Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid

Murotsu, Tomoaki; Matsubara, Kouki; Sugisaki, Hiroyuki; Takanami, Mituru

doi:10.1016/0378-1119(81)90135-9

Cited by 175 publications

(98 citation statements)

References 23 publications

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“…Direct repeats occur in other replication systems: R6K (Stalker et al, 1979), miniF (Tolun and Helinski, 1981;Murotsu et al, 1981) EMBO Workshop, 1982) and lambda (Grosschedl and Hobom, 1979;Moore et al, 1979 . Preliminary experiments (Thomas, unpublished) indicate the existence of transcription in a direction opposite to replication through the groups of five and three direct repeats.…”

Section: Resultsmentioning

confidence: 99%

Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2.

Thomas¹,

Cross²,

Hussain³

et al. 1984

The EMBO Journal

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Broad host-range plasmid RK2 is able to replicate in a controlled manner in most Gram negative bacterial species. To analyze the elements of its control mechanism, we have measured the copy number in Escherichia coli of mini-RK2 replicons isogenic except for defined deletions in regions adjacent to the vegetative replication origin, ortVRK2, which have previously been implicated in copy number control because of their expression of plasmid incompatibility. The results indicate that while the previously defined 700-bp HaeII oriVRK2 fragment carries one copy control element (copA), a second (copB) lies at least partly outside this fragment towards the tetracycline resistance genes of RK2. Deletions affecting both these regions give a mini replicon with a copy number of 35-40 compared with 4-7 for parental RK2. Further incompatibility experiments indicate that targets for both incA (copA) and incB (copB) lie within the 700-bp HaeII oriVRK2 fragment.

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Section: Resultsmentioning

confidence: 99%

Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2.

Thomas¹,

Cross²,

Hussain³

et al. 1984

The EMBO Journal

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“…Sedimentation coefficients of RepE were also determined by ultracentrifugation and shown to be 4.1 S for RepE+ and 2.8 S for RepE54. Since the molecular mass of RepE monomer predicted from the DNA sequence is %29 kDa (14), the wild-type RepE must be a dimer as reported (11)(12)(13) and RepE54 must be a monomer. Evidently, RepE54 cannot form stable dimers under the conditions employed.…”

Section: Methodsmentioning

confidence: 94%

“…RepE exhibits two major functions: initiation of DNA replication from ori2 (initiator function) and autogenous repression of repE transcription (repressor function) (7,8). These functions of RepE require its binding to the four 19-bp direct repeat sequences (iterons) found within ori2 and to the repE promoter/operator, which contains an inverted repeat sequence (9)(10)(11)(12)(13); the half-sequence (10 bp) of the latter is similar (8-bp matches) to the 19-bp repeats (14). RepE has been purified as a dimer in several laboratories (11)(12)(13) and its binding to ori2 iterons and the operator was demonstrated by using DNase I footprinting (9,10) and gel-retardation (10-13) assays.…”

mentioning

confidence: 99%

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

Ishiai

Wada

Kawasaki

et al. 1994

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

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Replication of mini F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherchia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaj-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dlmeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization ofRepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication.The mini-F plasmid, derived from the F (fertility) factor, replicates as a low-copy plasmid (one to two copies per host chromosome) in Escherichia coli (1, 2). The plasmid-encoded RepE initiator protein plays an essential and a specific role in initiating replication from the origin, ori2 (3-6). RepE exhibits two major functions: initiation of DNA replication from ori2 (initiator function) and autogenous repression of repE transcription (repressor function) (7,8). These functions of RepE require its binding to the four 19-bp direct repeat sequences (iterons) found within ori2 and to the repE promoter/operator, which contains an inverted repeat sequence (9-13); the half-sequence (10 bp) of the latter is similar (8-bp matches) to the 19-bp repeats (14). RepE has been purified as a dimer in several laboratories (11-13) and its binding to ori2 iterons and the operator was demonstrated by using DNase I footprinting (9, 10) and gel-retardation (10-13) assays. Thus, RepE dimers have been thought to be involved in binding to both DNA regions, but the structural basis for each of the specific functions of RepE remained undetermined.Besides RepE, several host factors including those involved in chromosomal DNA replication are required for mini-F plasmid replication. In particular, the heat shock oa factor (v.32), which is essential for transcription of repE (8,15), and a subset of heat shock proteins (DnaJ, DnaK, and GrpE) actively...

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“…It contains two origins of replication, a high-copynumber P1 origin under the control of the lac promoter and a low-copy-number mini-F origin. Mini-F replication is controlled by the RepE protein that binds to DNA direct repeats in the origin to control plasmid replication and copy number (41). pBDJ200 carries the lacI q allele, encodes ampicillin resistance, replicates via the high-copy-number ColE1 origin of replication, and carries a truncated repE replication gene and RepE DNA binding sites.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional Organization and Function of Invasion Genes within Salmonella enterica Serovar Typhimurium Pathogenicity Island 1, Including the prgH , prgI , prgJ , prgK , orgA , orgB , and orgC Genes

2000

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Salmonella enterica serovar Typhimurium initiates infection of a host by inducing its own uptake into specialized M cells which reside within the epithelium overlaying Peyer's patches. Entry of Salmonella into intestinal epithelial cells is dependent upon invasion genes that are clustered together in Salmonella pathogenicity island 1 (SPI-1). Upon contact between serovar Typhimurium and epithelial cells targeted for bacterial internalization, bacterial proteins are injected into the host cell through a type III secretion system that leads to internalization of the bacteria. Previous work has established that the prgH, -I, -J, and -K and orgA genes reside in SPI-1, and the products of these genes are predicted to be components of the invasion secretion apparatus. We report that an error in the published orgA DNA sequence has been identified so that this region encodes two small genes rather than a single large open reading frame. These genes have been designated orgA and orgB. Additionally, an opening reading frame downstream of orgB, which we have designated orgC, has been identified and partially characterized. Previously published work has indicated that the prgH, -I, -J, and -K genes are transcribed from a promoter distinct from that used by the gene immediately downstream, orgA. Here, we present experiments indicating that orgA expression is driven by the prgH promoter. In addition, using reverse transcriptase PCR analysis, we have found that this polycistronic message extends downstream of prgH to include a total of 10 genes. To more fully characterize this invasion operon, we demonstrate that the prgH, prgI, prgJ, prgK, orgA, and orgB genes are each required for invasion and secretion, while orgC is not essential for the invasive phenotype.Salmonella infections are an important health problem in both the developing and developed world (9,32,34). Pathogenic Salmonella species cause infections that range in severity from self-limiting gastroenteritis to life-threatening systemic dissemination (45). After entry into a host, the bacteria establish infection by attaching to and invading specialized M cells associated with Peyer's patches in the small intestine (5,23,26). Following M-cell invasion and destruction, host-restricted Salmonella species cause localized destruction of the intestinal epithelium (gastroenteritis). In contrast, passage of hostadapted Salmonella species through M cells allows rapid dissemination to the mesenteric lymph nodes and then to the liver and spleen, where unchecked growth causes death (25).A critical determinant in the development of Salmonella disease is the ability of the bacteria to invade cells. Salmonella enterica serovar Typhimurium mutants that are unable to invade tissue culture cells are defective in their ability to invade and destroy M cells (26,43). This defect severely limits the ability of the bacteria to initiate infection and reduces their virulence in mice (14,24,43). Genes required for Salmonella internalization into mammalian cells have been identified (4, 7, ...

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Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid

Cited by 175 publications

References 23 publications

Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2.

Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2.

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

Transcriptional Organization and Function of Invasion Genes within Salmonella enterica Serovar Typhimurium Pathogenicity Island 1, Including the prgH , prgI , prgJ , prgK , orgA , orgB , and orgC Genes

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