Synthesis of bacilysin by Bacillus subtilis branches from prephenate of the aromatic amino acid pathway.

Previously characterized mutants of Bacillus subtilis 168 were analyzed for the accumulation of the antibiotic bacilysin in culture broths and for the presence of bacilysin synthetase in cell extracts. All aro mutants tested were deficient in bacilysin biosynthesis but had synthetase activity. Mutants with lesions in tyrA and pheA produced normal levels of bacilysin, suggesting that prephenate is the primary metabolic precursor of bacilysin.     

Isolation of new variants of surfactin by a recombinant Bacillus subtilis

A recombinant Bacillus subtilis MI113(pC115), carrying a gene responsible for the production of surfactin and iturin A cloned from B. subtilis RB14C, produced new surfactin variants, in addition to the already reported surfactin, when MI113(pC115) was cultured in solid-state fermentation of soybean curd residue (okara) as a substrate. All variants isolated by HPLC were characterized. Received: 18 December 1996 / Received revision: 20 February 1997 / Accepted: 28 February 1997     

枯草芽孢杆菌内切木聚糖酶的分离纯化与鉴定

目的：建立一种简便、快速的木聚糖酶分离和提取方法。方法：采用活性聚丙烯酰胺凝胶电泳和均质提取法相结合,分离纯化枯草芽孢杆菌(Bacillus subtilis)固体培养基发酵产物中的木聚糖酶,进一步用薄层色谱和高压液相色谱对木聚糖酶进行鉴定。结果：采用活性聚丙烯酰胺凝胶电泳和均质提取法相结合,从枯草芽孢杆菌(Bacillus subtilis)固体培养基发酵产物中分离得到了两种内切木聚糖酶,酶解桦木木聚糖的产要产物以木二糖和木三糖为主。结论：活性聚丙烯酰胺凝胶电泳和均质提取法相结合是一种新的分离纯化木聚糖酶的简便、有效方法。     

Differential amino acid requirements for sporulation in Bacillus subtilis

The amino acid requirements for sporulation were studied by use of auxotrophic mutants of Bacillus subtilis 168. Cells were grown to T(0) in medium containing the test amino acid and were then transferred to a minimal medium lacking that amino acid. Omission of leucine caused no reduction in sporulation. Omission of methionine, lysine, and phenylalanine appeared to cause reduced levels of sporulation, and sporulation was completely inhibited when isoleucine, tryptophan, and threonine were omitted. The amino acids in this third class showed a sequence of requirements, with tryptophan required earlier than isoleucine, which in turn was required earlier in the sporulation process than threonine. Isoleucine omission did not affect the early sporulation functions of extracellular protease formation or septum formation, but prevented the increased levels of protein synthesis and oxygen consumption that normally accompany early sporulation stages. Isoleucine did not appear to be metabolized to other compounds in significant amounts during sporulation. The role of isoleucine in the sporulation process remains unclear.     

Isolation and Characterization of a Proteinaceous Protease Inhibitor from Bacillus subtilis

A proteinaceous protease inhibitor which might have an intracellular role in modulating protease activity during sporulation was isolated from B. subtilis IFO 3027 by trichloroacetic acid and ethanol precipitations, and column chromatographies on SP-Sephadex, DEAE-Sephadex, DE AE-Toyopearl and Sephadex G-75.

The molecular weight of the inhibitor was estimated by gel filtration and SDS polyacrylamide gel electrophoresis to be about 16,000. The isoelectric point was determined as pH 4.8. The inhibitor is an acid and thermostable protein. The-amino acid sequence in the amino terminal region was determined to be (Met)-Glu-Asn-Gln-Glu-Val-Val-Leu-X-X-Asp-Ala-Ile-Gln-Glu- ··· (X, unidentified).

In addition to cytoplasmic serine proteases of the inhibitor-producing strain, the inhibitor inhibits various microbial serine proteases.     

Tn10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin

Transposon mutagenesis was employed to isolate the gene(s) related with the biosynthesis of dipeptide antibiotic in Bacillus subtilis PY79 (a prototrophic derivative of the standard 168 strain). The blocked mutants were phenotypically selected from the transposon library by bioassay and the complete loss of biosynthetic ability was verified through ESI-mass spectrometry analysis. Four different bacilysin nonproducer mutants (Bac(-)::Tn10(ori-spc)) were isolated from the transposon library. The genes involved in bacilysin biosynthesis were identified as thyA (thymidilate synthetase), ybgG (unknown; similar to homocysteine methyl transferase) and oppA (oligopeptide permease), respectively. The other blocked gene was yvgW (unknown; similar to heavy metal-transporting ATPase); however, backcross studies did not verify its involvement in bacilysin biosynthesis. This gene, on the other hand, appeared to be necessary for efficient sporulation and transformation. Opp involvement was significant as it suggested that bacilysin biosynthesis is under or a component of the quorum sensing pathway which has been shown to be responsible for the establishment of sporulation, competence development and onset of surfactin biosynthesis. For verification, it was necessary to check the involvement of peptide pheromones (PhrA or PhrC) internalized by the Opp system and response regulator ComA as the essential components of this global control. phrA, phrC and comA deleted mutants of PY79 were thus constructed and the latter two genes were shown to be essential for bacilysin biosynthesis.     

Isolation and characterization of a novel extracellular metalloprotease from Bacillus subtilis.

We have isolated and characterized two minor extracellular proteases from culture supernatants of a strain of Bacillus subtilis containing deletion mutations of the genes for the extracellular proteases subtilisin (apr) and neutral protease (npr) and a minor extracellular protease (epr) as well as intracellular serine protease-I (isp-1). Characterization studies have revealed that one of these enzymes is the previously described protease bacillopeptidase F. The second enzyme, the subject of this report, is a novel metalloprotease, which we designate Mpr. Mpr is a unique metalloprotease that has been purified to apparent homogeneity by using both conventional and high-performance liquid chromatography procedures. Mpr has a molecular mass of approximately 28 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a basic isoelectric point of 8.7. The enzyme showed maximal activity against azocoll at pH 7.5 and 50 degrees C. Mpr was inhibited by dithiothreitol and a combination of beta-mercaptoethanol and EDTA. Activity was moderately inhibited by beta-mercaptoethanol and EDTA alone as well as by cysteine and citrate and only marginally by phosphoramidon 1,10-phenanthroline and N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine. Mpr was not inhibited by phenylmethylsulfonyl fluoride. In addition, Mpr showed esterolytic but not collagenolytic activities. Our studies suggest that Mpr is a secreted metalloprotease containing cysteine residues that are required for maximal activity.     

Aromatic amino acid biosynthesis: gene-enzyme relationships in Bacillus subtilis

Single step mutants of Bacillus subtilis which required either one or all of the aromatic amino acids for growth were isolated. The relevant gene defect was determined for each mutant by enzyme assays in vitro. A mutant deficient in each enzyme step of aromatic amino acid biosynthesis was found with the exceptions of the shikimate kinase and the phenylalanine and tyrosine transaminases. Representative mutants carrying the defective genes were mapped by deoxyribonucleic acid mediated transformation by reference to the aromatic amino acid gene (aro) cluster and, alternately, to any of the other unlinked aro genes. The genes coding for dehydroquinate synthetase, 3-enol pyruvylshikimate 5-phosphate synthetase, one form of chorismate mutase, and prephenate dehydrogenase are linked to the aro cluster. Except for the previously identified linkage between the genes of 3-deoxy-d-arabino heptulosonic acid 7-phosphate synthetase and one species of chorismate mutase, the other genes involved in this pathway are neither linked to the aro cluster nor to each other.