The purine efflux pump PbuE in Bacillus subtilis modulates expression of the PurR and G-box (XptR) regulons by adjusting the purine base pool size.

In Bacillus subtilis, the expression of genes encoding enzymes and other proteins involved in purine de novo synthesis and salvage is affected by purine bases and phosphoribosylpyrophosphate (PRPP). The transcription of the genes belonging to the PurR regulon is negatively regulated by the PurR protein and PRPP. The expression of the genes belonging to the G-box (XptR) regulon, including the pbuE gene, is negatively regulated by a riboswitch-controlled transcription termination mechanism. The G-box regulon effector molecules are hypoxanthine and guanine. pbuE encodes a purine base efflux pump and is now recognized as belonging to a third purine regulon. The expression of the pbuE gene is positively regulated by a riboswitch that recognizes adenine. Here we show that the expression of pbuE'-lacZ transcriptional fusions are induced by adenine to the highest extent in mutants which do not express a functional PbuE pump. In a mutant defective in the metabolism of adenine, the ade apt mutant, we found a high intracellular level of adenine and constitutive high levels of PbuE. A growth test using a purine auxotroph provided further evidence for the role of PbuE in lowering the intracellular concentration of purine bases, including adenine. Purine analogs also affect the expression of pbuE, which might be of importance for the protection against toxic analogs. In a mutant that overexpresses PbuE, the expression of genes belonging to the PurR regulon was increased. Our findings provide further evidence for important functions of the PbuE protein, such as acting as a pump that lowers the purine base pool and affects the expression of the G-box and PurR regulons, including pbuE itself, and as a pump involved in protection against toxic purine base analogs.

Definition of a second Bacillus subtilis pur regulon comprising the pur and xpt-pbuX operons plus pbuG, nupG (yxjA), and pbuE (ydhL).

In Bacillus subtilis expression of genes or operons encoding enzymes and other proteins involved in purine synthesis is affected by purine bases and nucleosides in the growth medium. The genes belonging to the PurR regulon (purR, purA, glyA, guaC, pbuO, pbuG, and the pur, yqhZ-folD, and xpt-pbuX operons) are controlled by the PurR repressor, which inhibits transcription initiation. Other genes are regulated by a less-well-described transcription termination mechanism that responds to the presence of hypoxanthine and guanine. The pur operon and the xpt-pbuX operon, which were studied here, are regulated by both mechanisms. We isolated two mutants resistant to 2-fluoroadenine in which the pur operon and the xpt-pbuX operon are expressed at increased levels in a PurR-independent manner. The mutations were caused by deletions that disrupted a potential transcription terminator structure located immediately upstream of the ydhL gene. The 5' part of the ydhL leader region contained a 63-nucleotide (nt) sequence very similar to the 5' ends of the leaders of the pur and xpt-pbuX operons. Transcripts of these regions may form a common tandem stem-loop secondary structure. Two additional genes with potential leader regions containing the 63-nt sequence are pbuG, encoding a hypoxanthine-guanine transporter, and yxjA, which was shown to encode a purine nucleoside transporter and is renamed nupG. Transcriptional lacZ fusions and mutations in the 63-nt sequence encoding the possible secondary structures provided evidence that expression of the pur and xpt-pbuX operons and expression of the ydhL, nupG, and pbuG genes are regulated by a common mechanism. The new pur regulon is designated the XptR regulon. Except for ydhL, the operons and genes were negatively regulated by hypoxanthine and guanine. ydhL was positively regulated. The derived amino acid sequence encoded by ydhL (now called pbuE) is similar to the amino acid sequences of metabolite efflux pumps. When overexpressed, PbuE lowers the sensitivity to purine analogs. Indirect evidence indicated that PbuE decreases the size of the internal pool of hypoxanthine. This explains why the hypoxanthine- and guanine-regulated genes are expressed at elevated levels in a mutant that overexpresses pbuE.

Roles of PucR, GlnR, and TnrA in regulating expression of the Bacillus subtilis ure P3 promoter.

Expression of the P3 promoter of the Bacillus subtilis ureABC operon is activated during nitrogen-limited growth by PucR, the transcriptional regulator of the purine-degradative genes. Addition of allantoic acid, a purine-degradative intermediate, to nitrogen-limited cells stimulated transcription of ure P3 twofold. Since urea is produced during purine degradation in B. subtilis, regulation of ureABC expression by PucR allows purines to be completely degraded to ammonia. The nitrogen transcription factor TnrA was found to indirectly regulate ure P3 expression by activating pucR expression. The two consensus GlnR/TnrA binding sites located in the ure P3 promoter region were shown to be required for negative regulation by GlnR. Mutational analysis indicates that a cooperative interaction occurs between GlnR dimers bound at these two sites. B. subtilis is the first example where urease expression is both nitrogen regulated and coordinately regulated with the enzymes involved in purine transport and degradation.

Transcription analysis of the Bacillus subtilis PucR regulon and identification of a cis-acting sequence required for PucR-regulated expression of genes involved in purine catabolism.

The PucR protein of Bacillus subtilis has previously been suggested to regulate the expression of 15 genes, pucABCDE, pucFG, pucH, pucI, pucJKLM, pucR, and gde, all of which encode proteins involved in purine catabolism. When cells are grown under nitrogen-limiting conditions, the expression of these genes is induced and intermediary compounds of the purine catabolic pathway affect this expression. By using pucR deletion mutants, we have found that PucR induces the expression of pucFG, pucH, pucI, pucJKLM, and gde while it represses the expression of pucR and pucABCDE. Deletions in the promoters of the five induced operons and genes combined with bioinformatic analysis suggested a conserved upstream activating sequence, 5'-WWWCNTTGGTTAA-3', now named the PucR box. Potential PucR boxes overlapping the -35 and -10 regions of the pucABCDE promoter and located downstream of the pucR transcription start point were also found. The positions of these PucR boxes are consistent with PucR acting as a negative regulator of pucABCDE and pucR expression. Site-directed mutations in the PucR box upstream of pucH and pucI identified positions that are essential for the induction of pucH and pucI expression, respectively. Mutants with decreased pucH or increased pucR expression obtained from a library of clones containing random mutations in the pucH-to-pucR intercistronic region all contained mutations in or near the PucR box. The induction of pucR expression under nitrogen-limiting conditions was found to be mediated by the global nitrogen-regulatory protein TnrA. In other gram-positive bacteria, we have found open reading frames that encode proteins similar to PucR located next to other open reading frames encoding proteins with similarity to purine catabolic enzymes. Hence, the PucR homologues are likely to exert the same function in other gram-positive bacteria as PucR does in B. subtilis.

Transcriptome analysis documents induced competence of Bacillus subtilis during nitrogen limiting conditions.

DNA microarrays were used to analyze the changes in gene expression in Bacillus subtilis strain 168 when nitrogen limiting (glutamate) and nitrogen excess (ammonium plus glutamate) growth conditions were compared. Among more than 100 genes that were significantly induced during nitrogen starvation we detected the comG, comF, comE, nin-nucA and comK transcription units together with recA. DNA was added to B. subtilis grown in minimal medium with glutamate as the sole nitrogen source and it was demonstrated that the cells were competent. Based on these observations we propose a simplification of previously designed one-step transformation procedures for B. subtilis strain 168.

Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source.

Bacillus subtilis can utilize the purine bases adenine, hypoxanthine and xanthine as nitrogen sources. The utilization of guanine as a nitrogen source is reported here. The first step is the deamination of guanine to xanthine catalysed by guanine deaminase (GDEase). To isolate mutants defective in GDEase activity, a collection of mutant strains was screened for strains unable to use guanine as a nitrogen source. The strain BFA1819 (yknA) showed the expected phenotype and no GDEase activity could be detected in this strain. A new name for yknA, namely gde, is proposed. The gde gene encodes a 156 amino acid polypeptide and was preceded by a promoter sequence that is recognized by the sigma(A) form of RNA polymerase. High levels of GDEase were found in cells grown with purines and intermediary compounds of the purine catabolic pathway as nitrogen sources. Allantoic acid, most likely, is a low molecular mass inducer molecule. The level of GDEase was found to be subjected to global nitrogen control exerted by the GlnA/TnrA-dependent signalling pathway. The two regulatory proteins of this pathway, TnrA and GlnR, indirectly and positively affected gde expression. This is the first instance of a gene whose expression is positively regulated by GlnR. The GDEase amino acid sequence shows no homology with the mammalian enzyme. In agreement with this are the different physiological roles for the two enzymes.

Catabolite repression of dra-nupC-pdp operon expression in Bacillus subtilis.

Expression of the Bacillus subtilis dra-nupC-pdp operon is subject to catabolite repression by glucose. It was shown that a cis-acting catabolite-responsive element (CRE) sequence located 64 bp downstream of the transcription-start site mediated catabolite repression of the dra-nupC-pdp operon as it does for many other B. subtilis genes. Point mutations in the CRE sequence caused the loss of catabolite repression of the operon. Catabolite repression of dra-nupC-pdp expression was relieved in a ccpA mutant and was found to be dependent on both HPr and the HPr-like protein Crh. Furthermore, a transcription-repair coupling factor, Mfd, was also found to be involved in the glucose repression of dra-nupC-pdp expression. By the use of in vitro gel mobility shift analysis, a specific HPr-P dependent binding of CcpA to the dra CRE site was demonstrated.

The yexA gene product is required for phosphoribosylformylglycinamidine synthetase activity in Bacillus subtilis.

The yexA gene encodes an 84 amino acid reading frame; in Bacillus subtilis it is positioned between the purC and purQ genes of the purine biosynthetic operon. Disruption of yexA resulted in a purine-auxotrophic phenotype. When yexA was expressed in trans it was able to complement a yexA mutation. Growth experiments and enzyme analysis of yexA mutant strains revealed a defective phosphoribosylformylglycinamidine synthetase (FGAM synthetase). In the organisms in which FGAM synthetase has been studied a single polypeptide is responsible for activity. In some organisms two separate genes - in B. subtilis the purL and purQ genes - encode polypeptides with similarity to the N-terminal and the C-terminal region, respectively, of the single-polypeptide FGAM synthetase. Thus, active FGAM synthetase in B. subtilis requires the yexA gene product in addition to the purL and purQ gene products. Open reading frames with sequence similarity to yexA are found in other Gram-positive organisms, in a cyanobacterium and in methanogenic archaea. The designation purS is proposed for this novel function in purine biosynthesis in B. subtilis.

Functional and genetic characterization of mcpC, which encodes a third methyl-accepting chemotaxis protein in Bacillus subtilis.

A 3135 bp DNA segment downstream of the spl gene on the Bacillus subtilis chromosome was cloned and its nucleotide sequence determined. An open reading frame capable of encoding a putative protein of 654 amino acids with a calculated molecular mass of 72.1 kDa was identified. The deduced amino acid sequence was similar to the McpA and McpB proteins of B. subtilis. McpA and McpB encode different methyl-accepting chemotaxis proteins (MCPs). A mutant strain containing an antibiotic resistance DNA cassette inserted into the region containing the MCP-like reading frame suffered a complete loss of taxis to the amino acids cysteine, proline, threonine, glycine, serine, lysine, valine and arginine. The open reading frame was designated mcpC. The wild-type and an mcpC mutant strain were analysed for their content of methylated proteins and it was found that mcpC encodes a methylated membrane protein that has previously been designated H3. These results show that mcpC encodes a third MCP in B. subtilis. The transcription start site upstream of the mcpC gene was determined by primer extension analysis and it was found to be preceded by a potential promoter sequence that is recognized by the sigma D form of RNA polymerase. The level of beta-galactosidase expressed from a transcriptional mcpC-lacZ fusion was increased threefold when cells entered the stationary phase. No beta-galactosidase could be detected in a sigD genetic background.