Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells.

Figurski, David H.; Pohlman, Robert F.; Bechhofer, David H.; Prince, Alice; Kelton, Christie

doi:10.1073/pnas.79.6.1935

Cited by 116 publications

(150 citation statements)

References 35 publications

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…19; this work), and its own expression (18), and it has a positive effect on expression of korC (20). Less is known about korB, which is required to control kilB2 (15,19), and korC, which regulates kilC (15,20). It is now evident that kil and kor functions are involved in the control of RK2 replication by regulating expression of the essential replication gene trfA (16,(21)(22)(23).…”

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confidence: 99%

“…RK2, a 56.4-kilobase-pair (kbp) self-transmissible IncP plasmid (3,4), has two genetic determinants essential to its replication (5)(6)(7)(8)(9)(10)(11)(12)(13)(14): an origin of replication, oriV, and a gene in the trfA operon that specifies a polypeptide needed to activate oriV. RK2 also encodes several other genes that are both common among and unique to plasmids of the IncP group: kil genes (kilA, kilBi, kilB2, and kilC), which are potentially lethal to Escherichia coli host cells, and kor genes (korA, korB, and korC), which prevent the lethal action (15,16). They form a regulatory network, in which korA is the central control element (16).…”

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confidence: 99%

“…They form a regulatory network, in which korA is the central control element (16). korA negatively controls kilA (15,17,18), kilBI (ref. 19; this work), and its own expression (18), and it has a positive effect on expression of korC (20).…”

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confidence: 99%

“…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).…”

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Gene control in broad host range plasmid RK2: expression, polypeptide product, and multiple regulatory functions of korB.

Bechhofer¹,

Kornacki²,

Firshein³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Introductionmentioning

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Gene control in broad host range plasmid RK2: expression, polypeptide product, and multiple regulatory functions of korB.

Bechhofer¹,

Kornacki²,

Firshein³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression.

Thomas¹,

Hussain²

1984

The EMBO Journal

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For replication, plasmid RK2 encodes a vegetative replication origin, oriV RK2, and a gene, trfA, whose polypeptide product(s) is essential for oriV RK2 activity. The trfA gene is transcribed as part of a polycistronic operon which also includes kilD. Transcription of this operon is negatively regulated by the products of the trfB/korD/korA and korB loci. Mini replicons previously studied in detail lack the korB locus and have copy numbers significantly higher than RK2 itself. Here we report that korB in trans expresses incompatibility towards RK2 replicons either when the korB gene dosage is high or when it is expressed from a strong foreign promoter. This incompatibility can be largely overcome if a trfA gene which is expressed from a foreign promoter, and is therefore not regulated by korB, is supplied in trans. When korB is introduced in cis to mini RK2 replicons the copy number is reduced to within the range estimated for parental RK2. Deletions in the oriV RK2 region which otherwise cause quite large increases in plasmid copy number have only a small effect when korB is present in cis. These results suggest that korB in combination with trfB may be the overriding copy number control element in RK2 reducing trfA expression to levels limiting for replication.

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Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon.

Gerdes¹,

Bech²,

Jørgensen³

et al. 1986

The EMBO Journal

269

201

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The parB region of plasmid R1 encodes two genes, hok and sok, which are required for the plasmid‐stabilizing activity exerted by parB. The hok gene encodes a potent cell‐killing factor, and it is regulated by the sok gene product such that cells losing a parB‐carrying plasmid during cell division are rapidly killed. Coinciding with death of the host cell, a characteristic change in morphology is observed. Here we show that the killing factor encoded by the hok gene is a membrane‐associated polypeptide of 52 amino acids. A gene located in the Escherichia coli relB operon, designated relF, is shown to be homologous to the hok gene. The relF gene codes for a polypeptide of 51 amino acids, which is 40% homologous to the hok gene product. Induced overexpression of the hok and relF gene products results in the same phenomena: loss of cell membrane potential, arrest of respiration, death of the host cell and change in cell morphology. The parB region and the relB genes were cloned into unstably inherited oriC minichromosomes. Whereas the parB region also conferred a high degree of genetic stability to an oriC minichromosome, the relB operon (with relF) did not; therefore the latter does not appear to ‘stabilize’ its replicon (the chromosome). The function of the relF gene is not known.

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Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells.

Cited by 116 publications

References 35 publications

Gene control in broad host range plasmid RK2: expression, polypeptide product, and multiple regulatory functions of korB.

Gene control in broad host range plasmid RK2: expression, polypeptide product, and multiple regulatory functions of korB.

The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression.

Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon.

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