Identification and isolation of glucose dehydrogenase genes of Bacillus megaterium M1286 and their expression in Escherichia coli

A gene encoding glucose dehydrogenase of Bacillus megaterium M1286 was isolated from a lambda-EMBL3 phage library. It is transcribed and translated in cells of the heterologous organism Escherichia coli by own control regions. The gene is located on a 1126-bp HindIII fragment. Its nucleotide sequence contains 220 bp in the 5' non-coding region, 783 bp in the coding region and 123 bp in the 3' non-coding region. The amino acid sequence, as deduced from the coding region, consists of 261 amino acids and is different from the known protein sequence of glucose dehydrogenase from B. megaterium M1286. [Jany, K. D., Ulmer, W., Fr?schle, M. & Pfleiderer, G. (1984) FEBS Lett. 165, 6-10]. By using this gene as a hybridization probe a second glucose dehydrogenase gene was isolated, which was also directly expressed in E. coli. Additionally a DNA region with extended sequence homology to the hybridization probe was identified. This work indicates the existence of at least two independent glucose dehydrogenase genes in B. megaterium M1286. Homologies in the primary structures of the two different glucose dehydrogenases of B. megaterium M1286 and of the corresponding Bacillus subtilis enzyme are discussed.     

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.     

Isolation of a tetracycline-resistance plasmid excised from a chromosomal DNA sequence in Bacillus subtilis

When Bacillus subtilis GSY908 (recE4-) (H. C. Spatz and T. A. Trautner, 1971, Mol. Gen. Genet. 113, 174-190) protoplasts were infected with Staphylococcus aureus plasmid pNS1 specifying tetracycline resistance (Tcr) (N. Noguchi et al., 1983, Gene 21, 105-112), which was modified such that it either could not replicate or did not carry a functional Tcr gene, a plasmid with a molecular weight of 3.1 X 10(6) (4.9 kb) was generated in Tcr phenotypes. This plasmid, named Tcr pNS1981, exhibited completely different restriction endonuclease cleavage patterns to pNS1 and showed only negligible sequence homology in hybridization experiments. Southern hybridization experiments revealed that pNS1981 arises by excision of a B. subtilis chromosomal DNA sequence. No sequence corresponding to pNS1 was detectable on the chromosome of pNS1981-maintaining B. subtilis. The production of pNS1981 was also observed in B. subtilis RM125 (r-Mm-Mrec+) (T. Uozumi et al., 1977, Mol. Gen. Genet. 152, 65-69.) with almost the same frequency as B. subtilis GSY908. Since the recipient B. subtilis Marburg 168 derivatives stated above are sensitive to Tc, the results indicate that information essential for Tcr is under negative regulatory control in the integrated state on the chromosome. Restriction endonuclease analysis suggested that pNS1981 is essentially the same as pBC16, formerly found in B. cereus (K. Bernhard, H. Schrempf, and W. Goebel, 1978, J. Bacteriol. 133, 897-903).     

Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis.

Penicillin G acylase gene from Bacillus megaterium ATCC 14945 has been isolated. Recombinant Escherichia coli clones were screened for clear halo forming activity on the lawn of Staphylococcus aureus ATCC 6538P using the enzymatic acylating reaction of 7-aminodeacetoxycephalosporanic acid (7-ADCA) and D-(alpha)-phenylglycine methylester. The gene was contained within a 2.8 kb DNA fragment and expressed efficiently when transferred from E. coli to Bacillus subtilis. A twenty times greater amount of enzyme was produced in B. subtilis transformant than that in B. megaterium. The purified enzyme from subcloned B. subtilis showed that the native enzyme consisted of two identical subunits, each with a molecular weight of 57,000. The enzyme was able to react on various cephalosporins, i.e., cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin and cephradine.     

Gene dosage effect on the expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium

Significant differences in expression of the delta-endotoxin genes cryA1 and cryA2 of Bacillus thuringiensis subsp. kurstaki were observed in B. subtilis and B. megaterium. The cryA1 gene was expressed when present on a high-copy-number (hcn) vector in B. megaterium but not in B. subtilis. The cryA2 gene was expressed in both hosts, but at a higher level in B. megaterium. Expression of the cryA2 gene in B. megaterium was better from a hcn vector than from a low copy number vector. Inhibition of sporulation was observed when the toxin genes were present on hcn plasmids in B. subtilis while no such effect was evident in B. megaterium. In addition, there was a significant reduction in copy numbers in both B. subtilis and B. megaterium when delta-endotoxin genes or a spoVG promoter-containing fragment of DNA were cloned into hcn plasmids.     

Cloning and nucleotide sequence of a plasmid-carried gene coding for a minor small, acid-soluble protein from Bacillus megaterium spores.

The gene (termed sspG) coding for a small, acid-soluble protein (SASP) from spores of Bacillus megaterium QMB1551, termed SASP-G, has been cloned, and its nucleotide sequence has been determined. SASP-G is a 42-residue protein containing 2 tryptophan and 11 lysine residues, including a hexalysine sequence, and is not homologous to any previously described SASP. The sspG gene appears to be an additional member of the sigma G regulon. No gene homologous to sspG is present in B. cereus T or B. subtilis 168. The reason for the absence of sspG from other Bacillus species appears to be that in B. megaterium, sspG is present only on a 111-kb plasmid; this plasmid is not present in B. cereus T or B. subtilis 168. The sspG gene is the first forespore-expressed gene found to be on a plasmid.     

枯草芽孢杆菌葡萄糖脱氢酶基因的克隆及其序列分析

根据Lampel报道的葡萄糖脱氢酶基因序列设计合成两条引物,以野生型枯草芽孢杆菌染色体DNA为模板,PCR扩增得到含有葡萄糖脱氢酶基因的大约780bp的DNA片段,将其克隆到pUC-T载体中。序列分析表明,克隆得到的葡萄糖脱氢酶基因含有783bp,编码261个氨基酸的蛋白质。得到的基因序列与文献报道的进行比较,其核苷酸同源率为75．5％,编码氨基酸序列的同源率为83．9％。     

Cloning, nucleotide sequencing, and genetic mapping of the gene for small, acid-soluble spore protein gamma of Bacillus subtilis.

The Bacillus subtilis gene (sspE) which codes for small acid-soluble spore protein gamma (SASP-gamma) was cloned, and its chromosomal location (65 degrees, linked to glpD) and nucleotide sequence were determined. The amino acid sequence of SASP-gamma is similar to that of SASP-B of Bacillus megaterium, but these sequences are not as highly conserved across species as are those of other SASPs. The SASP-gamma gene is transcribed only in sporulation in parallel with other SASP genes and gives a single mRNA that is approximately 340 nucleotides long. The results of hybridization of an sspE gene probe to Southern blots of B. subtilis DNA suggested that there is only a single gene coding for the SASP-gamma type of protein in B. subtilis. This was confirmed by introducing a deletion mutation into the cloned sspE gene and transferring the deletion into the B. subtilis chromosome, with concomitant loss of the wild-type gene. This sspE deletion strain sporulated well, but lacked the SASP-gamma type of protein.     

Complete Sequence of Bacillus subtilis Plasmid p1414 and Comparison with Seven Other Plasmid Types Found in Russian Soil Isolates of Bacillus subtilis

We determined the complete sequence of a cryptic 7949-bp plasmid isolated from naturally occurring Bacillus subtilis found in Russian soil from Moscow. We found 15 putative open reading frames (ORFs), all of which were preceded by a ribosome binding site. One encodes the gene (rep) which should be essential for vegetative rolling circle replication (RCR). The putative double-stranded origin as well as a palT1-like single-stranded origin was also identified. The predicted product of another ORF showed similarity to a moblization protein while a third showed similarity to a ubiquitous family of small proteins whose members have so far been associated with stress response. We used fragments with these latter ORFs to probe representatives of seven other groups of cryptic RCR plasmids from geographically related B. subtilis isolates. All plasmids carried the mob function, suggesting a common ancestor for the rep/mob region but the putative hsp was present only on some of the plasmids. This suggests that the putative hsp gene is not an essential plasmid component and may therefore be present as a phenotypic marker-perhaps providing response to stress. This adds weight to the growing evidence that these small Bacillus plasmids may not be cryptic but may provide an adaptive advantage for the host in its natural environment.     

Proteolytic processing of the protease which initiates degradation of small, acid-soluble proteins during germination of Bacillus subtilis spores.

Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.