A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDB and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.

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The Salmonella typhimurium gene prsA, which encodes phosphoribosylpyrophosphate synthetase, has been cloned, and the nucleotide sequence has been determined. The amino acid sequence derived from the S. typhimurium gene is 99% identical to the derived Escherichia coli sequence and 47% identical to two rat isozyme sequences. Strains containing plasmid-borne prsA have been used to overproduce and purify the enzyme. The promoter for the S. typhimurium prsA gene was identified by deletion analysis and by similarity to the promoter for the E. coil prsA gene. The location of the prsA promoter results in a 416-base-pair 5' untranslated leader in the prsA transcript, which was shown by deletion to be necessary for maximal synthesis of phosphoribosylpyrophosphate synthetase. The S. typhimurium leader contains a 115-base-pair insert relative to the E. coli leader. The insert appears to have no functional significance.The enzyme phosphoribosylpyrophosphate (PRPP) synthetase catalyzes a reaction at a key junction in intermediary metabolism. The enzyme diverts ribose 5-phosphate from energy generation by the pentose phosphate pathway to biosynthesis via the intermediate PRPP. PRPP is utilized in the biosynthesis of pyridine nucleotide coenzymes, the amino acids histidine and tryptophan, and the pyrimidine and purine nucleotides. From 70 to 80% of the carbon flow through PRPP synthetase is directed to nucleotide and nucleic acid synthesis (15).The amount of PRPP synthetase activity in enteric bacteria is mediated at two levels: enzyme inhibition and regulation of synthesis. ADP is a potent inhibitor which binds at an allosteric site as well as competitively with ATP at the active site (5, 22). The ADP inhibition of PRPP synthetase at the allosteric site requires occupation of the active site. Thus, in the presence of ribose 5-phosphate, the activity of PRPP synthetase is mediated by the ratio of ADP to ATP. A pyrimidine, probably UDP or UTP, represses the synthesis of the enzyme 2-to 10-fold (17,26). A Salmonella typhimurium rpoBC mutant which showed derepressed levels of aspartate transcarbamylase (pyrBI) and orotate phosphoribosyltransferase (pyrE), enzymes that have been shown to be regulated by attenuation mechanisms, also had a derepressed level of PRPP synthetase (8). This observation suggests that an attenuation mechanism may also function to regulate the gene encoding PRPP synthetase (prsA).The catalytic mechanism of S. typhimurium PRPP synthetase has been studied extensively, whereas the genes encoding the Escherichia coli and rat enzymes have been cloned and sequenced (6,7,23). To initiate molecular genetic studies and to examine the regulation of S. typhimurium PRPP synthetase expression, the gene encoding the S. typhimurium PRPP synthetase has been cloned and the nucleotide sequence has been determined. MATERIALS AND METHODSMicrobiology and molecular biology. The strains used are listed in Table 1. Strain SB139 was produced from TR5878 * Corresponding author. and SB179 was produced from JL1002 by transduction with ...

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (P bio ) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative P bio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminatorlike sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.Biotin biosynthesis in Escherichia coli is regulated at the level of transcription by a classical repressor-operator mechanism (5,10,12). The repressor is encoded by the birA gene, and when complexed with its corepressor, biotinoyl-5Ј-AMP, BirA binds to an operator that overlaps the promoters for divergent bioA and bioBCDF operons and represses transcription from both promoters (reviewed in reference 10). Binding is cooperative and involves two holorepressor monomers binding to the two palindromic half sites of the operator (1). Transcription of the bio genes is controlled from this bidirectional promoteroperator sequence, and the detection of additional, internal promoters has not been reported. This regulatory model has been confirmed by several methods, including isolation of mutations in the operator region or the birA gene that deregulated biotin production (4,5,9,15,17,21), DNA binding studies of purified BirA to the E. coli bio operon, and DNA protection (5,11,23).In gram-positive bacteria, biotin synthesis has been studied extensively in two species, Bacillus sphaericus (14,16,22) and, more recently, Bacillus subtilis (8,6,7). In B. sphaericus, the genes are located in two separate operons, bioXWF and bioDAYB. Characterization of the regulatory apparatus controlling expression of these genes included the isolation of constitutive biotin-producing mutations that map either to a conserved 15-bp inverted repeat preceding the two operons (i.e., possible operator mutants) or to another site(s) on the chromosome unlinked to either operon (25). Recently, our research group has cloned and sequenced the biotin biosynthetic genes of B. subtilis (7,8). B. subtilis contains at least six bio genes that are organized in a single operon, bioWAFDB Iorf2; a seventh open reading frame (orf2) of unknown function is located at the end of the bio operon. Four of the genes, bioA, -B, -D, and -F, show strong similarity to genes of the same name from B. sphaericus and E. coli, and bioW shows strong similarity to bioW of B. sphaericus. The bioI gene encodes a cytochrome P-450-like enzyme that appears to be involved ...

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