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Bacterial species belonging to the genus Pseudobutyrivibrio are important members of the rumen microbiome contributing to the degradation of complex plant polysaccharides. Pseudobutyrivibrio xylanivorans MA3014 was selected for genome sequencing to examine its ability to breakdown and utilize plant polysaccharides. The complete genome sequence of MA3014 is 3.58 Mb, consists of three replicons (a chromosome, chromid, and plasmid), has an overall G + C content of 39.6%, and encodes 3,265 putative protein-coding genes (CDS). Comparative pan-genomic analysis of all cultivated and currently available P. xylanivorans genomes has revealed a strong correlation of orthologous genes within this rumen bacterial species. MA3014 is metabolically versatile and capable of growing on a range of simple mono- or oligosaccharides derived from complex plant polysaccharides such as pectins, mannans, starch, and hemicelluloses, with lactate, butyrate, and formate as the principal fermentation end products. The genes encoding these metabolic pathways have been identified and MA3014 is predicted to encode an extensive range of Carbohydrate-Active enZYmes with 78 glycoside hydrolases, 13 carbohydrate esterases, and 54 glycosyl transferases, suggesting an important role in solubilization of plant matter in the rumen. [ABSTRACT FROM AUTHOR]

The rumen bacterium Butyrivibrio proteoclasticus B316 has a 4.4-Mb genome composed of four replicons (approximately 3.55 Mb, 361, 302 and 186 kb). Mutagenesis of B316 was performed with the broad host-range conjugative transposon Tn 916 to screen for functionally important characteristics. The insertion sites of 123 mutants containing a single copy of Tn 916 were identified and corresponded to 53 different insertion points, of which 18 (34.0%), representing 39 mutants (31.7%), were in ORFs and 12 were where transposition occurred in both directions (top and bottom DNA strand). Up to eight mutants from several independent conjugation experiments were found to have the same integration site. Although transposition occurred in all four replicons, the number of specific insertion sites, transposition frequency and the average intertransposon distance between insertions varied between the four replicons. In silico analysis of the 53 insertion sites was used to model a target consensus sequence for Tn 916 integration into B316. A search of the B316 genome using the modelled target consensus sequence (up to two mismatches) identified 39 theoretical Tn 916 insertion sites (19 coding, 20 noncoding), of which nine corresponded to Tn 916 insertions identified in B316 mutants during our conjugation experiments. [ABSTRACT FROM AUTHOR]

The outer membrane protein, intimin ( eae), which mediates bacterial attachment to epithelial cells, is associated with enteropathogenic Escherichia coli and some Shiga toxin-producing E. coli. The eae subtype of E. coli strains isolated from healthy cattle and sheep was identified using a rapid PCR-restriction fragment length polymorphism (RFLP) method to produce profiles that were compared with those generated in silico. The 139 eae-positive E. coli strains were separated into 11 different PCR-RFLP profiles. The most common eae PCR-RFLP type was β (23.7%), followed by ζ (20.1%), θ (16.5%), ι (12.2%), κ (8.6%), ℇ (7.2%), γ (2.9%), ν and β2 (2.2%) and ι2 (1.4%). Four isolates did not yield a PCR-RFLP amplification product but complete sequencing of the eae gene matched subtype ρ. Two different eae variants were isolated from the same swab from 18 different animals and subtype ι was the most ‘promiscuous’, being isolated with four other eae subtypes from seven separate animals. None of the eae-positive STEC were subtype γ, which is associated with STEC serogroup O157. This method allowed the rapid identification of eae subtypes and indicates that forage-fed animals possessed a wide diversity of bacterial eae subtypes with a low frequency of eae subtype γ. [ABSTRACT FROM AUTHOR]

PCR primers were designed to amplify the gene that encodes bovicin 255 from Streptococcus gallolyticus LRC0255 and the bacteriocin genes from Butyrivibrio fibrisolvens strains AR10 and OR79A (bviD and bvi79A) in order to screen for their incidence in rumen and caecal B. fibrisolvens and Streptococcus bovis-like isolates from New Zealand and North American ruminants. None of the B. fibrisolvens-like strains (n=34) isolated from New Zealand or North America had the genes encoding for butyrivibriocins AR10 (bviD) or OR79 (bvi79A). However, seven S. bovis isolates from New Zealand ruminants and three from North American animals had the bovicin 255 gene. Sequence comparison of cloned bovicin 255 PCR products indicated a 92.9–95.7% similarity to that of the corresponding bovicin 255 gene sequence of S. gallolyticus. Four of the New Zealand bovicin 255 positive S. bovis isolates were from the caecal contents of the same sheep and had identical PFGE profiles. Two other S. bovis isolates sharing the same PFGE profile were isolated from a separate animal from the same flock. PFGE analysis of the North American strains indicated that all three were closely related as two of three had identical PFGE profiles with the remaining isolate differing only by a single band position. The 16S rRNA gene sequences of the 10 isolates were at least 99.8% identical to S. bovis. All 10 S. bovis isolates having the gene for bovicin 255 produced bacteriocin activity that inhibited the growth of Peptostreptococcus anaerobius D1 in a deferred antagonism plating (DAP) assay. Certain S. bovis isolates obtained from ruminants have bacteriocin activity associated with a distinct bovicin 255 gene sequence but it appears that bacteriocin production by the rumen anaerobe B. fibrisolvens may be uncommon in strains isolated from cattle and sheep in New Zealand. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]