A DNA fragment from Bacillus natto IF03936 has been cloned which enhances the production of both extraceilular alkaline and neutral proteases in Bacillus subtilis. The DNA sequence analysis around the gene responsible for the hyperproduction, prtR, revealed one open reading frame (comprising 60 amino acid residues) which was bounded by potential transcriptional and translational regulatory signals in its preceding and following regions. This open reading frame was not homologous to the published sequences of the structural genes of the two proteases. The calculated molecular weight (7,109) of the polypeptide predicted from the DNA sequence is much smaller than those of the two proteases, indicating that the gene product is distinct from those enzymes. In-frame fusion between the N-terminal region of the coding sequence and the lacZ gene of Eschenichia coli demonstrated that the coding region was indeed translated in vivo. By deletion analysis it was suggested that prtR was the structural gene for the 60-amino-acid polypeptide. Cells carrying a prtR plasmid secreted both proteases 40 to 400 times more than the cells carrying the vector alone. Furthermore, it was found that prtR also enhanced the production of levansucrase by 1 or 2 orders of magnitude. There was no difference, however, in the amount of the other extracellular enzymes such as oa-amylase, RNase, and alkaline phosphatase. These results indicate that prtR is specffic for the hyperproduction of the proteases and levansucrase.

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Studies were performed on the prtR gene which enhances the production of the Bacillus subtilis extracellular proteases and levansucrase, but not the a-amylase, RNase, and alkaline phosphatase. To investigate the mode of action of prtR, the Escherichia coli bla gene was placed under the control of two promoters. One was the promoter of the alkaline protease gene (aprE), and the other was the promoter of B. subtilis dihydrofolate reductase gene (dfrA). Expression of the bla gene was enhanced by prtR only when the apr promoter was used. From these results, it was concluded that the apr promoter or its vicinity was the target of pftR and that prtR does not affect the process after transcription. The mRNA levels of aprE and nprE (the neutral proteae gene) were significantly increased by prtR, but the half-life of the aprE mRNA was not affected. These results show that the prtR gene product enhances protease production by increasing the rate of transcription initiation.

~~Thiamin caused depression of growth, a marked decrease in cellular vitamin B6 content and cytochrome oxidase activity in Saccharomyces yeasts growing in a vitamin B,-free medium under aerobic conditions but had practically no effect in Kluyveromyces, Schizosaccharomyces and Candida spp. Pyridoxine added concomitantly with thiamin permitted the thiaminsensitive yeasts to grow normally with increased activity of cytochrome oxidase. 6-Aminolaevulinate also caused the increase in cytochrome oxidase activity but growth was only partially improved by the addition of this precursor of haem biosynthesis. These phenomena were similar to those found previously in Saccharomyces carlsbergensis strain 4228 (ATCC 9080) (Nakamura et al., 198 1). Thiamin-sensitive yeasts accumulated thiamin more than 24-fold when compared with the thiamin-insensitive cells. Thiamin transported into the thiamin-sensitive yeasts was recovered only in the non-esterified form. Thiamin added to the growth medium was also accumulated by growing cells of the thiamin-sensitive yeasts especially during the lag and early exponential phases of growth. Pyridoxine did not affect either thiamin accumulation or the intracellular form of the transported thiamin.

An extracellular-protease-deficient mutant, ME 142, was isolated from Bacillus subtilis as a spontaneous erythromycin-resistant (Eryr) clone. This mutant showed conditional sporulation and only sporulated normally in the absence of erythromycin. In the presence of the antibiotic, sporulation was greatly reduced. Production of extracellular proteases by ME 142 also exhibited conditional deficiency, possibly due to pleiotropic effects of the sporulation deficiency. The production of protease was 2-10% that of the wild-type level in the presence of erythromycin. ME 142 showed poor competence for transformation even in the absence of erythromycin; however, derivatives of ME142 were isolated which had the same Eryr phenotype but which exhibited normal competence. One such mutant, ME 162, was used as a host for the secretion of Escherichia coli /I-lactamase. The amount of p-lactamase in the culture supernatants of ME 162 increased significantly when the cells were cultured with erythromycin, suggesting that proteolysis of the P-lactamase in the supernatants of ME 162 was greatly reduced as compared to that in the supernatants of the wild-type strain.