Detection of genes for membrane-bound nitrate reductase in nitrate-respiring bacteria and in community DNA

A nested PCR primed by four degenerate oligonucleotides was developed for the specific amplification of sequences from the narG gene encoding the membrane-bound nitrate reductase. This approach was used to amplify fragments of the narG gene from five Pseudomonas species previously shown to be able to express the membrane-bound nitrate reductase and from community DNA extracted from a freshwater sediment. Amino acid sequences encoded by the narG fragments were compared to one another, and to the corresponding regions of related enzymes. This comparison indicates that the amplification protocols are specific for their intended targets. Sequences amplified from community DNA were tightly clustered, which may indicate a degree of homogeneity in the sediment community. The PCR primers and amplification protocols described will be useful in future studies of nitrate respiring populations.     

Hydroquinone degradation via reductive dehydroxylation of gentisyl-CoA by a strictly anaerobic fermenting bacterium

Anaerobic degradation of hydroquinone was studied with the fermenting bacterium strain HQGö1. The rate of hydroquinone degradation by dense cell suspensions was dramatically accelerated by addition of NaHCO₃. During fermentation of hydroquinone in the presence of ¹⁴C-Na₂CO₃ benzoate was formed as a labelled product, indicating an initial ortho-carboxylation of hydroquinone to gentisate. Gentisate was activated to the corresponding CoA-ester in a CoA ligase reaction at a specific activity of 0.15 mol x min^–1 x mg protein^–1. Gentisyl-CoA was reduced to benzoyl-CoA with reduced methyl viologen as electron donor by simultaneous reductive elimination of both the ortho and meta hydroxyl group. The specific activity of this novel gentisyl-CoA reductase was 17 nmol x min^–1 x mg protein^–1. Further degradation to acetate was catalyzed by enzymes which occur also in other bacteria degrading aromatic compounds via benzoyl-CoA.     

Degradation of hydroquinone,gentisate, and benzoate by a fermenting bacterium in pure or defined mixed culture

From a methanogenic fixed-bed reactor fed with hydroquinone as sole energy and carbon source, a rodshaped bacterium was isolated in pure culture which could degrade hydroquinone and gentisate (2,5-dihydroxybenzoate). In syntrophic coculture with either Desulfovibrio vulgaris or Methanospirillum hungatei, also benzoate could be degraded. Other substrates such as sugars, fatty acids, alcohols, and cyclohexane derivatives were not degraded. Sulfate, sulfite, or nitrate were not used as external electron acceptor. The isolate was a Gram-negative, non-motile, nonsporeforming strict anaerobe; the guanine-plus-cytosine content of the DNA was 53.2±1.0 mol%. In pure culture, hydroquinone was degraded to acetate and benzoate, probably via an intermediate carboxylation. In syntrophic mixed cultures, all three substrates were converted completely to acetate. Phenol was never detected as a fermentation product.     

Aerobic nitrate respiration in a nitrite-oxidising bioreactor

The ability of heterotrophic bacteria in a nitrite-oxidising bioreactor to respire with nitrate as an electron acceptor was examined. Approximately 70% of 1000 heterotrophic isolates were able to express a nitrate reductase. A detailed survey of 15 isolates showed that five expressed the azide-insensitive nitrate reductase encoded by the napA gene. A two-round PCR amplification of the napA gene using degenerate PCR primers and DNA sequence analysis of these products confirmed the presence of this gene in the positive isolates. Partial 16S rDNA products and napA products were amplified from the biomass in the bioreactor and denaturing gradient gel electrophoresis of these products identified 21 distinct ribotypes and 12 distinct napA sequences. The results show that the ability to respire with nitrate as an electron acceptor under aerobic conditions is widespread among the heterotrophic population of this bioreactor.     

ABA分解代谢及其代谢关键酶——8'-羟化酶

脱落酸(ABA)在植物的生长发育和环境胁迫响应等过程中具有重要作用。ABA合成与分解代谢的动态平衡共同调控植物内源ABA水平。ABA8′位甲基羟基化途径是高等植物内源ABA代谢的主要途径;8′-羟化酶是该代谢途径的关键酶,属于P450酶系。生物化学和基因组学研究表明,拟南芥CYP707A家族基因编码8′-羟化酶,该基因家族广泛存在于高等植物中,调控植物内源ABA代谢,介导ABA相关的生理生化过程。本文综述了ABA分解代谢的基本途径,详细概述了ABA8′位甲基羟基化途径及该代谢途径的关键酶8′-羟化酶。同时介绍了8′-羟化酶编码基因-CYP707A家族基因的生物学特征和功能。     

Generation of anticalins with specificity for a nonsymmetric phthalic acid ester

A set of engineered lipocalins, so-called anticalins, that bind benzyl butyl phthalate, a potential pollutant of environmental and food samples or medical plastic ware, has been generated. To this end, the synthesis of a derivative of the target analyte carrying an activatable carboxylate group at the end of an aliphatic spacer arm was established. This compound was covalently coupled to amino-functionalized paramagnetic beads. Using phage display technology three variants were selected from a random library of the bilin-binding protein (BBP), a prototypic lipocalin, which exhibit binding activity toward the nonsymmetric phthalic acid ester. These anticalins (denominated PhtA, PhtB, and PhtC) possess dissociation constants of 9.1, 6.2, and 11.6 microM, respectively. Specificity for the binding of other phthalic acid esters was studied. No cross-reactivity was found for diethyl phthalate, while binding to dibutyl phthalate was observed with higher dissociation constants. Interestingly, two differing types of binding behavior were observed among the three selected anticalins. Sequence comparison of these engineered lipocalins with the wild-type BBP revealed that all of the 16 randomized positions carried an amino acid exchange and that a certain sequence pattern had been selected, thus pointing toward a peculiar mode of structural interaction. Our data suggest that the generation of anticalins may provide an alternative to antibodies for the creation of stable receptor proteins against haptens with bioanalytical relevance.     

DMSO respiration by the anaerobic rumen bacterium Wolinella succinogenes

The anaerobic rumen bacterium Wolinella succinogenes was able to grow by respiration with dimethylsulphoxide (DMSO) as electron acceptor and formate or H₂ as electron donors. The growth yield amounted to 6.7 g and 6.4 g dry cells/mol DMSO with formate or H₂ as the donors, respectively. This suggested an ATP yield of about 0.7 mol ATP/mol DMSO. Cell homogenates and the membrane fraction contained DMSO reductase activity with a high K _m (43 mM) for DMSO. The electron transport from H₂ to DMSO in the membranes was inhibited by 2-(heptyl)-4-hydroxyquinoline N-oxide, indicating the participation of menaquinone. Formation of DMSO reductase activity occurred only during growth on DMSO, presence of other electron acceptors (fumarate, nitrate, nitrite, N₂O, and sulphur) repressed the DMSO reductase activity. DMSO can therefore be used by W. succinogenes as an acceptor for phosphorylative electron transport, but other electron acceptors are used preferentially.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DMNH ₂ Reduced DMN - DMS Dimethylsulphide (CH₃)₂S - DMSO Dimethylsulphoxide (CH₃)₂SO - HQNO 2-(Heptyl)-4-hydroxyquinoline-N-oxide - TMAO Trimethylamine-N-oxide - Y _s Growth yield for substrate S     

厌氧甲烷氧化微生物物质代谢与能量代谢研究进展

甲烷既是一种温室气体,也是一种潜在的能源物质,其源与汇的平衡对地球化学循环及工程应用均有重要意义。厌氧甲烷氧化(anaerobic oxidation of methane,AOM)过程是深海、湿地和农田等自然生境中重要的甲烷汇,在缓解温室气体排放方面发挥了巨大作用。AOM微生物的中枢代谢机制及其能量转化途径则是介导厌氧甲烷氧化耦合其他物质还原的关键所在。因此,本文从电子受体多样性的视角,主要分析了硫酸盐型,硝酸盐/亚硝酸盐型,金属还原型厌氧甲烷氧化微生物的生理生化过程及环境分布,并对近些年发现的新型厌氧甲烷氧化进行了梳理;重点总结了厌氧甲烷氧化微生物细胞内电子传递路径以及胞外电子传递方式;根据厌氧甲烷氧化微生物环境分布及反应特征,就其生态学意义及在污染治理与能源回收方面的潜在应用价值进行了展望。本综述以期深化对厌氧甲烷氧化过程的微生物学认知,并为其潜在的工程应用方向提供新的思路。     

Peptidases and amino acid catabolism in lactic acid bacteria

The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.     

Aerobic and anaerobic metabolism of squalene by a denitrifying bacterium isolated from marine sediment

The aerobic and anaerobic metabolism of the isoprenoid alkene squalene was investigated in a new type of marine denitrifying bacterium, strain 2sq31, isolated from marine sediment. Strain 2sq31 was identified as a species of Marinobacter. Under denitrifying conditions, the strain efficiently degraded squalene; of 0.7 mmol added per liter of medium, 77% was degraded within 120 days under anoxic conditions with nitrate as electron acceptor. Tertiary diols and methyl ketones were identified as metabolites, and an anaerobic pathway was suggested to explain the formation of such compounds. The first step in anaerobic degradation of squalene by strain 2sq31 involves hydration of double bonds to tertiary alcohols. Under oxic conditions, the degradation of squalene by strain 2sq31 was rapid and involved oxidative splitting of the C-10/C-11 or C-14/C-15 double bonds, in addition to the pathways observed under denitrifying conditions.     

Aerobic and anaerobic degradation of a range of alkyl sulfides by a denitrifying marine bacterium.

A pure culture of a bacterium was obtained from a marine microbial mat by using an anoxic medium containing dimethyl sulfide (DMS) and nitrate. The isolate grew aerobically or anaerobically as a denitrifier on alkyl sulfides, including DMS, dimethyl disulfide, diethyl sulfide (DES), ethyl methyl sulfide, dipropyl sulfide, dibutyl sulfide, and dibutyl disulfide. Cells grown on an alkyl sulfide or disulfide also oxidized the corresponding thiols, namely, methanethiol, ethanethiol, propanethiol, or butanethiol. Alkyl sulfides were metabolized by induced or derepressed cells with oxygen, nitrate, or nitrite as electron acceptor. Cells grown on DMS immediately metabolized DMS, but there was a lag before DES was consumed; with DES-grown cells, DES was immediately used but DMS was used only after a lag. Chloramphenicol prevented the eventual use of DES by DMS-grown cells and DMS use by DES-grown cells, respectively, indicating separate enzymes for the metabolism of methyl and ethyl groups. Growth was rapid on formate, acetate, propionate, and butyrate but slow on methanol. The organism also grew chemolithotrophically on thiosulfate with a decrease in pH; growth required carbonate in the medium. Growth on sulfide was also carbonate dependent but slow. The isolate was identified as a Thiobacillus sp. and designated strain ASN-1. It may have utility for removing alkyl sulfides, and also nitrate, nitrite, and sulfide, from wastewaters.     

Characterization of a carbofuran-degrading bacterium and investigation of the role of plasmids in catabolism of the insecticide carbofuran

A bacterium capable of using the carbamate insecticide carbofuran as a sole source of carbon and energy, was isolated from soil. The ability to catabolise carbofuran phenol, produced by cleavage of the carbamate ester linkage of the insecticide, was lost at very high frequency when the bacterium was grown in the absence of carbofuran. Plasmid analyses together with curing and mating experiments indicated that the presence of a large plasmid (pIH3, >199 kb) was required for the degradation of carbofuran phenol.Abbreviations Rif^r Rifampicin resistant - Rif^s Rifampicin sensitive - CFH⁺ Carbofuran hydrolase activity present - CFH^- Carbofuran hydrolase activity absent - CFP⁺ ability to degrade carbofuran phenol present - CFP^- ability to degrade carbofuran phenol absent - MS mineral salts medium. MSCF minimal mineral salts medium containing 0.25 mM carbofuran as sole source of carbon and energy - YP MS medium containing 5 g/l yeast extract and 5 g/l Bactopeptone. YPCF as above but with the addition of 1 mM carbofuran - EPTC S-ethyl-N,N-dipropylthiocarbamate - 2,4-D 2,4-dichlorophenoxyacetic acid - NAG N-acetylglucosamine - 3-HB 3-hydroxybutyrate     

The anaerobic decomposition of benzoic acid during methane fermentation

Anaerobic rupture of the benzoic acid ring was investigated. Carbon 4 was converted primarily to carbon dioxide. Following ring rupture during methane fermentation, propanoic acid is an intermediate, and carbon 4 of benzoate becomes its carboxyl.Contribution No. 1285-j, Division of Biology, Kansas State University, Manhattan, KS 66506. This work was supported in part by funds from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506. Paper II of this series is Fina and Fiskin (1960)     

Members of the genus Arthrobacter grow anaerobically using nitrate ammonification and fermentative processes: anaerobic adaptation of aerobic bacteria abundant in soil

Members of the genus Arthrobacter are usually regarded as obligate aerobic bacteria. The anaerobic growth and energy metabolism of two Arthrobacter species were investigated. Arthrobacter globiformis utilized both nitrate ammonification and lactate, acetate and ethanol producing fermentation processes for anaerobic growth. Only nitrate supported anaerobic growth of Arthrobacter nicotianae. Anaerobically induced respiratory nitrate reductase activity was detected in both strains. Neither of the tested strains used the alternative electron acceptors fumarate, dimethylsulfoxide or trimethylamine-N-oxide.     

The specific functions of menaquinone and demethylmenaquinone in anaerobic respiration with fumarate,dimethylsulfoxide, trimethylamine N-oxide and nitrate by Escherichia coli

The respiratory activities of E. coli with H₂ as donor and with nitrate, fumarate, dimethylsulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as acceptor were measured using the membrane fraction of quinone deficient strains. The specific activities of the membrane fraction lacking naphthoquinones with fumarate, DMSO or TMAO amounted to 2% of those measured with the membrane fraction of the wild-type strain. After incorporation of vitamin K₁ [instead of menaquinone (MK)] into the membrane fraction deficient of naphthoquinones, the activities with fumarate or DMSO were 92% or 17%, respectively, of the activities which could be theoretically achieved. Incorporation of demethylmenaquinone (DMK) did not lead to a stimulation of the activities of the mutant. In contrast, the electron transport activity with TMAO was stimulated by the incorporation of either vitamin K₁ or DMK. Nitrate respiration was fully active in membrane fractions lacking either naphthoquinones or Q, but was 3% of the wild-type activity, when all quinones were missing. Nitrate respiration was stimulated on the incorporation of either vitamin K₁ or Q into the membrane fraction lacking quinones, while the incorporation of DMK was without effect. These results suggest that MK is specifically involved in the electron transport chains catalyzing the reduction of fumarate or DMSO, while either MK or DMK serve as mediators in TMAO reduction. Nitrate respiration requires either Q or MK.Abbreviations DMK demethylmenaquinone - MK menaquinone - Q ubiquinone - DMSO dimethylsulfoxide - TMAO trimethylamine N-oxide - DMS dimethylsulfide - TMA trimethylamine - BV benzylviologen     

l-Phenylalanine catabolism and l-phenyllactic acid production by a phototrophic bacterium,Rubrivivax benzoatilyticus JA2

A phototrophic bacterium (Rubrivivax benzoatilyticus JA2) grows at the expense of l-phenylalanine as sole source of nitrogen but not as carbon source. Near stoichiometric yields of l-phenylpyruvic acid (0.4 mM) and l-phenyllactate (0.4 mM) were observed from l-phenylalanine (0.9 mM consumed). Aminotransfarase and dehydrogenase activities involved in the formation of l-phenylpyruvic acid and l-phenyllactate were demonstrated unequivocally in Rubrivivax benzoatilyticus JA2. Growth conditions and carbon sources had an influence on l-phenyllactate production. The process yielded a maximum of 0.92 mM l-phenyllactate from l-phenylalanine (1 mM) when fructose served as carbon source for R. benzoatilyticus JA2.     

Aerobic and anaerobic metabolism of 6,10,14-trimethylpentadecan-2-one by a denitrifying bacterium isolated from marine sediments.

This report describes the metabolism of 6,10,14-trimethylpentadecan-2-one by a denitrifying bacterium (Marinobacter sp. strain CAB) isolated from marine sediments. Under aerobic and denitrifying conditions, this strain efficiently degraded this ubiquitous isoprenoid ketone. Several bacterial metabolites, 4,8,12-trimethyl-tridecan-1-ol, 4,8,12-trimethyltridecanal, 4,8,12-trimethyltridecanoic acid, Z-3,7-dimethylocten-2-oic acid, Z-3,7,11-trimethyldodecen-2-oic acid, and 6,10,14-trimethylpentadecan-2-ol, were formally identified, and different pathways were proposed to explain the formation of such isoprenoid compounds.     

Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms

Strain SB^T is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SB^T produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SB^T in coculture with Desulfovibrio strain G11. Strain SB^T grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 ± 1 g cell dry mass per mol of crotonate. Strain SB^T did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SB^T was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SB^T in the δ-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SB^T was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SB^T and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus. Received: 2 June 1998 / Accepted: 16 November 1998     

Estimating fermentative amino acid catabolism in the small intestine of growing pigs

Fermentative catabolism (FAAC) of dietary and endogenous amino acids (AA) in the small intestine contributes to loss of AA available for protein synthesis and body maintenance functions in pigs. A continuous isotope infusion study was performed to determine whole body urea flux, urea recycling and FAAC in the small intestine of ileal-cannulated growing pigs fed a control diet (CON, 18.6% CP; n=6), a high fibre diet with 12% added pectin (HF, 17.7% CP; n=4) or a low-protein diet (LP, 13.4% CP; n=6). ¹⁵N-ammonium chloride and ¹³C-urea were infused intragastrically and intravenously, respectively, for 4 days. Recovery of ammonia at the distal ileum was increased by feeding additional fibre when compared with the CON (P<0.05) but was not affected by dietary protein (0.24, 0.39 and 0.14 mmol nitrogen/kg BW/day for CON, HF and LP, respectively; P<0.05). Lowering protein intake reduced urea flux (25.3, 25.7 and 10.3 mmol nitrogen/kg BW/day; P<0.01), urinary urea excretion (14.4, 15.0 and 6.2 mmol N/kg BW/day; P<0.001) and urea recycling (12.1, 11.3 and 3.23 mmol nitrogen/kg BW/day; P<0.01) compared with CON. There was a rapid reduction in ¹⁵N-ammonia enrichment in digesta along the small intestine suggesting rapid absorption of ammonia before the distal ileum and lack of uniformity of enrichment in the digesta ammonia pool. A two-pool model was developed to determine possible value ranges for nitrogen flux in the small intestine assuming rapid absorption of ammonia. Maximum estimated FAAC based on this model was significantly lower when dietary protein content was decreased (32.9, 33.4 and 17.4 mmol nitrogen/kg BW/day; P<0.001). There was no impact of dietary fibre on estimates of small intestine nitrogen flux (P>0.05) compared with CON. The two-pool model developed in the present study allows for estimation of FAAC but still has limitations. Quantifying FAAC in the small intestine of pigs, as well as other non-ruminants and humans, offers a number of challenges but warrants further investigation.