Mechanistic Analysis Of A Two-Component Transduction System PilS-PilR In Regulating Antifungal HSAF Biosynthesis In Lysobacter Enzymogenes

HSAF(heat-stable antifungal factor)produced by Lysobacter enzymogenes OH 11 is a broad-spectrum antifungal secondary antibiotic with new chemical structure and mode of action.In our earlier study,we have shown that a two-component transduction system,called PilS-PilR evolves to control HSAF biosynthesis via a c-di-GMP pathway.Available earlier reports suggest that PilS-PilR is widespread in bacterial genus,with a conserved function in regulating type Ⅳ-pilus mediated twitching motility,wherein PilS encodes a histidine kinase response for receiving extra-/intra-sitmuli to undergo an autho-phosphorylation,followed to transfer the phosphor group to the cognate response regulator PilR to achive functional out.Finding that regulation of PilS-PilR in HSAF biosynthesis enables this two-component system to have a previously uncharacterized function that is to control production of bioactive natural product in bacteria.The objective of this study is explore how the PilS-PilR system evolves to control HSAF biosynthesis via a c-di-GMP pathway by using L.enzymogenes OH11 as a working model.The main findings of this study includes:Considering the fact that intracellular c-di-GMP metabolism is determined by a group of GGDEF-domain proteins responsible for c-di-GMP synthesis and EAL or HD-GYP domain proteins for degradation,we thus selected 8 such protein genes with a known fuction in regulating HSAF biosynthesis according to our earlier reports,to explore whether any of them is directly controlled by PilR at transcription level.For this purpose,the bacterial one-hybrid method was applied to screen whether PilR could establish a direct interation with any of test gene promoter regions.Via such an approach,we identified that PilR could directly bind to the promoter of lchD,whose product contains a single GGDEF dmain.In agreement,overexpression of lchD in the wild-type OH 11 significantly impaired HSAF yiled,suggesting that PilR seems to directly bind to the lchD promoter to control LchD-mediated intracellular c-di-GMP levels,leading to regulate HSAF production.To test the above hypothesis,we further validated the direct interaction of PilR-His fusion protein with the lchD promoter via both electrophoretic mobility shift assay and microscale thermophoresis(MST).Results of quantitative RT-PCR assay showed that mutation of pilR caused a remarkable increase of lchD transcript,further revealing that direct binding of PilR on the lchD promoter is likely to inhibit the lchD transcription.In consistence,deletion of lchD or chromosomal substitution of the LchD enzymatically active motif from GGDEF to GGAAF at the background of the pilR mutation indeed rescued the HSAF production and the HSAF biosynthesis operon gene expression,while inactivation of other GGDEF-domain genes at the pilR mutations failed to achieve such a rescue.To further expore how PilS-PilR connects LchD to form a circuit to fine-tune regulation of HSAF biosynthesis in response to stimuli,we test the potential physical interaction between PilS and LchD via both bacterial two-hybrid and MST assays,and results showed that cytoplasmic domain of PilS physically interacted with the LchD-GGDEF domain,with a moderate binding affinity(Kd,3.6 μM)that is consistent with physical concentration of protein levels in vivo.Further truncation assays revealed that the catalytic ATP-binding domain of PilS is required for PilS-LchD interaction.Finally,we explored whether the phosphorylation process of PilS-PilR is key for HSAF biosynthesis.To achieve this,the predicted key amino residues,H328 and D55 responsible for PilS auto-phosphorylation and phosphor transfer to PilR,respectively,were substitulted to alanine(A)at choromosal via site-direct mutagenesis and double-crossover homologous recombination.HSAF quantification assays showed that individual alanine substitution at H328 or D55 casued a significant decrease on HSAF production.Moreover,we further found that the parental PilR seems to localize two polars of cells,while chaning D55 into A55 led to PilR localization on single polar of cells.It is also observed that the parental PilS appears to localize the inner-membrane of cells,while PilS with a H328 substitution by A328 blocked this membrane localization.These results collectively suggest that the phosphorylation process of PilS-PilR is key both for their regulation on HSAF biosynthesis and their protein localization in cells.