Structure, function, and regulation of the kilB locus of promiscuous plasmid RK2

Abstract

The kil-kor regulon of the self-transmissible, broad-host-range plasmid RK2 is a unique network with eight coregulated operons. Among the genes encoded by the kil-kor regulon are trfA, which encodes the replication initiator, and several kil loci (kiL4, kilB, WidC, and WilE), each of which is lethal to the host cell in the absence of appropriate negative regulatory elements encoded by the korA, korB, korC, and korE determinants. We have proposed that the functions of the kil loci are related to RK2 maintenance or host range. Here, we report the nucleotide sequence of a 2.44-kb region that includes the lethal kilB determinant. We identified the first three genes of the kilB operon (designated klbA, k1bB, and klbC), and we determined by deletion analysis that the host-lethal phenotype requires klbB. The predicted amino acid sequence of the 34,995-Da klbA product reveals a potential ATP-binding fold. The klbB product is predicted to be a membrane protein with a molecular mass of 15,012 Da with homology to the RK2 KlaC membrane protein encoded by the kiL4 operon. The amino acid sequence of the 12,085-Da IdbC product contains a perfect match to the leucine zipper motif common to eukaryotic regulatory proteins. Primer extension analysis revealed unambiguously that transcription of the kilB operon begins 46 nucleotides upstream of kibA. No transcription was initiated from the sequence previously presumed by other investigators to be the kilB promoter. The abundance of kilB transcripts is reduced in the presence of KorB, consistent with the prediction that KorB acts at the level of transcription. A degenerate KorB-binding site that contains a perfect half-palindrome overlaps the kilB promoter, but this site is insufficient for regulation by KorB. The region containing a KorB-binding site located 183 bp upstream of the transcriptional start is required for regulation by KorB, indicating that KorB acts at a distance to regulate transcription of kilB. Our studies with the mutant plasmid pRP101, a transfer-defective derivative of the RK2-like plasmid RP4, demonstrated that the kiB operon includes the conjugal transfer and surface exclusion genes of the Tra2 region. Nucleotide sequence analysis revealed that the transposon Tn7 insertion in pRP101 is located in the kdbC gene, and complementation analysis showed that this mutation has a strong polar effect on the expression of genes for conjugal transfer and surface exclusion located several kilobases downstream. A idbA mutant was constructed and found to be both transfer defective and complementable, thus demonstrating a requirement for the kibA product in plasmid transmissibility. These results have demonstrated a role for the kilB operon in conjugal transfer. The kil-kor regulon of RK2 is the only known example of plasmid-mediated coregulation of replication and transfer.The self-transmissible plasmids of incompatibility group P (IncP) are remarkable for autonomous replication in a wide variety of gram-negative bacterial hosts (17,67,98). In addition, the range of organisms...

Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids

“…The Tra2 operon encodes at least 14 genes (trbB through trbO). The kilB locus includes trbB and trbC (58). (Note that trbB, trbC, and trbD were previously designated kibA, klbB, and klbC, respectively [58].)…”

“…One primer (35-mer) was specific for kilC RNA (described above); the second primer was specific for RNA initiated from the constitutive bla promoter on pRK21660. The level of bla-specific cDNA served as an internal control (58 Fig. 2.…”

“…To assay for the effects of korA and korC on the relative abundance of kilC transcripts, primer extension analysis was done with two primers in the reaction mixture, as previously described (29,58). One primer (35-mer) was specific for kilC RNA (described above); the second primer was specific for RNA initiated from the constitutive bla promoter on pRK21660.…”

“…The transcription start point for the kilC operon was determined by primer extension analysis, as previously described (29,58). A 300-bp fragment containing the kilC promoter was inserted into pCH8, which carries a transcriptional terminator downstream of the cloning site, to produce pRK21660 (described above).…”

“…RNA was extracted from exponential-phase cultures. An oligonucleotide primer (35-mer) complementary to the transcribed region downstream of the promoter in pRK21660 (58) was end labeled with [y-32P]ATP, annealed to the RNA, and extended by avian myeloblastosis virus reverse transcriptase. The cDNA products were separated by electrophoresis in polyacrylamide sequencing gels and visualized by autoradiography.…”