Cloning and nucleotide sequence of the highly thermostable neutral protease gene from Bacillus stearothermophilus

The gene (nprM) for the highly thermostable neutral protease of Bacillus stearothermophilus MK232 was cloned in Bacillus subtilis using pTB53 as a vector. The nucleotide sequence of nprM and its flanking regions was determined. The DNA sequence revealed only one large open reading frame, composed of 1656 base pairs and 552 amino acid residues. A Shine-Dalgarno (SD) sequence was found 12 bases upstream from the translation start site (ATG). A possible promotor sequence (TTTTCC for the -35 region and TATTGT for the -10 region), which was nearly identical to the promoter for another thermostable neutral protease gene, nprT, was also found about 40 bases upstream of the SD sequence. The deduced amino acid sequence contained a signal sequence in its amino-terminal region. The sequence of the first five amino acids of purified extracellular protease completely matched residues 237-241 of the open reading frame. This suggests that the enzyme is translated as a large polypeptide containing a pre-pro structure as is known for other neutral proteases. The amino acid sequence of the extracellular form of this protease (316 amino acids, molecular mass 34,266 Da) was identical to that of the thermostable neutral protease (thermolysin) from Bacillus thermoproteolyticus except for two amino acid substitutions (Asp37 to Asn37 and Glu119 to Gln119). The G + C content of the coding region of nprM was 42 mol%, while that of the third letter of the codons was lower (36 mol%). This extremely low content is an exceptional case for genes from thermophiles. When the protease genes, nprM and nprT, were cloned on pTB53 in B. subtilis, the expression of nprM was about 20 times higher than that of nprT. The reason for the difference between the two systems is discussed.     

Cloning and sequencing of Serratia protease gene.

The gene encoding an extracellular metalloproteinase from Serratia sp. E-15 has been cloned, and its complete nucleotide sequence determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature protein of the Serratia protease consists of 470 amino acids with a molecular weight of 50,632. The G+C content of the coding region for the mature protein is 58%; this high G+C content is due to a marked preference for G+C bases at the third position of the codons. The gene codes for a short pro-peptide preceding the mature protein. The Serratia protease gene was expressed in Escherichia coli and Serratia marcescens; the former produced the Serratia protease in the cells and the latter in the culture medium. Three zinc ligands and an active site of the Serratia protease were predicted by comparing the structure of the enzyme with those of thermolysin and Bacillus subtilis neutral protease.     

Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus.

The gene (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrio proteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coli produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl)acryloyl]-L-alanylphenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long 'pro' sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified from cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the 'pro' and mature enzyme downstream from a Bacillus promoter and signal sequence.     

Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene.

The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.     

Bacillopeptidase F of Bacillus subtilis: purification of the protein and cloning of the gene.

We have purified a minor extracellular serine protease from Bacillus subtilis. Characterization of this enzyme indicated that it was most likely the previously reported enzyme bacillopeptidase F. The amino-terminal sequence of the purified protein was determined, and a "guess-mer" oligonucleotide hybridization probe was constructed on the basis of that sequence. This probe was used to identify and clone the structural gene (bpr) for bacillopeptidase F. The deduced amino acid sequence for the mature protein (496 amino acids) was preceded by a putative signal sequence of 30 residues and a putative propeptide region of 164 amino acids. The bpr gene mapped near pyrD on the chromosome and was not required for growth or sporulation.     

Gene encoding a minor extracellular protease in Bacillus subtilis.

The gene for a minor, extracellular protease has been identified in Bacillus subtilis. The gene (epr) encoded a primary product of 645 amino acids that was partially homologous to both subtilisin (Apr) and the major internal serine protease (ISP-1) of B. subtilis. Deletion analysis indicated that the C-terminal 240 amino acids of Epr were not necessary for activity. This C-terminal region exhibited several unusual features, including a high abundance of lysine residues and the presence of a partially homologous sequence of 44 amino acids that was directly repeated five times. The epr gene mapped near sacA and was not required for growth or sporulation.     

Molecular cloning, nucleotide sequence, and expression of the structural gene for alkaline serine protease from alkaliphilic Bacillus sp. 221.

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus subtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6-61.7%). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

Cloning and sequencing of the Escherichia coli gyrA gene coding for the A subunit of DNA gyrase

The gene gyrA of Escherichia coli, which encodes the A subunit of DNA gyrase (topoisomerase II), has been cloned and a region of approximately 3300 base-pairs sequenced. An open reading frame of 2625 nucleotides coding for a protein of 97,000 Mr is located. The peptide weight of the subunit predicted from this open reading frame is in close agreement with previously published estimates of that of the A subunit. There is a "TATAAT" promoter motif located 44 bases upstream from the first "ATG" of the open reading frame. The amino acid sequence derived from the nucleotide sequence is about 50% homologous with that derived from the Bacillus subtilis gyrA gene sequence, with several regions showing greater than 90% homology.