Transient transcriptional activation of the Incl1 plasmid anti-restriction gene (ardA) and SOS inhibition gene (psiB) early in conjugating recipient bacteria.

The ardA gene of the enterobacterial plasmid CollbP-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.

Distribution of restriction enzyme recognition sequences on broad host range plasmid RP4: molecular and evolutionary implications.

IncP alpha plasmids, exemplified by RP4, are remarkable for their broad host range. They contain strikingly few cleavage sites for many commonly used type II restriction enzymes but an overabundance of sites for certain enzymes that target G + C-rich sequences. To identify factors responsible for these distributions, the recently compiled nucleotide sequence of RP4 was analysed to determine the frequency of tetra- and hexanucleotide motifs in the 49 kb plasmid backbone. This is defined as the sectors encoding basic plasmid functions. The overabundant restriction targets in RP4 are concentrated in the backbone and contain overlapping copies of CGGC/GCCG, identified as the most abundant tetranucleotide motif in the plasmid. Motif frequencies in the RP4 backbone are shown to be similar to those in Pseudomonas aeruginosa, a natural host of RP4, with the notable exception that a number of 6-bp palindromes are underrepresented in the plasmid. It is proposed that 6-bp palindromes were counterselected as type II restriction enzyme recognition sequences. Conjugative transfer of RP4 and R751 (IncP beta) is unusually sensitive to restriction compared to enterobacterial plasmids of the IncFII and IncI1 groups, implying that IncP plasmids experienced particularly strong selection for loss of restriction targets. Pseudomonas spp. of rRNA homology group I specify many type II restriction enzymes that target 6-bp palindromes and are candidates for the evolutionary hosts of IncP alpha plasmids.

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.