Microbial Recombination and Gene Transfer Questions and Answers

Answer: C

Correct answer: Virulence plasmid

A virulence plasmid carries genes that make its host more pathogenic - adhesins, invasion determinants, toxins or iron-scavenging systems. The Ent plasmids encoding enterotoxins in Escherichia coli, the pXO1 and pXO2 plasmids of Bacillus anthracis and the invasion plasmids of Shigella are all examples.

An F factor is a fertility plasmid concerned with conjugation and confers no virulence, while a metabolic plasmid encodes degradative or biosynthetic enzymes that widen the range of substrates the cell can use. The classification is by the function the accessory genes serve.

Answer: B

Correct answer: the F plasmid has integrated into the bacterial chromosome

An Hfr cell - high frequency of recombination - is one in which the F plasmid has integrated into the chromosome by homologous recombination at an insertion sequence. The consequence is that conjugation now transfers chromosomal genes. Transfer begins at the point of integration and proceeds linearly, so genes nearest that point enter the recipient first. Because the mating pair almost always separates before the whole chromosome has passed, the recipient usually receives only a portion, and rarely the F plasmid itself - which is why an Hfr cross seldom converts the recipient to a donor, unlike an F+ cross. The name records the high rate of chromosomal recombinants such crosses produce. Its experimental value is interrupted mating: breaking the mating at intervals and testing which markers have arrived gives their order and spacing along the chromosome, measured in minutes of transfer time. The whole E. coli chromosome takes about 100 minutes.

Answer: B

Correct answer: DNA is transferred from F+ to F- cells

In an F⁺ × F⁻ mating the DNA moves from donor to recipient - from F⁺ to F⁻. The F⁺ cell makes the sex pilus, contact is established, and one strand of the F plasmid is nicked at oriT and transferred; both cells then synthesise the complementary strand, so the recipient becomes F⁺.

Transfer is therefore one-way, defined by which cell carries the F factor. The F⁻ cell is not incapable of conjugating - it is a perfectly good recipient - it simply lacks the plasmid that encodes the machinery, so it cannot act as donor.

Answer: D

Correct answer: a bacteriophage genome is integrated into the bacterial genome

In lysogeny the phage genome is integrated into the bacterial chromosome as a prophage and is replicated passively with it at every cell division. No new virions are made while this lasts, and the lysogenic cell is immune to superinfection by the same phage until induction triggers the lytic cycle.

The other options describe different processes: transfer of bacterial DNA by a phage is transduction, uptake of double-stranded DNA from the environment is transformation, and it is transformation, not lysogeny, that extracellular nucleases would block, since lysogeny involves no naked DNA in the medium.

Answer: C

Correct answer: a bacteriophage

Transduction is the transfer of bacterial DNA from one cell to another by a bacteriophage, discovered by Zinder and Lederberg in 1952. It occurs in two forms. In generalised transduction, a phage in the lytic cycle packages a fragment of host DNA into a capsid by mistake, so any gene may be carried; the resulting particle injects bacterial rather than phage DNA into the next cell. In specialised transduction, a temperate phage integrated as a prophage excises imprecisely and carries the adjacent host genes with it - so only genes flanking the integration site can be transferred, which for phage lambda in E. coli means the gal and bio genes. The distinction is worth holding: generalised transduction moves any gene at random, specialised transduction moves particular genes only. It is used experimentally for fine-scale genetic mapping and for moving mutations between strains.

Answer: C

Correct answer: Transduction

Transduction is the mode of bacterial recombination that uses a viral vector. A bacteriophage packages a fragment of host DNA into its capsid during assembly, and that particle injects bacterial rather than viral DNA into the next cell it infects, where it may recombine into the recipient's chromosome.

The DNA is protected inside the capsid throughout, so the process is unaffected by nucleases in the medium. Transformation uses naked DNA taken from the surroundings and conjugation needs direct cell contact; assimilation is not a mechanism of gene transfer.

Answer: A

Correct answer: Lysogeny

Lysogeny is the relationship in which the viral genome is maintained and replicated along with the host chromosome while no new virions are produced. The integrated phage genome is called a prophage, and the bacterium carrying it is a lysogen, which passes it on to every daughter cell.

The prophage represses its own lytic genes and can confer new properties on the host - diphtheria and cholera toxins are both prophage-encoded. Lysis is the opposite outcome, in which progeny virions are made and the cell bursts; transformation and conjugation are bacterial gene-transfer mechanisms, not host-virus relationships.

Answer: A

Correct answer: a cell takes up free DNA from its surroundings

Transformation is the uptake of naked DNA from the environment by a bacterial cell, which then incorporates it - usually by homologous recombination into the chromosome. It was demonstrated by Frederick Griffith in 1928, whose experiment showed that heat-killed virulent pneumococci could convert live avirulent ones into virulent form. The transforming principle was identified as DNA by Avery, MacLeod and McCarty in 1944, which was among the decisive pieces of evidence that DNA rather than protein carries heredity. A cell able to take up DNA is said to be competent. Competence arises naturally in species such as Streptococcus pneumoniae, Bacillus subtilis and Haemophilus influenzae, and can be induced artificially by treatment with calcium chloride followed by heat shock, or by electroporation - the methods on which routine cloning depends. The second option describes transduction.

Answer: A

Correct answer: the ability to act as a donor in conjugation

The F or fertility plasmid carries the tra genes, which encode the sex pilus and the machinery of DNA transfer, so a cell possessing it can act as a donor in conjugation. The states are worth distinguishing. An F+ cell carries the plasmid free in the cytoplasm and transfers a copy of it to an F− recipient, which thereby becomes F+ - the plasmid spreads through a population like an infection. An Hfr cell has the plasmid integrated into the chromosome and transfers chromosomal genes in a fixed linear order. An F’ cell carries a plasmid that has excised imprecisely, taking a few chromosomal genes with it. The Hfr state is the basis of interrupted mating experiments, in which conjugation is broken off at intervals and the order in which markers appear gives their order on the chromosome - the first genetic map of E. coli was constructed this way. Antibiotic resistance is carried by R plasmids instead.

Answer: C

Correct answer: join DNA fragments by forming phosphodiester bonds

DNA ligase seals a break in the sugar-phosphate backbone by forming a phosphodiester bond between an adjacent 3’ hydroxyl and 5’ phosphate. In cloning it is what joins an insert to a vector after both have been cut. Its natural role is in replication and repair: it joins the Okazaki fragments of the lagging strand, and seals the nick left after excision repair. The enzyme most used in the laboratory is T4 DNA ligase, from bacteriophage T4, which will join blunt ends as well as sticky ones - the E. coli enzyme requires a sticky-ended join. The distractors are the other enzymes of the toolkit. A restriction endonuclease cuts at a recognition sequence; a helicase unwinds the helix; a methyltransferase adds methyl groups, which is how a cell protects its own DNA from its restriction enzymes. Ligase and restriction enzyme together are often described as the scissors and glue of recombinant DNA work.