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...

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903φkan, carries a cryptic kangene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903φkan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903φkaninsertion mutants were readily selected in Escherichia coliby their resistance to kanamycin. We used IS903φkan to isolate three catalase-deficient mutants of the periodontal pathogenActinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903φkan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903φkan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903φkan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.

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