Mechanism of expression of the overlapping genes of Bacillus subtilis aspartokinase II

The mechanism of expression of the overlapping genes that encode the alpha and beta subunits of aspartokinase II of Bacillus subtilis was studied by specific mutagenesis of the cloned coding sequence. Escherichia coli or B. subtilis VB31 (aspartokinase II-deficient), transformed with plasmids carrying either a deletion of the translation start site and about one-half of the coding region for the larger alpha subunit or a frameshift mutation early in the alpha subunit coding region, produced the smaller beta subunit in the absence of alpha subunit synthesis, indicating that beta subunit is not derived from alpha subunit and that its synthesis does not depend on the alpha subunit translation initiation site. The beta subunit translation start site was identified by oligonucleotide-directed mutagenesis of the putative translation start codon. Modification of the nucleotide sequence encoding methionine residue 247 of the alpha subunit from ATG to either TTA or AAT (but not GTG) abolished beta subunit synthesis but had no effect on the production of alpha subunit. This observation is consistent with peptide chain initiation by N-formylmethionine, which specifically requires an ATG or GTG sequence, and indicates that translation of the beta subunit starts at a site corresponding to Met247 of the alpha subunit. Initial studies on the function of the aspartokinase II subunits, using E. coli as a heterologous host, showed that beta subunit was not essential for the expression of the catalytic function of aspartokinase, measured in vitro and in vivo, nor for its allosteric regulation by L-lysine. Whether the beta subunit has a function specific to B. subtilis needs to be explored in a homologous expression system.     

Cloning and structure of the gene for the subunits of aspartokinase II from Bacillus subtilis

A library of Bacillus subtilis DNA in lambda Charon 4A (Ferrari, E., Henner, D.J., and Hoch, J.A. (1981) J. Bacteriol. 146, 430-432) was screened by an immunological procedure for DNA sequences encoding aspartokinase II of B. subtilis, an enzyme composed of two nonidentical subunits arranged in an alpha 2 beta 2 structure (Moir, D., and Paulus, H. (1977a) J. Biol. Chem. 252, 4648-4654). A recombinant bacteriophage was identified that harbored an 18-kilobase B. subtilis DNA fragment containing the coding sequences for both aspartokinase subunits. The coding sequence for aspartokinase II was subcloned into bacterial plasmids. In response to transformation with the recombinant plasmids, Escherichia coli produced two polypeptides immunologically related to B. subtilis aspartokinase II with molecular weights (43,000 and 17,000) indistinguishable from those found in enzyme produced in B. subtilis. Peptide mapping by partial proteolysis confirmed the identity of the polypeptides produced by the transformed E. coli cells with the B. subtilis aspartokinase II subunits. The size of the cloned B. subtilis DNA fragment could be reduced to 2.9 kilobases by cleavage with PstI restriction endonuclease without affecting its ability to direct the synthesis of complete aspartokinase II subunits, irrespective of its orientation in the plasmid vector. Further subdivision by cleavage with BamHI restriction endonuclease resulted in the production of truncated aspartokinase subunits, each shortened by the same extent. This suggested that a single DNA sequence encoded both aspartokinase subunits and provided an explanation for the earlier observation that the smaller beta subunit of aspartokinase II was highly homologous or identical with the carboxyl-terminal portion of the alpha subunit (Moir, D., and Paulus, H. (1977b) J. Biol. Chem. 252, 4655-4661). A map of the gene for B. subtilis aspartokinase II is proposed in which the coding sequence for the smaller beta subunit overlaps in the same reading frame the promoter-distal portion of the coding sequence for the alpha subunit.     

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.     

Expression of the gene for Bacillus subtilis aspartokinase II in Escherichia coli

The gene coding for the subunits of aspartokinase II from Bacillus subtilis has been identified in a B. subtilis DNA library and cloned in a bacterial plasmid (Bondaryk, R. P., and Paulus, H. (1984) J. Biol. Chem. 259, 585-591). The introduction of a plasmid carrying the aspartokinase II gene into an auxotrophic Escherichia coli strain lacking all three aspartokinases restored its ability to grow in the absence of L-lysine, L-threonine, and L-methionine. The B. subtilis aspartokinase gene could thus be functionally expressed in E. coli and substitute for the E. coli aspartokinases. Measurement of aspartokinase levels in extracts of aspartokinaseless E. coli transformed with the B. subtilis aspartokinase II gene revealed an enzyme level comparable to that in a genetically derepressed B. subtilis strain. In spite of the high level of aspartokinase, the growth of the transformed E. coli strain was severely inhibited by the addition of L-lysine but could be restored by also adding L-homoserine. This apparently paradoxical sensitivity to lysine was due to the allosteric inhibition of B. subtilis aspartokinase II by that amino acid, a property which was also observed in extracts of the transformed E. coli strain. The synthesis and degradation of the aspartokinase II subunits were measured by labeling experiments in E. coli transformed with the B. subtilis aspartokinase II gene. In contrast to exponentially growing cells of B. subtilis which contained equimolar amounts of the aspartokinase alpha and beta subunits, the transformed E. coli strain contained a 3-fold molar excess of beta subunit. Pulse-chase experiments showed that the disproportionate level of beta subunit was not due to more rapid turnover of alpha subunit, both subunits being quite stable, but presumably to a more rapid rate of synthesis. After the addition of rifampicin, the synthesis of alpha subunit declined much more rapidly than that of beta subunit, indicating that the two subunits were translated independently from mRNA species that differ in functional stability. In conjunction with the results described in the preceding paper which demonstrated that the aspartokinase subunits are encoded by a single DNA sequence, these observations imply that the alpha and beta subunits of B. subtilis aspartokinase II are the products of in-phase overlapping genes.     

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.     

Chromosomal location of the Bacillus subtilis aspartokinase II gene and nucleotide sequence of the adjacent genes homologous to uvrC and trx of Escherichia coli

The aspartokinase II (ask) operon of Bacillus subtilis consists of two in-phase overlapping genes that encode the two subunits of the lysine-sensitive isoenzyme of aspartokinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4). Transduction mapping of the ask operon, inactivated by recombinational insertion of a cat marker, indicates a chromosomal location (about 253 degrees) between leuA and aroG. ask is thus remote from aecB, eliminating aecB as a possible locus for the structural gene of aspartokinase II, but close to aecA and uvrB. The nucleotide sequence of a 2 kb DNA fragment just upstream of the ask operon was determined and found to contain two open reading frames. The deduced amino acid sequence of the distal reading frame exhibits extensive homology with Escherichia coli thioredoxin and that of the proximal one, which overlaps with the ask promoter, is homologous to the deduced product of the E. coli uvrC gene. Insertional mutagenesis of the proximal open reading frame led to a mitomycin-sensitive phenotype, consistent with a role in DNA repair. In conjunction with the data of M. Petricek, L. Rutberg & L. Hederstedt [FEMS Microbiology Letters 61, 85-88] our results define the nucleotide sequence of an 8.8 kb segment of the B. subtilis chromosome near 253 degrees and the following order of genes: trx-uvrB-ask-orfX-sdhC-sdhA-sdhB-orfY++ +-gerE.     

Nucleotide sequence of the Bacillus subtilis phoR gene.

The nucleotide sequence of phoR, the positive and negative regulatory gene for alkaline phosphatase and phosphodiesterase formation in Bacillus subtilis, was determined. The sequence data predicted an open reading frame of 1,740 base pairs (579 amino acids) which overlaps the 5 base pairs of the preceding phoP coding sequence. The deduced amino acid sequence was significantly homologous with that of the Escherichia coli phoR gene product, which is the sensory element for the pho regulon.     

Nucleotide sequence of the sucrase gene of Bacillus subtilis

The sucrase gene (sacA) and part of the sacP locus, which corresponds to a membrane component of the phosphotransferase system (PTS) of sucrose transport of Bacillus subtilis, were previously cloned on a 2.1-kb EcoRI DNA fragment. Genes sacA and sacP were localized on this DNA fragment and the nucleotide sequence of the 2.1-kb DNA fragment was determined. A 1440-bp open reading frame (480 codons) was identified coding for a deduced polypeptide of Mr54827, which corresponds to that of purified sucrase. The amino acid sequence shares homology with that of yeast invertase (SUC2 gene product). The sacA gene and the preceding sacP gene seem to belong to the same operon.     

Nucleotide sequence of the Bacillus subtilis developmental gene spoVE

We have determined the nucleotide sequence of a 1159 bp DNA fragment containing the spoVE locus of Bacillus subtilis. The locus contained a single open reading frame of 293 codons. On the basis of the predicted amino acid sequence, the product of the spoVE gene is believed to be a protein with an Mr of 31,539. The amino-terminal portion of the spoVE gene was used to construct a translational fusion with the lacZ' gene. The hybrid spoVE-lacZ' gene was shown to be expressed in Escherichia coli and, therefore, it seems reasonable to conclude that the proposed open reading frame for the spoVE gene does indeed function in vivo.     

Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex.

The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of Bacillus subtilis was determined. This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB. The predicted molecular weights for the Fp and Ip subunits, 65,186 (585 amino acids) and 28,285 (252 amino acids), agreed with the values determined independently for the labeled Fp and Ip antigens, although it appeared that the B. subtilis Fp was not functional after expression of the sdhA gene in Escherichia coli. Both subunits closely resembled the corresponding Fp and Ip subunits of the succinate dehydrogenase (SDH) and fumarate reductase of E. coli in size, composition, and amino acid sequence. The sequence homologies further indicated that the B. subtilis SDH subunits are equally related to the SDH and fumarate reductase subunits of E. coli but are less closely related than are the corresponding pairs of E. coli subunits. The regions of highest sequence conservation were identifiable as the catalytically significant flavin adenine dinucleotide-binding sites and cysteine clusters of the iron-sulfur centers.     

Nucleotide sequence of sporulation locus spoIIA in Bacillus subtilis

We have determined a sequence of 2073 bp from two recombinant plasmids carrying the whole spoIIA locus from Bacillus subtilis, the expression of which is required for spore formation. The sequence contains three long open reading frames (ORFs), each of them being preceded by a ribosome binding site. These three putative proteins (mol. wts 13100, 16300 and 22200) are likely to be expressed and are probably encoded on the same mRNA. The stop codon of ORF1 overlaps with the start codon of ORF2 suggesting that there might be translational coupling between the two ORFs. Although some known promoter sequences were found, the only one upstream from the first open reading frame is about 260 bp from it.     

Nucleotide sequence of a cellulase gene of Bacillus subtilis

The nucleotide sequence of an endolytic cellulase gene of Bacillus subtilis was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme. The mature protein appeared to be extended by a signal sequence of 36 amino acids. The putative AUG initiation codon was preceded by a sigma 43-type promoter of B. subtilis and an AAGGAGG sequence, typical of procaryotic ribosomal binding sites. Partial homology of amino acid sequences was found between B. subtilis cellulase and an alkalophilic Bacillus cellulase.     

Nucleotide sequence of threonine tRNA from Bacillus subtilis.

A threonine tRNA was purified from Bacillus subtilis W168 by a combined use of column chromatographic systems. The nucleotide sequence was determined to be pG-C-C-G-G-U-G-U-A-G-C-U-C-A-A-U-D-G-G-D(U)-A-G-A-G-C-A-A-C-U-G-A-C-U-mo5U-G-U-t6A-A-psi-C-A-G-U-A-G-m7G-U-U-G-G-G-G-G-T-psi-C-A-A-G-U-C-C-U-C-U-U-G-C-C-G-G-C-A-C-C-AOH, where about 40 % of D20 remained unmodified as U20. It consists of 76 nucleotides including a new minor nucleoside, 5-methoxyuridine (mo5U), which occupies the wobble position of anticodon.