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The korB gene of broad host-range plasmid RK2 prevents host-cell lethality by kiLB and negatively controls RK2 replication. We precisely mapped the limits of korB to a region near korA, an autoregulated gene involved in control of several RK2 genes. The following results show that korA and korB are cotranscribed from the korA promoter: (i) Mutants deleted for the korA promoter fail to express korB, even with korA function supplied in trans; (ii) the korA promoter is nonessential to korB if a heterologous promoter is present; and (iii) RNA produced in vivo has both korA-and korB-specific sequences. Analysis of polypeptides synthesized from wild-type and mutant korB plasmids in maxicells revealed that korB encodes a 52-kDa polypeptide, whose activity is extremely sensitive to changes in its carboxyl terminus but relatively unaffected by replacement of its amino terminus. The minimal korB-encoding region allowed us to identify two new regulatory functions, both of which duplicate previously known functions of korA. First, korB alone was found to control the kilI component of kilB, thus resolving the paradox of korAindependent control of kiLB. Second, analysis of polypeptides from the korA-korB region in the presence and absence ofkorB, and studies with the korA promoter fused to the chloramphenicol acetyltransferase structural gene (cat) showed that korB, like korA, autoregulates expression of the korA-korB operon. We suggest that korA and korB gene products act as corepressors in the control of certain RK2 genes.Plasmids of incompatibility group P (IncP) replicate in greatly diverse Gram-negative bacterial hosts (1, 2 (26) and concentrations of antibiotics used for selection (15) have been described.Manipulation and Analysis of Nucleic Acids. Methods for preparation and gel electrophoresis of plasmid DNA are detailed elsewhere (27). Enzymes and DNA linkers were purchased. 32P-labeled DNA fragments were prepared by nick-translation with DNA polymerase I (28). Extraction, fractionation, and hybridization analysis of RNA from E. coli were done as described (17,29,30).Analysis of Polypeptides. Plasmid-encoded polypeptides were selectively labeled with a mixture of 14C-labeled amino acids (ICN) in maxicells (25), separated by electrophoresis through 12% NaDodSO4/polyacrylamide gels (with 5% stacking gel), and visualized by autoradiography essentially by a published protocol (11).RESULTS AND DISCUSSION Location of korB on RK2. Interruption of the Sst II site in the 50'-56.4' region of RK2 causes loss of korB function (15). We therefore cloned the 3

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The kil-kor regulon of IncP plasmid RK2 is a complex regulatory network that includes genes for replication and conjugal transfer, as well as for several potentially host-lethal proteins encoded by the kiL4, kiB, and kilC loci. While kilB is known to be involved in conjugal transfer, the functions of kiL4 and kUC are unknown. The coregulation of kiL4 and kUC with replication and transfer genes indicates a possible role in the maintenance or broad host range of RK2. In this work, we found that a fourth kil locus, designated kilE, is located in the kb 2.4 to 4.5 region of RK2 and is regulated as part of the kil-kor regulon. The cloned kilE locus cannot be maintained in Escherichia coli host cells, unless korA or korC is also present in trans to control its expression. The nucleotide sequence of the kilE region revealed two potential multicistronic operons. The kleA operon consists oftwo genes, kHeA and kieB, predicted to encode polypeptide products with molecular masses of 8.7 and 7.6 kDa, respectively. The kkeC operon contains four genes, kHeC, kieD, kleE, and kieF, with predicted products of 9.2, 8.0, 12.2, and 11.3 kDa, respectively. To identify the polypeptide products, each gene was cloned downstream of the phage T7 +10 promoter and expressed in vivo in the presence of T7 RNA polymerase. A polypeptide product of the expected size was observed for all six Hde genes. In addition, kdeF expressed a second polypeptide of 6 kDa that most likely results from the use of a predicted internal translational start site. The kkA and kieC genes are each preceded by sequences resembling strong &o70 promoters. Primer extension analysis revealed that the putative keA and ideC promoters are functional in E. coli and that transcription is initiated at the expected nucleotides. The abundance of transcripts initiated in vivo from both the klA and kieC promoters was reduced in cells containing korA or korC. When korA and korC were present together, they appeared to act synergistically in reducing the level of transcripts from both promoters. The kieA and kdeC promoter regions are highly homologous and contain two palindromic sequences (A and C) that are the predicted targets for KorA and KorC proteins. DNA binding studies showed that protein extracts from korA-containing E. colt cells specifically retarded the electrophoretic mobility of DNA fragments containing palindrome A. Extracts from korC-containing cells altered the mobility of DNA fragments containing palindrome C. These results show that KorA and KorC both act as repressors of the kHeA and kdeC promoters. In the absence of korA and korC, expression of the cloned kleA operon was lethal to E. colt cells, whereas the cloned kieC operon gave rise to slowly growing, unhealthy colonies. Both phenotypes depended on at least one structural gene in each operon, suggesting that the operons encode genes whose products interact with critical host functions required for normal growth and viability. Thus, the kiL4, kilC, and kilE loci of RK2 constitute a cluster of at least 10 genes th...

Broad-host-range plasmid RK2 encodes several kil operons (kiLA, kiLB, ki4C, kilE) whose expression is potentially lethal to Escherichia coli host cells. The kil operons and the RK2 replication initiator gene (trfA) are coregulated by various combinations of kor genes (korA, korB, korC, korE). This regulatory network is called the kU-kor regulon. Presented here are studies on the structure, product, and expression of korC. Genetic mnpping revealed the precise location of korC in a region near transposon Tnl. We determined the nucleotide sequence of'this region and identified the korC structural gene by analysis of korC mutants. Sequence analysis predicts the korC product to be a polypeptide of 85 amino acids with a molecular mass of 9,150 daltons. The KorC polypeptide was identified in vivo by expressing wild-type and mutant korC allel,s from a bacteriophage T7 RNA' polymerase-dependent promoter. The predicted structure of KorC polypeptide has a net positive charge and a helix-turn-helix region similar to those of known DNA-binding proteins. These properties 'are consistent with the repressorlike function of KorC protein, and we discuss the evidence that KorA'and KorC proteins act as corepressors in-the control of the kilC and kilE operons. Finally, we show that koiC is expressed from the bla promoters within the upstream transposon Tnl, suggesting that insertion of Tnl interrupted a plasmid operon that may have originally included korC and kilC.Plasmids of incompatibility group P (IncP) can replicate in many different species of gram-negative bacteria (17,44,80). The genetic and molecular basis for this extensive host range is not yet understood. However, studies on the IncP plasmid RK2 (30) have revealed an unusual system of genetic interactions involved in the control of plasmid replication and maintenance (22, 80).Two genetic determinants are required for RK2 replication: oriV, the origin of unidirectional replication, and trfA, a gene that encodes a polypeptide needed for initiation of replication at oriV (20,33,41,46,47,53,54,59,64,66,74,78,81). The trfA operon is controlled as part of a complex regulatory network. This network, designated the kil-kor regulon, also includes several potentially host-lethal kil operons (kilA, kilB, kilC, kilE) whose functions are unknown (21,48,62; J. Kornacki, C. Chang, and D. Figurski, unpublished data). The kil operons and the trfA operon are negatively regulated by various kor genes (korA, korB, korC, korE) (4,5,21,55,60,72,76,77,(86)(87)(88) Kornacki et al., unpublished data). The regulation of trfA by kor genes directly links the kil-kor regulon to control of plasmid replication. Furthermore, coregulation of the trfA and kil operons hints that the kil determinants may be involved in plasmid maintenance or host range.A distinctive feature of the kil-kor regulon is that the operons are regulated by combinations of kor genes (Fig. 1). korA and korB functions inhibit expression of the trfA operon (55, 60), the kilA operon (4, 21, 87, 88), and the korA-korB operon (5, 72, ...