Two closely linked genes encoding thioredoxin and thioredoxin reductase in Clostridium litorale

The genes encoding thioredoxin and thioredoxin reductase of Clostridium litorale were cloned and sequenced. The thioredoxin reductase gene (trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequence showed 44% identity to the corresponding protein from Escherichia coli. The gene encoding thioredoxin (trxA) was located immediately downstream of trxB. TrxA and TrxB were each encoded by two gene copies, both copies presumably located on the chromosome. Like other thioredoxins from anaerobic, amino-acid-degrading bacteria investigated to date by N-terminal amino acid sequencing, thioredoxin from C. litorale exhibited characteristic deviations from the consensus sequence, e.g., GCVPC instead of WCGPC at the redox-active center. Using heterologous enzyme assays, neither thioredoxin nor thioredoxin reductase were interchangeable with the corresponding proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gly-31 in C. litorale thioredoxin was substituted with Trp (the W in the consensus sequence) by site-directed mutagenesis. The mutant protein was expressed in E. coli and was purified to homogeneity. Enzyme assays using the G31W thioredoxin revealed that Gly-31 was not responsible for the observed incompatibility with the E. coli thioredoxin reductase, but it was essential for activity of the thioredoxin system in C. litorale. Received: 19 September 1996 / Accepted: 21 May 1997     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Nucleotide sequence of the celA gene encoding a cellodextrinase of Ruminococcus flavefaciens FD-1

Summary The nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) fromRuminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region inEscherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CeIA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other oganisms. Two lysozymetype active sites were found in the amino-terminal third of the enzyme. InE. coli the cloned CeIA protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposonphoA mutagenesis experiments indicated that CeIA is secreted by a mechanism other than a leader peptide.Abbreviations CMCase carboxymethylcellulase - celA gene coding for CeIA - CelA cellodextrinase - ORF open reading frame - phoA gene encoding alkaline phosphatase - pNPC p-nitrophenyl--d-cellobioside     

Phospholipase D fromStreptomyces antibioticus: Cloning,sequencing, expression,and relationship to other phospholipases

The extracellular phospholipase D (PLD) gene fromStreptomyces antibioticus was cloned, sequenced, and expressed inEscherichia coli. Analysis of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein was expressed in an insoluble form inE. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCI and 2-mercaptoethanol, subsequent dialysis restored the PLD activity. Comparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larger precursor with a 47-amino-acid N-terminal extension to the mature enzyme of 509 amino acids. The amino acid sequence of the S.antibioticus PLD was extensively compared with other PLDs and phospholipase C (PLC). The deduced amino acid sequence of the cloned PLD was highly homologous to PLDs from S. acidomyceticus andStreptomyces sp., and contained a conserved region with S.chromofuscus PLD. From comparisons of the structural similarity and properties of the various PLDs, a classification of PLDs into two subgroups has been proposed and the highly conserved region designated tentatively region X_PLD, which may be important in the catalytic function, has been identified. The homology comparison between our PLD and phosphatidylinositol-specific phospholipase C (PI-PLC) is also discussed.     

Cloning,characterization and insertional inactivation of theLactobacillus helveticus D(−) lactate dehydrogenase gene

A plasmid, designated pSUW100, encoding the D(-)lactate dehydrogenase [D(-)-LDH; NAD⁺ oxidoreductase, EC 1.1.1.28] fromLactobacillus helveticus CNRZ32 was identified from a genomic library by complementation ofEscherichia coli FMJ39. The D(-)LDH gene was localized by Tn5 mutagenesis and subcloning to a 1.4-kb region of pSUW100. A 2-kbDraI fragment of pSUW100 encoding D(-)LDH activity was subcloned and its nucleotide sequence determined. Analysis of this sequence identified a putative 1,014-bp D(-) LDH open reading frame that encodes a polypeptide of 337 amino acid residues with a deduced molecular mass of 38 kDa. The distribution of homology to the CNRZ32 D(-)LDH gene in several lactic acid bacteria was determined by Southern hybridization using an internal fragment of the D(-)LDH gene as a probe. Hybridization was detected in leuconostocs and pediococci but not in lactococci orLactobacillus casei. An integration plasmid was constructed from pSA3 and a 0.60-kb internal fragment of the D(-)LDH gene. This plasmid was used to construct a D(-)LDH-negative derivative ofL. helveticus CNRZ 32 by gene disruption; this derivative was determined as producing only L(+)lactic acid. No significant difference in growth or total lactic acid production was observed between CNRZ32 and its D(-)LDH mutant.     

Fusion expression of mutated cecropin CMIV in E. coli

A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. coli HB101. The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product. The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.     

A wound-inducible gene fromSalix viminalis coding for a trypsin inhibitor

A gene designatedswin1.1 has been isolated by screening aSalix viminalis genomic library with a heterologous probe,win3 fromPopulus. The region sequenced included the entire coding sequence for a protein with 199 amino acids plus the promoter and terminator. At the 5 end of the coding region is a sequence that encodes a hydrophobic region of 25–30 amino acids, that could form a signal peptide. A putative TATAA box and polyadenylator sequence were identified. Introns were absent. The gene product showed similarities with serine protease inhibitors from the Kunitz family and especially withwin3 from wounded leaves ofPopulus. Southern blot analysis indicated thatswin1.1 is a member of a clustered gene family,swin1. An oligonucleotide corresponding to the putative hypervariable region to-wards the carboxyl end when used as a probe in Southern hybridization showed high specificity forswin1.1. Expression of theswin1.1 gene was enhanced in wounded leaves. Theswin1.1 coding region without the signal sequence was highly expressed inEscherichia coli and the protein showed inhibitory activity against trypsin but at most slight activity against the other proteases tested. A systemically induced protein, SVTI, with inhibitor activity against trypsin, was isolated fromSalix leaves by affinity chromatography on a column of trypsin-Sepharose 4B and N-terminal sequenced. It corresponded with the translatedswin1.1 gene at 16 of the 19 amino acid sites, suggesting that SVTI is encoded by another member of theswin1 gene family.     

Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins,purification and biochemical properties

Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for two thioredoxins (m and h) of Chlamydomonas reinhardtii. Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acids (15 101 Da) containing a transit peptide of 34 amino acids with a very high content of Ala and Arg residues. The sequence for thioredoxin h codes for a 113 amino acid protein with a molecular mass of 11817 Da and no signal sequence. The thioredoxin m gene contains a single intron and seems to be more archaic in structure than the thioredoxin h gene, which is split into 4 exons. The cDNA sequences encoding C. reinhardtii thioredoxins m and h have been integrated into the pET-3d expression vector, which permits efficient production of proteins in Escherichia coli cells. A high expression level of recombinant thioredoxins was obtained (up to 50 mg/l culture). This has allowed us to study the biochemical/biophysical properties of the two recombinant proteins. Interestingly, while the m-type thioredoxin was found to have characteristics very close to the ones of prokaryotic thioredoxins, the h-type thioredoxin was quite different with respect to its kinetic behaviour and, most strikingly, its heat denaturation properties.Abbreviations DTT dithiothreitol - FBPase Fructose 1,6-biphosphate phosphatase - FTR ferredoxin-thioredoxin reductase - IPTG isopropyl thiogalactoside - NADP-MDH NADPH-dependent malate dehydrogenase - NMR nuclear magnetic resonance - NTR NADPH-dependent thioredoxin reductase Dedicated to the memory of Claude Crétin     

Molecular characterization of a cDNA encoding caffeoyl-coenzyme A 3-O-methyltransferase ofStellaria longipes

A cDNA clone encodingS-adenosyl-L-methionine:trans-caffeoyl-CoA 3-O-methyl-transferase (EC 2.1.1.104; CCoAOMT) fromStellana longipes Goldie (long-stalked chick-weed) was isolated and studied. Structural analysis of both the nucleotide sequence and the predicted amino acid sequence suggests that our cloned sequence encoded a CCoAOMT enzyme ofStellaria longipes, which shared overall structural similarity with other plant CCoAOMTs but exhibited certain distinct characteristics. Southern blot hybridization and cloning analyses indicating a small CCoAOMT gene family in theStellana longipes genome and the absence of introns in the coding region of the cDNA-corresponding gene. Sequence variations in the coding region were found among three genotypes from geographically isolated populations. Higher levels of CCoAOMT mRNA were detected in stems and leaves than in roots. The cDNA-encoded protein expressed inEschendia coli was shown to utilize caffeoyl-CoA, but not caffeic acid or 5-hydroxy ferulic acid, as its substrate.     

Identification of Streptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin

A 50 kb region of DNA fromStreptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) fromStreptomyces griseus. Cultures ofEscherichia coli containing plasmids with ORF2, on a 2.1 kbBamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-d-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase fromStreptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism. Dedicated to Professor Satoshi Ōmura, a pioneer in the field of antibiotics, on the occasion of his 60th birthday     

Structure of the beta-1,3-1,4-glucanase gene of Bacillus macerans: homologies to other beta-glucanases

Summary The nucleotide sequence of an 852 base pair (bp) DNA fragment containing the entire gene coding for thermostable beta- 1,3-1,4-glucanase ofBacillus macerans has been determined. ThebglM gene comprises an open reading frame (ORF) of 711 by (237 codons) starting with ATG at position 93 and extending to the translational stop codon TAA at position 804. The deduced amino acid sequence of the mature protein shows 70% homology to published sequences of mesophilic beta- 1,3-1,4-glucanases fromB. subtilis andB. amyloliquefaciens. The sequence coding for mature beta-glucanase is preceded by a putative signal peptide of 25 amino acid residues, and a sequence resembling a ribosome-binding site (GGAGG) before the initiation codon. By contrast with the processed protein, the N-terminal amino acid sequence constituting the putative leader peptide bears no or only weak homology to signal peptides of mesophilicBacillus endo-beta-glucanases. TheB. macerans signal peptide appears to be functional in exporting the enzyme to the periplasm inE. coli. More than 50% of the whole glucanase activity was localized in the periplasmic space and in the supernatant. Whereas homology to endo-1,4-beta-glucanases is completely lacking, a weak amino acid homology between the sequence surrounding the active site of phage T4 lysozyme and a sequence spanning residues 126 through 161 ofB. macerans endo-beta-glucanase could be identified.     

AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)⁺ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP maltose binding protein - PCR polymerase chain reaction - Stress70c the cytosolic member of the 70 kDA family of stress-related proteins     

Cloning and Expression of the Anti tumor Glycoprotein Gene of Streptococcus pyogenes Su in Escherichia coli

The structural gene for the streptococcal acid glycoprotein (SAGP), an antitumor glycoprotein produced by Streptococcus pyogenes Su, was cloned in Escherichia coli, and the nucleotide sequence was determined. The deduced amino acid sequence of SAGP was composed of 410 amino acids, and was highly homologous with colicin E1, a bacterial antimicrobial protein. The SAGP gene was subcloned into an expression plasmid and was expressed in E. coli. The gene product was demonstrated to have almost the same molecular weight and an immuno-chemical property with those of the native SAGP.     

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

Molecular Cloning and Expression of Proteinaceous α-Amylase Inhibitor Gene from Streptomyces nitrosporeus in Escherichia coli

The proteinaceous α-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous α-amylase inhibitors.     

Cloning and expression of a full-length glutamate decarboxylase gene fromLactobacillus brevis BH2

A bacterium (BH2) that was found to produce a large amount of γ-aminobutyric acid (GABA) was isolated fromKimchi, a traditional fermented food in Korea. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that BH2 belonged to the genusLactobacillus brevis. Under controlled conditions in MRS broth (Difco) with 5% monosodium glutamate, this strain produced GABA at a concentration of 194 mM with a 73% GABA conversion rate after 48 h. A full-length glutamate decarboxylase (gad) gene was cloned by the rapid amplification of cDNA ends (RACE) PCR. The open reading frame (ORF) of thegad gene was composed of 1,407 nucleotides and encoded a protein (468 amino acids) with a predicted molecular weight of 53.5 kDa. The deduced amino acid sequence of GAD fromL. brevis showed 97.5 and 82.7% identities to theL. brevis OPK-3 GAD andL. plantarum WCFS1 GAD, respectively. Thegad gene was expressed inEscherichia coli cells and the expression was confirmed by SDS-PAGE analysis and enzyme activity studies.     

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene

Summary The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely –35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.     

Identification, cloning, nucleotide sequence and chromosomal map location of hns, the structural gene for Escherichia coli DNA-binding protein H-NS

Summary Beginning with a synthetic oligonucleotide probe derived from its amino acid sequence, we have identified, cloned and sequenced the hns gene encoding H-NS, an abundant Escherichia coli 15 kDa DNA-binding protein with a possible histone-like function. The amino acid sequence of the protein deduced from the nucleotide sequence is in full agreement with that determined for H-NS. By comparison of the restriction map of the cloned gene and of its neighboring regions with the physical map of E. coli K12 as well as by hybridization of the hns gene with restriction fragments derived from the total chromosome, we have located the hns gene oriented counterclockwise at 6.1 min on the E. coli chromosome, just before an IS30 insertion element.     

Nucleotide sequence of the Escherichia coli hisD gene and of the Escherichia coli and Salmonella typhimurium hisIE region

Summary In this paper we report the nucleotide sequence of the hisD gene of Escherichia coli and of the hisIE region of both E. coli and Salmonella typhimurium. The hisD gene codes for a bifunctional enzyme, l-histidinol: NAD⁺ oxidoreductase, of 434 amino acids with a molecular mass of 46,199 daltons. We established that the hisIE region of both S. typhimurium and E. coli is composed of a single gene and not, as previously believed, of two separate genes. The derived amino acid sequence indicates that the hisIE gene codes for a bifunctional protein of 203 amino acids with an approximate molecular mass of 22,700 daltons. We also determined the nucleotide sequence of a deletion mutant in S. typhimurium which abolishes the hisF and hisI functions but retains the hisE function. We deduced that the mutant produces a chimeric protein fusing the aminoterminal region of the upstream hisF gene to the carboxylterminal domain of the hisIE gene which encodes for the hisE function. In view of these results the structural and functional organization of the histidine operon in enteric bacteria needs to be revised. The operon is composed of only 8 genes and the pathway leading to the biosynthesis of the amino acid requires 11 enzymatic steps.     

Cloning and Characterization of the Gene Encoding Pyrroloquinoline Quinone-dependent Poly (vinyl alcohol) Dehydrogenase of Pseudomonas sp. Strain VM15C

A gene library of poly (vinyl alcohol) (PVA)-degrading Pseudomonas sp. strain VM15C was constructed in Escherichia coli with the vector pUC18. Screening of this library with a chromogenic PVA dehydrogenase assay resulted in the isolation of a clone that carries the gene (pdh) for the PVA dehydrogenase, and the entire nucleotide sequence of its structural gene was determined. The gene encodes a protein of 639 amino acid residues (68,045 Da) and in the deduced amino acid sequence, some putative functional sites, a signal sequence, a heme c-binding site, and a PQQ-binding site, were detected. The amino acid sequence showed low similarity to other types of quinoprotein dehydrogenases. PVA dehydrogenase expressed in E. coli clones required PQQ. Ca²⁺, and Mg²⁺ stimulated the activity. PVA-dependent heme c reduction occurred with exogenous PQQ in cell extracts of the E. coli clone. The PVA dehydrogenase in the E. coli clone was localized in the cytoplasm.