Abstract:[Background] The quorum sensing signal molecule AI-2 is spontaneously cyclized from 4,5-dihydroxy-2,3-pentanedione (DPD), which is derived from S-adenosylhomocysteine (SAH) via catalysis by S-ribosylhomocysteinase (LuxS) and S-adenosylhomocysteine nucleosidase (Pfs, also known as MtnN). AI-2 regulates a variety of physiological processes including chemotaxis, bioluminescence, and biofilm formation of bacteria. However, the effect of AI-2 on the salt adaptability of Halobacillus halophilus has not been reported. [Objective] To synthesize AI-2 in vitro and reveal its effect on the salt adaptability of H. halophilus. [Methods] The relationship between luxS transcript level in H. halophilus and salt concentration was assessed by real-time quantitative PCR. Then, multiple sequence alignments of LuxS, MtnN-1, MtnN-2, and MtnN-3 were performed to identify the key amino acid sites, and the four proteins were heterologously expressed and purified for the synthesis of AI-2 in vitro. Finally, luxS was knocked out by homologous recombination, and the effect of luxS on the salt adaptability of H. halophilus was studied through salt stress assay, intracellular compatible solute content determination, and biofilm formation assay. [Results] The transcript level of luxS in H. halophilus increased with the elevation in salt concentration and was positively regulated by the Cl– concentration. The growth curve and bioluminescence assay showed that as H. halophilus grew, the AI-2 activity in the supernatant of the culture medium reached the maximum at 15 h, and it enhanced with the increase in salt concentration. The in vitro enzymatic reaction assay showed that LuxS collaborated with MtnN-1 or MtnN-2 to catalyze the synthesis of AI-2. The luxS-deleted mutant of H. halophilus was successfully constructed by homologous recombination. Under a low salt concentration (0.5 mol/L NaCl), there were no significant differences in the growth curves between the wild type, luxS-deleted mutant, and complementary strain. However, under high salt (3.5 mol/L NaCl), the luxS-deleted mutant grew slow, while the complementary strain demonstrated a growth trend similar to that of the wild type. Furthermore, the deletion of luxS led to decreases in the survival rate, intracellular compatible solute content, and biofilm formation of H. halophilus under salt stress, whereas the complementary strain could recover to the levels close to those of the wild type. [Conclusion] H. halophilus can synthesize AI-2, and its luxS is positively regulated by the Cl– concentration and plays an important role in regulating salt adaptability. The findings provide a basis for further research on the regulatory mechanism of salt adaptability in H. halophilus.
