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Commensal butyrate-producing bacteria in the Firmicutes phylum are abundant in the human intestine and are important for maintaining health. However, understanding of the metabolism and host interaction of these bacteria is limited by the lack of genetic modification techniques. Here we establish a protocol enabling the transfer of autonomously-replicating shuttle vectors by conjugative plasmid transfer from an Escherichia coli donor into representatives of an important sub-group of strictly anaerobic human colonic Firmicutes. Five different plasmid shuttle vectors were tested, each carrying a different origin of replication from Gram-positive bacteria. Plasmid pMTL83151 (pCB102 replicon) were successfully transferred into two strains of Eubacterium rectale, while pMTL83151 and pMTL82151 (pBP1 replicon) were transferred into Roseburia inulinivorans A2-194. Plasmids that carried a Streptococcus bovis JB1 glycoside hydrolase family 16 β-(1,3-1,4)-glucanase gene were constructed and conjugated into Roseburia inulinivorans A2-194 and Eubacterium rectale T1-815, resulting in successful heterologous expression of this introduced enzymatic activity in these two strains of butyrate-producing Firmicutes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

During screening of human gut isolates in search of novel antimicrobials, the butyrate-producing strain Roseburia faecis M72/1 was found to produce an inhibitory substance active against Bacillus subtilis. Partial purification of the antimicrobial was achieved and activity found to be heat labile. Our findings suggest that R. faecis M72/1 produces a proteinaceous inhibitor whose production may be triggered by trypsin in the gastrointestinal tract., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Recent molecular analyses suggest that bacteria related to strains SS2/1 and SSC/2, previously reported to be distantly related to Anaerostipes caccae NCIMB 13811(T), represent one of the ten most abundant phylotypes detected in adult human faecal samples. These two strains were isolated as d-lactate-utilizing bacteria from faecal samples of a healthy individual. We show here that they share >99.9% similarity in 16S rRNA gene sequence with a new butyrate-producing isolate recovered from a colonic biopsy of a Crohn's disease patient, and also with the sequence reported recently for Eubacterium hadrum ATCC 29173(T). Biochemical profiling using API Rapid ID 32A and API ZYM test systems confirmed a close phenotypic similarity to E. hadrum ATCC 29173(T), but also indicated that the description of this species should be expanded to include the ability to produce butyrate from d-lactate and acetate. Phylogenetic analysis confirmed an affinity between E. hadrum and members of the genus Anaerostipes (92.3-94.2% sequence similarity) belonging to the family Lachnospiraceae (formerly Clostridium cluster XIVa). Based on phylogenetic, phenotypic and chemotaxonomic evidence it is proposed that E. hadrum be transferred to the genus Anaerostipes with the name Anaerostipes hadrus comb. nov. The type strain of A. hadrus comb. nov. is =ATCC 29173(T) (=DSM 3319(T) = VP 82-52(T))., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4., (Copyright 2001 Academic Press.)

Plasmid pJW1 from Selenomonas ruminantium subsp. lactilytica strain JW13 has been cloned in Escherichia coli vector pBluescriptSK(-) and completely sequenced. The plasmid is only 1410 bp with an overall GC content of 42.2%. Computer analysis of sequence data revealed a single open reading frame (ORF1, 146 amino acids, MW 16,525.5 Da) encoding a putative replication protein which is similar to the Rep protein of Ruminobacter amylophilus plasmid pRAO1. ORF1 is followed by a long AT-rich (75%) region and a region abundant in direct and inverted repeats. Comparison of DNA sequences revealed the presence of a short (<250 bp) DNA segment which is highly conserved between several small S. ruminantium plasmids including pJDB21., (Copyright 2000 Academic Press.)

Freshly harvested whole cells from cultures of P. bryantii B(1)4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantii B(1)4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation with P. bryantii showed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens gave little increase in overall pentose utilization suggesting that external P. bryantii xylanases are as effective as the cloned R. flavefaciens enzymes in releasing products that can be utilised by P. bryantii cells. The xylanase system of P. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.