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

Abstract

The bacterial second messenger cyclic diguanylate (c-di-GMP) modulates plankton-to-biofilm lifestyle transition of Pseudomonas 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 P. putida, including PP_0681, PP_0788, PP_4519 (lapE), PP_5222 (cyaA), and PP_5586. Except lapE encoding an outer membrane pore protein and cyaA 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 PP_0788 and cyaA and promote transcription of PP_0681, lapE, and PP_5586. Both in vitro and in vivo 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 lapE 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 cyaA 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 P. putida. IMPORTANCE c-di-GMP/FleQ promotes the plankton-to-biofilm lifestyle transition at the transcriptional level via FleQ in Pseudomonas 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 lapE 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 P. putida and P. aeruginosa remains unknown. Our result concerning transcriptional inhibition of cyaA by c-di-GMP/FleQ reveals the mechanism underlying the decrease of cAMP content by c-di-GMP in P. putida.

Transcriptomic analysis of Pseudomonas ogarae F113 reveals the antagonistic roles of AmrZ and FleQ during rhizosphere adaption

“…putida compared to the wild-type strain. In agreement with this work, a significant number of those genes were related to cell motility and secretion processes, defence mechanisms, amino acid transport/metabolism, carbohydrate transport/metabolism, and signal transduction [68].…”

“…aeruginosa with the finding of 89 activated genes and 249 repressed genes by this TF [4]. Regarding FleQ, Xiao et al (2021) [68] showed a similar number of up- and down-regulated genes, 172 and 272 respectively, in a fleQ mutant in P. putida compared to the wild-type strain.…”

Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ‐FleN complex in Pseudomonas putida

“…Pseudomonas putida KT2440 could use K1-T6SS to eradicate a broad range of bacteria, including E. coli and some phytopathogens (Bernal et al, 2017). Previous studies and our recent transcriptome data suggested that c-di-GMP and FleQ regulated K1-T6SS transcription in P. putida (Wang et al, 2017a;Wang et al, 2017b;Xiao et al, 2021). However, the exact mechanism of FleQ-FleN-c-di-GMP complex regulating T6SS remains unclear.…”

“…Previous studies and our recent transcriptome data suggested that c‐di‐GMP and FleQ regulated K1‐T6SS transcription in P . putida (Wang et al ., 2017a; Wang et al ., 2017b; Xiao et al ., 2021). However, the exact mechanism of FleQ‐FleN‐c‐di‐GMP complex regulating T6SS remains unclear.…”

The two‐component system TarR–TarS is regulated by c‐di‐GMP/FleQ and FliA and modulates antibiotic susceptibility in Pseudomonas putida

“…AAN69951), we found that tarR-tarS transcription was decreased in both wild-type strain with high-level c-di-GMP and fleQ deletion mutant (Xiao et al, 2021). We further compared tarR-tarS transcription in fleQ mutant and wild-type using qRT-PCR.…”