Gene-enzyme relationships of the purine biosynthetic pathway in Bacillus subtilis

Summary The gene-enzyme relationship has been established for most of the steps of the purine de novo biosynthetic pathway in Bacillus subtilis. The synthesis of inosine monophosphate (IMP) involves ten steps, and the branching from IMP to AMP and to guanosine monophoshate (GMP) synthesis both require two steps. To avoid confusion in the nomenclature of the pur genes we have adopted the Escherichia coli system for B. subtilis. The two genes specifying the enzymes catalysing the conversion of IMP to succinyl-AMP (purA), and the conversion of IMP to xanthosine monophosphate (guaB), occur as single units whilst the other purine genes are clustered at 55 degrees on the B. subtilis linkage map. Based on transformation and transduction studies, and on complementation studies using B. subtilis pur genes cloned in plasmids, the arrangement of some of the clustered genes has been determined relative to outside markers. The following gene order has been established: pbuG-purB-purF-purM-purH-purD-tre. Three other genes were also found to be located in the cluster, guaA, purL and purE/C. However, we were not able to find their exact location. When the purF, purM, purD and purB genes of B. subtilis are present in plasmids they are capable of directing the synthesis in E. coli of phosphoribosylpyrophosphate amidotransferase (purF), aminoimidazole ribonucleotide synthetase (purM), glycinamide ribonucleotide synthetase (purD) and adenylosuccinate lyase (purB), respectively.     

Regulation of Salmonella typhimurium pyr gene expression: effect of changing both purine and pyrimidine nucleotide pools

The synthesis of the pyrimidine biosynthetic enzymes is repressed by the pyrimidine nucleotide end-products of the pathway. However, purine nucleotides also play a role. In this study, I have measured expression of the pyr genes (pyrA-E) in Salmonella typhimurium strains harbouring mutations that permit manipulation of the intracellular pools of both pyrimidine and purine nucleotides. The results identify the effectory purine compound as being a guanine nucleotide; it is probably GTP, but it may be GDP or GMP. The synthesis of carbamoylphosphate synthase, encoded by pyrA, and particularly dihydroorotase, encoded by pyrC, and dihydroorotate dehydrogenase, encoded by pyrD, is stimulated by the guanine nucleotide, while the synthesis of aspartate transcarbamoylase, encoded by pyrBI, and orotate phosphoribosyltransferase, encoded by pyrE, is inhibited by guanine nucleotides. The regulatory pattern of each pyr gene is discussed in relation to present knowledge on gene structure and regulatory mechanism.     

Regulation of sigma B levels and activity in Bacillus subtilis.

The sigB operon of Bacillus subtilis encodes sigma B plus three additional proteins (RsbV, RsbW, and RsbX) that regulate sigma B activity. Using an anti-sigma B monoclonal antibody to monitor the levels of sigma B protein, PSPAC to control the expression of the sigB operon, and a ctc-lacZ reporter system to monitor sigma B activity, we observed that the rsbV and rsbW products control sigma B activity at the ctc promoter independently of their effects on sigma B levels. In contrast, RsbX was found to have no effect on expression of ctc when the sigB operon was controlled by PSPAC. The data are consistent with RsbV and RsbW being regulators of sigma B activity and RsbX acting primarily as a negative regulator of sigB operon expression. Evidence that stationary-phase induction of the sigma B-dependent ctc promoter is accomplished by a reduction in RsbW-dependent inhibition of sigma B activity is also presented. In addition, Western blot (immunoblot) analyses of sigB operon expression demonstrated that sigma B accumulation is coupled to the synthesis of its primary inhibitor (RsbW). This finding is consistent with RsbW and sigma B being present within the cell in equivalent amounts, a circumstance that would permit RsbW to directly influence sigma B activity by a direct protein-protein interaction.     

Regulation of glutamate dehydrogenase in Bacillus subtilis.

The activity of the nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase in Bacillus subtilis was influenced by the carbon source, but not the nitrogen source, in the growth medium. The highest specific activity for this enzyme was found when B. subtilis was grown in a minimal or rich medium that contained glutamate as the carbon source. It is proposed that glutamate dehydrogenase serves a catabolic function in the metabolism of glutamate, is induced by glutamate, and is subject to catabolite repression.     

Cloning and characterization of the Bacillus subtilis hemEHY gene cluster, which encodes protoheme IX biosynthetic enzymes.

Mutations that cause a block in a late step of the protoheme IX biosynthetic pathway, i.e., in a step after uroporphyrinogen III, map at 94 degrees on the Bacillus subtilis chromosomal genetic map. We have cloned and sequenced the hem genes at this location. The sequenced region contains six open reading frames: ponA, hemE, hemH, hemY, ORFA, and ORFB. The ponA gene product shows over 30% sequence identity to penicillin-binding proteins 1A of Escherichia coli, Streptococcus pneumoniae, and Streptococcus oralis and probably has a role in cell wall metabolism. The hemE gene was identified from amino acid sequence comparisons as encoding uroporphyrinogen III decarboxylase. The hemH gene was identified by enzyme activity analysis of the HemH protein expressed in E. coli. It encodes a water-soluble ferrochelatase which catalyzes the final step in protoheme IX synthesis, the insertion of ferrous iron into protoporphyrin IX. The function of the hemY gene product was not elucidated, but mutation analysis shows that it is required for a late step in protoheme IX synthesis. The hemY gene probably encodes an enzyme with coproporphyrinogen III oxidase or protoporphyrinogen IX oxidase activity or both of these activities. Inactivation of the ORFA and ORFB genes did not block protoheme IX synthesis. Preliminary evidence for a hemEHY mRNA was obtained, and a promoter region located in front of hemE was identified. From these combined results we conclude that the hemEHY gene cluster encodes enzymes for the synthesis of protoheme IX from uroporphyrinogen III and probably constitutes an operon.     

Regulation of the purine catabolic enzymes in Neurospora crassa.

Neurospora crassa can utilize purines and their metabolic products as a nitrogen source. Regulation of the five enzymes required for uric acid metabolism was studied. The first three enzymes of this catabolic pathway are controlled in a complex manner that involves both induction and repression. Both uricase and allantoicase were induced by uric acid while allantoinase was induced by either uric acid or allantoin. Synthesis of all three of these enzymes was repressed by the end product, ammonia. The ure-2 mutant, which is urease deficient and cannot derive ammonia from purines, shows a hyperinducibility of these same three enzymes. The last two enzymes of the pathway, ureidoglycollase and urease, were found to be constitutive.