Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

The nucleotide sequence of the rodC operon of Bacillus subtilis

The rodC1 mutation of Bacillus subtilis is a temperature-sensitive marker which affects the levels of teichoic acid synthesis and the cellular morphology. We have determined the nucleotide sequence of the bicistronic operon which contains the rodC gene and the nucleotide sequence of the rodC1 mutant allele. The temperature-sensitive phenotype of the rodC mutant is the result of a single base-pair change. A cytosine to thymine transition in the non-coding strand results in the replacement of a serine residue in the wild-type protein with a phenylalanine residue in the mutant protein. The other gene in the operon, the rodD gene, appears to be equivalent to the gtaA gene which encodes uridine diphosphate-glucose poly-(glycerol phosphate) alpha-glucosyl transferase, an enzyme involved in techoic acid synthesis. This is the first nucleotide sequence analysis of both the wild-type and mutant alleles of a morphogene in B. subtilis.     

Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon

The levanase gene (sacC) of Bacillus subtilis is the distal gene of a fructose-inducible operon containing five genes. The complete nucleotide sequence of this operon was determined. The first four genes levD, levE, levF and levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of Escherichia coli. The levD and levE gene products are homologous to the N and C-terminal part of the enzyme IIIMan, respectively, whereas the levF and levG gene products have similarities with the enzymes IIMan. Surprisingly, the polypeptides encoded by the levD, levE, levF and levG genes are not involved in mannose uptake, but form a fructose phosphotransferase system in B. subtilis. This transport is dependent on the enzyme I of the phosphotransferase system (PTS) and is abolished by deletion of levF or levG and by mutations in either levD or levE. Four regulatory mutations (sacL) leading to constitutive expression of the lavanase operon were mapped using recombination experiments. Three of them were characterized at the molecular level and were located within levD and levE. The levD and levE gene products that form part of a fructose uptake PTS act as negative regulators of the operon. These two gene products may be involved in a PTS-mediated phosphorylation of a regulator, as in the bgl operon of E. coli.     

Phosphoenolpyruvate: sugar phosphotransferase system from the hyperthermophilic Thermoanaerobacter tengcongensis

Thermoanaerobacter tengcongensis is a thermophilic eubacterium that has a phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS) of 22 proteins. The general PTS proteins, enzyme I and HPr, and the transporters for N-acetylglucosamine (EIICB(GlcNAc)) and fructose (EIIBC(Fru)) have thermal unfolding transitions at ～90 °C and a temperature optimum for in vitro sugar phosphotransferase activity of 65 °C. The phosphocysteine of a EIICB(GlcNAc) mutant is unusually stable at room temperature with a t(1/2) of 60 h. The PEP binding C-terminal domain of enzyme I (EIC) forms a metastable covalent adduct with PEP at 65 °C. Crystallization of this adduct afforded the 1.68 ? resolution structure of EIC with a molecule of pyruvate in the active site. We also report the 1.83 ? crystal structure of the EIC-PEP complex. The comparison of the two structures with the apo form and with full-length EI shows differences between the active site side chain conformations of the PEP and pyruvate states but not between the pyruvate and apo states. In the presence of PEP, Arg465 forms a salt bridge with the phosphate moiety while Glu504 forms salt bridges with Arg186 and Arg195 of the N-terminal domain of enzyme I (EIN), which stabilizes a conformation appropriate for the in-line transfer of the phosphoryl moiety from PEP to His191. After transfer, Arg465 swings 4.8 ? away to form an alternative salt bridge with the carboxylate of Glu504. Glu504 loses the grip of Arg186 and Arg195, and the EIN domain can swing away to hand on the phosphoryl group to the phosphoryl carrier protein HPr.     

Nucleotide sequence of the Bacillus subtilis tryptophan operon

In Bacillus subtilis, tryptophan biosynthesis is one of the most thoroughly characterized biosynthetic pathways. Recombinant DNA methodology has permitted a rapid characterization of the tryptophan (trp) gene cluster at the molecular level. In this report the nucleotide sequence of the six structural genes together with the intercistronic regions and flanking regulatory regions are presented.     

Nucleotide sequence of the Bacillus subtilis ribosomal RNA operon, rrnB

The primary sequence of DNA covering a complete ribosomal RNA (rRNA) operon from Bacillus subtilis, designated rrnB has been elucidated. Following a set of tandem promoters, rrnB encodes: (i) a 16S and a 23S rRNA determinant with no tRNA spacer region in between; (ii) a 5S rRNA determinant; and (iii) 21 contiguous tRNA species; before (iv) two characteristic terminator hairpins are found. More than 7 kb are included within this operon, which maps between aroG and thr5 on the B. subtilis chromosome. This represents the first report of the entire sequence of an rRNA operon from B. subtilis or from any Gram-positive organism.     

The nucleotide sequence of proline tRNAmo5UGG from Bacillus subtilis

The nucleotide sequence of a proline tRNA from Bacillus subtilis W168 was determined to be pC-G-G-G-A-A-G-U-A-G-C-U-C-A-G- C-U-U-G-G-D-A-G-A-G-C-A-C-A-U-G-G-psi-U-mo5U-G-G-m1G-A-C-C-A-U-G-G -G-m7G-U-C-G-C-A-G-G-T-psi-C-G-A-A-U-C-C-U-G-U-C-U-U-C-C-C-G-A-C-C- AOH, by the analysis of the unlabeled preparation and by post-labeling technique. This tRNAPro contained 5-methoxyuridine (mo5U) which is specifically distributed in bacillaceae at the wobble position of the anticodon.     

Cloning and nucleotide sequence of the Bacillus subtilis ansR gene, which encodes a repressor of the ans operon coding for L-asparaginase and L-aspartase.

Previous work has shown that expression of the Bacillus subtilis ans operon which codes for L-asparaginase and L-aspartase, is both increased and made insensitive to repression by NH4+ by the aspH1 mutation. In current work, the gene in which the aspH1 mutation resides has been identified and sequenced; this gene, termed ansR, is immediately upstream of, but transcribed in the opposite direction from, the ans operon. The promoter region of ansR contains -10 and -35 sequences similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigma A, and ansR appears to be monocistronic. The ansR gene codes for a 116-residue protein, but the aspH1 mutant allele has an additional guanine residue at codon 55, resulting in generation of a truncated polypeptide of only 58 residues. Insertional inactivation of ansR resulted in a phenotype identical to that of the aspH1 mutant. The predicted amino acid sequence of the ansR gene product (AnsR) was homologous to that of the repressor of B. subtilis prophage PBSX, and a helix-turn-helix motif, characteristic of many DNA-binding proteins, was present in the AnsR amino-terminal region. These results suggest that ansR codes for a repressor of the ans operon.     

The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon.

The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.     

Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis

We have determined the nucleotide sequence of a 3706 bp stretch of Bacillus subtilis chromosomal DNA that complements all known spoVA mutations. The sequence contains five consecutive large open reading frames capable of encoding proteins of molecular weights ranging from approximately 15000 to 36000. Analysis using integrational plasmids suggests that the region is likely to be transcribed as a single mRNA. A novel form of complementation analysis, based on derivatives of bacteriophage phi 105 carrying the cloned spoVA locus, has been used to define four distinct complementation groups among the eight previously characterized spoVA mutations. The spoVA locus is the largest polycistronic sporulation operon yet characterized.