Identification of c-di-GMP/FleQ-Regulated New Target Genes, Including , Encoding Adenylate Cyclase, in Pseudomonas putida.

The bacterial second messenger cyclic diguanylate (c-di-GMP) modulates plankton-to-biofilm lifestyle transition of species through its transcriptional regulatory effector FleQ. FleQ regulates transcription of biofilm- and flagellum-related genes in response to c-di-GMP. Through transcriptomic analysis and FleQ-DNA binding assay, this study identified five new target genes of c-di-GMP/FleQ in , including , , (), (), and Except encoding an outer membrane pore protein and encoding an adenylate cyclase, the functions of the other three genes encoding hypothetical proteins remain unknown. FleQ and c-di-GMP coordinately inhibit transcription of and and promote transcription of , , and Both and assays show that FleQ binds directly to promoters of the five genes. Further analyses confirm that LapE plays a central role of in the secretion of adhesin LapA and that c-di-GMP/FleQ increases transcription, thereby promoting adhesin secretion and biofilm formation. The adenylate cyclase CyaA is responsible for synthesis of another second messenger, cyclic AMP (cAMP). FleQ and c-di-GMP coordinate to decrease the content of cAMP, suggesting that c-di-GMP and FleQ coregulate cAMP by modulating expression. Overall, this study adds five new members to the c-di-GMP/FleQ-regulated gene family and reveals the role of c-di-GMP/FleQ in LapA secretion and cAMP synthesis regulation in c-di-GMP/FleQ promotes the plankton-to-biofilm lifestyle transition at the transcriptional level via FleQ in species. Identification of new target genes directly regulated by c-di-GMP/FleQ helps to broaden the knowledge of c-di-GMP/FleQ-mediated transcriptional regulation. Regulation of by c-di-GMP/FleQ guarantees highly efficient LapA secretion and biofilm formation. The mechanism of negative correlation between c-di-GMP and cAMP in both and remains unknown. Our result concerning transcriptional inhibition of by c-di-GMP/FleQ reveals the mechanism underlying the decrease of cAMP content by c-di-GMP in .