sigmaA-type Promoter Research Articles

Cloning, sequencing and analysis of dnaK ‐dnaJ gene cluster of Bacillus megaterium

The DNA fragment of heat shock genes (hrcA-grpE-dnaK-dnaJ) containing complete hrcA-grpE-dnaK operon and the transcription unit of dnaJ was cloned, sequensed and analyzed from Bacillus megaterium RF5. The sequence of hrcA, grpE and dnaJ were first time reported, and their coding products exibit 60%, 63% and 81% of identities to the homologs of B. subtilis. A sigmaA-type promoter of Gram-positive bacteria (PA1) and a terminator were located upstream of the hrcA and downstream of dnaK, and a Controlling inverted repeat of chaperone expression element (CIRCE) was identified between PA1 and hrcA. Another sigmaA-type promoter (PA2) and a terminator were found upstream and downstream of dnaJ, indicating B. megaterium has a transcription unit containing a single gene dnaJ. The structure of dnaJ transcription unit is more similar to that of Listeria monocytogenes than other species of Bacillus. A partial protein-based phylogenetic tree, derived from Gram-positive bacteria using HrcA sequence, indicated a closer phylogenetic relationship between B. megaterium and Geobacillus species than other two Bacillus species.

Construction of a new reporter system to study the NaCl-dependent dnaK promoter activity of Lactobacillus sanfranciscensis

A reporter system was developed to study gene expression in Lactobacillus sanfranciscensis. It was based on the Escherichia coli/Lactobacillus shuttle vector pLP3537 and the melA gene encoding alpha-galactosidase originating from Lactobacillus plantarum. melA was functionally expressed in E. coli and L. sanfranciscensis, and activity was easily monitored in vivo as well as in vitro by applying an optimized enzyme assay. The reporter system was validated by demonstrating the induction of the dnaK operon of L. sanfranciscensis by NaCl stress. The complete operon, which was composed of hrcA, grpE, dnaK, and dnaJ, was sequenced. A 299-bp sequence upstream of this operon, including a putative sigmaA-type promoter and a single conserved Controlling Inverted Repeat of Chaperone Expression element, was amplified. This amplicon was cloned directly upstream of melA. Both reporter enzyme activity and Northern hybridization analyses of dnaK and melA revealed a transcriptional induction, reaching its maximum when the culture was exposed to 0.75 M NaCl.

Bacillus subtilis CPx-type ATPases: characterization of Cd, Zn, Co and Cu efflux systems.

Metal ion homeostasis requires the balanced expression of metal ion uptake systems, when metals are limiting, and corresponding efflux or storage systems, when metals are in excess. CPx-type ATPases are a family of membrane-bound transporters that often function to export toxic metals from cells. The Bacillus subtilis genome encodes three CPx-type ATPases: zosA, yvgW and yvgX. We demonstrate that yvgW and yvgX encode CadA and CopA, respectively, and that these genes function in metal ion resistance. A cadA mutant was sensitive to Cd(II), Zn(II) and Co(II), but not copper. Transcription of cadA initiates from a single, sigmaA-type promoter and was induced by Cd(II), Zn(II), and Co(II). The adjacent copZA operon is expressed as a bicistronic transcript from a sigmaA-type promoter and is selectively induced by copper. Mutation of either copZ, encoding a metallochaperone, or copA sensitizes the cells to copper but not to other metal ions.

In vivo and in vitro processing of the Bacillus subtilis transcript coding for glutamyl-tRNA synthetase, serine acetyltransferase, and cysteinyl-tRNA synthetase

In Bacillus subtilis, the adjacent genes gltX, cysE, and cysS encoding respectively glutamyl-tRNA synthetase, serine acetyl-transferase, and cysteinyl-tRNA synthetase, are transcribed as an operon but a gltX probe reveals only the presence of a monocistronic gltX mRNA (Gagnon et al., 1994, J Biol Chem 269:7473-7482). The transcript of the gltX-cysE intergenic region contains putative alternative secondary structures forming a p-independent terminator or an antiterminator, and a conserved sequence (T-box) found in the leader of most aminoacyl-tRNA synthetase and many amino acid biosynthesis genes in B. subtilis and in other Gram-positive eubacteria. The transcription of these genes is initiated 45 nt upstream from the first codon of gltX and is under the control of a sigmaA-type promoter. Analysis of the in vivo transcript of this operon revealed a cleavage site immediately downstream from the p-independent terminator structure. In vitro transcription analysis, using RNA polymerases from Escherichia coli, B. subtilis, and that encoded by the T7 phage, in the presence of various RNase inhibitors, shows the same cleavage. This processing generates mRNAs whose 5'-end half-lives differ by a factor of 2 in rich medium, and leaves putative secondary structures at the 3' end of the gltX transcript and at the 5' end of the cysE/S mRNA, which may be involved in the stabilization of these mRNAs. By its mechanism and its position, this cleavage differs from that of the other known transcripts encoding aminoacyl-tRNA synthetases in B. subtilis.

In vivo and in vitro processing of the Bacillus subtilis transcript coding for glutamyl-tRNA synthetase, serine acetyltransferase, and cysteinyl-tRNA synthetase.

In Bacillus subtilis, the adjacent genes gltX, cysE, and cysS encoding respectively glutamyl-tRNA synthetase, serine acetyl-transferase, and cysteinyl-tRNA synthetase, are transcribed as an operon but a gltX probe reveals only the presence of a monocistronic gltX mRNA (Gagnon et al., 1994, J Biol Chem 269:7473-7482). The transcript of the gltX-cysE intergenic region contains putative alternative secondary structures forming a p-independent terminator or an antiterminator, and a conserved sequence (T-box) found in the leader of most aminoacyl-tRNA synthetase and many amino acid biosynthesis genes in B. subtilis and in other Gram-positive eubacteria. The transcription of these genes is initiated 45 nt upstream from the first codon of gltX and is under the control of a sigmaA-type promoter. Analysis of the in vivo transcript of this operon revealed a cleavage site immediately downstream from the p-independent terminator structure. In vitro transcription analysis, using RNA polymerases from Escherichia coli, B. subtilis, and that encoded by the T7 phage, in the presence of various RNase inhibitors, shows the same cleavage. This processing generates mRNAs whose 5'-end half-lives differ by a factor of 2 in rich medium, and leaves putative secondary structures at the 3' end of the gltX transcript and at the 5' end of the cysE/S mRNA, which may be involved in the stabilization of these mRNAs. By its mechanism and its position, this cleavage differs from that of the other known transcripts encoding aminoacyl-tRNA synthetases in B. subtilis.

Transcriptional analysis of the Streptococcus mutans hrcA, grpE and dnaK genes and regulation of expression in response to heat shock and environmental acidification

The dental pathogen Streptococcus mutans persists and causes diseases in highly dynamic environments and gains a selective ecological advantage in environmental conditions that frequently exceed the limits for growth of the organism, particularly with regard to environmental pH. The goal of this study was to begin a molecular genetic analysis of a major stress protein, DnaK/Hsp70, to begin to understand how stress responses are regulated in this lactic acid bacterium and to establish a relationship between dnaK gene expression and exposure to acidic environments. Cloning and nucleotide sequence analysis revealed that the dnaK gene is preceded by, and is in an operon-like arrangement with, the hrcA and grpE genes, although intergenic spacing was unlike that described in other bacteria. An inverted repeat (a CIRCE element) was identified by sequence analysis and, using primer extensions, a heat shock-responsive, sigmaA-type promoter, P1, 5' to the hrcA gene, and a sigmaB-type promoter, 5' to the grpE translational start site, were identified. No promoters were detected between grpE and dnaK. A strain carrying a strongly polar insertion in the hrcA gene had markedly diminished levels of dnaK mRNA, indicating that dnaK was transcribed as part of an operon from P1, and to a lesser extent from P2. Results from physiological manipulation of S. mutans in continuous chemostat culture demonstrated that steadystate levels of S. mutans dnaK mRNA and DnaK protein were (i) increased in response to acid shock; (ii) elevated in acid 'adapted' cells; and (iii) induced in response to alkali shock of acid 'adapted' cells. In all cases, increased amounts of dnaK mRNA could be correlated with enhanced transcription from P1. This study provides the first detailed analysis of the expression of a heat shock gene from an oral isolate, and the evidence provided suggests that sigmaB-like promoters may also be involved in class I heat shock gene expression in some Gram-positive organisms.

Bacillus subtilis ComK negatively regulates degR gene expression.

Exoprotease production in Bacillus subtilis is positively regulated by a two-component regulatory system, DegS-DegU, and, in addition, by several minor factors. The product of the degR gene belongs to the latter group and exerts a positive effect in a DegS-DegU dependent manner. We found that transcription of degR was prevented by disruption of either mecA or mecB but was relieved by further disruption of comK. Disruption of comK alone, however, did not alter degR transcription. We also found that the mecA mutation requires a region around position -52 relative to the transcription start point of degR to exert its effect. We have shown previously that the transcription of degR is driven by an alternative sigma factor, sigmaD. When degR expression was directed by an artificially constructed sigmaA-type promoter, disruption of mecA/B did not result in any significant decrease in degR expression, indicating that the negative effect of the mec mutations acts through sigmaD-driven transcription. It has been demonstrated that MecA and MecB negatively regulate ComK, and mecA/B null mutations cause enhanced expression of comK. From these results we conclude that ComK negatively regulates degR expression by preventing sigmaD-driven transcription of degR, possibly through interaction with the control region.

Transcriptional regulation of the Bacillus subtilis glucitol dehydrogenase gene.

The regulatory region of the Bacillus subtilis glucitol dehydrogenase (gutB) gene was divided into three subregions: a promoter, an upstream positive regulatory region, and a downstream negative regulatory region. Data from primer extension, deletion, and site-directed mutagenesis analyses were consistent with two possible models for the gutB promoter. It is either a sigma A-type promoter with an unusually short spacer region (15 bp) or a special sigma A promoter which requires only the hexameric -10 sequence for its function. Sequence carrying just the promoter region (from -48 to +6) failed to direct transcription in vivo. An upstream regulatory sequence was essential for glucitol induction. When this sequence was inserted in a high-copy-number plasmid, an effect characteristic of titration of a transcriptional activator was seen. Downstream from the promoter, there is an imperfect, AT-rich inverted repeat sequence. Deletion of this element did not lead to constitutive expression of gutB. However, the induced gutB expression level was enhanced three- to fourfold.

The response of a Bacillus subtilis temperature-sensitive sigA mutant to heat stress.

The mutant sigA allele of Bacillus subtilis DB1005 was confirmed to be temperature sensitive (ts) and transferable among strains of B. subtilis by chromosomal transformation and gene conversion. This ts sigA allele had a pleiotropic effect on gene expression of DB1005. The induction of certain heat shock proteins in DB1005 was markedly less significant than that observed in the wild-type strain (DB2) under heat stress. In contrast, some proteins required for coping with oxidative stress and glucose starvation were induced abruptly in DB1005 but not in DB2. Heat induction of the groEL gene in vivo at both transcription and translation levels was much lower in DB1005 than in DB2. Besides, the putative sigma A-type promoter from the groESL operon of B. subtilis was able to be transcribed by the reconstituted sigma A RNA polymerase in vitro at both 37 and 49 degrees C. These results strongly suggest that the expression of the groEL gene of B. subtilis under heat stress is regulated at least in part by sigma A at the level of transcription. Our results also showed that DB1005 did not respond too differently from the wild type to ethanol stress, except after a relatively long exposure.

Nucleotide sequence of the gene for an alkaline endoglucanase from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis.

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M(r) of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M(r) of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.