80 generations) in 100% of both E. coli DH5α and S. Typhimurium SL1344 host cells (Table 1). These data indicate that none of the six selected pCT genes are individually responsible for the short term maintenance and successful vertical transfer of this plasmid, as their inactivation did not impact on the inheritance of pCT. The pndACB operon is homologous to

known and characterised systems in other plasmids, CHIR98014 order such as R64, R483, p026-vir, ColIb-P9 and pO113, with protein identity between 91% and 100%. Furuya and Komano (1996) showed that when the pndACB operon, similar to that found on the IncI plasmid R64 was inactivated, R64 was rapidly lost from the bacterial population, therefore it was required for maintenance of R64 over a similar time period [24]. Based on protein homology, plasmid pCT was found to encode a putative parB-like nuclease gene which shares 100% identity to a previously characterised ParB

protein in p026-vir. However, the putative parB gene on pCT shares no significant homology to the parB DNA sequences ACY-1215 solubility dmso from other IncI plasmids, such as R64 and CoIIb-P9. We found that the recombinant pCT plasmid carrying the inactivated putative parB gene also showed no significant difference in stability when compared to the wild-type plasmid. This was in contrast to work by others with plasmid P1, which showed that an intact parB is essential for the stable partitioning of P1 [25]. Our data with pCT indicated that neither pndACB nor the putative parB genes are individually essential for pCT stability under conditions tested suggesting they may not be expressed under such conditions; may work in conjunction with other elements; or are non-essential for stability due to the presence of other currently unidentified genes or gene regions. These data also suggest that broad conclusions about gene function SAHA HDAC clinical trial cannot be extrapolated from data obtained with other plasmids. Table 1 Comparison of recombinant plasmids with wildtype pCT plasmid Gene inactivated on pCT Stability Conjugation to an E. colirecipient Conjugation to a Salmonellarecipient Bacterial host growth

kinetics Biofilm formation Competitive index when PRKACG co-cultured with WT pCT Sigma factor::aph = = = = = 1.00 pilS::aph = ↓ ↓ = = 1.00 traY::aph = UD UD = = 0.99 rci::aph = = ↓ = = 0.99 pndACB::aph = = = = = 1.00 parB::aph = ND ND = = ND =, the same as wild-type (WT) pCT; ↓, reduced rate when compared to pCT; ND, not determined; UD, Undetectable. The relative contribution of each conjugation pilus in pCT horizontal transfer To investigate the contribution of the two conjugation pilus genes (tra and pil) in the dissemination of pCT, the effects of inactivating the major structural protein genes of each pilus (traY and pilS) were assessed. Inactivation of traY prevented pCT transfer both in liquid and on solid surfaces (Figure 2) confirming the essential role of the tra locus for pCT conjugation under both conditions [26].