Studies on the molecular biology and ecology of butyrate-producing human colonic bacteria

The role of butyrate in the metabolism and development of a healthy colonic epithelium and thus in the prevention of colonic diseases is well documented, but the ecology of butyrate-producing bacteria is little understood. Dietary polysaccharides that are not digested in the small intestine are a major source of energy for the commensal bacteria resident in the large intestine. Resistant starch makes up a large proportion of dietary substrate reaching the human colon and is also reported to be butyrogenic. Recent studies have shown that low G+C% Gram-positive bacteria, which include butyrate-producing isolates, are among the most abundant components of the adult colonic microbiota. Here, in vitro growth experiments demonstrated that certain new butyrate producing isolates (Roseburia spp./Butyrivibrio fibrisolvens) were able to utilise starch as a sole energy source, with a growth preference for high amylopectin starches. Active starch-degrading enzyme were cell-associated in these strains and were visualised using zymogram analysis and found to be of high molecular weight. An amylase gene from Butyrivibrio fibrisolvens 16.4 was sequenced following polymerase chain reaction (PCR) amplification with degenerate oligonucleotides and chromosome walking. The putative amylase consists of an open reading frame of 4002 bp encoding a protein of 1333 amino acids with a calculated Mr of 144,470. Analysis of the multidomain organisation of this enzyme indicated that it is bound to the cell wall, with its putative catalytic domain and repeated starch-binding modules protruding into the extracellular matrix. This work also describes the use of an in vitro continuous culture fermentor system and human microbiota-associated mice (in vivo) to assess the potential of certain butyrate-producing anaerobes for probiotic and/or prebiotic applications.EThOS - Electronic Theses Online ServiceGBUnited Kingdo