Molecular cloning, sequencing, and expression of the genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca

The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase genes under control of their own promoter. Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10-18 base pairs. The sequential three open reading frames from the second to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (gamma), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.     

Cloning and sequencing of a gene encoding D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 and expression of the gene in Escherichia coli

The gene encoding the D-aminoacylase of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was cloned and its complete nucleotide sequence was identified. The D-aminoacylase structural gene consists of 1452 nucleotides and encodes 484 amino acid residues. The molecular weight of D-aminoacylase was calculated to be 51,918. This value agreed well with the apparent molecular weight of 52,000 found for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The N-terminal amino acid sequence (NH2-SQSDSQPFDLLRAG-) predicted by the nucleotide sequence exactly matched those of the purified D-aminoacylase both from Alcaligenes A-6 and from cloned Escherichia coli (E. coli), with the exception of the removal of the N-terminal methionine processed after translation. The purified recombinant enzyme showed almost the same enzymatic properties as the native enzyme from Alcaligenes A-6. Alcaligenes A-6 D-aminoacylase showed 25-29% homology with L-aminoacylases from Bacillus stearothermophilus, porcine and humans.     

Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase

The cDNA encoding vanillyl-alcohol oxidase (EC 1.1.3.7) was selected from a cDNA library constructed from mRNA isolated from Penicillium simplicissimum CBS 170.90 grown on veratryl alcohol by immunochemical screening. The vaoA-cDNA nucleotide sequence revealed an open reading frame of 1680 base pairs encoding a 560-amino acid protein with a deduced mass of 62,915 Da excluding the covalently bound FAD. The deduced primary structure shares 31% sequence identity with the 8alpha-(O-tyrosyl)-FAD containing subunit of the bacterial flavocytochrome p-cresol methyl hydroxylase. The vaoA gene was isolated from a P. simplicissimum genomic library constructed in lambdaEMBL3 using the vaoA-cDNA as a probe. Comparison of the nucleotide sequence of the vaoA gene with the cDNA nucleotide sequence demonstrated that the gene is interrupted by five short introns. Aspergillus niger NW156 prtF pyrA leuA cspA transformed with the pyrA containing plasmid and a plasmid harboring the complete vaoA gene including the promoter and terminator was able to produce vaoA mRNA and active vanillyl-alcohol oxidase when grown on veratryl alcohol and anisyl alcohol. A similar induction of the vaoA gene was found for P. simplicissimum, indicating that similar regulatory systems are involved in the induction of the vaoA gene in these fungi. Introduction of a consensus ribosome binding site, AGAAGGAG, in the vaoA-cDNA resulted in elevated expression levels of active vanillyl-alcohol oxidase from the lac promoter in Escherichia coli TG2. The catalytic and spectral properties of the purified recombinant enzyme were indistinguishable from the native enzyme.     

N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression

The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.     

Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin-dependent Na+ pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli

1. The primary sodium-ion pump glutaconyl-CoA decarboxylase (GCD) from Acidaminococcus fermentans is composed of four subunits: GCDA, the carboxytransferase (65 kDa), GCDB, the carboxylyase (36 kDa), GCDC, the biotin carrier (24 kDa) and GCDD (14 kDa) of unknown function. A genomic library of A. fermentans was screened with an antiserum raised against whole GCD. A clone giving the strongest reaction in an immunoassay contained a 12-kbp genomic fragment from A. fermentans and was analysed further. An oligonucleotide deduced from the N-terminus of GCDA was used for probing the corresponding gene gcdA. It is 1761 bp in length and encodes for a protein of 64.3 kDa. Both partial amino acid sequences obtained from GCDA, the N-terminus as well as an internal tryptic peptide, were detected in the open reading frame (ORF) of gcdA. 2. Sequencing of the flanking regions revealed three adjacent ORF (ORF1-3) which do not code for any of the peptide sequences known of the other GCD subunits. The ORF downstream of gcdA (ORF3) is followed by hgdA and hgdB coding for 2-hydroxyglutaryl-CoA dehydratase, the preceding enzyme of the pathway of glutamate fermentation. Our results suggest that at least these three genes of the hydroxyglutarate pathway are organised in an operon and that the genes of the other GCD subunits from which peptide sequences are known (GCDB and GCDC) are not located adjacent to gcdA. 3. gcdA was amplified from genomic DNA using the polymerase chain reaction and cloned into the expression vector pJF118HE. Active GCDA subunit (up to 2.8 nkat/mg protein), catalysing the biotin-dependent formation of crotonyl-CoA from glutaconyl-CoA, was obtained in cell-free extracts of Escherichia coli DH5 alpha by moderately inducing the tac promoter of pJF118HE with 25-100 microM isopropyl-1-thio-beta-D-galactoside. Strong induction (1 mM isopropyl-1-thio-beta-D-galactoside) led to the formation of inclusion bodies from which GCDA could not be reactivated. The apparent Km = 51 mM for free biotin of the expressed GCDA subunit with V = 1.9 nkat/mg protein is similar to that of butanol-treated GCD composed of GCDA and GCDC (apparent Km = 40 mM). Biocytin was found to be a somewhat better carboxy acceptor for the expressed GCDA subunit (apparent Km = 13 mM; V = 1.0 nkat/mg protein). 4. Native GCD and expressed GCDA were treated with 2 mM N-ethylmaleimide showing different kinetics of inactivation: GCD lost half of its activity within 6 min, whereas expressed GCDA required 21 min.     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans

The gene chiA, which codes for endochitinase, was cloned from a soilborne Enterobacter agglomerans. Its complete sequence was determined, and the deduced amino acid sequence of the enzyme designated Chia_Entag yielded an open reading frame coding for 562 amino acids of a 61-kDa precursor protein with a putative leader peptide at its N terminus. The nucleotide and polypeptide sequences of Chia_Entag showed 86.8 and 87.7% identity with the corresponding gene and enzyme, Chia_Serma, of Serratia marcescens, respectively. Homology modeling of Chia_Entag's three-dimensional structure demonstrated that most amino acid substitutions are at solvent-accessible sites. Escherichia coli JM109 carrying the E. agglomerans chiA gene produced and secreted Chia_Entag. The antifungal activity of the secreted endochitinase was demonstrated in vitro by inhibition of Fusarium oxysporum spore germination. The transformed strain inhibited Rhizoctonia solani growth on plates and the root rot disease caused by this fungus in cotton seedlings under greenhouse conditions.     

Cloning, expression, and sequencing of a protease gene from Bacteroides forsythus ATCC 43037 in Escherichia coli

We have isolated and characterized an N-benzoyl-Val-Gly-Arg-p-nitroanilide-specific protease gene, designated prtH, from Bacteroides forsythus ATCC 43037. Nucleotide sequencing of the DNA insert from the clone (hereafter referred to as clone FST) revealed that the protease activity corresponded to an open reading frame consisting of 1,272 bp coding for a 47.8-kDa protein. When plasmid pFST was used as a probe in Southern hybridization, Sau3AI-digested chromosomal DNA of B. forsythus ATCC 43037 as well as the chromosomal DNAs of the isolated strains Ta4, TR5, and YG2 showed 0.6- and 0.8-kb hybridizing bands. The cell-free extracts of clone FST showed hemolytic activity on human blood cells. The hydrolytic activity of cell extracts of the pFST clone was inhibited by p-toluenesulfonyl-L-lysine chloromethyl ketone hydrochloride, leupeptin, N-ethylmaleimide, iodoacetic acid, iodoaceteamide, and EDTA.     

Seryl-tRNA synthetase from the extreme halophile Haloarcula marismortui--isolation, characterization and sequencing of the gene and its expression in Escherichia coli

A longitudinal characterization of immune cell subpopulations (lymphocytes, CD4+ and CD8+ cells), of routine haematological parameters and of immunoglobulin serum levels was carried out in newborn Macaca fascicularis starting from 1 week up to 1 year of life. In neonates, the percentage of CD4+ lymphocytes is almost double, while the percentage of CD8+ cells is lower than that found in adult monkeys (> 5-years old). An inverted trend in the percentage of the two T-lymphocyte subpopulations was observed during the weeks following birth, with a progressive increase of circulating CD8+, paralleled by a decrease of CD4+ cell number. Consequently, the CD4/CD8 ratio slowly decreases, even if, at 12 months of life, it is still higher than that found in adult animals. Several differences were also noted between young and adult monkeys with regard to the total number of circulating CD4+ and CD8+ cells. Haematological parameters did not show consistent differences with respect to adult values. The plasma IgG level is high at birth, then decreases until 6 months of life, while the IgM and IgA values are very low during the first weeks of life but increase in the following period. Our data showed that variations of immunological (CD4+, CD8+ cells) patterns and of some haematological parameters in M. fascicularis are dependent on age. These variations should be therefore considered whenever young animals are used in experimental protocols.     

Cloning and sequencing of a gene from the archaeon Pyrococcus furiosus with high homology to a gene encoding phosphoenolpyruvate synthetase from Escherichia coli

A gene from the hyperthermophilic archaeon Pyrococcus furiosus, strain Vc1 (DSM 3638), contains an 817-amino-acid open reading frame which shows 42% identity to the phosphoenolpyruvate (PEP) synthetase of Escherichia coli. This putative P. furiosus PEP synthetase is slightly larger than the E. coli enzyme, the region between residues 58 and 89 being absent from the latter.     

Molecular cloning of levan fructotransferase gene from Arthrobacter nicotinovorans GS-9 and its expression in Escherichia coli

The gene encoding an extracellular levan fructotransferase, designated the lft gene, was cloned from the genomic DNA of Arthrobacter nicotinovorans GS-9, and expressed in Escherichia coli. It was found that a single open reading frame consisted of 1554 base pairs that encoded a polypeptide composed of a signal peptide of 33 amino acids and a mature protein of 484 amino acids (M(r) 53,152), and it was also found that a putative ribosome-binding site was present in the upstream from the ORF. The primary structure had no significant similarity with those of inulin fructotransferases, but had low similarity to the catalytic regions of other fructosylhydrolases. The expression of the lft gene was increased on a plasmid, pLFT-BB1, in which the lft gene was fused with alpha-peptide of the lacZ gene of pUC18. An E. coli transformant carrying pLFT-BB1 expressed six times as much activity of levan fructotransferase as that of the original strain, A. nicotinovorans GS-9.     

Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants

Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA. Endonuclease III is encoded by the nth gene. Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics. The gene nei is located at 16 min on the E. coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins. A nei partial deletion replacement mutant was constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis. nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo). Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type. Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay. In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type.     

Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.     

Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression

The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli. The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E. coli chromosome.     

The F420H2-dehydrogenase from Methanolobus tindarius: cloning of the ffd operon and expression of the genes in Escherichia coli

Acute desensitization of contraction and its relative mechanisms have been studied in smooth muscle cells isolated from guinea pig stomach. Desensitization was induced by pre-exposure of the cells to one of the excitatory neuropeptides linked to the phospholipase C intracellular cascade, i.e., cholecystokinin (CCK), gastrin-releasing peptide, and Substance P. Desensitization was homologous after a 30-s pre-exposure and heterologous if pre-exposure lasted for 5 min or longer. Homologous desensitization was studied in a more detailed way after pre-exposure to CCK. Preincubation with increasing concentrations of CCK (10 pM-1 microM) induced a progressive rightward shift of the dose-response curves associated with both a decrease in potency (ED50 4.5 pM-2.2 nM) and a maximum response that were not related to a modification of response kinetics. After brief pre-exposure to 1 nM CCK (Dmax), an inhibition of contraction was observed in response to an identical dose of CCK (45.1 +/- 8.6%), the decreased response being associated with an inhibition of inositol phosphates and [Ca++]i mobilization. Both inositol trisphosphate (InsP3)-induced contraction and [Ca++]i mobilization were inhibited to a lesser extent than CCK-induced responses. Any longer pre-exposure of cells to one of the above-mentioned neuropeptides caused heterologous desensitization, with an observed inhibition of contraction in response to all tested agonists (CCK, 60.3 +/- 5.9%; gastrin-releasing peptide: 56.7 +/- 3. 5%; Substance P, 60.6 +/- 6.5%). A similar decrease was observed in InsP3-induced contractions resulting in a desensitization of the InsP3 response as well. Full recovery of contractile responses appeared within 30 min from the end of preincubation, thus indicating that degradation of membrane receptors did not occur. Although pre-exposure of the cells to protein kinase C inhibitor GF109203X did not modify CCK-induced homologous desensitization, it blocked CCK-induced heterologous desensitization. This study demonstrates that excitatory phospholipase C-coupled enteric neuropeptides induce a time-dependent homologous as well as heterologous desensitization of smooth muscle contraction occurring at receptor and postreceptor levels.     

Molecular cloning and sequencing of three granulocytic Ehrlichia genes encoding high-molecular-weight immunoreactive proteins

Granulocytic Ehrlichia was isolated from canine blood obtained from animals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.