Molecular Analysis of the Enterococcus faecalis Serotype 2 Polysaccharide Determinant

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Abstract
We previously described a 15-kb genetic cluster consisting of 11 open reading frames ( cps2A to cps2K ) of Enterococcus faecalis FA2-2 that is responsible for the production of the serotype 2 capsular polysaccharide. By using transcriptional fusions to a promoterless lac Z gene, we identified two independent promoters related to the expression of the polysaccharide. Both transcription initiation sites were mapped by primer extension. Reverse transcription-PCR (RT-PCR) demonstrated the transcriptional linkage of genes present in both transcripts. Real-time RT-PCR quantification of transcripts revealed maximum transcription during log phase growth, an observation confirmed by promoter fusion studies. The heterologous expression of this pathway in Escherichia coli caused reactivity with E. faecalis type 2 antiserum, thus demonstrating the essential role of this pathway in the synthesis of the type-specific polysaccharide.