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Bacteriocin-like activity of Butyrivibrio fibrisolvens JL5 and its effect on other ruminal bacteria and ammonia production

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 2002年第3期68卷 1040-1046页

作者： Rychlik, JL Russell, JB Cornell Univ Microbiol Sect Ithaca NY 14853 USA USDA ARS Ithaca NY 14853 USA

When ruminal bacteria from a cow fed hay were serially diluted into an anaerobic medium that had only peptides and amino acids as energy sources, little growth or ammonia production was detected at dilutions greater than 10(-6). The 10(-8) and 10(-9) dilutions contained bacteria that fermented carbohydrates, and some of these bacteria inhibited Clostridium sticklandii SR, an obligate amino acid-fermenting bacterium. Phylogenetic analysis indicated that the most active isolate (JL5) was closely related to Butyrivibrio fibrisolvens B835. Strain JL5 inhibited B. fibrisolvens 49 and a variety of other gram-positive organisms, but it had little effect on most gram-negative ruminal bacteria. Strain JL5 did not produce a bacteriocin-like inhibitory substance (BLIS) until it reached the late log or stationary phase. The JL5 BLIS did not cause the lysis of B. fibrisolvens 49, but the intracellular potassium level, the ATP level, the electrical potential, and the viability decreased rapidly. The JL5 BLIS also caused marked decreases in the viability and cellular potassium level of C sticklandii SR. The membrane potential and intracellular ATP level also declined. The BLIS was degraded very slowly by pronase E, but it could be precipitated with 60% ammonium sulfate and dialyzed (3,500-Da cutoff). The BLIS could be separated from other peptides by polyacrylamide gel electrophoresis, and C sticklandii SR overlays indicated that the molecular size of this compound was approximately 3,600 Da. Based on these results, it appeared that the JL5 BLIS was a pore-forming peptide. Because carbohydrate-fermenting ruminal bacteria could inhibit the growth of obligate amino acid-fermenting bacteria, BLIS may play a role in regulating ammonia production in vivo.

关键词：氨基酸类/代谢氨/代谢抗生作用细菌厌氧/分类细菌厌氧/遗传学细菌厌氧/生长和发育细菌厌氧/代谢细菌素类/代谢细菌素类/药理学梭菌属/药物作用梭菌属/生长和发育梭菌属/代谢集落计数微生物培养基发酵分子序列数据消化链球菌属/药物作用消化链球菌属/生长和发育消化链球菌属/代谢瘤胃/微生物学动物

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Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第3期88卷 458-466页

作者： Donaghy, JA Bronnenmeier, K Soto-Kelly, PF McKay, AM Dept Agr No Ireland Food Sci Div Microbiol Belfast BT9 5PX Antrim North Ireland Queens Univ Belfast Dept Food Sci Microbiol Belfast BT7 1NN Antrim North Ireland Tech Univ Munich Lehrstuhl Mikrobiol D-8000 Munich Germany

Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the K-m and V-max values for the hydrolysis of methyl ferulate were 0.04 mmol l(-l) and 131 mu mol min(-1) mg(-1), respectively;the respective values for methyl coumarate were 0.86 mmol l(-l) and 18 mu mol min(-1) mg(-1.) The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l(-l), the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.

关键词：厌氧生活羧酸酯水解酶类/化学羧酸酯水解酶类/分离和提纯羧酸酯水解酶类/代谢梭菌属/酶学梭菌属/生长和发育培养基底物特异性木聚糖类/代谢

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Evaluation of 16s rRNA and cellular fatty acid profiles as markers of human intestinal bacterial growth in the chemostat

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第4期89卷 668-677页

作者： Hopkins, MJ Macfarlane, GT Univ Dundee MRC Microbiol & Gut Biol Grp Dundee Scotland

Chemostats were used to study the effects of carbon and nitrogen limitation and specific growth rate on 16S rRNA synthesis and cellular fatty acid (CFA) profiles in four human intestinal bacteria (Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Clostridium bifermentans and Cl. difficile). Cellular fatty acid synthesis varied with dilution rate and nutrient availability in different species, but these cellular constituents were relatively stable phenotypic characteristics in Bact. thetaiotaomicron, where branched chain and hydroxy CFA were good taxonomic markers. Conversely, CFA in the Gram-positive bacteria varied markedly with changes in growth environment. For example, in chemostats, cyclopropane CFA were only synthesized in Cl. bifermentans and Cl. difficile under N-limited conditions. Similarly, Dimethyl acetal (DMA) fatty acids in Bif. adolescentis were primarily produced during N-limited growth, and this was inversely related to dilution rate. At low growth rates, 16S rRNA concentrations (mug rRNA per ml culture) correlated with viable bacterial counts, but were more closely related to specific growth rate when expressed as a function of cell mass (mug rRNA per mg dry weight bacteria). However, this did not reveal differences in bacterial population size and rRNA concentration in C-limited cultures. Thus, at low dilution rates, C limitation strongly reduced rRNA synthesis in Cl. bifermentans, despite viable cell counts being similar to those in N-limited cultures. These results indicate that, while 16S rRNA is a useful indicator of microbial activity, cell growth rate does not necessarily relate to rRNA concentration under all nutritional conditions. Consequently, bowel habit and diet will affect both CFA and rRNA content in bacteria isolated from intestinal samples, and this should be taken into consideration when interpreting such data measurements.

关键词：拟杆菌属/遗传学拟杆菌属/生长和发育拟杆菌属/代谢二裂菌属/遗传学二裂菌属/生长和发育二裂菌属/代谢梭菌属/遗传学梭菌属/生长和发育梭菌属/代谢难辨梭菌/遗传学难辨梭菌/生长和发育难辨梭菌/代谢结肠/微生物学培养基脂肪酸类/代谢肠/微生物学 RNA 细菌/分析 RNA 核糖体 16S/分析人类

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Duplicated Clostridium thermocellum cellobiohydrolase gene encoding cellulosomal subunits S3 and S5

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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 1999年第6期51卷 852-859页

作者： Zverlov, VV Velikodvorskaya, GA Schwarz, WH Kellermann, J Staudenbauer, WL Tech Univ Munich Inst Microbiol D-80290 Munich Germany Russian Acad Sci Inst Mol Genet Moscow 123182 Russia Max Planck Inst Biochem D-82152 Martinsried Germany

The upstream region of the cellobiohydrolase gene cbhA of Clostridium thermocellum F7 was sequenced. It was found that this region contains the previously sequenced gene celK encoding an enzyme closely related to CbhA (cellulosomal subunit S3). The presence of a putative transcription terminator in the 524-bp intergenic region indicates that celK and cbhA are not cotranscribed as an operon. Sequence comparison between the two cellobiohydrolases revealed high sequence conservation in the catalytic domain and in the N-terminal cellulose-binding domain (CBD) homologous to CBD family IV, which binds specifically to amorphous cellulose and soluble cellooligosaccharides. In contrast to CbhA, CelK lacks a family III CBD capable of binding to crystalline cellulose. By partial amino acid sequence determination CelK was shown to be identical to cellulosomal subunit S5. CelK and CbhA were found to be members of subfamily E1 of cellulase family E (glycosylhydrolase family 9). Sequence comparison of catalytic domains of family E1 cellulases with C. thermocellum CelD, a family E1 endoglucanase of known three-dimensional structure, revealed a significant variation in the lengths of substrate-binding loops connecting the helices of the (alpha/alpha)(6) barrel fold. The extended loops of CelK and CbhA might form an active-site tunnel, as found in the catalytic domains of fungal cellobiohydrolases.

关键词：氨基酸序列碱基序列结合部位催化域纤维素酶/化学纤维素酶/遗传学纤维素1 4-β-纤维二糖苷酶克隆分子梭菌属/酶学梭菌属/遗传学梭菌属/生长和发育基因细菌基因重复分子序列数据物理染色体图序列比对序列分析 DNA

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Antibiotic therapy attenuates colitis in interleukin 10 gene-deficient mice

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GASTROENTEROLOGY 2000年第6期118卷 1094-+页

作者： Madsen, KL Doyle, JS Tavernini, MM Jewell, LD Rennie, RP Fedorak, RN Univ Alberta Div Gastroenterol Dept Med Edmonton AB T6G 2C2 Canada Univ Alberta Dept Lab Med & Pathol Edmonton AB T6G 2C2 Canada Univ Alberta Dept Med Microbiol Edmonton AB T6G 2C2 Canada

Background & Aims: Interleukin (IL)-10 gene-deficient mice, raised under germfree conditions, do not develop colitis, implying a role for bacteria. This study mapped the appearance of luminal colonic bacteria and, using antibiotic treatment, determined their association with colitis in IL-10 gene-deficient mice, Methods: Mice were treated with ciprofloxacin or with neomycin and metronidazole, The intestine was harvested for histological scoring and bacterial assessment. Results: At 2 weeks of age, before the development of colitis, IL-10 gene-deficient mice demonstrated an earlier appearance of Streptococcus and Clostridium sp., and had a greater proportion (P < 0.01) of bacteria adherent to the colonic mucosa, This pattern of increased adherent bacteria persisted for the 12 weeks of study. Treatment of mice before the onset of colonic inflammation, with either antibiotic regime, reduced mucosal adherent bacteria and prevented colitis (P < 0.01). In contrast, treatment of established colitis with neomycin and metronidazole did not reduce adherent bacterial levels, yet was more efficacious (P < 0.05) in treating established colitis than ciprofloxacin, which did reduce adherent colonic bacteria. Conclusions: In the IL-10 gene-deficient mouse model, the appearance and number of mucosal adherent colonic bacteria are altered before the onset of colitis. Antibiotics both prevent and treat the colitis through correction of this primary bacterial alteration.

关键词：年龄因素抗菌药/药理学拟杆菌属/药物作用拟杆菌属/生长和发育环丙沙星/药理学梭菌属/药物作用梭菌属/生长和发育结肠炎/药物疗法结肠炎/遗传学结肠炎/病理学肠杆菌科/药物作用肠杆菌科/生长和发育肠杆菌科感染/药物疗法肠杆菌科感染/遗传学肠杆菌科感染/病理学肠球菌属/药物作用肠球菌属/生长和发育白细胞介素10/遗传学肠黏膜/微生物学肠黏膜/病理学乳杆菌属/药物作用乳杆菌属/生长和发育纵向研究甲硝唑/药理学小鼠近交系小鼠基因敲除新霉素/药理学链球菌属/药物作用链球菌属/生长和发育动物小鼠

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Influence of glucose on glycerol metabolism by wild-type and mutant strains of Clostridium butyricum E5 grown in chemostat culture

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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 2001年第2期55卷 226-233页

作者： Malaoui, H Marczak, R Univ Henri Poincare Lab Biochim Bacteries Gram Fac Sci F-54506 Vandoeuvre Les Nancy France

In order to improve the yield of 1,3-propanediol (1,3-PPD) in Clostridium butyricum E5, we carried out cofermentation experiments on glucose/glycerol mixtures in chemostat culture. The results showed the influence of the ratio of the two carbon substrates on the production of the required diol. The progressive increase of glucose in culture medium containing a given concentration of glycerol made it possible to highlight the deviation of carbon flow from the oxidative towards the reducing pathway, in order to maintain the oxidation/reduction balance in the cell. The conversion of glycerol into 1,3-PPD thus increased from 0.63 mol mol(-1), without the addition of glucose, to a maximum of 0.89 mol mol(-1) for a molar glucose/glycerol ratio of 0.2 for the wild-type strain. The same experiments carried out with the mutant MD strain, which is resistant to allyl alcohol, led to similar results but with a maximum of 0.84 mol mol(-1) for a glucose/glycerol molar ratio of 0.1. Beyond a molar ratio of 0.2, the biosynthesis of enzymes for the glycerol metabolism was less subject to catabolic repression by glucose in the mutant MD strain than in the wild-type strain.

关键词：醇氧化还原酶类/代谢生物反应器梭菌属/酶学梭菌属/遗传学梭菌属/生长和发育梭菌属/代谢培养基发酵葡萄糖/代谢甘油醛-3-磷酸脱氢酶类/代谢甘油/代谢突变 NAD/代谢氧化还原丙二醇类/代谢糖醇脱氢酶类/代谢

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Microbial sulfate reduction with acetate: process performance and composition of the bacterial communities in the reactor at different salinity levels

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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 2001年第6期55卷 787-793页

作者： Muthumbi, W Boon, N Boterdaele, R De Vreese, I Top, EM Verstraete, W State Univ Ghent Fac Agr & Appl Biol Sci Lab Microbial Ecol & Technol B-9000 Ghent Belgium

Microbial sulfate reduction with acetate as carbon source and electron donor was investigated at salinity levels between 0.53 and 1.48%. The experiment was carried out in a 2.3-l upflow anaerobic sludge blanket reactor inoculated with granular methanogenic sludge. A pH of 8.3, a temperature of 32 +/-1 degreesC and a chemical oxygen demand (COD)/SO42--S ratio of 2 were maintained in the reactor throughout the experiment. Sulfate reduction and the composition of the dominant bacterial communities in the reactor were monitored. The results showed that a maximal conversion rate for SO42--S of 14 g l(-1) day(-1) and a conversion efficiency of more than 90% were obtained at a salinity level of 1.26-1.39%. A further increase in the salinity level led to reactor instability. Denaturant gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from total bacterial DNA extracted from the inoculum and reactor sludge showed that salinity level had an impact on the composition of the bacterial communities in the reactor. However, no clear relationship was found between reactor performance and the composition of the dominant bacterial communities in the reactor.

关键词：乙酸盐类/代谢厌氧生活细菌/分类细菌/遗传学细菌/生长和发育细菌/代谢生物反应器梭菌属/分类梭菌属/遗传学梭菌属/生长和发育梭菌属/代谢 DNA 细菌/分析 DNA 细菌/遗传学 DNA 核糖体/分析 DNA 核糖体/遗传学电泳聚丙烯酰氨凝胶氢离子浓度分子序列数据氧化还原假单胞菌属/分类假单胞菌属/遗传学假单胞菌属/生长和发育假单胞菌属/代谢 RNA 核糖体 16S/遗传学氯化钠/代谢硫酸盐类/代谢温度

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Bacteria associated with butterfly stain of Chilean tepa

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CURRENT MICROBIOLOGY 2000年第2期40卷 86-90页

作者： McLemore, JB Tainter, FH McElreath, SD Clemson Univ Dept Forest Resources Clemson SC 29634 USA

Butterfly stain, common in Chilean tepa (Laureliopsis philippiana [Looser] Schodde) trees, appears in cross-section of the stem as a series of partially overlapping orange-brown arcs that in early stages resembles a butterfly. Bacterial isolates from stained tepa wood samples cultured under aerobic and anaerobic conditions were identified using several commercially available identification systems. Pseudomonas sp. were most common in the cultures handled aerobically, and Clostridium sp. were found in the cultures handled anaerobically. A pectinolytic Bacillus sp. and a Gram-negative rod-shaped bacterium that emitted a strong naphthalene-like odor similar to that of stained wood were also isolated. Not all isolates could be identified. This is the first report of Clostridium from tepa.

关键词：不动杆菌属/生长和发育不动杆菌属/分离和提纯细菌需氧/生长和发育细菌需氧/分离和提纯细菌厌氧/生长和发育细菌厌氧/分离和提纯智利梭菌属/生长和发育梭菌属/分离和提纯集落计数微生物色素类生物学假单胞菌属/生长和发育假单胞菌属/分离和提纯树/微生物学

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Bacterial spore inhibition and inactivation in foods by pressure, chemical preservatives, and mild heat

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JOURNAL OF FOOD PROTECTION 2000年第11期63卷 1503-1510页

作者： Shearer, AEH Dunne, CP Sikes, A Hoover, DG Univ Delaware Dept Anim & Food Sci Newark DE 19717 USA USA Natick Res Dev & Engn Ctr Natick MA 01760 USA

Sucrose laurates, sucrose palmitate, sucrose stearates, and monolaurin (Lauricidin) were evaluated for inhibitory effects against spores of Bacillus sp., Clostridium sporogenes PA3679, and Alicyclobacillus sp. in a model agar system. The combined treatment of sucrose laurate, high hydrostatic pressure, and mild heat was evaluated on spores of Bacillus and Alicyclobacillus in foods. The minimum inhibitory concentrations of the sucrose esters were higher than that of Lauricidin for all spores tested in the model agar system, but Lauricidin was not the most readily suspended in the test media. The sucrose laurates and sucrose palmitate were more effective and more readily suspended than the sucrose stearates. A combined treatment of sucrose laurate (less than or equal to1.0%), 392 megaPascals (MPa) at 45 degreesC for 10 to 15 min provided 3- to 5.5-log(10) CFU/ml reductions from initial populations of 10(6) CFU/ml for Bacillus subtilis 168 in milk, Bacillus cereus 14579 in beef, Bacillus coagulans 7050 in tomato juice (pH 4.5), Alicyclobacillus sp. N1089 in tomato juice (pH 4.5), and Alicyclobacillus sp. N1098 in apple juice. The most notable change in the appearance of the products was temporary foaming during mixing of the sucrose laurate in the foods. The effect of sucrose laurate appeared to be inhibitory rather than lethal to the spores. The inhibitory effects observed on Bacillus and Alicyclobacillus spores by the combined treatment of pressure, mild heat, and sucrose laurate appear promising for food applications where alternatives to high heat processing are desired.

关键词：芽孢杆菌属/药物作用芽孢杆菌属/生长和发育饮料/微生物学梭菌属/药物作用梭菌属/生长和发育集落计数微生物食品微生物学食品防腐剂/药理学甘油酯类/药理学热温度氢离子浓度流体静力压月桂酸盐类/药理学乳/微生物学单脂肪酸甘油酯类孢子细菌/药物作用孢子细菌/生长和发育蔗糖/类似物和衍生物蔗糖/药理学表面活性剂/药理学动物牛

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