3a). Expression was reduced further Epacadostat in vivo when only AI-2 was provided, and the lowest comEA transcription was observed when neither autoinducer was provided (Fig 3a). Likewise, a similar pattern was observed with the purified autoinducers in the crab-shell microcosm assay with the autoinducer-deficient V. choleraeΔcqsAΔluxS mutant. We suspect that the slightly lower levels of comEA expression observed when the autoinducers were produced by V. cholerae (Fig. 2a) compared with the results with purified autoinducers

(Fig. 3a) may perhaps reflect lower levels of autoinducer synthesis and/or secretion in artificial sea water, conditions under which autoinducer production has not been quantified. Finally, by providing exogenous, purified CAI-1 and AI-2 to the chitinous biofilm (as described in Materials and methods), the autoinducer-deficient strain selleckchem was capable of taking up

DNA with a transformation efficiency similar to V. cholerae strains that produced their own autoinducers (Fig. 3b). Based on our results with the QS mutants and purified autoinducers (Figs 2 and 3), we hypothesized that V. cholerae might also sense and respond to autoinducers irrespective of their origin, including autoinducers derived from other Vibrios within in a mixed-species biofilm. We reasoned that a mixed-species consortium may more closely reflect conditions in environmental biofilms that are unlikely to be mono-species in composition (Hall-Stoodley et al., 2004; Wintermute & Silver, 2010). To demonstrate the feasibility of a mixed-species, crab-shell microcosm assay, the V. cholerae autoinducer-deficient recipient (ΔcqsAΔluxS) was co-cultured on chitinous crab shells with V. cholerae autoinducer-proficient donor strains that were HapR− (and thus QS−) but still capable of producing both autoinducers, only CAI-1, only AI-2, or neither autoinducer. MYO10 The autoinducer-deficient V. cholerae recipient responded to both autoinducers derived from V. cholerae HapR− autoinducer donors within the biofilm and efficiently acquired extracellular DNA. Maximal transformation

frequency occurred when the V. cholerae autoinducer recipient was provided with both autoinducers, while the response to only CAI-1 or only AI-2 was reduced. An autoinducer donor unable to produce either autoinducer promoted the lowest transformation frequency (Fig. 4). Similar results were obtained with several additional V. cholerae isolates that served as the CAI-1 and AI-2 donor (data not shown). These results validated that in the crab-shell microcosm autoinducers derived from donor V. cholerae cells could promote comEA expression in a V. cholerae recipient; thus we monitored DNA uptake in the V. choleraeΔcqsAΔluxS autoinducer-deficient strain, co-cultured in a mixed biofilm with different Vibrio species serving as autoinducer donors. Indeed, in these mixed-species biofilms, V.