17.6 Genetic Transfer & Mapping in Bacteria and Bacteriophages

Genetic Transfer & Mapping in Bacteria and Bacteriophages

Quick Answer: Bacteria transfer genes by transformation (free DNA uptake), transduction (phage-mediated), and conjugation (cell-to-cell via an F plasmid). Hfr interrupted-mating maps chromosomal genes by time of entry. Complementation tests distinguish mutations in the same gene vs. different genes. Per the PA-CAT Bulletin of Information, rev. 20240815, genetic technologies and microbial gene transfer appear within the Genetics content area.

1. Transformation

Transformation is the uptake of naked, extracellular DNA by a competent cell. Griffith's 1928 experiment showed that heat-killed smooth (S) pneumococcus could transform live rough (R) pneumoccus into virulent S in mice — the "transforming principle." Avery, MacLeod, and McCarty (1944) identified DNA (not protein or RNA) as that principle, a landmark in establishing DNA as the genetic material.

Natural competence is a regulated physiological state in species like Streptococcus pneumoniae, Haemophilus influenzae, and Bacillus subtilis, often induced by starvation or quorum sensing. Artificial competence is induced in the lab by calcium chloride treatment and heat shock (chemical transformation) or by electroporation (electric pulses creating membrane pores) — the routine method for introducing plasmids into E. coli.

Transformed DNA integrates by homologous recombination into the chromosome (for linear fragments) or persists as a plasmid (for circular DNA with an origin of replication).

2. Conjugation and the F Plasmid

Conjugation, discovered by Lederberg and Tatum (1946), requires physical contact via a sex pilus encoded by the F (fertility) plasmid. Contact triggers transfer of a single-stranded copy of F from donor to recipient; the complementary strand is synthesized in the recipient, yielding a double-stranded F. The donor retains its F.

• F+ × F−: the donor transfers a copy of F; the recipient becomes F+. No chromosomal genes are transferred — only plasmid genes.
• Hfr (High-frequency recombination) strain: F is integrated into the bacterial chromosome by a single crossover between F and the chromosome. Transfer begins at the F origin and proceeds into the chromosome in a fixed direction. Because the mating bridge usually breaks before the whole chromosome (which would take ~100 minutes in E. coli) transfers, genes closest to the origin enter first and most frequently.
• F′ (F prime): F excises imprecisely, carrying adjacent chromosomal genes with it. Transfer of an F′ to a recipient creates a merodiploid (partial diploid) — one chromosomal copy and one plasmid copy of those genes. F′ strains are the workhorse of complementation tests in bacteria.

Interrupted Mating and Time-of-Entry Mapping

The Wollman and Jacob (1950s) interrupted-mating experiment is the classical Hfr map. An Hfr donor (e.g., str^R azi^R ton^S lac^+ gal^+) is mixed with an F− recipient (str^S azi^S ton^R lac− gal−) and samples are blender-agitated at timed intervals to break the mating pairs. Recombinants are selected on medium that kills unrecipiented donors (e.g., streptomycin) and scored for donor markers.

Entry time = distance from the origin; longer times = farther along the chromosome. This produces a time-of-entry map in minutes (the E. coli map totals ~100 min). Different Hfr strains integrate F at different sites and orientations, so comparing several Hfr strains assembles the circular map.

3. Transduction

Transduction, discovered by Zinder and Lederberg (1952), is phage-mediated DNA transfer.

• Generalized transduction: during the lytic cycle, a phage accidentally packages a random fragment of bacterial DNA instead of viral DNA inside the phage head. On infecting a new cell, the fragment injects and can recombine. Any bacterial gene can be transferred. Example: P22 in Salmonella, P1 in E. coli. Used for cotransduction mapping — two genes packaged in the same phage head are close together; cotransduction frequency is inversely related to the distance between them (the Wu formula: distance ≈ 1 − (cotransduction frequency)^(1/3)).
• Specialized (restricted) transduction: during induction of a lysogenic prophage from the host chromosome, imprecise excision carries adjacent bacterial genes along with phage DNA. E. coli phage λ transfers the gal or bio genes flanking its attB attachment site. Only genes near the attachment site are transferred, hence "specialized." The resulting transducing phage is often defective (missing some phage genes) and requires a helper phage.

Phage Life Cycles and Phage Mapping

Bacteriophages follow one of two pathways after infection:

• Lytic cycle: phage replicates, lyses the cell, releases progeny (virulent phage, e.g., T4).
• Lysogenic cycle: phage DNA integrates as a prophage and is replicated with the host (temperate phage, e.g., λ). Stress (UV, mitomycin C) induces the prophage to excise and enter the lytic cycle.

In phages, recombination mapping uses mixed infection of a bacterium with two phage mutants. Recombinant plaques (wild-type or double-mutant) appear at a frequency proportional to the distance between the mutations — analogous to eukaryotic RF. Benzer's rII work on T4 used this to define the gene as a unit of function and to map mutations within a single gene (fine-structure mapping), showing that genes are linear arrays of mutable sites and that the genetic map is colinear with the protein.

Complementation Tests

A complementation test asks whether two independently isolated mutations causing the same phenotype affect the same gene or different genes. Set up in the trans configuration: one mutation on each homolog (or, in bacteria, one chromosomal and one F′-borne).

• Mutations in the same gene (allelic): the trans heterozygote m1/m2 shows the mutant phenotype → no complementation (because neither copy encodes a functional protein).
• Mutations in different genes: the trans heterozygote m1/+ ; +/m2 shows wild-type → complementation (each homolog supplies a working copy of the gene the other lacks).

The cis-trans test defines the cistron — the functional gene unit — as the region within which mutations fail to complement in trans. Benzer's rII complementation work established that the rII region contains two cistrons (rIIA and rIIB).

A critical distinction: complementation tests whether two mutations are in the same functional unit (genes), whereas recombination tests whether two mutations are at the same physical site (can recombine only if at different sites). Complementation does not require recombination — it works in a heterozygote.

PA-CAT Application

Per the PA-CAT Bulletin of Information, rev. 20240815, genetic technologies and microbial genetics appear within the Genetics content area. Distinguish the three transfer mechanisms by whether they require cell contact (conjugation), a phage vector (transduction), or free DNA (transformation), and know that Hfr transfers chromosomal (not F) genes in a time-ordered gradient. Recognize that generalized transduction can move any gene while specialized transduction is limited to genes flanking the prophage attachment site. For complementation, remember the key rule: mutations in the same gene fail to complement in trans; mutations in different genes complement.