Molecular Cloning And Functional Analysis Of Antibiotics Synthesis Regulatory Gene In Pseudomonas Fluorescens FD6

Pseudomonas fluorescens FD6, a biocontrol bacterium originally isolated from the canola rhizosphere in Fujian Province and showed an effective protection against tomato grey mould and peach brown rot. Strain FD6 produces a couple of antifungal compounds, including 2, 4-diacetylphloroglucinol (2,4-DAPG), pyrrolnitrin (PRN) and pyoluterrin (PLT). Previous research had confirmed that biosynthesis of these three antibiotics were positively regulated by the sensor kinase GacS.RetS (Regulator of exopolysaccharide and type III secretion), a sensor kinase located in cell membrane, regulates the expression of a broad range of genes. A 3767 bp fragment containing the retS gene was obtained using PCR and analyzed the function of retS through gene deletion and complementation. The results of this study showed that the inactivation of retS gene resulted in a markedly increased production of antibiotics PRN,2,4-DAPG and PLT compared with the wild-type FD6. The FD6AretS strain exhibited a 8.4-fold increases in PRN production compared to those of wild-type FD6 and greater than one fold increase in 2, 4-DAPG and PLT production. These findings indicated that retS has negative effect on the production of antibiotics PRN,2,4-DAPG and PLT. And retS gene negatively controlled the expression of prnA, phlA and plt A genes at the transcriptional level. At the same time, retS gene negatively regulated the transcription of the small RNAs rsmX, rsmY and rsmZ. However, the phenotypes caused by retS gene inactivation were obviously reversed in the retSgacS double mutant and exhibited similar level with that of gacS single mutant. Collectively, these results suggest that down-regulation of RetS on the production of antibiotics relied on the GacS protein. Furthermore, and also affected the bacterial growth, motility, biofilm formation and colonization in strain FD6. In this current study, retS is an important regulatory element in strain FD6.In this paper we also cloned another 2496 bp vfr (Virulence factor regulator) gene fragment from strain FD6 using PCR and analyzed the effect of vfr on biocontrol-related traits in strain FD6 in the same way. The vfr mutant exhibited over two-fold increases in biosynthesis of antibiotics PRN,2,4-DAPG and PLT, suggesting that vfr negatively regulated the production of these three antibiotics. To explore the possible regulatory mechanism of vfr, lacZ-fusion constructs were used to analyze the relationship among them. We found that vfr modulated the production of antibiotics PRN,2,4-DAPG and PLT in a Gac/Rsm-independent mechanism, whereas vfr suppressed significantly the expression of rsmA,fleQ and rpoS genes. Meanwhile, vfr gene negatively controlled prnA, phlA and pltA genes at the transcriptional level and also had influence on the bacterial growth, motility ability, biofilm formation and colonization FD6. Collectively, our findings suggested that vfr is also an important regulatory element involved in the expression of biocontrol factors in strain FD6.