Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis.

We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation.     

Multiple active forms of a novel serine protease from Bacillus subtilis

Summary We have cloned and sequenced a gene (epr) encoding a novel serine protease from Bacillus subtilis. Several active forms of the enzyme with molecular masses between 40 and 34 kDa were found in the medium of B. subtilis cultures containing the epr gene cloned on a plasmid. Deletions at the 3 end of the gene, removing up to 240 amino acids of the reading frame, abolished the expression of the larger species but did not affect the expression of the 34 kDa enzyme. The C-terminal third of the protein is therefore not required for protease activity. The size variation of the active forms expressed by the complete epr gene appears to be the result of partial removal of the C-terminus either by processing or degradation. Thus, the epr gene consists of two domains, one encoding a serine protease homologous to subtilisin and the other a C-terminus of unknown function.Parts of this work were presented at the Fourth International Conference on Genetics and Biotechnology of Bacilli, San Diego, 1987     

Nucleotide sequence and promoter region for the neutral protease gene from Bacillus stearothermophilus.

The thermostable neutral protease gene nprT of Bacillus stearothermophilus was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1,644 bases and 548 amino acid residues. A Shine-Dalgarno sequence was found 9 bases upstream from the translation start site (ATG), and the deduced amino acid sequence contained a signal sequence in its amino-terminal region. The sequence of the first 14 amino acids of purified extracellular protease completely matched that deduced from the DNA sequence starting at GTC (Val), 687 bases (229 amino acids) downstream from ATG. This suggests that the protease is translated as a longer polypeptide. The amino acid sequence of the extracellular form of this protease (319 amino acids) was highly homologous to that of the thermostable neutral protease from Bacillus thermoproteolyticus but less homologous to the thermolabile neutral protease from Bacillus subtilis. A promoter region determined by S1 nuclease mapping (TTTTCC for the -35 region and TATTTT for the -10 region) was different from the conserved promoter sequences recognized by the known or factors in bacilli. However, it was very homologous to the promoter sequence of the spo0B gene from B. subtilis. The guanine-plus-cytosine content of the coding region of the nprT gene was 58 mol%, while that of the third letter of the codons was much higher (72 mol%).     

TmrB protein, responsible for tunicamycin resistance of Bacillus subtilis, is a novel ATP-binding membrane protein.

tmrB is the gene responsible for tunicamycin resistance in Bacillus subtilis. It is predicted that an increase in tmrB gene expression makes B. subtilis tunicamycin resistant. To examine the tmrB gene product, we produced the tmrB gene product in Escherichia coli by using the tac promoter. TmrB protein was found not only in the cytoplasm fraction but also in the membrane fraction. Although TmrB protein is entirely hydrophilic and has no hydrophobic stretch of amino acids sufficient to span the membrane, its C-terminal 18 amino acids could form an amphiphilic alpha-helix. Breaking this potential alpha-helix by introducing proline residues or a stop codon into this region caused the release of this membrane-bound protein into the cytoplasmic fraction, indicating that the C-terminal 18 residues were essential for membrane binding. On the other hand, TmrB protein has an ATP-binding consensus sequence in the N-terminal region. We have tested whether this sequence actually has the ability to bind ATP by photoaffinity cross-linking with azido-[alpha-32P]ATP. Wild-type protein bound azido-ATP well, but mutants with substitutions in the consensus amino acids were unable to bind azido-ATP. These C-terminal or N-terminal mutant genes were unable to confer tunicamycin resistance on B. subtilis in a multicopy state. It is concluded that TmrB protein is a novel ATP-binding protein which is anchored to the membrane with its C-terminal amphiphilic alpha-helix.     

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.     

Cloning, nucleotide sequence, and expression of Achromobacter protease I gene

Achromobacter protease I (API) is a lysine-specific serine protease which hydrolyzes specifically the lysyl peptide bond. A gene coding for API was cloned from Achromobacter lyticus M497-1. Nucleotide sequence of the cloned DNA fragment revealed that the gene coded for a single polypeptide chain of 653 amino acids. The N-terminal 205 amino acids, including signal peptide and the threonine/serine-rich C-terminal 180 amino acids are flanking the 268 amino acid-mature protein which was identified by protein sequencing. Escherichia coli carrying a plasmid containing the cloned API gene overproduced and secreted a protein of Mr 50,000 (API') into the periplasm. This protein exhibited a distinct endopeptidase activity specific for lysyl bonds as well. The N-terminal amino acid sequence of API' was the same as mature API, suggesting that the enzyme retained the C-terminal extended peptide chain. The present experiments indicate that API, an extracellular protease produced by gram-negative bacteria, is synthesized in vivo as a precursor protein bearing long extended peptide chains at both N and C termini.     

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.     

Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02

Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had molecular masses of 42,000 Da and 28,000 Da, respectively. The first 20 amino acids of the N-terminal sequence are AQSVPYGISQ IKAPALHSQG. The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are different from those of other proteases. The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate, and PMSF as well as aprotinin inhibited its fibrinolytic activity. These results indicated QK-1 was a plasmin-like serine protease and QK-2 a subtilisin family serine protease. Therefore, these enzymes were designated subtilisin QK. The sequence of a DNA fragment encoding subtilisin QK contained an open reading frame of 1149 base pairs encoding 106 amino acids for signal peptide and 257 amino acids for subtilisin QK, which is highly similar with that of a fibrinolytic enzyme, subtilisin NAT (identities 96.8%). Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are identical to the active site of nattokinase (NK) produced by B. subtilis natto.     

Characterization of a novel otubain-like cysteine protease of Cryptosporidium parvum

Otubains are a recently discovered family of cysteine proteases that participate in the ubiquitin pathway. Here, we partially characterized the biochemical properties of a cysteine protease of Cryptosporidium parvum, which is closely related to otubains. The gene encoding otubain-like cysteine protease of C. parvum (CpOTU) contained the aspartate, cysteine and histidine residues that form the catalytic triad of otubains. The modified ubiquitin-associated domain and LxxL motif were identified in CpOTU. The recombinant CpOTU showed the isopeptidase activity at neutral pH values and its activity was effectively inhibited by ubiquitin aldehyde, N-ethylmaleimide and iodoacetic acid. Interestingly, CpOTU had an unusual C-terminal extension of 217 amino acids compared to mammalian otubains, and the C-terminal extension is essential for the activity of the enzyme. Expression of CpOTU peaked in the oocyst stage of the parasite, which suggested its potential physiological role for the oocyst stage.     

Cloning and Sequence Analysis of a Protease-encoding Gene from the Marine Bacterium Alteromonas sp. Strain O-7

The gene (aprI) encoding alkaline serine protease (AprI; subtilase) from Alteromonas sp. strain O-7 was cloned and sequenced. The nucleotide sequence of aprI has been identified. The deduced amino acid sequence indicated that aprI codes for a precursor of 715 amino acids and the precursor is composed of four regions including a signal peptide, an N-terminal pro-region, a mature protease region and a C-terminal extension region of 215 amino acids as previously described for aprII [H. Tsujibo et al., Gene, 136, 247–251 (1993)]. The amino acid sequence of the mature AprI (AprI-M) showed high sequence homology with those of other class I subtilases. The C-terminal region was characterized by a repeat of 94 amino acids residues, which showed about 50% similarity with those of the C-terminal pro-region of several known proteases from Gram-negative bacteria.     

Identification of the protease and the turnover signal responsible for cell cycle-dependent degradation of the Caulobacter FliF motor protein

Flagellar ejection is tightly coupled to the cell cycle in Caulobacter crescentus. The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release. Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly. To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF. Cell cycle-dependent turnover of FliF requires an intact clpA gene, suggesting that the ClpAP protease is required for removal of the MS ring protein. Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously. However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability. This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence. The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF. Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms.     

Molecular cloning and sequencing of the gene for a halophilic alkaline serine protease (halolysin) from an unidentified halophilic archaea strain (172P1) and expression of the gene in Haloferax volcanii.

The gene of a halophilic alkaline serine protease, halolysin, from an unidentified halophilic archaea (archaebacterium) was cloned and its nucleotide sequence was determined. The deduced amino acid sequence showed that halolysin consists of 411 amino acids, with a molecular weight of 41,963. The highest homology was found with thermitase from Thermoactinomyces vulgaris. Halolysin has a long C-terminal extension of approximately 120 amino acids which has not been found in other extracellular subtilisin type serine proteases. The gene, hly, was expressed in another halophilic archaea, Haloferax volcanii, in a medium containing 18% salts by using a plasmid shuttle vector which has a novobiocin resistance determinant as a selectable marker.     

Cloning, characterization, and inactivation of the Bacillus brevis lon gene.

A gene of Bacillus brevis HPD31 analogous to the Escherichia coli lon gene has been cloned and characterized. The cloned gene (B. brevis lon gene) encodes a polypeptide of 779 amino acids with a molecular weight of 87,400 which resembles E. coli protease La, the lon gene product. Fifty-two percent of the amino acid residues of the two polypeptides were identical. The ATP-binding sequences found in E. coli protease La were highly conserved. The promoter of the B. brevis lon gene resembled that recognized by the major RNA polymerase of Bacillus subtilis and did not contain sequences homologous to the E. coli heat shock promoters. The B. brevis lon gene was inactivated by insertion of the neomycin resistance gene. A mutant B. brevis carrying the inactivated lon gene showed diminished ability for the degradation of abnormal polypeptides synthesized in the presence of puromycin.     

Peptide carrier potentiality of Bacillus subtilis levansucrase.

A synthetic oligodeoxynucleotide encoding the vasopressin peptide was ligated to the 3' terminal codon of sacB, the structural gene of levansucrase. This gene fusion was integrated into the chromosome of a Bacillus subtilis strain able to overproduce levansucrase. The extracellular production of the hybrid protein, consisting of the whole levansucrase primary sequence plus the nine amino acids of the vasopressin peptide added at the C-terminal end, represented 50-55% of that found for the wild-type levansucrase (20 mg l-1). The purified hybrid protein displayed the same conformational stability, protease insensitivity and enzymic properties as the wild-type levansucrase. However, the rate and the yield of the unfolding-folding transition at the pH and temperature used for bacterial growth were lower in the case of the hybrid protein; the latter also required a higher iron concentration to be completely folded.     

Epr plays a key role in DegU-mediated swarming motility of Bacillus subtilis

Epr, a minor extracellular protease, is involved in the swarming motility of Bacillus subtilis . It does so by providing essential signals required for swarming. It has also been demonstrated that DegU is required for swarming and that it occurs at very low levels of DegU∼P and is inhibited at high levels of DegU∼P. In this study, we show that maximal epr expression is observed at very low concentrations of DegU∼P, whereas it is repressed at high DegU∼P. A parallel effect of DegU∼P levels on swarming motility is also observed, where very low levels of DegU∼P support swarming and excessive DegU∼P abolishes swarming. We further demonstrate that the defect of swarming motility in a degU ⁻ strain can be rescued, albeit incompletely, by increased expression of an exogenous epr gene. We also show that an additional extracellular factor(s), apart from epr , regulated by DegU, is required for robust swarming.     

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.     

epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus capitis and Staphylococcus aureus.

Staphylococcus capitis EPK1 produces a glycylglycine endopeptidase, ALE-1 (M. Sugai, T. Fujiwara, T. Akiyama, M. Ohara, H. Komatsuzawa, S. Inoue, and H. Suginaka, J. Bacteriol. 179:1193-1202, 1997), which hydrolyzes interpeptide pentaglycine chains of cell wall peptidoglycan of S. aureus. Characterizations of the enzyme activity and cloning of ale-1 revealed that ALE-1 is very similar to prolysostaphin produced by S. simulans bv. staphylolyticus. Strain EPK1 is resistant to lysis by ALE-1 and by lysostaphin. A gene that renders the cells resistant to glycylglycine endopeptidase (epr) was found 322 bp upstream of and in the opposite orientation to ale-1. The deduced amino acid sequence of epr showed similarities to FemA and FemB, which have been characterized as factors essential for methicillin resistance of S. aureus. Inactivation of either femA or femB causes decreased resistance to methicillin, increased resistance to lysostaphin, and decreased glycine content in the interpeptide chains of peptidoglycan. Therefore, femAB is suggested to be involved in the addition of glycine to pentapeptide peptidoglycan precursor. S. aureus with epr on a multicopy plasmid had phenotypes similar to those of femAB mutants except that it did not alter resistance level to methicillin. These results suggest that epr and femAB belong to the protein family involved in adding amino acids to the pentapeptide peptidoglycan precursor and that epr is involved in the addition of serine to the pentapeptide.