Both the neutral protease gene (nprS) and its transcriptional activator gene (nprA) from Bacillus stearothermophilus TELNE were cloned in Bacillus subtilis by using pTB53 as a vector plasmid. The presence of the nprA gene enhanced protease synthesis by about fivefold. The nucleotide sequences of nprS and its flanking regions were determined. nprS was composed of 1,653 base pairs and 551 amino acid residues. A Shine-Dalgarno (SD) sequence was found 9 bases upstream from the translation start site (ATG). The deduced amino acid sequence was very similar to that of another thermostable neutral protease gene, nprM (M. Kubo and T. Imanaka, J. Gen. Microbiol. 134:1883-1892, 1988). the amino acid sequence of the extracellular neutral protease NprS was completely identical to that of NprM. By deletion analysis and substitution of the original promoter with a foreign promoter, it was found that the nprA gene existed upstream of nprS. It was also found that a possible target region (palindromic sequence) of the gene product of nprA existed near the promoter sequence of nprS. The nucleotide sequences of nprA and its flanking regions were determined. The DNA sequence revealed only one large open reading frame, composed of 1,218 base pairs (406 amino acids; molecular weight, 49,097). The SD sequence was found 4 bases upstream from the translation start site (GTG). A possible promoter sequence (TTGAAG for the -35 region and AATTTT for the -10 region) was also found about 20 bases upstream of the SD sequence. The nprA gene was separated from nprS by a typical terminator sequence. By constructing an in-frame fusion between the lacZ gene and the 5' region of the nprA gene, it was demonstrated that the coding region of nprA was indeed translated in vivo. Three palindromic sequences, which were highly homologous with a possible target region by NprA, were also found in the 5' region of the nprA gene. This suggests that eh expression of nprA is autoregulated. From the time course of the production of NprA-LacZ fusion protein, it was indicated that nprA was expressed in late log phase, whereas nprS was expressed in the stationary phase. The NprA protein had consensus regions homologous to the DNA recognition domains of DNA-binding proteins but showed no sequence homology with any other regulatory proteins for protease production. It is inferred that NprA protein binds to the upstream region of nprS promoter and activates transcription of nprS. A new regulatory mechanism by the nprA-nprS genes is discussed.
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