Interaction Between Indole-3-acetic Acid And Quorum Sensing Signallings In Serratia Plymuthica

Quorum sensing（QS） system mediated by N-acyl-L-homoserine lactones（AHLs） is widespread in Gram-negative bacteria which can produce and release chemical signal molecules called autoinducers in response to population density that influence bacterial physiology and behavior by activating or repressing specific target gene expression. AHL-mediated cell-to-cell signalling has been shown to play a central role in diverse processes such as virulence factor、motility、antibiotic secretion and biofilm development. An endophytic strain Serratia plymuthica G3 isolated from the stems of Triticum aestivum L, can produce an array of antimicrobial exoproducts, including chitinase, protease, antibiotic, to suppress phytopathogens. Moreover, it also produces the plant hormone indole-3-acetic acid to promote plant growth. Our previous studies have established that there are three AHL-mediated luxIR-type QS homologues constituting complicated QS regulatory networks in strain G3, composed of SplIR、Sps IR two QS systems and a Lux R homolog SptR. Additionally, bioinformatic analysis identified two copies of ipdC gene to synthesize IAA through IPyA pathway. In this study, with focus on the interaction between indole-3-acetic acid and quorum sensing signallings in G3. Firstly, using gene replacement and homologous recombination strategy, several luxIR homologous mutants including the double mutant in splI & spsI, and mutants deficient in splR or spsR, as well as the ipdC₁-2 double mutant were constructed and verified, followed by phenotypic analysis to investigate their biological functions. On the other hand, systemic analysis of the reciprocal regulation within the QS network, and between IAA & QS signallings, apart from their auto-regulation were performed using methods of lux-based promoter transcriptional fusions. The main results achieved in this study are as follows:（1） A spsI/splI double mutant, single mutant defective in splR、or sps R, and ipdC₁-2 double mutant were constructed, and phenotypic analysis showed that mutation in genes dsccribed above resulted in significant alteration in AHL signal synthesis, protease activity, swimming motility and the production of IAA and acetoin to some extent by strain G3.（2） Assay of lux-based transcriptional fusions indicated that splI was positive autoregulation, and positively controlled the transcription of spsI、splR、spsR、sptR; Likewise, spsI was positively autoregulated, besides positive regulation of the transcription of splI、splR、spsR、sptR. Inversely, splR was autoregulated negatively, but positively regulated the transcription of splI 、spsI、spsR; spsR was also positively auto-regulated, in addition to positive control of the expression of splI 、spsI、splR at the transcriptional level. However, both splR and spsR had no impact on the transcription of sptR. These findings implyed a complicated reciprocal regulation within the different QS regulators in S. plymuthica G3.（3） Furthermore, analysis of lux-based transcriptional fusions showed that both ipdC genes were positive autoregulation, whilst positively regulated by the IAA precursor tryptophan（Trp） and the end product（IAA）.（4） Finally, analysis the interplay between IAA and AHL signaling demonstrated that both splIR, spsIR and sptR were negatively regulated by ipdC₁ and ipdC₂ at the transcriptional level. Reciprocally, both splIR and spsIR also negatively regulated the transcription of ipdC₁ and ipdC₂ indicating a highly close interconnections bewteen the IAA and AHL signal pathways in S. plymuthica G3 which has not been documented before.The results presented here have gained more insights into the interactive mechanisms between quorum sensing network and IAA signaling pathway, which would help with genetic manipulation of biocontrol agents for improvement of their efficiency and stability in management of plant diseases.