elements and “complex transposons”): discrete segment of DNA which is capable of moving itself (or a copy of itself) from one chromosomal location to another;
• cannot self-replicate except as part of another self-replicating molecule (i.e., the bacterial chromosome, virus, or plasmid)
• Typically encode one or more proteins mediates transposition (transposase). Transposition is not dependent upon regions of extensive homology and does not require host recombination machinery.
• May transpose and become permanently integrated into the bacterial chromosome.
• “Insertion Sequences” (IS elements) are transposons that simply encode transposase. Play a role in genome evolution by inactivating genes into which they transpose, or turning on expression of adjacent genes.
• “Complex transposons” carry additional genes such as those encoding antibiotic resistance, toxins, adhesins and other virulence factors.

—Bacteriophage conversion. Certain virulence genes are carried on bacteriophage and are not a “normal” component of the respective bacterial genome, but rather have become lysogenized and the bacteriophage genome is stably maintained by the bacterium.

—Plasmids: autonomously replicating, usually circular, extrachromosomal DNA’s ranging in size from a couple genes to a few percent the size of the bacterial chromosome. Often they can be transferred from one bacterium to another by conjugation or transduction. Plasmids can carry virulence genes and genes conferring antibiotic resistance.

—“Pathogenicity Islands”: large segments of DNA present in the chromosome of some, but not all strains of a particular bacterial species. PIs encode genes that contribute to the virulence of these isolates. Bacterial isolates that lack the PI may be avirulent, or have a somewhat different disease-causing potential. Their origin is unknown

Question 2

Discuss how spontaneous mutation and selection can interact to determine the genetic composition of bacterial populations.

Answer 2

A. Spontaneous Mutation. Single base changes, deletions and insertions occur spontaneously within a population. Under appropriate selective pressure (e.g., a patient receiving streptomycin), the preferential growth of a pre-existing mutant within a population is selected.

Question 3

Transformation

Answer 3

• Uptake of naked DNA (likely plasmids or chromosomal fragments released from lysing cells) by competent adjacent cells.
• Cells may become competent for DNA uptake at only certain points in the growth cycle via specialized proteins to mediate the uptake, or can be induced to uptake DNA with CaCl2 and low temps
• Can occur between members of the same or different species –> acquisition of new genes and genetic potential of a given species.

Question 4

Transduction

Answer 4

• Mediated by a bacteriophage which transfers segments of DNA (2 - 200 genes) from one cell to another
• Virus adheres to cell and injects DNA
• Cell undergoes lytic or lysogenic cycle
• In lytic cycle, cell replicates viral DNA and packages into new phage capsids. However, 1/1000 times an error occurs and a plasmid or bacterial DNA is packaged instead (up to 1-2% of genome). This DNA is transmitted to a new cell and voila: transduction
• In lysogenic cycle, viral DNA inserts into bacterial genome –> prophage (recombined DNA)
• Cell may switch to lytic stage in times of stress (repressor proteins control this)
• Phage may insert DNA that encodes for a new phenotypic trait (e.g. toxin production). This is lysogenic conversion, wherein the genes controlling the new phenotypic trait are found ONLY as a component of the phage genome (not normally in bacterial genome)

Question 5

Conjugation

Answer 5

Genetic transfer dependent upon physical contact between the donor and recipient cells, mediated by certain types of bacterial plasmids

Conjugative plasmids, such as transmissible drug resistance plasmids, are self-transmissible. These plasmids mediate their own transfer, transfer of other plasmids, and occasionally chromosomal genes from donor to recipient cells. Plasmids may acquire new genetic material, especially by transposition.

The F plasmid is the prototype self-transmissible plasmid. It contains genetic information encoding the following traits:
- Autonomous replication of the plasmid DNA
- Synthesis of sex pili (F pili)
- Conjugative transfer of F DNA to recipient (F- ) cells
- Ability to integrate into the bacterial chromosome
Transfer of F DNA is coupled to a special round of DNA synthesis which is initiated at a site called oriT (the origin of transfer). The DNA is transferred as a single strand through a conjugation bridge

Hfr mating type are F+ populations that can transfer sections of chromosomal DNA to F- recipients and produce recombinants at very high frequencies (hence Hfr)

• In these cells, the sex factor is linearly inserted / integrated into the bacterial chromosome (its not on a plasmid). When transfer replication is initiated at oriT, within the integrated sex factor, both F DNA and the adjacent covalently attached chromosomal DNA sequences pass into the recipient cell.
• Transfer is unidirectional and shows a marked polarity (genes close to the integrated F) are transferred more efficiently than genes which are located farther away).

Conjugative transposons are mobile elements which mediate conjugation between pairs of cells, in which the transferred DNA is the conjugative transposon itself. Once transferred, the element transposes to the chromosome of the recipient cell. Therefore these elements encode both transfer (tra) genes and transposition genes. Conjugative transposons may encode antibiotic resistance, especially resistance to tetracycline (tetM), and are possibly responsible for the widespread dissemination of the tetM resistance gene.