Differentiation of lethal and nonlethal, kor-regulated functions in the kilB region of broad host-range plasmid RK2

Ayres, Elaine K.; Saadi, Soheyla; Schreiner, Helen; Thomson, Valeri J.; Figurski, David H.

doi:10.1016/0147-619x(91)90006-i

Cited by 14 publications

(9 citation statements)

References 44 publications

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“…We tested the possibility that plasmids with the newly defined kilB region failed to produce transformants of the kor°strain because of transcriptional read-through from a strong promoter, similar to the phenomenon we previously observed for the trfA (5) and kiA (28) promoters. A transcriptional terminator was positioned downstream of the kiEB region on pRK2709A36 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…They were found to have possible Shine-Dalgarno sequences (86) for E. coli and Pseudomonas aeruginosa and to exhibit the G+C content and codon structures characteristic of previously identified RK2 genes ( Table 2). These putative genes were named klbA, klbB, and klbC (kib is pronounced klib [from kilB]), in accordance with our previously established nomenclature for genes of the kilB locus (5,28). A fourth ORF was identified in the region between the kilB and trfA promoters (Fig.…”

Section: ____ F T L E Q Y V E a G I M T R E Q Y E V I K S A V A A H Rmentioning

confidence: 99%

“…Promoter-containing regions were cloned into pCH8. A synthetic DNA primer (35-mer, 5' -AAGCTTGCATGCCTGCAGGTCGACTCTAGAG GATC-3'), complementary to a region of the transcribed polylinker downstream of the cloned promoters, was used to determine the transcriptional start site. Another synthetic DNA primer (bla, 5'-TCTGGGTGAGCAAAAACAGGAAG GCAAAATGCCGC-3'), complementary to the bla transcript, was used as an internal control in each of the primer extension reactions.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 1993

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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...

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Section: Resultsmentioning

confidence: 99%

Section: ____ F T L E Q Y V E a G I M T R E Q Y E V I K S A V A A H Rmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 1993

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“…We have adopted a uniform nomenclature for genes of the various kil loci: kla (e.g., klaA, kiaB, and klaC), klb (e.g. klbA [2]), klc, and kle designate genes of kilA, kilB, kilC, and kilE, respectively.…”

Section: Methodsmentioning

confidence: 99%

Structural, molecular, and genetic analysis of the kilA operon of broad-host-range plasmid RK2

et al. 1991

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The kil loci (kilA, kilB, kilC, and kilE) of incompatibility group P (IncP), broad-host-range plasmid RK2 were originally detected by their potential lethality to Escherichia coli host cells. Expression of the kil determinants is controlled by different combinations of kor functions (korA, korB, korC, and korE). This system of regulated genes, known as the kil-kor regulon, includes trfA, which encodes the RK2 replication initiator. The functions of the kil loci are unknown, but their coregulation with an essential replication function suggests that they have a role in the maintenance or host range of RK2. In this study, we have determined the nucleotide sequence of a 3-kb segment of RK2 that encodes the entire kilA locus. The region encodes three genes, designated klaA, klaB, and klaC. The phage T7 RNA polymerase-dependent expression system was used to identify three polypeptide products. The estimated masses of klaA and klaB products were in reasonable agreement with the calculated molecular masses of 28,407 and 42,156 Da, respectively. The klaC product is calculated to be 32,380 Da, but the observed polypeptide exhibited an apparent mass of 28 kDa on sodium dodecyl sulfatepolyacrylamide gels. Mutants of klaC were used to confirm that initiation of translation of the observed product occurs at the first ATG in the klaC open reading frame. Hydrophobicity analysis indicated that the KlaA and KlaB polypeptides are likely to be soluble, whereas the KlaC polypeptide was predicted to have four potential membrane-spanning domains. The only recognizable promoter sequences in the kilA region were those of the kilA promoter located upstream of klaA and the promoter for the korA-korB operon located just downstream of a rho-independent terminatorlike sequence following klaC. The transcriptional start sites for these promoters were determined by primer extension. Using isogenic sets of plasmids with nonpolar mutations, we found that klaA, klaB, and klaC are each able to express a host-lethal (Kil') phenotype in the absence of kor functions. Inactivation of the kilA promoter causes loss of the lethal phenotype, demonstrating that all three genes are expressed from the kilA promoter as a multicistronic operon. We investigated two other phenotypes that have been mapped to the kilA region of RK2 or the closely related IncP plasmids RP1 and RP4: inhibition of conjugal transfer of IncW plasmids (fiwB) and resistance to potassium tellurite. The cloned kilA operon was found to express both phenotypes, even in the presence of korA and korB, whose functions are known to regulate the kilA promoter. In addition, mutant and complementation analyses showed that the kilA promoter and the products of all three kla genes are necessary for expression of both phenotypes. Therefore, host lethality, fertility inhibition, and tellurite resistance are all properties of the kilA operon. We discuss the possible role of the kilA operon for RK2.Bacterial plasmids of incompatibility group P (IncP) are distinguished by their extraordinary host range (1...

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“…We observed the polypeptide products for all six genes and mapped the transcriptional start sites for both operons. Genetic studies showed (i) that each operon is deleterious to E. coli host cells in the absence of kor functions and (ii) that the deleterious effects of each operon are prevented by korA (3,20). This system uses a three-letter genetic designation consisting of "ki" (for kid) followed by a letter indicating the specific kil locus.…”

Section: The Kile Locus Of Incp Plasmid Rk2 5079mentioning

confidence: 99%

kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus

1993

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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...

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Differentiation of lethal and nonlethal, kor-regulated functions in the kilB region of broad host-range plasmid RK2

Cited by 14 publications

References 44 publications

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

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

Structural, molecular, and genetic analysis of the kilA operon of broad-host-range plasmid RK2

kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus

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