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

ABSTRACT: The kil-kor regulon was first identified on the broad-host-range IncP alpha plasmid RK2 by the presence of multiple kil loci (kilA, kilB, kilC, and recently kilE) that are lethal to Escherichia coli host cells in the absence of regulation by kor functions in various combinations. Whereas the kilB operon is required for mating-pair formation during conjugation, the functions encoded by the other kil loci are not known. They are not essential for replication or conjugal transfer, but their coregulation with replication and transfer genes indicates that they are likely to be important for RK2. In this report, we describe molecular and genetic studies on kilC. We determined the nucleotide sequence of the kilC region, which is located between the origin of vegetative replication (oriV) and transposon Tn1 on RK2. Primer extension analysis identified the transcriptional start site and showed that a sequence corresponding to a strong sigma 70 promoter is functional. The abundance of RNA initiated from the kilC promoter is reduced in the presence of korA and korC, as predicted from genetic analysis of kilC regulation. The first gene of the kilC operon (klcA) is sufficient to express the host-lethal phenotype of the kilC determinant in the absence of korA and korC. By comparing RK2 to the related IncP alpha plasmids pUZ8 and R995, we determined that the Tn1 transposon in RK2 interrupts a gene (klcB) immediately downstream of klcA. Thus, the kilC determinant is normally part of an autoregulated operon of three genes: klcA, klcB, and korC. klcA is predicted to encode a 15,856-Da polypeptide that is related to the ArdB antirestriction protein of the IncN plasmid pKM101, suggesting a role for klcA in the broad host ranges of IncP alpha plasmids. The predicted product of the uninterrupted klcB gene is a polypeptide of 51,133 Da that contains a segment with significant similarity to the RK2 regulatory proteins KorA and TrbA. Located 145 bp upstream of the kilC promoter is a 10th copy of the 17-bp oriV iteron sequence in inverted orientation relative to that of the other nine iterons of oriV. Iteron 10 is identical to the "orphan" iteron 1, and both have identical 6-bp flanking sequences that make them likely to be strong binding sites for the TrfA replication initiator protein. The locations and relative orientation of orphan iterons 10 and 1 raise the possibility that these iterons promote the formation of a DNA loop via protein-protein interactions by bound TrfA and lead us to propose that they demarcate the functional origin of replication. This analysis of the kilC region and our previous studies on the other kil loci of RK2 have revealed that the region between oriV and the korABF operon in wild-type IncP alpha plasmids is saturated by the kilC, kilE, and kilA loci arranged in four kor-regulated operons encoding a total of 12 genes.

SUBMITTER: Larsen MH

PROVIDER: S-EPMC196341 | biostudies-other | 1994 Aug

REPOSITORIES: biostudies-other

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

Larsen M H MH Figurski D H DH

Journal of bacteriology 19940801 16

The kil-kor regulon was first identified on the broad-host-range IncP alpha plasmid RK2 by the presence of multiple kil loci (kilA, kilB, kilC, and recently kilE) that are lethal to Escherichia coli host cells in the absence of regulation by kor functions in various combinations. Whereas the kilB operon is required for mating-pair formation during conjugation, the functions encoded by the other kil loci are not known. They are not essential for replication or conjugal transfer, but their coregul ...[more]

PMID: 7519596

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Project description:In Lactococcus lactis, the his operon contains all the genes necessary for histidine biosynthesis. It is transcribed from a unique promoter, localized 300 bp upstream of the first gene. The region corresponding to the untranslated 5' end of the transcript, named the his leader region, displays the typical features of the T box transcriptional attenuation mechanism which is involved in the regulation of many amino acid biosynthetic operons and tRNA synthetase genes in gram-positive bacteria. Here we describe the regulation of transcription of the his operon by the level of histidine in the growth medium. In the absence of histidine, two transcripts are present. One covers the entire operon, while the other stops at a terminator situated about 250 bp downstream of the transcription start point. DNA sequences implicated in regulation of the his operon were identified by transcriptional fusion with luciferase genes and site-directed mutagenesis. In addition to the previously defined sequences necessary for effective T-box-mediated regulation, new essential regions were identified. Eighteen percent of the positions of the his leader region were found to differ in seven distantly related strains of L. lactis. Analysis of the variable positions supports the folding model of the central part of the his leader region. Lastly, in addition to the T-box-mediated regulation, the operon is regulated at the level of initiation of transcription, which is repressed in the presence of histidine. An operator site, necessary for full repression, overlaps the terminator involved in the T box attenuation mechanism. The functionality of the operator is altered on plasmids with low and high copy numbers, suggesting that supercoiling may play a role in the expression of the his operon. The extents of regulation at the levels of initiation and attenuation of transcription are 6- to 8-fold and 14-fold, respectively. Together, the two levels of control allow a 120-fold range of regulation of the L. lactis operon by histidine.

Project description: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 (kilA, kilB, kilC, and kilE), 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, klbB, 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 kilA operon. The amino acid sequence of the 12,085-Da klbC 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 klbA. 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 kilB 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 klbC 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 klbA mutant was constructed and found to be both transfer defective and complementable, thus, demonstrating a requirement was constructed and found to be both transfer defective and complementable, thus demonstrating a requirement for klbA 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.

Dataset Information

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

Publications

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

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