An upstream activating sequence containing curved DNA involved in activation of the Clostridium perfringens plc promoter

Matsushita, Chieko; Matsushita, Osamu; Katayama, Seiichi; Minami, Junzaburo; Takai, Kenichi; Okabe, Akinobu

doi:10.1099/00221287-142-9-2561

Cited by 26 publications

(17 citation statements)

References 17 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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“…Phased A-tracts inserted upstream of the Ϫ35 hexamer in synthetic hybrid promoters have also been reported to increase transcription (10-14). Because phased A-tracts result in macroscopic DNA curvature [i.e., intrinsic bends whose angle increases with the number of A-tracts (15, 16)], the effects of A-tracts on transcription often have been attributed to direct effects of DNA bending, even though a mechanism for such an effect was not clear (4,5,7,8,(10)(11)(12)(13)(14)(17)(18)(19).Upstream sequences are not as extensively conserved as the Ϫ10, Ϫ35 hexamers in E. coli promoters (1) and were not considered as an RNAP recognition element until recently (9,20). However, an A-tract positioned at about Ϫ40 (21, 22) or AϩT-rich sequences at about Ϫ40 and Ϫ50 (23) were noted in subsets of promoters.…”

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confidence: 99%

Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase α subunit

Aiyar

Gourse²,

Ross³

1998

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

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Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine-and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the ␣ subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP ␣ subunit. The A-tract sequence was protected by wild-type RNAP but not by ␣-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the ؊35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP ␣CTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.Promoters used by the major form of Escherichia coli RNA polymerase (RNAP), E 70 , generally contain two hexamers located about 10 and 35 bp upstream of the start site of transcription, which are recognized by the 70 subunit (1). In addition, sequences upstream of the Ϫ35 hexamer in some promoters in E. coli as well as in other bacterial species increase transcription in the absence of accessory proteins (2-9). These upstream sequences are generally AϩT-rich, and some contain multiple A-tracts in phase with the DNA helical repeat (phased A-tracts). Phased A-tracts inserted upstream of the Ϫ35 hexamer in synthetic hybrid promoters have also been reported to increase transcription (10-14). Because phased A-tracts result in macroscopic DNA curvature [i.e., intrinsic bends whose angle increases with the number of A-tracts (15, 16)], the effects of A-tracts on transcription often have been attributed to direct effects of DNA bending, even though a mechanism for such an effect was not clear (4,5,7,8,(10)(11)(12)(13)(14)(17)(18)(19).Upstream sequences are not as extensively conserved as the Ϫ10, Ϫ35 hexamers in E. coli promoters (1) and were not considered as an RNAP recognition element until recently (9,20). However, an A-tract positioned at about Ϫ40 (21, 22) or AϩT-rich sequences at about Ϫ40 and Ϫ50 (23) were noted in subsets of promoters. Some conservation of alternating A-and T-tracts was also noted in the upstream region of Bacillus subtilis promoters (24). Whereas many promoters co...

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“…difficile and phospholipase C in C . perfringens were reported at supraoptimal temperatures [31–33]. We tested the impact of high temperature on BoNT production for batch culture grown C .…”

Section: Resultsmentioning

confidence: 99%

Heat shock and prolonged heat stress attenuate neurotoxin and sporulation gene expression in group I Clostridium botulinum strain ATCC 3502

et al. 2017

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Foodborne pathogenic bacteria are exposed to a number of environmental stresses during food processing, storage, and preparation, and in the human body. In order to improve the safety of food, the understanding of molecular stress response mechanisms foodborne pathogens employ is essential. Many response mechanisms that are activated during heat shock may cross-protect bacteria against other environmental stresses. To better understand the molecular mechanisms Clostridium botulinum, the causative agent of botulism, utilizes during acute heat stress and during adaptation to stressfully high temperature, the C. botulinum Group I strain ATCC 3502 was grown in continuous culture at 39°C and exposed to heat shock at 45°C, followed by prolonged heat stress at 45°C to allow adaptation of the culture to the high temperature. Growth in continuous culture was performed to exclude secondary growth phase effects or other environmental impacts on bacterial gene transcription. Changes in global gene expression profiles were studied using DNA microarray hybridization. During acute heat stress, Class I and III heat shock genes as well as members of the SOS regulon were activated. The neurotoxin gene botA and genes encoding the neurotoxin-associated proteins were suppressed throughout the study. Prolonged heat stress led to suppression of the sporulation machinery whereas genes related to chemotaxis and motility were activated. Induced expression of a large proportion of prophage genes was detected, suggesting an important role of acquired genes in the stress resistance of C. botulinum. Finally, changes in the expression of a large number of genes related to carbohydrate and amino acid metabolism indicated remodeling of the cellular metabolism.

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An upstream activating sequence containing curved DNA involved in activation of the Clostridium perfringens plc promoter

Cited by 26 publications

References 17 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

Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase α subunit

Heat shock and prolonged heat stress attenuate neurotoxin and sporulation gene expression in group I Clostridium botulinum strain ATCC 3502

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