Regulation of a common amidotransferase subunit.

In Bacillus subtilis the trpX locus specifies a glutamine-binding protein designated subunit X, which forms a complex with subunit E to constitute the anthranilate synthase enzyme aggregate (EX) and subunit A to constitute the p-aminobenzoate synthase enzyme aggregate (AX). Subunit X confers upon these enzyme complexes the ability to utilize glutamine as a substrate. The trpX locus has been examined to determine its map position and control. (i) The trpX locus was found to be cotransformed with the lysS and pabA loci. The results of three-factor transformation analyses suggest the following order of these markers: lysS-sul-trpX-pabA. (ii) Mutation to constitutivity of the tryptophan operon resulted in a 50- to 60-fold increase in the level of subunit X when the mutant contained functional trE and abA gene products; however, in the absence of subunit E there was only a 4- to 5-fold increase in the glutamine-binding protein. (iii) Formation of subunit X was derepressed under conditions that allow for the derepression of the trpE and/or pabA loci. (iv) Subunit X synthesis was derepressed to a greater extent in mutants that contain a functional trpE gene product than in mutants that contain a nonsense mutation in the trpE locus. These results are consistent with the hypothesis that the trpE and pabA gene products affect the expression and control of the trpX locus.     

Nucleotide sequence of the Bacillus subtilis trpE and trpD genes

Several overlapping portions of the tryptophan (trp) operon of Bacillus subtilis have been cloned into plasmid pBR322. The nucleotide sequence of the region comprising the trpE and trpD genes and a portion of the trpC gene has been determined. When the deduced amino acid sequences of these genes are compared with their counterparts in Escherichia coli, several regions of striking homology are seen. The probable initiation codons for the trpE, D and C genes are each preceded by a recognizable Shine-Dalgarno sequence. The coding sequences for the trpE and trpD genes and for the trpD and trpC genes overlap slightly, leaving no intercistronic regions between the genes.     

Differences in the genetic structure of Bacillus subitilis strains carrying the trpE26 mutation and strain 168.

It was previously shown that in strains of Bacillus subtilis bearing the trpE26 mutation a chromosome segment (from trpD to ilvA) is translocated to a position near the thr region. Further PBS1-mediated transduction data have now revealed that these strains also possess an inversion of part of the chromosome from the origin of replication, down to the tre locus on one side and the cysB locus on the other. These data concern evidence of linkage of tre-12- to markers in the translocation (hisH2, tyrA1, and metB3) as well as linkage of the cysB3 marker to thi-86, gly-133, and catA. They explain the previously observed absence of linkage of markers in the translocated segment to cysB3. The model proposed for the formation of merodiploids in trpE26 strains, which calls for the fusion of two genetic elements, is not incompatible with this new finding.     

Genetic Studies Relating to the Production of Transformed Clones Diploid in the Tryptophan Region of the Bacillus subtilis Genome

Transformation experiments with Bacillus subtilis strains carrying trpE26 (the marker responsible for the detection of merodiploid clones after transformation or transduction) have established the precise position of this marker on the "aromatic region" of the chromosome, at the distal end of the anthranilate synthetase locus. Integration efficiency of the mutant allele (trpE26) seems to be very low. Co-transfer of markers situated on either side of it is almost nil when both donor and recipient carry this mutation. The "exclusion" of trpE26 does not, however, affect recombination frequencies for nearby markers. To explain these facts we considered the hypothesis of a preferential breakage of the deoxyribonucleic acid (DNA) at the trpE26 site or that of an insertion mutation. These studies have also demonstrated the establishment of physical linkage of a marker from the exogenote (hisH2) to a resident marker (tyrA1) in stable and unstable merodiploid clones, thus confirming integration of the donor DNA segment into a genetic structure of the recipient. Furthermore, duplication was shown in merodiploid clones (through reversion and transformation) for a locus of the recipient (tyrA) which was not involved in the initial transformation. This suggests that the diploid condition in this region extends beyond the transformed area. Interpretation of the genetic constitution of these partial diploids calls for postulation of the existence of long duplications, a second (incomplete) chromosome, or an episome-like element.     

The structure of the trpE, trpD and 5' trpC genes of Bacillus pumilus

The nucleotide (nt) sequences of the Bacillus pumilus trpE, trpD and 5' portions of trpC genes have been determined. Genetic analysis suggested the presence of an internal promoter upstream from the trpC gene, yet no typical consensus sequences were found. The nt and amino acid sequence homologies between the B. pumilus, Bacillus subtilis and Escherichia coli trp genes are presented.     

Construction of a promoter-probe vector for a Bacillus subtilis host by using the trpD+ gene of Bacillus amyloliquefaciens.

The trp gene cluster of Bacillus amyloliquefaciens was found to be structurally similar to that of the Enterobacteriaceae. The translation termination codon of the putative trpE gene and the initiation codon for the putative trpD gene overlap at the trpE-trpD junction, and a promoter for the putative trpC gene is suggested to exist. A promoter-probe vector of Bacillus subtilis, pFTB281, was constructed with a DNA fragment of B. amyloliquefaciens, complementing the trpC and trpD mutations of B. subtilis, a 42-base-pair DNA fragment of M13mp7, and the larger EcoRI-PvuII fragment of pUB110, which confers an autonomous replication function and the kanamycin-resistance phenotype to the chimeric plasmid. pFTB281 has BamHI, EcoRI, and SalI cloning sites in the 5'-upstream portion of the protein-coding region of the putative trpD gene, and the insertion of a certain DNA fragment at any of these sites allowed the plasmid to transform a trpD mutant of B. subtilis to the TrpD+ phenotype. DNA fragments showing the promoter function for the trpD gene were obtained from B. amyloliquefaciens and Saccharomyces cerevisiae chromosomes and rho 11 and lambda phage DNAs, but rarely from the DNAs of Escherichia coli and pBR322.     

Genetic Structure and DNA Sequences at Junctions Involved in the Rearrangements of Bacillus Subtilis Strains Carrying the Trpe26 Mutation

Studies on the region upstream to ribosomal operon rrnD of Bacillus subtilis led to the characterization of two of the four chromosomal junctions involved in the rearrangements (a translocation and an inversion) of the strains carrying the trpE26 mutation. Genetic analysis, by integrative mapping, showed linkage of rrnD to cysB and hisA (both on segment A) in the trpE26-type strains. Physical analysis showed that the region upstream to rrnD is now linked to the trpE-ilvA chromosome segment as demonstrated by analyzing restriction site-polymorphism between 168 and trpE26-type strains. Similar experiments confirmed the previous genetic data on linkage in these areas in strains carrying novel rearrangements derived from the trpE26-type strains: stable merodiploids and inversions. The nucleotide sequence of the area 5' to rrnD in both types of strains (168 and trpE26), the region downstream of the citG gene and the region carrying the trpE26 mutation (made available to us by D. Henner) provided evidence for the molecular basis of the differences in structure, allowed the identification of the break points and revealed the presence of a polypurine region upstream to rrnD as seen in other systems in B. subtilis. No extensive homology was found between pairs of junctions so far sequenced. The models proposed by C. Anagnostopoulos for the role of DNA sequences of intrachromosomal homology involved in the transfer of the trpE26 mutation and the formation of novel arrangements require therefore reevaluation.     

Cloning and characterization of a Bacillus subtilis gene encoding a homolog of the 54-kilodalton subunit of mammalian signal recognition particle and Escherichia coli Ffh.

By using a DNA fragment of Escherichia coli ffh as a probe, the Bacillus subtilis ffh gene was cloned. The complete nucleotide sequence of the cloned DNA revealed that it contained three open reading frames (ORFs). Their order in the region, given by the gene product, was suggested to be ORF1-Ffh-S16, according to their similarity to the gene products of E. coli, although ORF1 exhibited no significant identity with any other known proteins. The orf1 and ffh genes are organized into an operon. Genetic mapping of the ffh locus showed that the B. subtilis ffh gene is located near the pyr locus on the chromosome. The gene product of B. subtilis ffh shared 53.9 and 32.6% amino acid identity with E. coli Ffh and the canine 54-kDa subunit of signal recognition particle, respectively. Although there was low amino acid identity with the 54-kDa subunit of mammalian signal recognition particle, three GTP-binding motifs in the NH2-terminal half and amphipathic helical cores in the COOH-terminus were conserved. The depletion of ffh in B. subtilis led to growth arrest and drastic morphological changes. Furthermore, the translocation of beta-lactamase and alpha-amylase under the depleted condition was also defective.     

Evidence for the Translocation of a Chromosome Sement in Bacillus subtilis Strains Carrying the trpE26 Mutation.

The replication order of markers was studied in Bacillus subtilis strains bearing the trpE26 mutation by the use of the density transfer technique. An important difference in this order was observed in comparison with that of strain 168 T-. All markers tested of a chromosome segment extending from trpD to ilvA replicated early, after purB6 and before thr-5. Two markers flanking this region, trpE8 and citK7, replicated late as usual. The results suggested that this segment was shifted in trpE26 strains to a region closer to the origin of replication. PBS-1-mediated transduction crosses corroborated this hypothesis and revealed the position of the translocated segment. (i) Linkage was demonstrated for markers in the segment (hisH2, tryA1, met B3, ilvA2) to thr-5 and ald; (ii) aroB2 and citK7 were found to be linked; and (iii) linkage of cysB3 to thr-5 was lost in trpE26 strains. These findings made it possible to account for the characteristics of the trpE26 mutation and to propose a model explaining the fact that all trpE26+ transformants or transductants are merodiploid. The model calls for fusion of two genetic elements: two independent chromosomes, or two arms of a replicating structure. The resulting chromosome bears a long tandem duplication. Most of the features of this system of merodiploid formation can be interpreted by use of this model: the segregation pattern of the diploids, the stabilization of the unstable clones, and the length of the duplicated region. A relatively stable diploid strain was also studied by the density transfer technique. The data show that it remained diploid for the region corresponding to the translocated segment and are in agreement with the structure predicted by the model.     

Identification of a sporulation locus in cloned Bacillus subtilis deoxyribonucleic acid.

A cloned deoxyribonucleic acid from the purA-cysA region of the Bacillus subtilis chromosome was shown to contain the spoVC locus, a gene whose product is required for sporulation. This is the first demonstration of a spo locus in cloned B. subtilis deoxyribonucleic acid.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: I. Sequence of trpG encoding the glutamine amidotransferase subunit

We have determined the DNA sequence of the distal 148 codons of trpE and all of trpG in Pseudomonas aeruginosa. These genes encode, respectively, the large and small (glutamine amidotransferase) subunits of anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. The sequenced region of trpE is homologous with the distal portion of E. coli and Bacillus subtilis trpE, whereas the trpG sequence is homologous to the glutamine amidotransferase subunit genes of a number of bacterial and fungal anthranilate synthases. The two coding sequences overlap by 23 bp. Codon usage in these Pseudomonas genes shows a marked preference for codons ending in G or C, thereby resembling that of trpB, trpA, and several other chromosomal loci from this species and others with a high G + C content in their DNA. The deduced amino acid sequence for the P. aeruginosa trpG gene product differs to a surprising extent from the directly determined amino acid sequence of the glutamine amidotransferase subunit of P. putida anthranilate synthase (Kawamura et al. 1978). This suggests that these two proteins are encoded by loci that duplicated much earlier in the phylogeny of these organisms but have recently assumed the same function. We have also determined 490 bp of DNA sequence distal to trpG but have not ascertained the function of this segment, though it is rich in dyad symmetries.      

Bacillus subtilis rRNA promoters are growth rate regulated in Escherichia coli.

rRNA promoters from the rrnB locus of Bacillus subtilis and from the rrnB locus of Escherichia coli were fused to the gene for chloramphenicol acetyltransferase (CAT). The level of expression of CAT in E. coli showed growth rate dependence when the CAT gene was linked to either E. coli or B. subtilis tandem promoters. The downstream promoter of the tandem Bacillus pair showed growth rate regulation, while the upstream promoter did not, whereas for the E. coli tandem promoters, only the upstream promoter was growth rate regulated.     

Isolation of a Bacillus subtilis transformant producing thermostable alpha-amylase by DNA from a thermophilic bacterium

A Bacillus subtilis transformant producing thermostable α-amylase was isolated using DNA from a thermophilic bacterium, Thermophile V2. The extracellular α-amylase did not crossreact with a rabbit antiserum against B. subtilis α-amylase. The structural gene for the thermostable α-amylase was integrated at a different locus from B. subtilis α-amylase. It was linked to pyrA. The transformant was not thermophilic, and its upper temperature for growth was similar to that of the host bacterium.     

Nucleotide sequence and analysis of a gene encoding anthranilate synthase component I in Spirochaeta aurantia.

A Spirochaeta aurantia DNA fragment containing the trpE gene and flanking chromosomal DNA was cloned, and the sequence of the trpE structural gene plus 870 bp upstream and 1,257 bp downstream of trpE was determined. The S. aurantia trpE gene codes for a polypeptide of 482 amino acid residues with a predicted molecular weight of 53,629 that showed sequence similarity to TrpE proteins from other organisms. The S. aurantia TrpE polypeptide is not more closely related to the other published spirochete TrpE sequence (that of Leptospira biflexa) than to TrpE polypeptides of other bacteria. Two additional complete open reading frames and one partial open reading frame were identified in the sequenced DNA. One of the complete open reading frames and the partial open reading frame are upstream of trpE and are encoded on the DNA strand opposite that containing trpE. The other open reading frame is downstream of trpE and on the same DNA strand as trpE. On the basis of the results of a protein sequence data base search, it appears that trpE is the only tryptophan biosynthesis gene in the sequenced DNA. This is in contrast to L. biflexa, in which trpE is separated from trpG by only 64 bp.