Structure of the Bacillus brevis metalloprotease gene

Primary structure of DNA fragment of 2355 b.p., encoding metalloprotease gene of Bac. brevis, had been determined. Open reading frame for a protein with size of 528 amino acid residues was found in this sequence. The encoding protein is homologous to metalloproteases of Bac. stearothermophilus, Bac. cereus, Bac. subtilis and Bac. amyloliquefaciens. The structure of Bac. brevis metalloprotease gene reveals that this enzyme is synthesised as pre-pro-protease with signal peptide and pro-region, which are cut during its synthesis. The proposed size of mature protease is 304 amino acid residues. The residues, essential for catalysis, binding of Zn ion and Ca ions were found on the basis of Bacilli metalloproteases structures comparison.     

Amino acid sequence of thermostable direct hemolysin produced by Vibrio parahaemolyticus

The complete amino acid sequence of the subunit of thermostable direct hemolysin, a dimeric protein composed of identical subunits isolated from Vibrio parahaemolyticus, was determined by sequencing BrCN-peptides, their tryptic peptides, and overlaps obtained by Achromobacter protease I digestion. The subunit consists of 165 amino acid residues with the sole disulfide bond between Cys 151 and Cys 161. It is deduced that the biologically active hemolysin is formed by noncovalent association of subunits which are not linked together by disulfide bonds. The primary structure of hemolysin elucidated in the present study is essentially the same as that deduced from the nucleotide sequence of a gene encoding the protein but differs in 9 amino acid residues, suggesting the possibility of the presence of multiple genes for the thermostable direct hemolysin in Vibrio parahaemolyticus.     

Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.

A novel proteinaceous protease inhibitor was isolated from the culture supernatant of Bacillus brevis HPD31. The protease inhibitor of B. brevis (designated BbrPI) was produced extracellularly in multiple forms having at least three different molecular weights. One of them, BbrPI-a, was purified to near homogeneity and only showed inhibitory activity toward serine proteases, such as trypsin, chymotrypsin, and subtilisin. BbrPI was presumed to form a trypsin-inhibitor complex in a molar ratio of 1:1. The inhibitor was found to be heat resistant at neutral and acidic pHs. The gene coding for BbrPI was cloned into Escherichia coli, and its nucleotide sequence was determined. The sequence suggested that BbrPI is produced with a signal peptide of 24 amino acid residues. The amino acid sequence of the protein deduced from the DNA sequence contained the amino acid sequences of amino termini of the inhibitors, a, b, and c, and their putative precursor determined chemically. The molecular weight of the precursor was about 33,000, and the molecular weights of inhibitors a, b, and c were about 22,000, 23,500, and 24,000, respectively. It is presumed that the secreted precursor protein, which is probably inactive, is cleaved by protease into several active protease inhibitor molecules. BbrPI shows no significant homology to the protease inhibitors described previously and is unique in not having any cysteine residues in its molecule.     

Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.

A novel proteinaceous protease inhibitor was isolated from the culture supernatant of Bacillus brevis HPD31. The protease inhibitor of B. brevis (designated BbrPI) was produced extracellularly in multiple forms having at least three different molecular weights. One of them, BbrPI-a, was purified to near homogeneity and only showed inhibitory activity toward serine proteases, such as trypsin, chymotrypsin, and subtilisin. BbrPI was presumed to form a trypsin-inhibitor complex in a molar ratio of 1:1. The inhibitor was found to be heat resistant at neutral and acidic pHs. The gene coding for BbrPI was cloned into Escherichia coli, and its nucleotide sequence was determined. The sequence suggested that BbrPI is produced with a signal peptide of 24 amino acid residues. The amino acid sequence of the protein deduced from the DNA sequence contained the amino acid sequences of amino termini of the inhibitors, a, b, and c, and their putative precursor determined chemically. The molecular weight of the precursor was about 33,000, and the molecular weights of inhibitors a, b, and c were about 22,000, 23,500, and 24,000, respectively. It is presumed that the secreted precursor protein, which is probably inactive, is cleaved by protease into several active protease inhibitor molecules. BbrPI shows no significant homology to the protease inhibitors described previously and is unique in not having any cysteine residues in its molecule.     

In vitro reconstitution of a hexagonal array with a surface layer protein synthesized by Bacillus subtilis harboring the surface layer protein gene from Bacillus brevis 47.

Bacillus brevis 47 contains two surface layer proteins, termed the outer wall protein and the middle wall protein (MWP), which form a hexagonal array in the cell wall. Introduction of the MWP structural gene into Bacillus subtilis by using a low-copy-number plasmid led to the synthesis of an immunoreactive polypeptide with a molecular mass almost the same as that of the MWP synthesized by B. brevis 47. Biochemical analysis indicated that most of the MWP synthesized by B. subtilis was localized in the cytoplasmic fraction. This was further confirmed by using immunogold electron microscopy. The amino-terminal amino acid sequence of the MWP purified from the cytoplasm of B. subtilis indicated that the MWP was precursor with a signal peptide of 23 amino acid residues to the amino terminus of the mature protein. The precursor of the MWP possessed the ability to reassemble in vitro on the B. brevis 47 peptidoglycan layer, resulting in the formation of almost the same hexagonal arrays as with the mature MWP purified from B. brevis 47, judging from images averaged at a resolution of about 2.5 nm. Furthermore, a center-to-center distance of the hexagonal lattice on the envelope reconstituted by using the precursor MWP was calibrated as 18.3 nm, which was almost identical to the value of 17.8 nm obtained with the mature protein.     

Nucleotide sequence of the phoS gene, the structural gene for the phosphate-binding protein of Escherichia coli.

phoS is the structural gene for the phosphate-binding protein, which is localized in periplasm and involved in active transport of phosphate in Escherichia coli. It is also a negative regulatory gene for the pho regulon, and the gene expression is inducible by phosphate starvation. The complete nucleotide sequence of the phoS gene was determined by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). The amino acid sequences at the amino termini of the pre-PhoS and PhoS proteins and at the carboxy terminus of the PhoS protein were determined by using the purified proteins. Furthermore, the amino acid sequence of enzymatically digested peptide fragments of the PhoS protein was determined. The combined data established the nucleotide sequence of the coding region and the amino acid sequence of the pre-PhoS and the PhoS proteins. The pre-PhoS protein contains an extension of peptide composed of 25 amino acid residues at the amino terminus of the PhoS protein, which has the general characteristics of a signal peptide. The mature PhoS protein is composed of 321 amino acid residues, with a calculated molecular weight of 34,422, and lacks the disulfide bond and methionine. The regulatory region of phoS contains a characteristic Shine-Dalgarno sequence at an appropriate position preceding the translational initiation site, as well as three possible Pribnow boxes and one -35 sequence. the nucleotide sequence of the regulatory region of phoS was compared with those of phoA and phoE, the genes constituting the pho regulon.     

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.     

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.     

Amino acid sequence of Streptomyces metallo-proteinase inhibitor from Streptomyces nigrescens TK-23

Streptomyces Metallo-Proteinase Inhibitor (S-MPI) consists of 102 amino acid residues, including one methionine and two disulfide bridges. The complete amino acid sequence of S-MPI, including two disulfide bridges, was determined by sequencing of tryptic and chymotryptic peptides of two fragments obtained by cyanogen bromide cleavage followed by reduction and S-pyridylethylation of the protein. Incubation of the inhibitor with thermolysin slowly cleaved one peptide bond, Cys(64)-Val(65), which might be a reactive site of S-MPI.     

Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene.

The lon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family of sigma 32-dependent heat shock genes. In this paper, we report the cloning and characterization of the lon gene from the gram-positive bacterium Bacillus subtilis. The nucleotide sequence of the lon locus, which is localized upstream of the hemAXCDBL operon, was determined. The lon gene codes for an 87-kDa protein consisting of 774 amino acid residues. A comparison of the deduced amino acid sequence with previously described lon gene products from E. coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all known bacterial Lon proteins. Like the E. coli lon gene, the B. subtilis lon gene is induced by heat shock. Furthermore, the amount of lon-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatment with puromycin. The potential promoter region does not show similarities to promoters recognized by sigma 32 of E. coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase E sigma A of B. subtilis. A second gene designated orfX is suggested to be transcribed together with lon and encodes a protein with 195 amino acid residues and a calculated molecular weight of 22,000.     

Characterization of the genes for the hexagonally arranged surface layer proteins in protein-producing Bacillus brevis 47: complete nucleotide sequence of the middle wall protein gene.

Bacillus brevis 47 contains two surface (S)-layer proteins, termed the outer wall protein (OWP) and the middle wall protein (MWP), which form a hexagonal array in the cell wall. The MWP and OWP genes are contained in the 9-kilobase-pair (kbp) BclI fragment and constitute an operon under coordinate control of their expression. The nucleotide sequence of a 3.8-kbp EcoRI-SacI fragment containing the entire MWP gene has been determined in this study. Together with the DNA sequence of the promoter region for the MWP-OWP gene operon (H. Yamagata, T. Adachi, A. Tsuboi, M. Takao, T. Sasaki, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 169:1239-1245, 1987) and that of the OWP gene (A. Tsuboi, R. Uchihi, R. Tabata, Y. Takahashi, H. Hashiba, T. Sasaki, H. Yamagata, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 168:365-373, 1986), the complete nucleotide sequence of the MWP-OWP gene operon has been determined. The MWP gene encodes a secretory precursor of the MWP, consisting of a total of 1,053 amino acid residues with a signal peptide of 23 amino acid residues at its amino-terminal end. Bacillus subtilis harboring the MWP gene synthesized an immunoreactive polypeptide with almost the same molecular weight as the authentic MWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid compositions deduced from the MWP and OWP genes were similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic amino acids compared with basic amino acids and in the very low content of sulfur-containing amino acids. The acidic nature of the MWP and OWP was confirmed by isoelectric focusing on polyacrylamide gels. In addition, circular dichroism spectra indicated that the S-layer proteins in B. brevis 47 were composed of approximately 30% beta-sheet and 5% alpha-helical structures, with the remainder of the polypeptide backbone being aperiodic in nature.     

褐飞虱乙酰胆碱酯酶基因全长cDNA的克隆及序列分析

利用反转录聚合酶链式反应(RT_PCR)结合快速扩增cDNA末端(RACE)技术克隆了褐飞虱Nilaparvata lugens 乙酰胆碱酯酶基因cDNA。该cDNA全长2 467 bp,包含一个1 938 bp的开放阅读框(GenBank登录号AJ852420); 在推导出的646个氨基酸残基的前体蛋白中, N端的前30个氨基酸残基为信号肽,随后的616个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为69 418 D。在一级结构中,形成催化活性中心的3个氨基酸残基(Ser242,Glu371和His485),以及在亚基内形成二硫键的6个半胱氨酸完全保守; 位于催化功能域的14个芳香族氨基酸中有10 个完全保守。该酶的氨基酸序列与黑尾叶蝉的同源性最高,达83%。对来自23种昆虫（包括褐飞虱）的30个乙酰胆碱酯酶的聚类分析显示,褐飞虱的乙酰胆碱酯酶与其中6个Ⅱ型乙酰胆碱酯酶（AChE2）同属一个支系; 此外,只存在于昆虫AChE2中的超变区及特异的氨基酸残基,也存在于褐飞虱的乙酰胆碱酯酶中。以上结果表明,所克隆的褐飞虱的乙酰胆碱酯酶基因是一个与黑腹果蝇的orthologous型基因同源的AChE2基因。     

Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth.

The lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.     

Cloning, sequencing and expression of a novel glutamate decarboxylase gene from a newly isolated lactic acid bacterium, Lactobacillus brevis OPK-3

Lactobacillus brevis OPK-3, having 84.292 mg/L/h of gamma-aminobutyric acid (GABA) productivity, was isolated from Kimchi, a traditional fermented food in Korea. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from the L. brevis OPK-3, using primers based on two highly conserved regions of GAD. A full-length GAD (LbGAD) clone was subsequently isolated through rapid amplification of cDNA ends (RACE) PCR. Nucleotide sequence analysis revealed that the open reading frame (ORF) consisted of 1401 bases and encoded a protein of 467 amino acid residues with a calculated molecular weight of 53.4 kDa and a pI of 5.65. The amino acid sequence deduced from LbGAD ORF showed 83%, 71%, and 60% identity to the Lactobacillus plantarum GAD, Lactococcus lactis GAD, and Listeria monocytogenes GAD sequences, respectively. The LbGAD gene was expressed in Escherichia coli strain UT481, and the extract of transformed E. coli UT481 contained an induced 53.4 kDa protein and had significantly enhanced GAD activity.     

The primary structure of thioredoxin from the filamentous cyanobacterium Anabaena sp. 7119

Thioredoxin from the cyanobacterium Anabaena 7119 serves as electron donor to ribonucleotide reductase and as a protein disulfide reductase. This small, heat-stable protein was found to have structural and functional similarities to thioredoxins from both bacterial and mammalian sources. We here report the complete primary structure of Anabaena thioredoxin. The structure was determined by analysis of peptides obtained after cleavage with cyanogen bromide, Staphylococcus aureus protease, and trypsin. The protein consists of 106 residues with the following amino acid sequence: Ser-Ala-Ala-Ala-Gln-Val-Thr-Asp- Ser-Thr-Phe-Lys-Gln-Glu-Val-Leu-Asp-Ser-Asp-Val-Pro-Val-leu-Val-Asp-Phe- Trp-Ala-Pro-Trp-Cys-Gly-Pro-Cys-Arg-Met-Val-Ala-Pro-Val-Val-Asp-Glu- Ile-Ala-Gln-Gln-Tyr-Glu-Gly-Lys-Ile-Lys-Val-Val-Lys-Val-Asn-Thr-Asp- Glu-Asn-Pro-Gln-Val-Ala-Ser-Gln-Tyr-Gly-Ile-Arg-Ser-Ile-Pro-Thr-Leu- Met-Ile-Phe-Lys-Gly-Gly-Gln-Lys-Val-Asp-Met-Val-Val-Gly-Ala-Val-Pro- Lys-Thr-Thr-Leu-Ser-Gln-Thr-Leu-Glu-Lys-His-Leu. The sequence of Anabaena thioredoxin shows a definite homology to the protein from Escherichia coli, with 49% residue identities occurring in the proteins when aligned at the active site disulfide.