alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.     

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases.

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.     

Cloning, sequence analysis, and expression in Escherichia coli of a streptococcal plasmin receptor.

Plasmin(ogen) receptors are expressed by many gram-positive and gram-negative bacteria. We previously isolated a plasmin receptor from a pathogenic group A streptococcal strain (C. C. Broder, R. Lottenberg, G. O. von Mering, K. H. Johnston, and M. D. P. Boyle, J. Biol. Chem. 266:4922-4928, 1991). The gene encoding this plasmin receptor, plr, was isolated from a lambda gt11 library of chromosomal DNA from group A streptococcal strain 64/14 by screening plaques with antibodies raised against the purified streptococcal plasmin receptor protein. The gene was subcloned by using a low-copy-number plasmid and stably expressed in Escherichia coli, resulting in the production of an immunoreactive and functional receptor protein. The DNA sequence of the gene contained an open reading frame encoding 335 amino acids with a predicted molecular weight of 35,787. Upstream of the open reading frame, putative promoter and ribosomal binding site sequences were identified. The experimentally derived amino acid sequences of the N terminus and three cyanogen bromide fragments of the purified streptococcal plasmin receptor protein corresponded to the predicted sequence encoded by plr. The deduced amino acid sequence for the plasmin receptor protein revealed significant similarity (39 to 54% identical amino acid residues) to glyceraldehyde 3-phosphate dehydrogenases.     

紫杆柽柳与其它物种脂质转运蛋白基因编码蛋白的同源性比较

根据从柽柳cDNA文库克隆获得的脂质转运蛋白(LTP)的部分序列,用RACE技术克隆出其全长cDNA序列.基因的5'非翻译区96bp,3'非翻译区222bp,开放阅读框285bp,编码94个氨基酸,预计蛋白的分子量为9.9 kD,等电点为8.02.此基因有8个位置保守的Cys残基及26个氨基酸的信号肽,为典型的植物脂质转运蛋白基因.其基因序列数据库(GenBank)登录号为AY574218(基因)和AAS79106(蛋白).     

Molecular cloning and nucleotide sequence of a gene for alkaline cellulase from Bacillus sp. KSM-635

A gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2.6 kb and 4.0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2.4 kb region which was indispensable for the production of cellulase, and the flanking, 1.1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a sigma 43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the Bacillus stearothermophilus peroxidase gene (perA).

The gene encoding a thermostable peroxidase was cloned from the chromosomal DNA of Bacillus stearothermophilus IAM11001 in Escherichia coli. The nucleotide sequence of the 3.1-kilobase EcoRI fragment containing the peroxidase gene (perA) and its flanking region was determined. A 2,193-base-pair open reading frame encoding a peroxidase of 731 amino acid residues (Mr, 82,963) was observed. A Shine-Dalgarno sequence was found 9 base pairs upstream from the translational starting site. The deduced amino acid sequence coincides with those of the amino terminus and four peptides derived from the purified peroxidase of B. stearothermophilus IAM11001. E. coli harboring a recombinant plasmid containing perA produced a large amount of thermostable peroxidase which comigrated on polyacrylamide gel electrophoresis with the B. stearothermophilus peroxidase. The peroxidase of B. stearothermophilus showed 48% homology in the amino acid sequence to the catalase-peroxidase of E. coli.     

Cloning and characterization of the iutA gene which encodes ferric aerobactin receptor from marine Vibrio species

The iutA gene from marine Vibrio species SD004, which encoded a ferric aerobactin receptor for the uptake of iron(III), was cloned onto a multicopy plasmid, pUC 18, in Escherichia coli. Identification of the positive clone was achieved on the basis of its deferrization activity and was detected as a halo formation on the chrome azurol S (CAS)-containing selective plate. Nucleotide sequence analysis of the cloned DNA fragment revealed an open reading frame (ORF) which encoded a polypeptide of 706 amino acid residues, and the deduced molecular mass of this polypeptide was 77.906 kD. The amino acid sequence showed a 41% homology with that of the lutA protein from E. coli. The cloned gene was iutA, which encoded the ferric aerobactin receptor. Another incomplete ORF was found 100 bp upstream of the iutA gene, which was homologous (31 out of 49 amino acids) with the C-terminal region of the luc D protein of E. coli. It is suggested that aerobactin biosynthesis and the transport genes are located tandemly on the Vibrio chromosome and may form an aerobactin operon.     

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.     

Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis.

A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7 beta-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The DNA sequence revealed only one large open reading frame composed of 1,902 bp corresponding to 634 amino acid residues. The deduced amino acid sequence contained a potential signal sequence in its amino-terminal region. Expression of the gene for glutaryl 7-ACA acylase was performed in both E. coli and Bacillus subtilis. The enzyme preparations purified from either recombinant strain of E. coli or B. subtilis were shown to be identical with each other as regards the profile of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were composed of a single peptide with the molecular size of 70 kDa. Determination of the amino-terminal sequence of the two enzyme preparations revealed that both amino-terminal sequences (the first nine amino acids) were identical and completely coincided with residues 28 to 36 of the open reading frame. Extracellular excretion of the enzyme was observed in a recombinant strain of B. subtilis.     

Amino acid sequence of pheromone-inducible surface protein in Enterococcus faecalis, that is encoded on the conjugative plasmid pPD1

The major pheromone-inducible protein, PD78, believed to contribute to bacterial conjugation, was purified from Enterococcus (formerly Streptococcus) faecalis cells containing the plasmid pPD1. A cloned EcoRI-BglII 3.6-kbp fragment of the plasmid pAM351(pPdl::Tn916) contained an open reading frame corresponding to 467 amino acid residues representing PD78. In a central region of the deduced protein, there is a repeated sequence of X-X-Pro that is repeated 15 times. This is analogous to the Gln-Gln-Pro repeat in the C-terminal region of TraD product encoded on the R100 plasmid in Escherichia coli.     

Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85 degrees C and the enzyme exhibited a high level of heat stability.     

Cloning, expression and sequencing the molybdenum-pterin binding protein (mop) gene of Clostridium pasteurianum in Escherichia coli

mop is the structural gene for the molybdenum-pterin binding protein, which is the major molybdenum binding protein in Clostridium pastuerianum. The mop gene was detected by immunoscreening genomic libraries of C. pastuerianum and identified by determining the nucleotide sequence of the cloned insert of clostridial DNA. The deduced amino acid sequence of an open reading frame proved to be identical to the first twelve residues of purified Mop. The DNA sequence flanking the mop gene contains promoter-like consensus sequences which are probably responsible for the expression of Mop in Escherichia coli. The deduced amino acid composition shows that the protein is hydrophobic, lacks aromatic and cysteine residues and has a calculated molecular weight of 7,038. The N-terminal amino acid sequence of Mop has sequence homology with DNA binding proteins. The pattern and type of residues in the N-terminal region suggest it forms the helix-turn-helix structure observed in DNA binding proteins. We propose that Mop may be a regulatory protein binding the anabolic source of molybdenum.     

Cloning and expression of a cDNA encoding the laccase from Schizophyllum commune

We cloned and analyzed the nucleotide sequence of a cDNA that encodes polyphenol oxidase (laccase) from the white-rot basidiomycete Schizophyllum commune. The nucleotide sequence of the full-length cDNA contained a 1554-base open reading frame that encoded a polypeptide of 518 amino acid residues, including a putative signal peptide of 16 residues. It contained four highly similar regions that are conserved in the deduced amino acid sequences of other laccases, including the region thought to be involved in copper binding. Aspergillus sojae strain 1860 (which has low protease levels) was transformed with the plasmid lacAL/pTPT, which contained the laccase gene under the control of the tannase promoter from Aspergillus oryzae. Laccase was secreted into the medium when transformants A1 and A2 were cultured in tannic acid-containing medium.     

Characterization of the Superoxide dismutase gene and its upstream region from Methanobacterium thermoautotrophicum Marburg

Abstract A gene ( sod ) encoding Superoxide dismutase (SOD) was isolated from the strictly anaerobic archaeon Methanobacterium thermoautotrophicum Marburg. Its identity was confirmed by functional complementation of an Escherichia coli mutant strain lacking SOD activity and by DNA sequence analysis of a cloned fragment. Upstream of sod , separated by a 5-bp intergenic region, lies the open reading frame orfk which potentially codes for a protein of 209 amino acid residues. The amino acid sequence for this presumptive product had a similarity coefficient of 55.5% to a subunit of the alkyl hydroperoxide reductase (encoded by the ahpC gene) from Salmonella typhimurium .     

Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli

A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp. aizawai IPL7 was cloned from a 78-kb plasmid. The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions. The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues. Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions. It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli. The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene. Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein.     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding monovalent cation-activated levodione reductase from Corynebacterium aquaticum M-13

The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35% identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH-binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two-column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K+, Na+, and NH4+, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.     

Cloning, sequencing, expression and characterization of the manganese superoxide dismutase gene from Vibrio alginolyticus.

The sodA gene coding for manganese superoxide dismutase from the marine microorganism Vibrio alginolyticus was cloned, sequenced and over-expressed in Escherichia coli using the pET20b (+) expression vector. The full-length gene was consisted of 603bp open reading frame, which encoded a polypeptide of 201 amino acid residues, with a calculated molecular weight of 22672Da. The deduced amino acid sequence of the sodA showed considerable homology to other Mn-SODs. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by the metal ion affinity chromatography. The recombinant VAMn-SOD resisted thermo-denaturation up to 60 degrees C and was insensitive to inhibitors such as H2O2, NaN3 and diethyldithiocarbamic acid.     

Nucleotide and deduced amino acid sequence of the E1 alpha subunit of human liver branched-chain alpha-ketoacid dehydrogenase

A 1552-bp cDNA for the E1 alpha subunit of branched-chain alpha-ketoacid dehydrogenase (BCKDH) was isolated from a human liver cDNA library. The cDNA contained a 1134-bp open reading frame that encoded 378 amino acid (aa) residues of the enzyme and 418 bp of 3'-untranslated sequence. The deduced amino acid sequence of the human protein shows 96% identity with that of the rat enzyme subunit. Those 117-aa residues surrounding the phosphorylation sites are completely conserved between man and rat. BCKDH E1 alpha showed considerable amino acid sequence similarity with pyruvate dehydrogenase E1 alpha, particularly in the region of the two principal phosphorylation sites of these proteins. Northern blots of human liver and skin fibroblasts demonstrated a single 1.8-kb mRNA band, with a higher level of E1 alpha mRNA in liver than in normal fibroblasts. Fibroblasts from a patient with thiamine-responsive maple syrup urine disease (MSUD) contained an mRNA of the same size and abundance as that of normal fibroblasts. Genomic DNA from normal and MSUD fibroblasts gave the same restriction maps on Southern blots, and the gene was approximately 10-kb in size.