C. difficle p-cresol production

To identify the cellular localisation of the HpdBCA decarboxylase complex, a plasmid based HpdB-SNAP-tag translational fusion was constructed using a C. difficile compatible plasmid (pMTL84151) under control of the hpdBCA promoter. The hpdB coding sequence omitting the stop codon, was fused via a linker to a SNAP-tag (Table 1). Confirmation of HpdB linked to the SNAP-tag was carried out via western blot, and mass spectrometry, in which we identified six peptides, four with 100% identity and two with 99% identity unique to HpdB. Localisation of HpdB was visualised by confocal microscopy in 630Δerm and the p-cresol deficient mutant (hpdC::CT) (carrying the HpdB-SNAP-tag fusion (PhpdB-CDS-SNAP), in the presence and absence of p-HPA) to induce HpdBCA production.