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ABSTRACT: The sigma-like factor YvrI and coregulator YvrHa activate transcription from a small set of conserved promoters in Bacillus subtilis. We report here that these two proteins independently contribute sigma-region 2 and sigma-region 4 functions to a holoenzyme-promoter DNA complex. YvrI binds RNA polymerase (RNAP) through a region 4 interaction with the beta-subunit flap domain and mediates specific promoter recognition but cannot initiate DNA melting at the -10 promoter element. Conversely, YvrHa possesses sequence similarity to a conserved core-binding motif in sigma-region 2 and binds to the N-terminal coiled-coil element in the RNAP beta'-subunit previously implicated in interaction with region 2 of sigma-factors. YvrHa plays an essential role in stabilizing the open complex and interacts specifically with the N-terminus of YvrI. Based on these results, we propose that YvrHa is situated in the transcription complex proximal to the -10 element of the promoter, whereas YvrI is responsible for -35 region recognition. This system presents an unusual example of a two-subunit bacterial sigma-factor.
Proceedings of the National Academy of Sciences 11/2009; 106(50):21323-8. · 9.68 Impact Factor
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ABSTRACT: YvrI is a recently identified alternative sigma factor in Bacillus subtilis that requires the coactivator YvrHa to activate transcription. Previously, a strain engineered to overproduce YvrI was found to overproduce oxalate decarboxylase (OxdC), and further analysis identified three YvrI-activated promoters preceding the yvrI-yvrHa, yvrJ, and oxdC-yvrL operons. Independently, proteome analyses identified OxdC as a highly abundant, cell wall-associated protein that accumulated under acidic growth conditions. We show here that the accumulation of OxdC in the cell wall proteome under acidic growth conditions is absolutely dependent on YvrI and is correlated with enhanced transcription of both the yvrI-yvrHa and the oxdC-yvrL operons. Conversely, OxdC accumulates to a high level even under nonacidic growth conditions in cells lacking YvrL, a negative regulator of YvrI/YvrHa-dependent transcription. These results indicate that YvrI and its associated coregulators YvrHa and YvrL are required for the regulation of OxdC expression by acid stress. The high-level accumulation of OxdC depends, in part, on a strong oxdC promoter. A regulatory sequence with similarity to an upstream promoter element (UP) was identified upstream of the oxdC promoter and is required for high-level promoter activity. Conservation of the YvrI/YvrHa/YvrL regulatory system among related species allowed us to deduce an expanded consensus sequence for the compositionally unusual promoters recognized by this new sigma factor.
Journal of bacteriology 01/2009; 191(3):931-9. · 3.94 Impact Factor
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ABSTRACT: We describe an in vitro transcription-based method called ROMA (run-off transcription-microarray analysis) for the genome-wide analysis of transcription regulated by sigma factors and other transcriptional regulators. ROMA uses purified RNA polymerase with and without a regulatory protein to monitor products of transcription from a genomic DNA template. Transcribed RNA is converted to cDNA and hybridized to gene arrays allowing for the identification of genes that are specifically activated by the regulator. We discuss the use of ROMA to define sigma factor regulons in Bacillus subtilis and its broad application to defining regulons for other transcriptional regulators in various species.
Methods 11/2008; 47(1):73-7. · 4.01 Impact Factor
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ABSTRACT: We have investigated the function of a cell envelope stress-inducible gene, yvrI, which encodes a 22.5 kDa protein that includes a predicted sigma(70) region 4 domain, but lacks an apparent region 2 domain. YvrI interacts with RNA polymerase and overexpression of YvrI results in induction of OxdC, an oxalate decarboxylase maximally expressed under low-pH conditions. We have used microarray-based analyses to define the YvrI regulon. YvrI is required for the transcription of three operons (oxdC-yvrL, yvrJ and yvrI-yvrHa) each of which is preceded by a highly similar promoter sequence. Activation of these promoters requires both YvrI and the product of the second gene in the yvrI-yvrHa operon, YvrHa. YvrI and YvrHa together allow recognition of the oxdC promoter, stimulate DNA melting and activate transcription by core RNA polymerase. Together, these results suggest that YvrI is a previously unrecognized sigma factor in Bacillus subtilis and that the 9.5 kDa YvrHa protein acts as a required co-activator of transcription. A yvrL deletion results in the upregulation of YvrI activity suggesting that YvrL is a negative regulator of YvrI-dependent transcription, possibly functioning as an anti-sigma factor.
Molecular Microbiology 07/2008; 69(4):954-67. · 5.01 Impact Factor
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ABSTRACT: We have investigated the function of a cell envelope stress-inducible gene, yvrI, which encodes a 22.5 kDa protein that includes a predicted σ70 region 4 domain, but lacks an apparent region 2 domain. YvrI interacts with RNA polymerase and overexpression of YvrI results in induction of OxdC, an oxalate decarboxylase maximally expressed under low-pH conditions. We have used microarray-based analyses to define the YvrI regulon. YvrI is required for the transcription of three operons (oxdC-yvrL, yvrJ and yvrI-yvrHa) each of which is preceded by a highly similar promoter sequence. Activation of these promoters requires both YvrI and the product of the second gene in the yvrI-yvrHa operon, YvrHa. YvrI and YvrHa together allow recognition of the oxdC promoter, stimulate DNA melting and activate transcription by core RNA polymerase. Together, these results suggest that YvrI is a previously unrecognized σ factor in Bacillus subtilis and that the 9.5 kDa YvrHa protein acts as a required co-activator of transcription. A yvrL deletion results in the upregulation of YvrI activity suggesting that YvrL is a negative regulator of YvrI-dependent transcription, possibly functioning as an anti-σ factor.
Molecular Microbiology 06/2008; 69(4):954 - 967. · 5.01 Impact Factor
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ABSTRACT: The ability to recognize and predict non-sigma54 promoters in the alphaproteobacteria is not well developed. In this study, 25 experimentally verified Sinorhizobium meliloti promoter sequences were compiled and used to predict the location of other related promoters in the S. meliloti genome. Fourteen candidate predictions were targeted for verification and of these at least 12 proved to be genuine promoters. As a result, the experimental identification of 12 novel promoters linked to genes rpoD, topA, rpmJ, trpS, ropB1, metC, rpsT, secE, trkH and three tRNA genes is reported. In all, 99 predicted and verified promoters are reported, including those linked with 13 tRNA genes, eight ribosomal protein genes and a number of other physiologically important or essential genes. On the basis of sequence conservation and a mutational analysis of promoter activity, the -35 and -10 consensus for these promoters is 5-CTTGAC-N17-CTATAT. This promoter structure, which seems to be widely conserved amongst several other genera in the alphaproteobacteria, shares significant similarity with, but is skewed by a 1 nt step from, the canonical Escherichia coli sigma70 promoter. Perhaps this difference is responsible for the observation that S. meliloti promoters are often poorly expressed in E. coli. In this regard, expression data from plasmid-borne gfp-reporter fusions to eight of the S. meliloti promoters verified in this work revealed that while these promoters were very active in S. meliloti and Agrobacterium tumefaciens only very low, near-background activity was detected in E. coli.
Microbiology 07/2006; 152(Pt 6):1751-63. · 3.06 Impact Factor
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ABSTRACT: The basic replication unit of many plasmids and second chromosomes in the alpha-proteobacteria consists of a repABC locus that encodes the trans- and cis-acting components required for both semiautonomous replication and replicon maintenance in a cell population. In terms of physical genetic organization and at the nucleotide sequence level, repABC loci are well conserved across various genera. As with all repABC-type replicons that have been genetically characterized, the 1.4 Mb pSymA and 1.7 Mb pSymB megaplasmids from the plant endosymbiont Sinorhizobium meliloti encode strong incompatibility (inc) determinants. We have identified a novel inc sequence upstream of the repA2 gene in pSymA that is not present on pSymB and not reported in other repABC plasmids that have been characterized. This region, in concert with the repA and repB genes, stabilizes a test plasmid indicating that it constitutes a partitioning (par) system for the megaplasmid. Purified RepB binds to this sequence and binding may be enhanced by RepA. We have isolated 19 point mutations that eliminate incompatibility, reduce RepB binding or the stabilization phenotype associated with this sequence and all of these map to a 16-nucleotide palindromic sequence centred 330 bp upstream of the repA2 gene. An additional five near-perfect repeats of this palindrome are located further upstream of the repA2 gene and we show that they share some conservation with known RepB binding sites in different locations on other repABC plasmids and to two sequences found on the tumour inducing plasmid of Agrobacterium tumefaciens. These additional palindromes also bind RepB but one of them does not display obvious incompatibility effects. A heterogenic distribution of par sequences demonstrates unexpected diversity in the structural genetic organization of repABC loci, despite their obvious levels of similarity.
Molecular Microbiology 04/2006; 59(5):1559-75. · 5.01 Impact Factor
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ABSTRACT: The predicted chromosomal origin of replication (oriC) from the alfalfa symbiont Sinorhizobium meliloti is shown to allow autonomous replication of a normally non-replicating plasmid within S. meliloti cells. This is the first chromosomal replication origin to be experimentally localized in the Rhizobiaceae and its location, adjacent to hemE, is the same as for oriC in Caulobacter crescentus, the only experimentally characterized alphaproteobacterial oriC. Using an electrophoretic mobility shift assay and purified S. meliloti DnaA replication initiation protein, binding sites for DnaA were mapped in the S. meliloti oriC region. Mutations in these sites eliminated autonomous replication. S. meliloti that expressed DnaA from a plasmid lac promoter was observed to form pleomorphic filamentous cells, suggesting that cell division was perturbed. Interestingly, this cell phenotype is reminiscent of differentiated bacteroids found inside plant cells in alfalfa root nodules.
Microbiology 03/2006; 152(Pt 2):443-55. · 3.06 Impact Factor
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ABSTRACT: Large extrachromosomal replicons in many members of the alpha-proteobacteria encode genes that are required for plant or animal pathogenesis or symbiosis. Most of these replicons encode repABC genes that control their replication and faithful segregation during cell division. In addition to its chromosome, the plant endosymbiont Sinorhizobium meliloti also maintains the 1.4 Mb pSymA and 1.7 Mb pSymB symbiotic megaplasmids both of which are repABC-type replicons. In all repABC loci that have been characterized, an apparently untranslated intergenic region between the repB and repC genes encodes a strong incompatibility determinant (referred to as incalpha). Here we report the isolation of mutations within the incalpha regions of pSymA and pSymB that eliminate incompatibility. These mutations map to and inactivate a promoter in the intergenic region that drives the expression of an approximately 56 nucleotide untranslated RNA molecule that mediates incompatibility. This gene, that we have named incA, is transcribed antisense to the repABC genes. Our analysis suggests that the incA gene is conserved in repABC loci from a diverse spectrum of bacteria.
Molecular Microbiology 02/2005; 55(2):611-23. · 5.01 Impact Factor
