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E L Barrett

Publications and source records attributed to E L Barrett.

At least 19 recordsLinked to original sources
PubMed1995

Sequence analysis of the phs operon in Salmonella typhimurium and the contribution of thiosulfate reduction to anaerobic energy metabolism.

The phs chromosomal locus of Salmonella typhimurium is essential for the dissimilatory anaerobic reduction of thiosulfate to hydrogen sulfide. Sequence analysis of the phs region revealed a functional operon with three open reading frames, designated phsA, phsB, and phsC, which encode peptides of 82.7, 21.3, and 28.5 kDa, respectively. The predicted products of phsA and phsB exhibited significant homology with the catalytic and electron transfer subunits of several other anaerobic molybdoprotein oxidoreductases, including Escherichia coli dimethyl sulfoxide reductase, nitrate reductase, and formate dehydrogenase. Simultaneous comparison of PhsA to seven homologous molybdoproteins revealed numerous similarities among all eight throughout the entire frame, hence, significant amino acid conservation among molybdoprotein oxidoreductases. Comparison of PhsB to six other homologous sequences revealed four highly conserved iron-sulfur clusters. The predicted phsC product was highly hydrophobic and similar in size to the hydrophobic subunits of the molybdoprotein oxidoreductases containing subunits homologous to phsA and phsB. Thus, phsABC appears to encode thiosulfate reductase. Single-copy phs-lac translational fusions required both anaerobiosis and thiosulfate for full expression, whereas multicopy phs-lac translational fusions responded to either thiosulfate or anaerobiosis, suggesting that oxygen and thiosulfate control of phs involves negative regulation. A possible role for thiosulfate reduction in anaerobic respiration was examined. Thiosulfate did not significantly augment the final densities of anaerobic cultures grown on any of the 18 carbon sources tested. on the other hand, washed stationary-phase cells depleted of ATP were shown to synthesize small amounts of ATP on the addition of the formate and thiosulfate, suggesting that the thiosulfate reduction plays a unique role in anaerobic energy conservation by S typhimurium.

Amino Acid Sequence↗
PubMed1991

Sequence analysis and expression of the Salmonella typhimurium asr operon encoding production of hydrogen sulfide from sulfite.

A chromosomal locus of Salmonella typhimurium which complements S. typhimurium asr (anaerobic sulfite reduction) mutants and confers on Escherichia coli the ability to produce hydrogen sulfide from sulfite was recently cloned (C. J. Huang and E. L. Barrett, J. Bacteriol. 172:4100-4102, 1990). The DNA sequence and the transcription start site have been determined. Analysis of the sequence and gene products revealed a functional operon containing three genes which have been designated asrA, asrB, and asrC, encoding peptides of 40, 31, and 37 kDa, respectively. The predicted amino acid sequences of both asrA and asrC contained arrangements of cysteines characteristic of [4Fe-4S] ferredoxins. The sequence of asrB contained a typical nucleotide-binding region. The sequence of asrC contained, in addition to the ferredoxinlike cysteine clusters, two other cysteine clusters closely resembling the proposed siroheme-binding site in biosynthetic sulfite reductase. Expression of lacZ fused to the asr promoter was repressed by oxygen and induced by sulfite. Analysis of promoter deletions revealed a region specific for sulfite regulation and a second region required for anaerobic expression. Computer-assisted DNA sequence analysis revealed a site just upstream of the first open reading frame which had significant homology to the FNR protein-binding site of E. coli NADH-linked nitrite reductase. However, asr expression by the fusion plasmid was not affected by site-specific mutations within the apparent FNR-binding site.

Amino Acid Sequence↗
PubMed1990

Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium.

Transposon Tn5 insertions causing anaerobic cysteine auxotrophy were isolated from a Salmonella typhimurium cysI parent (auxotrophic under aerobic but not anaerobic conditions). Insertions in one mutant group appeared to be in cysG. A second group of insertions, designated asr (anaerobic sulfite reduction), were located near map unit 53 on the S. typhimurium chromosome. They did not cause aerobic or anaerobic auxotrophy in a cys1+ background but did prevent dissimilatory sulfite reduction. Plasmids containing asr DNA cloned from wild-type S. typhimurium conferred anaerobic prototrophy and the ability to produce hydrogen sulfide from sulfite on an Escherichia coli cys1 mutant.

Anaerobiosis↗
PubMed1989

Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase.

Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions (E. L. Barrett and G. W. Chang, J. Gen. Microbiol., 115:513-516, 1979). Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (less than 0.33%). It was also prevented by the presence of 0.1 mM nitrate, which argues against a strictly biosynthetic role for anaerobic sulfite reduction. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (10(-7)M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10(-7)M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADPH and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. Two high-molecular-weight proteins separated by gel filtration (Mr 360,000 and 490,000, respectively) were required for maximal activity with NADH. Indirect evidence, including in vitro complementation experiments with a cysG mutant extract, suggested that the 360,000-Mr component contains siroheme and is the terminal reductase. This component was further purified to near homogeneity and was found to consist of a single subunit of molecular weight 67,500. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function.

Anaerobiosis↗
PubMed1988

Formate-nitrate respiration in Salmonella typhimurium: studies of two rha-linked fdn genes.

Localized mutagenesis was used to obtain rha-linked mutations in Salmonella typhimurium, resulting in defects in the nitrate reductase-linked formate dehydrogenase (FDHN). The fdn mutants obtained fell into two groups which differed in several respects. Group I isolates lacked FDHN activity under all conditions examined and exhibited wild-type levels of the hydrogenase-linked formate dehydrogenase (FDHH). Group II isolates appeared defective in FDHN only when freshly prepared extracts were assayed; restoration of both FDHN and formate-nitrate reduction activity occurred on incubation of extracts for 2 to 3 h. Protease inhibitors prevented restoration. Group II isolates were also characterized by a conditional FDHH activity; this activity was absent unless the growth medium designed to optimize wild-type FDHH was altered either by lowering glucose concentration or by adding thiosulfate. Cotransduction of fdn with rha ranged from 4 to 22% for the group I isolates and from 20 to 40% for the group II isolates. Temperature-sensitive isolates from both groups synthesized FDHN activity with altered thermostability. In vitro complementation occurred in mixed extracts of amber mutants of the two respective classes. The results are consistent with two distinct rha-linked fdn genes, for which we suggest using the designations fdnB (group I) and fdnC (group II).

Anaerobiosis↗
PubMed1987

The phs gene and hydrogen sulfide production by Salmonella typhimurium.

Salmonella typhimurium produces H2S from thiosulfate or sulfite. The respective pathways for the two reductions must be distinct as mutants carrying motations in phs, chlA, and menB reduced sulfite, but not thiosulfate, to H2S, and glucose repressed the production of H2S from thiosulfate while it stimulated its production from sulfite. The phs and chlA mutants also lacked a methyl viologen-linked thiosulfate reductase activity present in anaerobically grown wild-type cultures. A number of hydroxylamine, transposon Tn10 insertion, and Mu d1(Apr lac) operon fusion mutants defective in phs were characterized. One of the hydroxylamine mutants was an amber mutant, as indicated by suppression of its mutation in a supD background. The temperature-sensitive phs mutants produced H2S and methyl viologen-linked thiosulfate reductase at 30 degrees C but not at 42 degrees C. The reductases in all such mutants grown at 30 degrees C were as thermostable as the wild-type enzyme and did not differ in electrophoretic relative mobility, suggesting that phs is not the structural gene for thiosulfate reductase. Expression of beta-galactosidase in phs::Mu d1(Apr lac) mutants was dependent on anaerobiosis and the presence of reduced sulfur. It was also strongly influenced by carbon source and growth stage. The results are consistent with a model in which the phs gene encodes a regulatory protein essential for the reduction of thiosulfate to hydrogen sulfide.

Genes, Bacterial↗
PubMed1986

Pseudomonas fluorescens biovar V: its resolution into distinct component groups and the relationship of these groups to other P. fluorescens biovars, to P. putida, and to psychrotrophic pseudomonads associated with food spoilage.

A numerical taxonomic analysis was performed to evaluate the appropriateness of a single biovar designation (biovar V) for all Pseudomonas fluorescens isolates negative for denitrification, levan production and phenazine pigmentation and to determine the relationship of biovar V strains to other taxa within the same Pseudomonas RNA homology group. Seventy-two strains assigned to P. fluorescens biovar V and four strains of P. fragi were characterized and the data subjected to a numerical taxonomic analysis along with comparable data for 17 previously characterized strains of this biovar and 89 P. putida strains. Seven distinct biovar V clusters containing three or more strains were revealed, and the carbon sources useful for their differentiation were identified. Cluster 1 (38 strains) closely resembled two atypical P. fluorescens I strains. It was also related to P. fluorescens biovar IV and to P. fragi. Cluster 2 (5 strains) was related to cluster 1. Cluster 3 (7 strains) was identical to a major group of meat spoilage psychrotrophic pseudomonads (P. lundensis). Cluster 4 (3 strains) was not related to any other group examined. Cluster 5 consisted of six isolates initially designated P. putida A along with four P. fluorescens biovar V strains all of which resembled P. putida more than they resembled the other P. fluorescens groups. Cluster 6 (16 strains) was distinct from the other biovar V clusters, but was closely related to P. fluorescens biovars I and II. Cluster 7 (3 strains) shared many characteristics with cluster 5. Separate P. fluorescens biovar designations are proposed for cluster 6 and for the combined clusters 1 and 2. A new P. putida biovar is proposed for the combined clusters 5 and 7.

Food Contamination↗
PubMed1985

Bacterial reduction of trimethylamine oxide.

Trimethylamine oxide, which is found in relatively high concentrations in the tissues of marine animals, serves as an electron acceptor in the anaerobic metabolism of a number of bacteria associated primarily with three environments: the marine environment (e.g. Alteromonas and Vibrio), the brackish pond (nonsulfur photosynthetic bacteria), and animal intestines (Enterobacteriaceae). Its reduction to trimethylamine by such bacteria can constitute a major spoilage reaction during the storage of marine fish. In the Enterobacteriaceae, anaerobic respiration with TMAO has been shown to support oxidative phosphorylation. Electron transport to TMAO in these bacteria involves flavin nucleotides, menaquinones, both b- and c-type cytochromes, and a molybdoenzyme reductase. Formate, hydrogen, lactate, and glycerol all serve as electron donors for TMAO respiration. Electrophoretically distinct constitutive and TMAO-induced reductases are synthesized by both E. coli and S. typhimurium. Electron transport to TMAO is repressed both by air and by nitrate. A number of genes involved in TMAO respiration have been mapped, but the structural gene for the inducible TMAO reductase has not yet been firmly established. Oxidative phosphorylation is also supported by TMAO reduction in Alteromonas. In this organism, which is nonfermentative, TMAO respiration resembles aerobic respiration in that intermediates of the TCA cycle are excellent electron donors. Alteromonas exhibits a requirement for NaCl for growth on TMAO and certain electron donors. As in the Enterobacteriaceae, air and nitrate both interfere with TMAO reduction. The role of TMAO reduction in the anaerobic metabolism of nonsulfur purple bacteria has not yet been resolved; it is not clear if TMAO serves simply as an accessory oxidant for fermentation or if TMAO reduction is associated with energy-yielding membrane-bound electron transport. Some of the confusion regarding this bacterial group stems from the fact that much of the work to date has involved parallel studies of TMAO and dimethyl sulfoxide reduction, and it is not yet known whether the two compounds are reduced by the same enzyme. Although our understanding of bacterial TMAO reduction lags far behind our knowledge of bacterial nitrate reduction, it is unlikely that this will always be the case.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteria↗
PubMed1984

Anaerobiosis, formate, nitrate, and pyrA are involved in the regulation of formate hydrogenlyase in Salmonella typhimurium.

Three groups of mutants defective in the fermentative production of gas were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). One group consisted of strains which lacked hydrogenase. The mutation site for this group was located in the vicinity of the known hyd gene. A second group consisted of mutants which lacked the formate dehyrogenase associated with hydrogenase. The mutation site was located in four of them. It was not in the vicinity of the previously described fhlD gene but was instead located at 93 min on the Salmonella map. The third mutant group, which consisted of strains that produced gas in triple sugar iron agar but not in nutrient agar supplemented with glucose, appeared to be pyrA mutants. The insertion site was located in the vicinity of pyrA , and they required arginine and pyrimidines for growth. Expression of the lac operon in the hyd mutants was induced by anaerobiosis. It was only slightly increased by the addition of formate under anaerobic conditions and slightly decreased by the addition of nitrate. Nitrate had no effect in an hyd ::Mu d strain that also carried a chlC::Tn10 insertion. Full expression of the lac operon in the fhl mutants required both formate and anaerobic conditions. The presence of nitrate in addition to formate resulted in activities about half those obtained in its absence, even in the fhl ::Mu d chlC::Tn10 double mutant. In the absence of formate, nitrate reduced expression only in the fhl ::Mu d single mutants. Expression of the lac operon among the pyrA mutants was repressed by arginine and cytosine and also by anaerobiosis. An explanation for the involvement of pyrA in aerobic and anaerobic energy metabolism is proposed.

Aerobiosis↗
PubMed1984

Map locations and functions of Salmonella typhimurium men genes.

Menaquinone (men) mutants of Salmonella typhimurium isolated on the basis of their inability to produce trimethylamine were characterized with respect to mutation site, the ability to cross-feed each other and be cross-fed by known Escherichia coli men mutants, and response to intermediates of the menaquinone biosynthetic pathway. Cross-feeding tests were based on the requirement of menaquinone for hydrogen sulfide production. Genotypes corresponding to the menA, B, C, D, and possibly E genes described in E. coli were all identified. Additional studies of deletions in the menBCD area revealed that this cluster lies between ack/pta and glpT, as in E. coli. The ack and pta mutants were also defective in the production of trimethylamine and failed to produce gas in the absence of added formate.

Chromosome Deletion↗
PubMed1983

Roles for menaquinone and the two trimethylamine oxide (TMAO) reductases in TMAO respiration in Salmonella typhimurium: Mu d(Apr lac) insertion mutations in men and tor.

Three groups of mutants defective in trimethylamine oxide (TMAO) reduction were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). Mutants were identified by their acidic reaction on a modified MacConkey-TMAO medium. Group I consisted of pleiotropic chlorate-resistant mutants which were devoid of TMAO reductase activity. None expressed the lac operon. Group II mutants were partially defective in TMAO reductase. Electrophoretic studies revealed that they lacked the inducible TMAO reductase, but retained the constitutive activity. The genotypic designation tor was suggested for these mutants. The tor mutation in one was located between 80 and 83 U on the S. typhimurium chromosome. Expression of the lac operon in these mutants was not affected by air, TMAO, or nitrate. Group III mutants reduced little or no TMAO in vivo, but their extracts retained full capacity to reduce it with methyl viologen. These mutants also failed to produce hydrogen sulfide from thiosulfate and could not grow anaerobically on glycerol-fumarate. Two subgroups were distinguished. Vitamin K5 restored wild-type phenotype in subgroup IIIa only; vitamin K1 restored wild-type phenotype in both IIIa and IIIb isolates. The genotypic designation men (menaquinone) was suggested for group III isolates. The mutation in IIIa mutants was cotransducible with glpT, which corresponds to the menBCD site in Escherichia coli. That in IIIb mutants was cotransducible with glpK, which corresponds to the menA site in E. coli. Expression of the lac operon in IIIa, but not IIIb, mutants was repressed by air. An additional mutant group isolated on the same medium consisted of strains defective in formate hydrogenlyase.

Chromosome Mapping↗
PubMed1983

Purification and properties of trimethylamine oxide reductase from Salmonella typhimurium.

The major inducible trimethylamine oxide reductase was purified from Salmonella typhimurium LT2. The molecular weights of the native enzyme were estimated to be 332,000 by gel filtration and 170,000 by nondenaturing disc gel electrophoresis. In sodium dodecyl sulfate-gel electrophoresis, the enzyme formed a single band of molecular weight 84,000. The isoelectric point was 4.28. Maximum activity was at pH 5.65 and 45 degrees C. Reduced flavin mononucleotide, but not reduced flavin adenine dinucleotide, served as an electron donor. The Km for trimethylamine oxide was 0.89 mM and Vmax was 1,450 U/mg of protein. The enzyme reduced chlorate with a Km of 2.2 mM and a Vmax of 350 U/mg of protein.

Chlorates↗
PubMed1983

Production of 2,3-butylene glycol from whey by Klebsiella pneumoniae and Enterobacter aerogenes.

Production of 2,3-butylene glycol from whey with Klebsiella pneumoniae and Enterobacter aerogenes was studied. Sterilization of the whey was unnecessary. Acid whey required neutralization, but sweet whey did not. Butylene glycol production was most efficient at 33 degrees C for Klebsiella pneumoniae and at 37 degrees C for Enterobacter aerogenes. Aeration significantly improved yields. Klebsiella pneumoniae produced more butylene glycol than did Enterobacter aerogenes in unsupplemented whey. The addition of 50 mM sodium acetate to whey increased the production of butylene glycol and acetoin by Enterobacter aerogenes; it also increased the production of glycol by Klebsiella pneumoniae, but the increase in this case was offset by a decrease of production of acetoin. Maximal yields of the glycol plus acetoin in whey were obtained in 48 to 64 h, but Enterobacter aerogenes required about 160 h for complete utilization of the lactose. Highest yields were about .3 M butylene glycol plus acetoin, which corresponds to the production of about 10 kg of glycol from 380 liters of whey.

Acetoin↗
PubMed1982

Salmonella typhimurium mutants defective in the formate dehydrogenase linked to nitrate reductase.

Six fdn mutants of Salmonella typhimurium defective in the formation of nitrate reductase-linked formate dehydrogenase (FDHN) but capable of producing both the hydrogenase-linked formate dehydrogenase (FDHH) and nitrate reductase were characterized. Results of phage P22 transduction experiments indicated that there may be three fdn genes located on the metE-metB chromosomal segment and distinct from all previously identified fdh and chl loci. All six FDHH+ FDHN- mutants were found to make FDHN enzyme protein which was indistinguishable from that of the wild type in electrophoretic studies. However, the results of the spectral studies indicated that all six mutants were defective in the anaerobic cytochrome b559 associated with FDHN. All contained the cytochrome b559 associated with nitrate reductase in amounts equal to or greater than the wild type. The results of the transduction experiments also indicated that the metE- metB segment of the Salmonella chromosome resembles that of Escherichia coli more than was originally thought.

Aldehyde Oxidoreductases↗
PubMed1982

Evidence of a second nitrate reductase activity that is distinct from the respiratory enzyme in Salmonella typhimurium.

Significant nitrate reductase activity was detected in mutants of Salmonella typhimurium which mapped at or near chlC and which were incapable of growth with nitrate as electron acceptor. The same mutants were sensitive to chlorate and performed sufficient nitrate reduction to permit anaerobic growth with nitrate as the sole nitrogen source in media containing glucose. The mutant nitrate-reducing protein did not migrate with the wild-type nitrate reductase in polyacrylamide electrophoretic gels. Studies of the electrophoretic mobility in gels of different polyacrylamide concentration revealed that the wild-type and mutant nitrate reductases differed significantly in both size and charge. The second enzyme also differed from the wild-type major enzyme in its response to repression by low pH and its lack of response to repression by glucose. The same mutants were found to be derepressed for nitrite reductase and for a cytochrome with a maximal reduced absorbance at 555 nm at 25 degrees C. This cytochrome was not detected in preparations of the wild type grown under the same conditions. Extracts of these mutants contained normal amounts of the b-type cytochromes which, in the wild type, were associated with nitrate reductase and formate dehydrogenase, respectively, although they could not mediate the oxidation of these cytochromes with nitrate. They were capable of oxidizing the derepressed 555-nm peak cytochrome with nitrate. It is suggested that these mutants synthesize a nitrate-reducing enzyme which is distinct from the chlC gene product and which is repressed in the wild type during anaerobic growth with nitrate.

Chlorates↗
PubMed1980

Evolution in Pseudomonas fluorescens.

The relationships among 93 strains of Pseudomonas fluorescens were investigated by (1) a numerical taxonomic analysis on the results of 150 phenotypic tests, (2) DNA hybridization studies using 16 reference strains, (3) quantitative microcomplement fixation studies using six reference strains with antibodies directed against the protein azurin. In general, the strains fell into distinct clusters. Assignment to these clusters on the basis of azurin immunological similarity showed 98% agreement with assignment based on DNA homology, suggesting that many genes will follow the same pattern. Of the strains that clustered on the basis of genotype (DNA, azurin) 88% also clustered on the basis of phenotype. The occasional noncongruency observed between the genotypic and phenotypic data may be due to the variable rates of phenotypic evolution. These results provide a perspective on the roles of horizontal and vertical transfer of genes in the evolution of this bacterial group.

Azurin↗
PubMed1980

Effect of moderately acidic pH on heat resistance of Clostridium sporogenes spores in phosphate buffer and in buffered pea puree.

The effect of pH in the range 5.0 to 7.0 on the thermal destruction of spores of Clostridium sporogenes putrefactive anaerobe 3679 was examined by three methods: a capillary tube method in which spores were suspended in phosphate buffers, a thermoresistometer method in which spores were suspended in buffered pea puree adjusted to the same set of pH values, and a thermal death time can method in which spores were again suspended in buffered pea puree. The results indicated that increasing acidity is, in general, accompanied by decreasing heat resistance, although the pH effect was more pronounced at the higher than at the lower processing temperatures. Certain pH values appear to be critical, as they produced, in all three sets of experiments, effects which would not be predicted by the overall relationship between acidity and spore heat resistance. Differences between heat resistance in phosphate buffer as compared with that in pea puree adjusted to the same pH were also noted. D-values in buffer were found to be lower than those in pea puree, except at the highest temperatures coupled with the lowest pH values. The differences between buffer D-value and pea puree D-value were found to increase with increasing pH and with decreasing temperature. On the other hand, at all pH values examined, z-values determined in buffer were somewhat higher than those determined in pea puree adjusted to the same pH.

Buffers↗