Molecular cloning and sequencing of the upstream region of the major Bacillus subtilis autolysin gene: a modifier protein exhibiting sequence homology to the major autolysin and the spoIID product.

The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking. Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M(r) of 76,725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein. Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M(r) of the mature form is 75,000. The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively. CwbA stimulated the major autolysin activity approximately threefold in vitro. These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D. R. & Glaser, L. (1975; Journal of Biological Chemistry 250, 1676-1682). In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase.     

Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene

We have cloned DNA fragments from Bacillus subtilis 168S into Escherichia coli, which produced a lytic zone on an agar medium containing B. subtilis cell wall. Sequencing of the fragments showed the presence of an open reading frame (ORF) which encodes a polypeptide of 272 amino acids with a molecular mass of 29919 Da. The deduced amino acid sequence showed considerable homology with that of the cell wall hydrolase gene of Bacillus sp. (Potvin, C., Leclerc, D., Tremblay, G., Asselin, A. & Bellemare, G. (1988). Molecular and General Genetics 214, 241-248). Accordingly, the gene was designated cwlA, for cell wall lysis. The N-terminal amino acid sequence of cwlA gene product prepared from a E. coli clone was AIKVVKNLVSKSKYGLKCPN, which is consistent with that of the deduced sequence starting from Ala at second position from the initiation codon of the cwlA gene. A presumed sigma A promoter and a rho-independent terminator were found upstream and downstream of the ORF, respectively. A chloramphenicol-resistance determinant integrated into the ORF was mapped by PBS1 transduction, which indicated the gene sequence dnaE-aroD-cwlA.     

Molecular cloning and nucleotide sequence of a cDNA clone coding for the cell attachment domain in human fibronectin

A cDNA clone coding for the cell attachment domain in human fibronectin has been isolated using synthetic oligonucleotides. Three sets of mixed tetradecamer oligonucleotides were synthesized based on amino acid sequences in the 108-amino acid cell attachment domain (Pierschbacher, M. D., Ruoslahti, E., Sundelin, J., Lind, P., and Peterson, P. A. (1982) J. Biol. Chem. 257, 9593-9597). One of these sets was made complementary to amino acids located near the COOH terminus of the cell attachment domain and synthesized as a mixture of 24 sequences. This oligonucleotide mixture was used to prime cDNA synthesis with mRNA prepared from a human fibrosarcoma as a template. A cDNA library was constructed with the oligonucleotide-primed sequences in the vector pBR322. Colonies that hybridized with the primer were isolated from the library and further identified by hybridization with oligonucleotides deduced from an amino acid sequence located 45 amino acid residues NH2-terminal of the primer sequence. One clone which hybridized to both probes was characterized in detail. The insert was 380 base pairs long and its nucleotide sequence agreed completely with the corresponding amino acid sequence of human plasma fibronectin, showing that the sequences for this region are identical in plasma fibronectin and fibronectin from a cell line. This clone should be useful for studies on the expression of fibronectins and may also allow for the production of the biologically active cell attachment domain of fibronectin in bacteria.     

Phenotypes of Bacillus subtilis mutants altered in the precursor-specific region of sigma E.

sigma E is a sporulation-specific sigma factor of Bacillus subtilis that is synthesized from an inactive precursor protein (P31). The structural gene (sigE) for P31 was reengineered by oligonucleotide-directed mutagenesis to encode sigma E directly. The sequence specifying the first amino acid of sigma E (GGC) was placed immediately downstream of the initiating codon (ATG) of P31. The resulting sigE allele (sigE delta 84) encodes a sigma E-like protein which differs from the "processed product" by a single Met residue at its amino terminus. B. subtilis strains which carried this allele were Spo- and contained no detectable sigma E. The sigE delta 84 allele generated a product in Escherichia coli which, by quantitative Western immunoblot analysis, was present at 10 to 20% of the level of product (P31) obtained from a wild-type allele. A sigma E-like product was also not detected in two B. subtilis strains with missense mutations in the sequence encoding the processed region of P31. These results suggest that sigma E is a highly labile protein that is stabilized during its synthesis by an element of the precursor sequence. A mutant allele (sigE delta 48) which made an active sigma E-like protein in B. subtilis was isolated. This gene specified a product in which five amino acids, not derived from the P31 processed region, were joined to P31 at a position eight amino acids upstream of the processing site. The sigE delta 48 product was not processed, but it activated the sigma E -dependent spoIID promoter in vivo. The sigE delta 48 product therefore lost both an essential target for processing and a region which inhibited sigma sigma E activity. Cells which carried sig E delta 48 were Spo-. The basis of the sigE delta 48-dependent defect in sporulation is unknown, but the sigma E delta 48 activity appeared to persist beyond the time in development (4 h after onset sporulation) when wild-type sigma E activity declines. Thus, it may interfere with the proper regulation of late sporulation genes.     

Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake

Alkaline cellulase-producing actinomycete strains were isolated from mud samples collected from East African soda lakes. The strains were identified as novel Streptomyces spp. by 16S rDNA sequence analysis. A cellulase gene (cel12A) from Streptomyces sp. strain 11AG8 was cloned by expression screening of a genomic DNA library in Escherichia coli. From the nucleotide sequence of a 1.5-kb DNA fragment, an open reading frame of 1,113 nucleotides was identified encoding a protein of 371 amino acids. From computer analysis of the sequence, it was deduced that the Cel12A mature enzyme is a protein of 340 amino acids. The protein contained a catalytic domain, a glycine-rich linker region, and a cellulose-binding domain of 221, 12, and 107 amino acids, respectively. FASTA analysis of the catalytic domain of Cel12A classified the enzyme as a family 12 endoglucanase and the cellulose-binding domain as a family IIa CBD. Streptomyces rochei EglS was determined as nearest neighbor with a similarity of 75.2% and 61.0% to the catalytic domain and the cellulose-binding domain, respectively. The cell2A gene was subcloned in a Bacillus high-expression vector carrying the Bacillus amyloliquefaciens amylase regulatory sequences, and the construct was transformed to a Bacillus subtilis host strain. Crude enzyme preparations were obtained by ultrafiltration of cultures of the Bacillus subtilis recombinant strain containing the 11AG8 cell2A gene. The enzyme showed carboxymethylcellulase (CMCase) activities over a broad pH range (5-10) with an optimum activity at pH 8 and 50 degrees C. The enzyme retained more than 95% of its activity after incubation for 30 min under these conditions.     

Cell-cycle-specific inhibition by chloramphenicol of septum fromation and cell division in synchronized cells of Bacillus subtilis.

The relationship between protein synthesis and processes of cell division was studied by using synchronized cells of Bacillus subtilis 168. The addition of chloramphenicol at the beginning of synchronous growth prevented septum formation and cell division, suggesting the requirement of protein synthesis for the processes of cell division. Experiments in which the drug was added to the cells at different cell ages showed that the protein synthesis required for the initiation of septum formation was completed at about 15 min and that the protein synthesis required for cell division was completed at about 45 min. By interpreting the result from the concept of the transition point for protein synthesis, it was suggested that the processes of cell division in B. subtilis require at least two kinds of protein molecules which are synthesized at distinct stages in the cell cycle. This was supported by the result of an experiment in which starvation and the readdition of a required amino acid to exponentially growing cells induced two steps of synchronous cell division. Further, the two transition points are in agreement with the estimations obtained by residual division after the inhibition of protein synthesis in asynchronous cells. The relationship of the timing between the completion of chromosome replication and the two transition points was also studied.     

Cloning and expression of subtilisin amylosacchariticus gene

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.     

Identification and characterization of novel cell wall hydrolase CwlT: a two-domain autolysin exhibiting n-acetylmuramidase and DL-endopeptidase activities

A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to dl-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a dl-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents "a new category of cell wall hydrolases."     

Identification of amino acid residues modified by two ATP analogs in Bacillus subtilis glutamine synthetase.

Bacillus subtilis glutamine synthetase was modified by two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-triphosphate (8-N3-ATP), each one containing either Mg2+ or Mn2+. The FSBA labeled peptide was monitored by measuring the characteristic absorbance of the 4-carboxybenzenesulfonyl (CBS) part at 243 nm. The 8-N3ATP photolabeled peptide could also be monitored by measuring its absorption at 310 nm. A single CBS-labeled tryptic peptide was obtained, spanning residues 89-91 from the N-terminal of the subunit polypeptide chain, and sequence analysis by Edman degradation revealed that CBS-arginine was at position 91. The amino acids photolabeled by 8-N3ATP at the ATP-binding site in B. subtilis GS were His-186, His-187, and Trp-424. These results suggested that these four amino acids constitute an ATP-binding active site located at the interface between two subunits. The region surrounding Trp-424, which varies among different prokaryotic enzymes, was considered to be involved in a catalytic or regulatory role in B. subtilis GS. Since the same amino acids were labeled when B. subtilis GS was modified with FSBA or 8-N3ATP in the presence of Mn2+ or Mg2+, no conformational difference between B. subtilis GS binding Mn(2+)-ATP and that binding Mg(2+)-ATP was detected by affinity labeling with ATP analogs.     

Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis

A tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1.1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10(-7) per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10(-8) per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9.5 and 11.0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.     

Genetic system constructed to overproduce and secrete proinsulin in<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

The first amino acid residue from a proinsulin gene was fused in frame with the last amino acid residue of the aprE signal peptide sequence from Bacillus subtilis, using an overlapping PCR methodology. For expression of the fused DNA the aprE regulatory region (aprERR) was used. A six-protease-deficient strain of B. subtilis with the hpr2 and degU32 mutations was constructed for overproduction of the recombinant protein. The production of proinsulin was carried out in a mineral medium which facilitated the purification of proinsulin. Samples were taken during growth and analyzed by RIA and Western blot. Proinsulin was overproduced (1 mg ml(-1)) and 90% was secreted into the culture medium 1 h after stationary phase began.     

Substrate requirements for regulated intramembrane proteolysis of Bacillus subtilis pro-sigmaK

During sporulation of Bacillus subtilis, pro-sigmaK is activated by regulated intramembrane proteolysis (RIP) in response to a signal from the forespore. RIP of pro-sigmaK removes its prosequence (amino acids 1 to 20), releasing sigmaK from the outer forespore membrane into the mother cell cytoplasm, in a reaction catalyzed by SpoIVFB, a metalloprotease in the S2P family of intramembrane-cleaving proteases. The requirements for pro-sigmaK to serve as a substrate for RIP were investigated by producing C-terminally truncated pro-sigmaK fused at different points to the green fluorescent protein (GFP) or hexahistidine in sporulating B. subtilis or in Escherichia coli engineered to coexpress SpoIVFB. Nearly half of pro-sigmaK (amino acids 1 to 117), including part of sigma factor region 2.4, was required for RIP of pro-sigmaK-GFP chimeras in sporulating B. subtilis. Likewise, pro-sigmaK-hexahistidine chimeras demonstrated that the N-terminal 117 amino acids of pro-sigma(K) are sufficient for RIP, although the N-terminal 126 amino acids, which includes all of region 2.4, allowed much better accumulation of the chimeric protein in sporulating B. subtilis and more efficient processing by SpoIVFB in E. coli. In contrast to the requirements for RIP, a much smaller N-terminal segment (amino acids 1 to 27) was sufficient for membrane localization of a pro-sigmaK-GFP chimera. Addition or deletion of five amino acids near the N terminus allowed accurate processing of pro-sigmaK, ruling out a mechanism in which SpoIVFB measures the distance from the N terminus to the cleavage site. A charge reversal at position 13 (substituting glutamate for lysine) reduced accumulation of pro-sigmaK and prevented detectable RIP by SpoIVFB. These results elucidate substrate requirements for RIP of pro-sigmaK by SpoIVFB and may have implications for substrate recognition by other S2P family members.     

Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis.

We analysed the Bacillus subtilis protein coding sequences termini, and compared it to other genomes. The analysis focused on signals, com-positional biases of nucleotides, oligonucleotides, codons and amino acids and mRNA secondary structure. AUG is the preferred start codon in all genomes, independent of their G+C content, and seems to induce less stable mRNA structures. However, it is not conserved between homologous genes neither is it preferred in highly expressed genes. In B.subtilis the ribosome binding site is very strong. We found that downstream boxes do not seem to exist either in Escherichia coli or in B.subtilis. UAA stop codon usage is correlated with the G+C content and is strongly selected in highly expressed genes. We found less stable mRNA structures at both termini, which we related to mRNA-ribosome and mRNA-release-factor interactions. This pattern seems to impose a peculiar A-rich nucleotide and codon usage bias in these regions. Finally the analysis of all proteins from B.subtilis revealed a similar amino acid bias near both termini of proteins consisting of over-representation of hydrophilic residues. This bias near the stop codon is partially release-factor specific.     

A new relaxed mutant of Bacillus subtilis.

A new relaxed mutant of Bacillus subtilis was isolated by screening Rifr clones for alterations in stringent control. The Rifr relaxed mutant which is described was found to contain a second-site mutation conferring a relaxed response to an energy source downshift and was partially relaxed after amino acid starvation. The new rel locus, called relG, was distinct from the two other known rel loci in B. subtilis, relA, and relC.     

Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases

Many peptide antibiotics in prokaryotes and lower eukaryotes are produced non-ribosomally by multi-enzyme complexes. Analysis of gene-derived amino acid sequences of some peptide synthetases of bacterial and fungal origins revealed a high degree of conservation (35-50% identity). The genes encoding those peptide synthetases are clustered into large operons with repetitive domains (about 600 amino acids), in the case of synthetases activating more than one amino acid. We used two 35-mer oligonucleotides derived from two highly conserved regions of known peptide synthetases to identify the surfactin synthetase operon in Bacillus subtilis ATCC 21332, a strain not accessible to genetic manipulation. We show that the derived oligonucleotides can be used not only for the identification of unknown peptide synthetase genes by hybridization experiments but also in sequencing reactions as primers to identify internal domain sequences. Using this method, a 25.8-kb chromosomal DNA fragment bearing a part of the surfactin biosynthesis operon was cloned and partial sequences of two internal domains were obtained.