For the C. difficile peptide fractions analysed in this investigation (Fig. 1), the number of unique proteins identified in a sample did not increase significantly after three replicate injections (Fig. S1) and therefore all peptide samples were injected and analysed three

separate times to maximize the overall protein identification. Stringent automated curation of the data set using provalt set with a FDR of 1% yielded a total of 560 uniquely identified peptides, corresponding to 107 uniquely identified proteins. The average MOWSE score was 240; the average number of peptides per protein was five and the average protein coverage was 24% (Tables S1 and S2). The proteins identified had widely varying physiochemical characteristics, with the most acidic protein being a conserved hypothetical protein (CD2522; pI 4.57) and the most basic being 50S ribosomal learn more protein L20 (pI 11.48). The lowest molecular mass protein identified was 50S ribosomal protein L36 (Mr 4277 Da) and the highest was a hypothetical protein (CD0590; Mr 197 241 Da). We could functionally categorize all except for three of the

proteins identified in this study according to the SubtiList functional category list (Graham et al., 2006a, b, 2007) (Table 1). The largest category of identified proteins was that involved Akt inhibitor in protein many synthesis (45.8%), followed by that involved in the metabolism of amino acids and related molecules (10.3%). Of the three ‘uncategorizable’ proteins identified, those encoded by CD2552 (iojap-like protein) and CD1711 may be part of the bacterial core genome, a concept proposed by Mulkidjanian et al. (2006) and further developed in the recent work of Callister et al. (2008). Homologues of these proteins are also found in other species of saccharolytic and fermentative clostridia, in addition to other known gut bacteria

including Roseburia intestinalis and Faecalibacterium prausnitzii (Aminov et al., 2006). The third, CD0590, encodes a conserved hypothetical protein that has an N-terminal Mg2+/GTP-binding motif as identified by blastp analysis. Interestingly, and in contrast to the other two hypothetical proteins identified in this study, CD0590 appears to be absent from all other Clostridia species and indeed yields no significant homology matches with any other organism in the NCBI database. The exception to this appears to be a protein encoded by the adjacent gene, CD0589, which shares significant homology and appears to represent a duplication of the N-terminal Mg2+/GTP-binding region of CD0590. All publicly available C. difficile genomes also appear to contain homologues of both CD0590 and CD0589. As regards a possible function for protein CD0590, O’Connor et al.