scophthalmi A102. The growth rate was reduced in V. scophthalmi A089_23 overexpressing luxR (black square) compared to the control strain (black triangle) (Figure 1a), while strain A102_6.2 expressing the lactonase (black square) had a longer lag phase with respect to the control strain A102_pACYC (black triangle) (Figure VX-765 research buy 1b). In contrast, quorum-sensing was shown to positively regulate biofilm formation in vitro since both luxR and luxS null mutants had altered biofilm formation (Figure 2). Noticeably, biofilm was only formed when bacteria were grown in MB medium in either the mutant
or the wild-type strains and abolished when bacteria were cultured in TSB2 (data not shown). MB medium is used to culture heterotrophic marine bacteria and mimics the marine salt concentration and, although TSB also allowed growth of the bacterium, for some reason the differences in salt concentration or in nutrient or carbohydrate contents exerted an effect on biofilm formation. In order to investigate a possible effect of catabolite repression, we supplemented MB with glucose 0.5% and 1% w/v which resulted in a decrease in biofilm formation. On the other hand, over-expression of luxR decreased the amount of biofilm, perhaps due to the decrease AZD6244 in the growth rate caused by the deregulation of luxR, as stated above. In the case of luxS buy CB-839 overexpression no differences were found between the over-expressed luxS and the control
strain carrying pMMB207 plasmid. Complementation of the A102 null luxS mutant strain with the pACYC184 plasmid reverted the strain to the wild type phenotype. Figure 2 Biofilm formation in the V. scophthalmi A102 strain cultured in MB; wt, wild type strain; ΔluxR , A102_56 strain; ΔluxS , mutant A102_73 strain; pMMB207, A102_90; pMMB207 ::luxR , A102_78 mutant; pACYC, A102_pACYC184; pACYC184:: luxS , A102_99 strain. The error bars indicate standard
deviation based on three independent assays with four replicates each one. Statistical analysis was performed by student’s t test. Similar results were obtained with the A089 mutant strains. Positive and Cyclin-dependent kinase 3 negative regulation of biofilm formation has been reported in other vibrio such as V. anguillarum and V. cholerae, respectively [16, 17]. Interestingly, in a recent study on quorum-sensing in V. ichthyoenteri (the most closely related species to V. scophthalmi), its luxS homologue was sequenced and a mutant for this gene constructed, but no functions were reported to be regulated by this gene . It has to be noted that neither the V. ichthyoenteri wild type, nor the luxS mutant formed biofilms in the microwell plates. Our results showed that luxS is involved in biofilm formation at least in vitro in V. scophthalmi. However, it is important to highlight that in our study the V. scophthalmi wild-type strain was only able to form significant biofilm when grown in MB, while TSB inhibited biofilm formation in vitro. Therefore, it would be interesting to assess if V.