Genetic Transplantation:
            <i>Salmonella enterica</i>
            Serovar Typhimurium as a Host To Study Sigma Factor and Anti-Sigma Factor Interactions in GeneticallyIntractable Systems

Karlinsey, Joyce E.; Hughes, Kelly T.

doi:10.1128/jb.188.1.103-114.2006

Cited by 27 publications

(26 citation statements)

References 46 publications

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“…In addition, the regions involved in promoter recognition, such as region 2.4 for Ϫ10 recognition (50% identity plus 25% similarity) and region 4.2 for Ϫ35 recognition (62% identity plus 17% similarity) show a higher level of conservation. There is also in vivo functional evidence of 28 conservation as Chlamydia 28 protein can complement a Salmonella enterica serovar Typhimurium 28 mutant in motility studies (13). In the Ϫ35 promoter element, our findings confirm the importance of the core Ϫ35 consensus sequence (TAAA) and provide experimental support for the extended Ϫ35 promoter that has been predicted from alignment of strongly transcribed 28 promoters from E. coli and Salmonella (12).…”

Section: Discussionsupporting

confidence: 75%

Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ ²⁸ RNA Polymerase

Yu¹,

Russo²,

Rounds³

et al. 2006

J Bacteriol

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28 RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial 28 promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the 28 -dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a ؊35 element recognized by chlamydial 28 RNA polymerase that resembles the consensus ؊35 sequence. Within the ؊10 element, however, chlamydial 28 RNA polymerase showed a striking preference for a CGA sequence at positions ؊12 to ؊10 rather than the longer consensus ؊10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli 28 RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the ؊10 promoter element recognized by 28 RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that 28 RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11-or 12-nt spacer equally well. Altogether, we found very similar results for 28 RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred 28 promoter that we defined in the context of the hctB promoter is TAAAGwwy-n 11/12 -ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n 11/12 is a spacer of 11 or 12 nt.

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Section: Discussionsupporting

confidence: 75%

Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ ²⁸ RNA Polymerase

Yu¹,

Russo²,

Rounds³

et al. 2006

J Bacteriol

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“…For FliC, overexpression from a multicopy vector would presumably allow secretion to be directed by amino acid and chaperone binding signals only. The simple technology of the bacteriophage recombination system to efficiently direct mutagenesis to a specific region of the chromosome such as the fliC 5ЈUTR is a powerful tool not only for the selection and screening of specific mutants from the natural chromosomal location but also for mutational changes resulting in no phenotypic differences from the wild type (16). This allows for a thorough analysis of any specific segment of a gene to be analyzed.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional and Translational Control of the Salmonella fliC Gene

et al. 2006

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The flagellin gene fliC encodes the major component of the flagellum in Salmonella enterica serovar Typhimurium. This study reports the identification of a signal within the 5 untranslated region (5UTR) of the fliC transcript required for the efficient expression and assembly of FliC into the growing flagellar structure. Primer extension mapping determined the transcription start site of the fliC flagellin gene to be 62 bases upstream of the AUG start codon. Using tetA-fliC operon fusions, we show that the entire 62-base 5UTR region of fliC was required for sufficient fliC mRNA translation to allow normal FliC flagellin assembly, suggesting that translation might be coupled to assembly. To identify sequence that might couple fliC mRNA translation to FliC secretion, the 5 end of the chromosomal fliC gene was mutagenized by PCR-directed mutagenesis. Single base sequences important for fliC-dependent transcription, translation, and motility were identified by using fliC-lacZ transcriptional and translational reporter constructs. Transcription-specific mutants identified the ؊10 and ؊35 regions of the consensus flagellar class 3 gene promoter. Single base changes defective in translation were located in three regions: the AUG start codon, the presumed ribosomal binding site region, and a region near the very 5 end of the fliC mRNA that corresponded to a potential stem-loop structure in the 5UTR. Motility-specific mutants resulted from base substitutions only in the fliC-coding region. The results suggest that fliC mRNA translation is not coupled to FliC secretion by the flagellar type III secretion system.

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“…Construction of an opsM mutant. A mutant containing a scrambled operon polarity suppressor site (designated opsM) was constructed by first replacing the ops region upstream of siiA with a tetRA element by allelic exchange using -red-mediated recombination (43,44). KMp124 and KMp125, the primers used for amplication of the tetRA cassette, have 40 bp of flanking homology to the untranslated region surrounding the ops region.…”

Section: Methodsmentioning

confidence: 99%

Coordinate Regulation of Salmonella Pathogenicity Island 1 (SPI1) and SPI4 in Salmonella enterica Serovar Typhimurium

et al. 2008

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Salmonella enterica serovar Typhimurium harbors five pathogenicity islands (SPI) required for infection in vertebrate hosts. Although the role of SPI1 in promoting epithelial invasion and proinflammatory cell death has been amply documented, SPI4 has only more recently been implicated in Salmonella virulence. SPI4 is a 24-kb pathogenicity island containing six open reading frames, siiA to siiF. Secretion of the 595-kDa SiiE protein requires a type I secretory system encoded by siiC, siiD, and siiF. An operon polarity suppressor (ops) sequence within the 5 untranslated region upstream of siiA is required for optimal SPI4 expression and predicted to bind the antiterminator RfaH. SiiE concentrations are decreased in a SPI1 mutant strain, suggesting that SPI1 and SPI4 may have common regulatory inputs. SPI1 gene expression is positively regulated by the transcriptional activators HilA, HilC, and HilD, encoded within SPI1, and negatively regulated by the regulators HilE and PhoP. Here, we show that mutations in hilA, hilC, or hilD similarly reduce expression of siiE, and mutations in hilE or phoP enhance siiE expression. Individual overexpression of HilA, HilC, or HilD in the absence of SPI1 cannot activate siiE expression, suggesting that these transcriptional regulators act in concert or in combination with additional SPI1-encoded regulatory loci to activate SPI4. HilA is no longer required for siiE expression in an hns mutant strain, suggesting that HilA promotes SPI4 expression by antagonizing the global transcriptional silencer H-NS. Coordinate regulation suggests that SPI1 and SPI4 play complementary roles in the interaction of S. enterica serovar Typhimurium with the host intestinal mucosa.

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Genetic Transplantation: Salmonella enterica Serovar Typhimurium as a Host To Study Sigma Factor and Anti-Sigma Factor Interactions in GeneticallyIntractable Systems

Cited by 27 publications

References 46 publications

Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ ²⁸ RNA Polymerase

Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ ²⁸ RNA Polymerase

Transcriptional and Translational Control of the Salmonella fliC Gene

Coordinate Regulation of Salmonella Pathogenicity Island 1 (SPI1) and SPI4 in Salmonella enterica Serovar Typhimurium

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Genetic Transplantation: Salmonella enterica Serovar Typhimurium as a Host To Study Sigma Factor and Anti-Sigma Factor Interactions in GeneticallyIntractable Systems

Cited by 27 publications

References 46 publications

Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ 28 RNA Polymerase

Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ 28 RNA Polymerase

Transcriptional and Translational Control of the Salmonella fliC Gene

Coordinate Regulation of Salmonella Pathogenicity Island 1 (SPI1) and SPI4 in Salmonella enterica Serovar Typhimurium

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Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ ²⁸ RNA Polymerase

Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ ²⁸ RNA Polymerase