H-073. A Family of Transcriptional Antitermination Factors Is Necessary for Synthesis of the Multiple Capsular Polysaccharides of Bacteroides fragilis

M. Chatzidaki-Livanis, M. J. Coyne, L. E. Comstock;
Channing Lab., Harvard Med. Sch., Boston, MA.

Bacteroides fragilis, one of the numerically dominant microorganisms of the human intestinal microbiota, synthesizes eight distinct capsular polysaccharides (PSA-PSH) and an extracellular polysaccharide. The expression of these surface polysaccharides is subject to phase variation dictated by invertible promoters upstream of the polysaccharide biosynthesis loci. The first gene of each of the eight PS biosynthesis loci are homologous to each other and have been designated upxY (where x= a-h depending on the PS biosynthesis locus). The eight UpxY products are 38-86% similar to each other and contain a NusG-like domain. Analysis of internal, nonpolar deletion mutants of three of these genes, upaY, upcY, and uphY revealed that each UpxY products is necessary for synthesis of its respective polysaccharide and that these products function in trans. Northern analysis demonstrated that the defect in polysaccharide synthesis in the upxY mutants is due to lack of transcription of the respective polysaccharide locus. We made transcriptional fusions of the PSA region using a xylE reporter plasmid to determine if the UpxY products are necessary for transcription initiation or a post-initiation event. These studies demonstrated that UpaY is not necessary for transcription initiation. We found that the defect occurs downstream of the promoter in the 126-bp untranslated region upstream of upaY and we identified a site where the majority of the PSA transcripts terminate in the absence of UpaY. Additional studies have confirmed the role of the UpxY products in transcriptional antitermination. The similarity of the 5’ untranslated regions of the polysaccharide transcripts has allowed us to determine what nucleotide sequences dictate the specificity of the UpxY products for their cognate region.