Role of the Upstream Region Containing an Intrinsic DNA Curvature in the Negative Regulation of the Phospholipase C Gene of <i>Clostridium perfringens</i>

Toyonaga, Tatsuo; Matsushita, Osamu; Katayama, Seiichi; Minami, Junzaburo; Okabe, Akinobu

doi:10.1111/j.1348-0421.1992.tb02060.x

Cited by 28 publications

(15 citation statements)

References 29 publications

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“…A 393-bp fragment (from Ϫ252 to ϩ142 with respect to the transcription start site) containing the single plc promoter (3), as well as a regulatory deoxyribosyladenine rich region that is involved in DNA bending (30,57), was used as the target DNA in gel mobility shift experiments. The results showed that when this DNA target was incubated with different amounts of VirR, no shifts in DNA mobility were observed ( Fig.…”

Section: Resultsmentioning

confidence: 99%

The VirR Response Regulator from Clostridium perfringens Binds Independently to Two Imperfect Direct Repeats Located Upstream of the pfoA Promoter

Cheung

Rood

2000

J Bacteriol

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Regulation of toxin production in the gram-positive anaerobe Clostridium perfringens occurs at the level of transcription and involves a two-component signal transduction system. The sensor histidine kinase is encoded by the virS gene, while its cognate response regulator is encoded by the virR gene. We have constructed a VirR expression plasmid in Escherichia coli and purified the resultant His-tagged VirR protein. Gel mobility shift assays demonstrated that VirR binds to the region upstream of the pfoA gene, which encodes perfringolysin O, but not to regions located upstream of the VirR-regulated plc, colA, and pfoR genes, which encode alpha-toxin, collagenase, and a putative pfoA regulator, respectively. The VirR binding site was shown by DNase I footprinting to be a 52-bp core sequence situated immediately upstream of the pfoA promoter. When this region was deleted, VirR was no longer able to bind to the pfoA promoter. The binding site was further localized to two imperfect direct repeats (CCCAGTTNTNCAC) by site-directed mutagenesis. Binding and protection analysis of these mutants indicated that VirR had the ability to bind independently to the two repeated sequences. Based on these observations it is postulated that the VirR positively regulates the synthesis of perfringolysin O by binding directly to a region located immediately upstream of the pfoA promoter and activating transcription.

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Section: Resultsmentioning

confidence: 99%

The VirR Response Regulator from Clostridium perfringens Binds Independently to Two Imperfect Direct Repeats Located Upstream of the pfoA Promoter

Cheung

Rood

2000

J Bacteriol

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“…None of the mutations seen in the different strains affects the enzyme activity significantly. The promoter of the C. perfringens plc gene is one of the strongest promoters (42). It is unique in that it possesses a TG motif at position Ϫ12 and an AT block at Ϫ43, both of which compensate for a Ϫ35 region with poor resemblance to the consensus sequence (5,31).…”

Section: Discussionmentioning

confidence: 99%

Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi

et al. 1995

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The phylogenetic interrelationships between strains of 5 toxin types (A to E) of Clostridium perfringens were examined by analysis of differences in the nucleotide sequences of phospholipase C genes (plc genes) among 10 strains, including 3 strains for which the plc gene sequences have been previously reported. perfringens phospholipase C enzymes were purified from cultures of Escherichia coli cells into which the encoding plc genes had been cloned. All of the enzymes showed the same specific activity. On the other hand, the level of plc transcripts differed greatly (up to 40-fold) from one C. perfringens strain to another. No significant difference in the nucleotide sequence of the plc promoter region was observed for any of the plc genes. These results suggest that the variation in phospholipase C activity among different strains is not due to mutation in the plc coding region but to that in an extragenic region. The evolution of C. perfringens phospholipase C is discussed on the basis of similarities and differences between clostridial plc genes.

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“…Although the promoter sequence was the same as that reported in strain 8-6 by other workers (16), the transcription start point was different from that of strain 8-6. Finally, the (dA-dT)n regions, which are thought to be involved in DNA bending and the regulation of plc gene expression (25), were present in the region adjacent to the plc promoter of strain 13.…”

Section: Vol 178 1996 Transcriptional Analysis Of Virr and Virs Mutmentioning

confidence: 99%

The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens

et al. 1996

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Extracellular toxin production in Clostridium perfringens is positively regulated by the two-component regulatory genes virR and virS. Northern (RNA) blots carried out with RNA preparations from the wild-type strain 13 and the isogenic virR and virS mutants TS133 and JIR4000 showed that the virR and virS genes composed an operon and were transcribed as a single 2.1-kb mRNA molecule. Primer extension analysis led to the identification of two promoters upstream of virR. Hybridization analysis of the mutants and their complemented derivatives showed that the virR/virS system positively regulated the production of alpha-toxin (or phospholipase C), theta-toxin (perfringolysin O), and kappa-toxin (collagenase) at the transcriptional level. However, the modes of regulation of these genes were shown to differ. The theta-toxin structural gene, pfoA, had both a major and a very minor promoter, with the major promoter being virR/virS dependent. The colA gene, which encodes the kappa-toxin, had two major promoters, only one of which was virR/virS-dependent. In contrast, the alpha-toxin structural gene, plc, had only one promoter, which was shown to be partially regulated by the virR and virS genes. Comparative analysis of the virR/virS-dependent promoters did not reveal any common sequence motifs that could represent VirR-binding sites. It was concluded that either the virR/virS system modulates its effects via secondary regulatory genes that are specific for each toxin structural gene or the VirR protein does not have a single consensus binding sequence.

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Role of the Upstream Region Containing an Intrinsic DNA Curvature in the Negative Regulation of the Phospholipase C Gene of Clostridium perfringens

Abstract: The phospholipase C (ƒ¿-

Cited by 28 publications

References 29 publications

The VirR Response Regulator from Clostridium perfringens Binds Independently to Two Imperfect Direct Repeats Located Upstream of the pfoA Promoter

The VirR Response Regulator from Clostridium perfringens Binds Independently to Two Imperfect Direct Repeats Located Upstream of the pfoA Promoter

Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi

The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens

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