Function of oxygen resistance proteins in the anaerobic,sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough

Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H(2)O(2)) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H(2)O(2) resistance may also be conferred by two other rbr genes in the D. vulgaris genome. Inhibition of Sod activity by azide and H(2)O(2), but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D. vulgaris. Thus, Sor plays a key role in oxygen defense of D. vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.     

Effect of sodium nitrite inhibition on intracellular thiol groups and on the activity of certain glycolytic enzymes in Clostridium perfringens.

Activities of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) (GAP-DH) and aldolase (EC 4.1.2.13) in cells of Clostridium perfringens that had been inhibited with sodium nitrite were investigated. A complete loss in GAP-DH activity and a 67% decrease in aldolase activity were observed when growth of C. perfringens was inhibited. There was also a 91% decrease in the concentration of free sulfhydryl groups of soluble cellular components. Dithiothreitol restored some activity to inactive GAP-DH from sodium nitrite-inhibited cells, indicating that a loss of reduced sulfhydryl groups was involved in the inactivation of the enzyme. The evidence presented suggests that sodium nitrite inhibition of C. perfringens may involve an interaction of sodium nitrite as nitrous acid with sulfhydryl-containing constituents of the bacterial cell.     

Cloning and characterization of a conjugated bile acid hydrolase gene from Clostridium perfringens.

The gene encoding a conjugated bile acid hydrolase (CBAH) from Clostridium perfringens 13 has been cloned and expressed in Escherichia coli, and its nucleotide sequence has been determined. Nucleotide and predicted amino acid sequence analyses indicated that the gene product is related to two previously characterized amidases, a CBAH from Lactobacillus plantarum (40% identity) and a penicillin V amidase from Bacillus sphaericus (34% identity). The product is apparently unrelated to a CBAH from C. perfringens for which N-terminal sequence information was determined. The gene product was purified from recombinant E. coli and used to raise antibody in rabbits. The presence of the protein in C. perfringens was then confirmed by immunoblot analysis. The protein was shown to have a native molecular weight of 147,000 and a subunit molecular weight of 36,100, indicating its probable existence as a tetramer. Disruption of the chromosomal C. perfringens CBAH gene with a chloramphenicol resistance cartridge resulted in a mutant strain which retained partial CBAH activity. Polyacrylamide gel electrophoresis followed by enzymatic activity staining and immunoblotting indicated that the mutant strain no longer expressed the cloned CBAH (CBAH-1) but did express at least one additional CBAH (CBAH-2). CBAH-2 was immunologically distinct from CBAH-1, and its mobility on native polyacrylamide gels was different from that of CBAH-1. Furthermore, comparisons of pH optima and substrate specificities of CBAH activities from recombinant E. coli and wild-type and mutant C. perfringens provided further evidence for the presence of multiple CBAH activities in C. perfringens.     

Effect of sodium nitrite inhibition on intracellular thiol groups and on the activity of certain glycolytic enzymes in Clostridium perfringens.

Activities of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) (GAP-DH) and aldolase (EC 4.1.2.13) in cells of Clostridium perfringens that had been inhibited with sodium nitrite were investigated. A complete loss in GAP-DH activity and a 67% decrease in aldolase activity were observed when growth of C. perfringens was inhibited. There was also a 91% decrease in the concentration of free sulfhydryl groups of soluble cellular components. Dithiothreitol restored some activity to inactive GAP-DH from sodium nitrite-inhibited cells, indicating that a loss of reduced sulfhydryl groups was involved in the inactivation of the enzyme. The evidence presented suggests that sodium nitrite inhibition of C. perfringens may involve an interaction of sodium nitrite as nitrous acid with sulfhydryl-containing constituents of the bacterial cell.     

Cloning and sequencing of beta toxin gene of Clostridium perfringens type C

A gene encoding beta toxin was amplified by polymerase chain reaction from C. perfringens type C isolate and cloned in pUC 19 vector. The nucleotide sequence was identical with C. perfringens type B beta toxin gene sequence. The Southern hybridization using labelled beta toxin gene probe revealed the presence of positive signals only in beta producing C. perfingens.     

Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid.

A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from the C. perfringens replicon pIP404 and the E. coli vector pUC18. The multiple cloning site and lacZ' gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E. coli. Both chloramphenicol and erythromycin resistance can be selected in C. perfringens and E. coli since pJIR418 carries the C. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes from C. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Five-gene cluster in Clostridium thermoaceticum consisting of two divergent operons encoding rubredoxin oxidoreductase- rubredoxin and rubrerythrin-type A flavoprotein- high-molecular-weight rubredoxin

A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C. thermoaceticum.     

甜菜夜蛾核多角体病毒sod基因的克隆及原核表达

甜菜夜蛾核型多角体病毒中国株(Spotoptera exigua MNPV—Z)超氧化物歧化酶基因(sod)业已被克隆及在大肠杆菌中进行了表达,证明了SeMNPV—Z的sod基因产物确有SOD活性,其活力单位约为291．19U／mL培养液。DNA测序结果表明SeMNPV-Z的sod基因编码151个氨基酸,与人的sodl基因的核苷酸的同源性为50％,与LdNPV、HaSNPV、HcNPV、AcNPV和BmNPV的sod基因的同源性分别为64％、63％、63％、65％、63％。     

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 .     

Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli]

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.     

An investigation of the prevalence of the toxigenic types of Clostridium perfringens in horses with anterior enteritis: preliminary results

Equine anterior enteritis is an acute syndrome with unknown aetiology, although salmonellosis and infection with Clostridium perfringens have both been suggested as potential causes. The main aim of this preliminary study was to compare the prevalence of toxigenic types of C. perfringens in clinically healthy horses and in horses with anterior enteritis. From horses admitted with colic at Phillip Leverhulme Large Animal Hospital in 1995-1996, samples of gastric reflux, small intestinal contents and faeces were taken for isolation of C. perfringens. Five of those horses were admitted as anterior enteritis cases, of which C. perfringens was isolated in pure culture in all five horses. Two of the anterior enteritis cases from which viable bacterial counts had been performed revealed 10(6) CFU/g faeces C. perfringens. Samples of gastric reflux and small intestinal contents submitted from one of these horses revealed 10(4) CFU/mL and 10(5) CFU/mL respectively. The number of C. perfringens observed in the gastric reflux was considered significant as the total volume removed was 12 L. The counts observed in faeces taken from horses admitted with anterior enteritis, were significantly higher than the <10(2) CFU/g faeces observed in faeces from healthy horses and horse presenting with colic and with other diagnoses. The major toxigenic types of C. perfringens in both healthy and diseased horses are being investigated using the polymerase chain reaction (PCR) to amplify target DNA sequences of the toxin genes. Primers have been designed from the published DNA sequences of the enterotoxin, alpha, beta, epsilon and iota toxin genes. PCR products obtained from NCTC strains of C. perfringens have been cloned and the sequenced, to verify that the amplicon sequence is correct. Initial typing suggests that C. perfringens type A is the predominant toxin type isolated from healthy horses and horses with colic with other diagnoses.C. perfringens strains isolated from horses with anterior enteritis are of type D.