The Enterococcus faecalis pyr operon is regulated by autogenous transcriptional attenuation at a single site in the 5' leader.

The 5' end of the Enterococcus faecalis pyr operon specifies, in order, the promoter, a 5' untranslated leader, the pyrR gene encoding the regulatory protein for the operon, a 39-nucleotide (nt) intercistronic region, the pyrP gene encoding a uracil permease, a 13-nt intercistronic region, and the pyrB gene encoding aspartate transcarbamylase. The 5' leader RNA is capable of forming stem-loop structures involved in attenuation control of the operon. No attenuation regions, such as those found in the Bacillus subtilis pyr operon, are present in the pyrR-pyrP or pyrP-pyrB intercistronic regions. Several lines of evidence demonstrate that the E. faecalis pyr operon is repressed by uracil via transcriptional attenuation at the single 5' leader termination site and that attenuation is mediated by the PyrR protein.

Sequence analysis of the Bacillus subtilis 168 chromosome region between the sspC and odhA loci (184 degrees-180 degrees).

The nucleotide sequence of 45,389 bp in the 184 degrees-180 degrees region of the Bacillus subtilis chromosome, containing the cge cluster, which is controlled by the sporulation regulatory protein GerE, was determined. Fifty-four putative ORFs with putative ribosome-binding sites were recognized. Seven of them correspond to previously characterized genes: cgeB, cgeA, cgeC, cgeD, cgeE, ctpA, and odhA. The deduced products of 25 ORFs were found to display significant similarities to proteins in the data banks. We have identified genes involved in detoxification, cell walls, and in the metabolism of biotins, purines, fatty acids, carbohydrates and amino acids. The remaining 22 ORFs showed no similarity to known proteins. Both an attachment site of the SPbeta prophage and 2 new putative DNA replication terminators were identified in this region.

An 8 kb nucleotide sequence at the 3' flanking region of the sspC gene (184 degrees) on the Bacillus subtilis 168 chromosome containing an intein and an intron.

As part of the Bacillus subtilis genome sequencing project, we determined the complete nucleotide sequence of an 8000-bp fragment downstream of the sspC gene (184 degrees) of the B. subtilis 168 chromosome. The sequence analysis shows that the sspC gene is located inside of the SP beta region, which differs from the current genetic map of B. subtilis 168. This region contains 12 putative ORFs (yojQ through yojZ and sspC). A homology search for the deduced products of the ORFs shows significant similarities to enzymes involved in deoxyribonucleotide metabolism: ribonucleotide reductase (Nrd) E, NrdF, thioredoxin and dUTPase. Interestingly, this DNA fragment includes two split genes, yojP containing conserved motifs of an intein and yojQ and yojS with an 808-bp intervening sequence for a putative intron structure. In addition, the yojR gene includes a putative new DNA replication terminator.

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Characterization of cis-acting mutations in the first attenuator region of the Bacillus subtilis pyr operon that are defective in pyrimidine-mediated regulation of expression.

A transcriptional attenuation mechanism for the regulation of pyr operon expression in Bacillus subtilis in which the PyrR regulatory protein binds pyr mRNA at three sites with similar sequences to cause transcription termination in response to elevated pyrimidine nucleotide pools has been proposed (R. J. Turner, Y. Lu, and R. L. Switzer, J. Bacteriol. 176:3708-3722, 1994). Twenty-seven mutants with cis-acting defects in the repression by pyrimidines of beta-galactosidase expression of a pyr-lacZ fusion-integrant were isolated as blue colonies on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) agar plates containing uracil and uridine after UV irradiation or treatment with mutagens or following mutD mutagenesis. These mutants showed normal repression of the chromosomal pyr operon by exogenous pyrimidines. Sequence analysis revealed 12 unique sites of mutation, which occurred in the conserved putative PyrR binding sequence (10 of the 12) or in the stem of the transcriptional terminator structure. These mutants strongly support the proposed model for regulation of the pyr operon.

Mutations in Bacillus subtilis PyrR, the pyr regulatory protein, with defects in regulation by pyrimidines.

The pyrimidine nucleotide biosynthetic (pyr) operon in Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which PyrR, a bifunctional pyr RNA-binding attenuation protein/uracil phosphoribosyltransferase, plays a crucial role. A convenient procedure for isolation of pyrR mutants with defects in the regulation of pyr operon expression is described. The selection is based on the selection of spontaneous mutations that convert the pyrimidine-sensitive growth of cpa strain (lacking arginine-repressible carbamyl phosphate synthetase) to pyrimidine resistance. Twelve such mutants were isolated and sequenced. All resulted from point mutations in the pyrR gene.

The pyrimidine biosynthesis operon of the thermophile Bacillus caldolyticus includes genes for uracil phosphoribosyltransferase and uracil permease.

A 3-kb DNA segment of the Bacillus caldolyticus genome including the 5' end end of the pyr cluster has been cloned and sequenced. The sequence revealed the presence of two open reading frames, pyrR and pyrP, located immediately upstream of the previously sequenced pyrB gene encoding the pyrimidine biosynthesis enzyme aspartate transcarbamoylase. The pyrR and pyrP genes encoded polypeptides with calculated molecular masses of 19.9 and 45.2 kDa, respectively. Expression of these ORFs was confirmed by analysis of plasmid-encoded polypeptides in minicells. Sequence alignment and complementation analyses identified the pyrR gene product as a uracil phosphoribosyltransferase and the pyrP gene product as a membrane-bound uracil permease. By using promoter expression vectors, a 650-bp EcoRI-HincII fragment, including the 5' end of pyrR and its upstream region, was found to contain the pyr operon promoter. The transcriptional start point was located by primer extension at a position 153 bp upstream of the pyrR translation initiation codon, 7 bp 3' of a sequence resembling a sigma A-dependent Bacillus subtilis promoter. This established the following organization of the ten cistrons within the pyr operon: promoter-pyrR-pyrP-pyrB-pyrC-pyrAa-pyrA b-orf2-pyrD-pyrF-pyrE. The nucleotide sequences of the region upstream of pyrR and of the pyrR-pyrP and pyrP-pyrB intercistronic regions indicated that the transcript may form two mutually exclusive secondary structures within each of these regions. One of these structures resembled a rho-independent transcriptional terminator. The possible implication of these structures for pyrimidine regulation of the operon is discussed.

Molecular characterization of pyrimidine biosynthesis genes from the thermophile Bacillus caldolyticus.

The genes encoding the six pyrimidine biosynthesis enzymes from the thermophile Bacillus caldolyticus were characterized by cloning and complementation in Escherichia coli, and by nucleotide sequence analysis. Nine cistrons are clustered within an 11 kb region of the chromosome, the gene order being: orf1-pyrB-pyrC-pyrAa-pyrAb-orf2-p yrD-pyrF-pyrE. This organization of the cluster is very similar to that of the pyr operon of Bacillus subtilis. Different parts of the B. caldolyticus cluster were cloned in two orientations in the expression shuttle vector pHPS9. Complementation studies in B. subtilis established that expression of the pyr genes was dependent on the vector-borne promoter, suggesting that they are part of an operon, and that the native promoter of the operon had not been cloned. The deduced amino acid sequence of the individual cistrons showed 49 to 78% identity with the corresponding B. subtilis cistrons. Measurements of the aspartate transcarbamylase (pyrB), orotidine monophosphate decarboxylase (pyrF) and orotate phosphoribosyltransferase (pyrE) levels in cells grown under different conditions indicated that expression of the operon is repressed 7-9-fold by addition of uracil to the growth medium. Based on the nucleotide sequence in the intercistronic region between orf1 and pyrB a regulatory mechanism involving transcriptional termination and antitermination is proposed to control expression of the operon.