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  • 1
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The complete nucleotide sequence of a small cryptic plasmid designated pRAO1, from the Gram-negative ruminal bacterium Ruminobacter amylophilus NIAH-3, was determined. The plasmid is a circular DNA molecule, 2140 bp in size, with a GC content of 40%. Computer-assisted analysis identified three open reading frames (ORFs), one of which, ORF3 (347 amino acids), displayed a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmid recombination of plasmids from Gram-positive bacteria. We proved the mobilization properties of pRAO1 in the Escherichia coli system using the coresident IncW broad-host-range conjugative plasmid R388. These data demonstrated, for the first time, the mobilization properties of small cryptic plasmids from Gram-negative inhabitants of the rumen.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Molecular diversity of rumen bacteria was analyzed by PCR amplification and sequencing of 16S rDNA clone libraries prepared from the rumen content of Holstein cows fasted for 16 h. A total of 84 clones, containing almost full size 16S rDNA sequences (about 1.5 kb long), were completely sequenced and subjected to an on line similarity search. Four sequences from the 84 clones closely resembled that of Butyrivibrio fibrisolvens and one clone was found to be related to Treponema bryantii. For 38% of the sequences, the similarity with database sequences was in the range of 90%–98% and for the remaining 56% the similarity was less than 90%. The bacterial community structure was also revealed by phylogenetic placement of sequences in relation to different fractions of rumen content. In the library from the rumen fluid, the sequences were affiliated with the following major phyla: low G+C Gram-positive bacteria (52.4%), Cytophaga-Flexibacter-Bacteroides (38.1%), Proteobacteria (4.7%) and Spirochaetes (2.4%). 2.4% had an uncertain affiliation. The vast majority of sequences from the rumen solids were found to be related to low G+C Gram-positive bacteria (71.4%) and the remaining sequences were placed within the Cytophaga-Flexibacter-Bacteroides (26.2%) and Spirochaetes (2.4%) phyla.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Molecular biology approaches were employed to examine the genetic diversity of bacteria from the Cytophaga/Flexibacter/Bacteroides (CFB) phylum in the rumen of cattle. By this means we were able to identify cultured strains that represent some of the larger CFB clusters previously identified only by PCR amplification and sequencing. Complete 16S rDNA sequences were obtained for 16 previously isolated rumen strains, including the type strains of Prevotella ruminicola, P. bryantii, P. brevis and P. albensis to represent a wide range of diversity. Phylogenetic analysis of cultured strains revealed the existence of three clusters of ruminal CFB: (i) a cluster of Prevotella strains, which have been found only in the rumen, including the two type strains, P. brevis GA33T and P. ruminicola 23T; (ii) Prevotella spp. that cluster with prevotellas from other ecological niches such as the oral cavity and which include the type strains, P. bryantii B14T and P. albensis M384T; (iii) two Bacteroides spp. strains clustering with B. forsythus of oral origin. In order to establish whether the cultivated isolates cover the whole range of ruminal CFB genetic diversity, 16S rRNA gene sequences were amplified and cloned from DNA extracted from the same rumen samples (one cow in Slovenia, one in Scotland and three in Japan). Sequencing and phylogenetic analysis of 16S rRNA genes confirmed the existence of two superclusters of ruminal Prevotella, one exclusively ruminal and the other including non-ruminal species. In the case of ruminal Bacteroides spp., however, phylogenetic analysis revealed the existence of three new superclusters, one of which has as yet no cultivable counterpart. Interestingly, these Bacteroides clusters were represented almost exclusively by clone libraries from the Japanese cattle and only three sequences were from the European cattle. This study agrees with previous analyses in showing that rumen Prevotella/Bacteroides strains exhibit a remarkable degree of genetic diversity and suggests that different strain groupings may differ greatly in their recovery by cultural methods. The most important conclusion, however, is that cultured strains can be identified that represent some of the larger clusters previously identified only by PCR amplification and sequencing.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The regulation of the recA gene expression in the obligately anaerobic rumen bacterium Prevotella ruminicola was investigated by monitoring the recA-specific transcript level. P. ruminicola recA forms a monocistronic unit, but no SOS-box sequences resembling those of Escherichia coli or Bacillus subtilis can be identified upstream of the recA coding region. At the same time, we observed a fivefold increase in the level of recA mRNA in response to DNA damaging agents, mitomycin C and methyl methanesulfonate, as well as under conditions of oxidative stress. No induction was detected when growth of P. ruminicola was arrested by shifting to acidic (pH 4.8) conditions. Primer extension experiment revealed the three very close transcriptional start sites for recA. The putative −10 and −35 RNA polymerase binding regions were proposed on the basis of transcript mapping. These regions bear very little similarity to the E. coli (σ70) and B. subtilis (σA) consensus sequences, as well as to the recognition sites of other minor σ-factors. Transcript mapping experiments in E. coli expressing P. ruminicola recA confirmed that the transcription machineries of these two bacteria recognize completely different regulatory sequences on the template to initiate transcription. Preliminary DNase I footprinting analysis data revealed that the region of imperfect dyad symmetry (AATTATAATCAATTATAAAT) found between the putative −10 region and the translation initiation codon may serve as an SOS-box-like regulatory sequence in P. ruminicola. This sequence bears no similarity to the known SOS-box sequences and, in particular, to that of E. coli and other Gram-negative bacteria.
    Type of Medium: Electronic Resource
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