Co-operative interactions control conjugative transfer of broad host-range plasmid RK2: full effect of minor changes in TrbA operator depends on KorB.
A network of circuits, with KorB and TrbA as key regulators, controls genes for conjugative transfer of broad host range plasmid RK2. To assess the importance of the TrbA regulon, mutational analysis was applied to the TrbA operator at the trbB promoter and then to other TrbA-regulated promoters in the tra region. All identified TrbA operators are submaximal; in the case of trbBp, a G to A transition that made the operator core a perfect palindrome increased repression by about 50% compared to the wild type. When this change was introduced into the RK2 genome, decreases in transfer frequency of up to three orders of magnitude were observed, with bigger effects when Escherichia coli was the donor compared to Pseudomonas putida. Western blotting showed a significant decrease in Trb protein levels. These effects were much greater than the effect of the mutation on repression by TrbA alone. When KorB was introduced into the reporter system, the effects were closer to those observed in the whole RK2 context. These results indicate that co-operativity, previously observed between TrbA and KorB, allows big changes in transfer gene expression to result from small changes in individual regulator activities.
Bingle
Lewis E H
LE
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Zatyka
Malgorzata
M
Manzoor
Susan E
SE
Thomas
Christopher M
CM
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003227:Conjugation, Genetic; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C053863:KorB protein, Plasmid RK2; D012097:Repressor Proteins; C076244:trbA protein, E coli