Brian M. Wilkins

DNA Processing and Replication during Plasmid Transfer between Gram-Negative Bacteria

This chapter concerns the processing and synthesis of plasmid DNA during its transmission between conjugating gram-negative bacteria, focusing on conjugation systems specified by plasmids isolated in or transferred experimentally to enterobacteria. This extensive collection of plasmids can be classified into more than 25 different incompatibility (Inc) groups (34), each of which is generally associated with a distinct conjugation system (158). Only a few of these systems have been investigated at the biochemical and molecular levels, but studies have identified unifying themes as well as an interesting diversity of enzymatic strategies for the conjugative processing of plasmid DNA.

Protein transfer into the recipient cell during bacterial conjugation: studies with F and RP4

Transfer of donor cell proteins to the recipient bacterium was examined in F‐ and RP4‐mediated conjugation. Transfer of a 120kD polypeptide, identified as the larger product of the plasmid DNA primase gene, was readily detected during RP4‐promoted conjugation. The protein was transmitted to the cytoplasm of the recipient, presumably complexed to the transferred ssDNA. F DNA was transferred without detectable association with any cytoplasmic tra protein or with the ssDNA‐binding protein encoded by the plasmid. However, a 92kD protein, possibly F TraD product, was transmitted to the membrane fraction of the recipient cell.

Distribution of the ardA family of antirestriction genes on conjugative plasmids.

The ardA gene of I1 plasmid ColIb-P9 was previously shown to alleviate DNA restriction by type I enzymes and to promote conjugative transmission of the unmodified plasmid to a restricting host. To clarify the ecological role of ardA, its distribution was determined on plasmids from 23 incompatibility groups using hybridization to the coding sequence as an assay. Hybridizing sequences, shown by nucleotide sequencing to have at least 60% identity with ardA, were detected on plasmids belonging to the I complex (IncB, I1 and K), the F complex (IncFV) and the IncN group. The ardA homologues were found to specify an antirestriction phenotype which was enhanced by genetic depression of the plasmid transfer system. ardA loci map in plasmid leading regions but show no consistent association with a particular type of origin-of-transfer or a leading region gene of the ssb (single-stranded DNA-binding protein), psiB (plasmid SOS inhibition) and hok (host killing) families. It may be significant that ardA+ plasmids are authentic enterobacterial plasmids and that type I restriction systems are associated historically with members of the Enterobacteriaceae.

DNA‐independent transport of plasmid primase protein between bacteria by the I1 conjugation system

The ColIb‐P9 (IncI1)‐encoded conjugation system supports transfer of the plasmid T‐strand plus hundreds of molecules of the Sog polypeptides determined by the plasmid primase gene. Here, we report that Sog primase is abundantly donated to the recipient cell from cells carrying a non‐transferable ColIb plasmid deleted of the nic site essential for DNA export. Such DNA‐independent secretion of Sog primase is typical of authentic conjugation, both in being blocked when the recipient cell specifies the entry exclusion function of ColIb and in requiring the thin I1 pilus encoded by the ColIb pil system under the mating conditions used. It is proposed that Sog polypeptides form a complex with the ColIb T‐strand during conjugation and aid DNA transport through processive secretion of the proteins into the recipient cell. Functional and genetic relationships between the ColIb conjugation system and other type IV secretion pathways are discussed.

Transient transcriptional activation of the IncI1 plasmid anti‐restriction gene (ardA) and SOS inhibition gene (psiB) early in conjugating recipient bacteria

The ardA gene of the enterobacterial plasmid ColIbP‐9 acts to alleviate restriction of DNA by type I systems, while psiB inhibits induction of the bacterial SOS response. Both genes are transferred early in a round of bacterial conjugation as part of the plasmid leading region. We report here that ardA and psiB are transcribed transiently after their conjugative transport into the recipient cell. Transcript levels, monitored by competitive reverse transcription–polymerase chain reaction (RT–PCR) amplification of RNA templates, started to increase about 5 min after the initiation of conjugation in a cell population and probably before the first round of plasmid transfer was completed. Genetic evidence is given that the expression of ardA and psiB is activated when the genes enter the recipient cell on the transferring plasmid strand. It is proposed that these and other leading region genes function to promote the establishment of the immigrant plasmid in the new host and are expressed by transcription from promoters active only in single‐stranded DNA.

Zygotic induction of plasmid ssb and psiB genes following conjugative transfer of Incl1 plasmid Collb-P9

The Incl1 conjugative plasmid Collb‐P9 carries a psiB gene that prevents induction of the SOS response in host bacteria. This locus is located 2.5 kb downstream of the ssb (single‐stranded DNA‐binding protein) gene in the leading region. This portion of Collb is strikingly similar to part of the leading region of the otherwise distinct F plasmid. Expression of psiB and ssb is increased when the host cell is exposed to an SOS‐inducing treatment or the Collb transfer system is derepressed. Moreover, expression of both genes on a derepressed plasmid is strongly enhanced in conjugatively infected recipient cells. Carriage of the psiB gene by Collb is shown to prevent a low level of SOS induction following conjugation. Plasmid ssb and psiB genes may function to promote installation of the replicon in the new cell.

Evasion of type I and type II DNA restriction systems by Incl1 plasmid Collb‐P9 during transfer by bacterial conjugation

Transmission of unmodified plasmid Collb‐P9 by bacterial conjugation is markedly resistant to restriction compared with transfer by transformation. One process allowing evasion of type I and II restriction systems involves conjugative transfer of multiple copies of the plasmid. A more specialized evasion mechanism requires the Ard (alleviation of restriction of DNA) system encoded by Collb. The ard gene is transferred early in conjugation and specifically alleviates DNA restriction by all known families of type I enzyme, including Eco K. Collb has no effect on EcoK modification but this activity is impaired by multicopy recombinant plasmids supporting overexpression of ard. Genetic evidence shows that Ard protects Collb from Eco K restriction following conjugative transfer and that this protection requires expression of the gene on the immigrant plasmid. It is proposed that carriage of ard facilitates transfer of Collb between its natural enterobacterial hosts and that the route of DNA entry is important to the restriction‐evasion mechanism.

Organization and regulation of the conjugation genes of IncI1 plasmid ColIb-P9

The IncI1 plasmid ColIb-P9 is among a group of related plasmids that encode the I1 type of conjugation system. The I1 system is known to include two morphologically distinct types of pilus, a DNA primase gene (sog) and an exclusion determinant (exc). Transposon mutagenesis and analysis of cloned fragments of ColIb were used to identify the location of these determinants with respect to an EcoRI restriction map. Also identified were the location of the origin of transfer (oriT) and a gene determining an EDTA-resistant nuclease, which is coordinately regulated with the transfer genes. The results indicate that the ColIb tra genes are separated into at least three Tra regions. The pleiotropic nature of transposon insertion mutations in two of these regions suggests that two positive regulators are required for expression of the transfer genes and evidence is also found for a trans-acting repressor. It is suggested that the I1 conjugation system may have evolved following fusion of two distinct types of conjugative plasmid.

Plasmid R16 ArdA Protein Preferentially Targets Restriction Activity of the Type I Restriction-Modification System EcoKI

The ArdA antirestriction protein of the IncB plasmid R16 selectively inhibited the restriction activity of EcoKI, leaving significant levels of modification activity under conditions in which restriction was almost completely prevented. The results are consistent with the hypothesis that ArdA functions in bacterial conjugation to allow an unmodified plasmid to evade restriction in the recipient bacterium and yet acquire cognate modification.

Expression of leading region genes on IncI1 plasmid ColIb-P9: genetic evidence for single-stranded DNA transcription.

The leading region of a plasmid is the first sector to enter the recipient cell in bacterial conjugation. This sector of IncI1 plasmid ColIb-P9 includes genes that are transcribed in a transient pulse early in the conjugatively infected cell to promote establishment of the immigrant plasmid. Evidence is presented that the burst of gene expression is regulated by a process which is independent of a repressor but dependent on the orientation of the genes on the unique plasmid strand transferred in conjugation. The nucleotide sequence of 11.7 kb of the leading region was determined and found to contain 10 ORFs; all are orientated such that the template strand for transcription corresponds to the transferred strand. The leading region contains three dispersed repeats of a sequence homologous to a novel promoter in ssDNA described by H. Masai & K. Arai (1997, Cell 89, 897-907). It is proposed that the repeats are promoters that form in the transferring strand of ColIb to support transient transcription of genes transferred early in conjugation.