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  • 1
    ISSN: 1432-072X
    Keywords: Synergistes jonesii ; Rumen bacteria ; Arginine metabolism ; Pyridinediol detoxification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The ruminal bacterium Synergistes jonesii strain 78-1, which is able to degrade the pyridinediol toxin in the plant Leucaena leucephala, was studied for its ability to utilise amino acids. The organism used arginine, histidine and glycine from a complex mixture of amino acids, and both arginine and histidine supported growth in a semi-defined medium. The products of (U-14C)-arginine metabolism were CO2 acetate, butyrate, citrulline and ornithine. The labelling pattern of end products from (U-14C)-histidine metabolism differed in that carbon also flowed into formate and propionate. Arginine was catabolised by the arginine deiminase pathway which was characterised by the presence of arginine deiminase, ornithine transcarbamylase and carbamate kinase. This is the first report of a rumen bacterium that uses arginine and histidine as major energy yielding substrates.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The current research was aimed at comparing proteolytic activities among ruminal Prevotella spp. Growth rates of Prevotella sp. 2202, Prevotella ruminicola D31d, P. brevis GA33, P. albensis M384, and P. bryantii B14 varied with N source, and no one N source produced the fastest growth in all species. Proteolytic activity was greatest with casein compared with peptides, AA, and NH4Cl in all species. Proteolytic activity of Prevotella sp. 2202, P. brevis GA33, and P. bryantii B14 was modulated by N source. With gelatin co-polymerized SDS-PAGE, the extracellular activities of the Prevotella spp. showed wide variation in number, size, and type of proteases. Prevotella sp. 2202 and P. albensis M384 produced metalloproteases of low molecular weight (40 kDa). P. ruminicola D31d produced one cysteine protease (100–200 kDa) and two metalloproteases (90–100 kDa). P. brevis GA33 generated a diffuse clearing zone (95–160 kDa) containing serine, cysteine, and metalloproteases. P. bryantii B14 produced a metalloprotease greater than 200 kDa in size. The molecular sizes provided are estimations and served only to differentiate among the bacterial species in this study. Large variations in proteolytic activities among species and the known genetic diversity of the Prevotella taxon suggested that targeting this bacterial assemblage for genetic manipulation in order to alter the bacterial impact on ruminal protein degradation would be difficult.
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  • 3
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The objective of this study was to characterize the extracellular proteolytic activity of Streptococcus bovis. Strains KEG, JB1, NCFB 2476, and K11.21.09.6C produced very similar large molecular weight (160–200 kDa) extracellular proteases that were specifically inhibited by PMSF, a serine protease inhibitor. Further experiments with S. bovis KEG indicated that cultures grown with casein as the sole added N source produced the greatest level of proteolytic activity, and the level of proteolytic activity was independent of growth rate. Clarified ruminal fluid (CRF) decreased proteolytic activity by 54% compared with cultures grown with casein alone, and addition of exogenous peptides and carbohydrates (CHO) to the CRF further reduced the level of proteolytic activity by 44% and 52%, respectively. These results suggested that the proteolytic activity of S. bovis KEG was modulated by available N source and that the proteolytic activity was present for reasons other than providing N for growth. The role of S. bovis in ruminal proteolysis requires further definition, but phenotypic similarity among some ruminal strains would suggest a common niche in ruminal proteolysis. The uniformity of proteolytic activities could make S. bovis a prime candidate for manipulation in ruminal proteolysis control strategies.
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  • 4
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract The complete nucleotide sequence of a cryptic plasmid designated pBAW301, from the Gram-positive ruminai bacterium Ruminococcus flavefaciens R13e2, has been determined. This plasmid is 1768 bp in size and has an overall G+C content of 43.5%. Computer analysis of the sequence data revealed an open reading frame, ORF1 (256 amino acids), which is similar to the Rep protein of the Bacillus borstelensis plasmid pHT926. ORF1 is preceded by Shine-Dalgarno and Escherichia coli—10 and —35 like sequences. Nine smaller open reading frames showed no significant homologies to known protein sequences. Analysis of replication intermediates and the nucleotide sequence indicate that the plasmid does not replicate by a rolling-circle mode of replication similar to other plasmids from Gram-positive bacteria. Moreover, sequences typical of theta replication origins were not found in the nucleotide sequence of pBAW301. These data suggest that this plasmid either replicates by an as yet undescribed mechanism, or represents a new class of theta replicating plasmids.
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  • 5
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract Ruminococcus flavefaciens has been hypothesized to produce cellulase constitutively. We have studied the effect of carbon source, either cellobiose or cellulose, on the production of cellulase in batch cultures of R. flavefaciens FD-1. Total CMCase and 14C-cellulase activity was approximately 2-fold higher in cellobiose grown cells than in cellulose grown cells, whereas p-nitrophenyl-β-d-cellobiosidase (PNPCase) activity was not affected by culture conditions. The addition of cellulose to cells growing on cellobiose did not alter the amount or rate of PNPCase and 14C-cellulase production. Northern blot analysis of mRNAs produced by R. flavefaciens FD-1 grown using either cellobiose or cellulose as the substrate indicated that two of the four β-glucanase genes cloned from R. flavefaciens FD-1 were only expressed in cells grown with cellulose as the substrate. Although the adherence of cells and cellulase enzyme to native cellulose can complicate interpretations of these data, the results indicate that cellulase synthesis by R. flavefaciens is differentially regulated by carbon source.
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  • 6
    ISSN: 1574-6976
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The degradation of plant cell walls by ruminants is of major economic importance in the developed as well as developing world. Rumen fermentation is unique in that efficient plant cell wall degradation relies on the cooperation between microorganisms that produce fibrolytic enzymes and the host animal that provides an anaerobic fermentation chamber. Increasing the efficiency with which the rumen microbiota degrades fiber has been the subject of extensive research for at least the last 100 years. Fiber digestion in the rumen is not optimal, as is supported by the fact that fiber recovered from feces is fermentable. This view is confirmed by the knowledge that mechanical and chemical pretreatments improve fiber degradation, as well as more recent research, which has demonstrated increased fiber digestion by rumen microorganisms when plant lignin composition is modified by genetic manipulation. Rumen microbiologists have sought to improve fiber digestion by genetic and ecological manipulation of rumen fermentation. This has been difficult and a number of constraints have limited progress, including: (a) a lack of reliable transformation systems for major fibrolytic rumen bacteria, (b) a poor understanding of ecological factors that govern persistence of fibrolytic bacteria and fungi in the rumen, (c) a poor understanding of which glycolyl hydrolases need to be manipulated, and (d) a lack of knowledge of the functional genomic framework within which fiber degradation operates. In this review the major fibrolytic organisms are briefly discussed. A more extensive discussion of the enzymes involved in fiber degradation is included. We also discuss the use of plant genetic manipulation, application of free-living lignolytic fungi and the use of exogenous enzymes. Lastly, we will discuss how newer technologies such as genomic and metagenomic approaches can be used to improve our knowledge of the functional genomic framework of plant cell wall degradation in the rumen.
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  • 7
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract Molecular studies of the rumen bacterium Ruminococcus flavefaciens are constrained by the lack of stable gene transfer systems. We report here on the characterization of RflFII, a restriction endonuclease isolated from R. flavefaciens FD-1. The enzyme is an isoschizomer of ScaI, and cleavage of the DNA is blunt-ended, between the internal TA dinucleotide sequence of 5′-AGTACT-3′. Chromosomal DNA preparations were used to demonstrate that adenine methylation of DNA within the sequence 5′-GTAC-3′ inhibits both RflFII and the restriction endonucleases RsaI and ScaI. Chromosomal DNA from R. flavefaciens FD-1 is also host modified to protect against cleavage by ScaI.
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  • 8
    ISSN: 0006-3592
    Keywords: methanogenic population dynamics ; anaerobic digesters ; solid waste ; biosolids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An aggressive start-up strategy was used to initiate codigestion in two anaerobic, continuously mixed bench-top reactors at mesophilic (37°C) and thermophilic (55°C) conditions. The digesters were inoculated with mesophilic anaerobic sewage sludge and cattle manure and were fed a mixture of simulated municipal solid waste and biosolids in proportions that reflect U.S. production rates. The design organic loading rate was 3.1 kg volatile solids/m3/day and the retention time was 20 days. Ribosomal RNA-targeted oligonucleotide probes were used to determine the methanogenic community structure in the inocula and the digesters. Chemical analyses were performed to evaluate digester performance. The aggressive start-up strategy was successful for the thermophilic reactor, despite the use of a mesophilic inoculum. After a short start-up period (20 days), stable performance was observed with high gas production rates (1.52 m3/m3/day), high levels of methane in the biogas (59%), and substantial volatile solids (54%) and cellulose (58%) removals. In contrast, the mesophilic digester did not respond favorably to the start-up method. The concentrations of volatile fatty acids increased dramatically and pH control was difficult. After several weeks of operation, the mesophilic digester became more stable, but propionate levels remained very high. Methanogenic population dynamics correlated well with performance measures. Large fluctuations were observed in methanogenic population levels during the start-up period as volatile fatty acids accumulated and were subsequently consumed. Methanosaeta species were the most abundant methanogens in the inoculum, but their levels decreased rapidly as acetate built up. The increase in acetate levels was paralleled by an increase in Methanosarcina species abundance (up to 11.6 and 4.8% of total ribosomal RNA consisted of Methanosarcina species ribosomal RNA in mesophilic and thermophilic digesters, respectively). Methanobacteriaceae were the most abundant hydrogenotrophic methanogens in both digesters, but their levels were higher in the thermophilic digester. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 342-355 1998.
    Additional Material: 2 Ill.
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