The Roles Of LuxS/AI-2 Quorum Sensing System In Bacterial Environmental Adaptation And Plant Growth Promoting Regulation In Rahnella Aquatilis HX2

Rahnella aquatilis strain HX2,is a rhizobacteria that could effectively promote plant growth.It has been found to produce indole acetic acid(IAA),pyrrolequinoline quinone(PQQ),and various organic acids,dissolve soil insoluble phosphorus,and survive in the roots of maize and grape.LuxS/AI-2 quorum sensing(QS)system is useded in bacteria intraspecific and interspecific communication.It is crucial in regulating bacterial physiological function,signal conduction,and enironmental adaptation,and bridges the interaction of bacteria and plant.The current sutdies of LuxS/AI-2 QS system mostly focused on the interaction mechanisms between pathogen and plant.However,how LuxS/AI-2 QS system regulates bacterial physiological metabolism and environment adaptation has not been reported.In this study,we obtained the HX2 genome sequence by whole-genome sequencing,revealed the adaptability of HX2 to stress,screened the optimum reference gene using quantitative real-time PCR,determined the differences of stress genes transcription in different stress conditions,analyzed the functions of the luxS,IsrK,and tqsA genes via gene knock out,complementation,and transcriptome experiments.(1)Genome sequence analysis showed that the HX2 genome is 5,656,799 bp.It contains one chromosome and two plasmids,with 4,942 protein-coding sequences.Phylogenetic analysis found that HX2 has close evolutionary relationship with the Rahnella sp.Y9602 and R.aquatilis CIP 78.65 strain.Comparative genomic analysis revealed that HX2 strain comprises of 245 essential genes,and ones that are involved in flagellar synthesis and chemotaxis,pyrrolequinoline quinone(PQQ)synthesis,organic acid synthesis,phosphatase and carbon-phosphorous lyase synthesis,nitrogen fixation,and IAA biosynthesis.As well as genes of 19 sets of two-component regulatory system,LuxS/AI-2 QS system,a variety of secretion system,selenium metabolism,and stress response genes.These functional genes are closely related to plant growth promotion,disease suppression,and environment adaptability.(2)The limit of acid tolerance is pH4.0,the limit of salt tolerance is 1.5M NaCl,the semi-lethal concentrations of paraquat and glyphosate is 2579 ?M and 79 mM,respectively.According to the transcription of fourteen internal control gene candidates in different treatments,we designated atpD as the optimal reference gene for subsequent real-time PCR analysis.Based on the expression of luxS,IsrK and tqsA gene which is responsible for AI-2 signal synthesis and transportation in different experimental treatments,we found that acidity,salinity,and oxidant affected the transcription of target genes,suggesting that LuxS/AI-2 QS system plays an important role in HX2 environmental adaptation.(3)Three mutants,HX2AluxS,HX2AIsrK.and HX2?tqsA were obtained by in-frame deletion disruptions of the luxS,IsrK,and tqs A gene through a PCR-based double crossover strategy.The complementary strains CHX2?luxS,CHX2?lsrK,and CHX2AtqsA were also constructed.Based on function analysis of all derivative strains,we found that LuxS/AI-2 QS system positively affects bacteria growth,AI-2 signal synthesis,biofilm formation,certain heavy metal tolerance,nano-selenium synthesis,and plant growth promotion.It also negatively affects swimming ability,chemotaxis,IAA synthesis,and other heavy metal tolerance.However,the swamming ability,phosphorus solubilizing capability,antibacterial substances synthesis,extracellular polysaccharide production,and lipopolysaccharide synthesis were not affected.(4)Transcriptome analysis of HX2?luxS and wild-type HX2 revealed 152 differentially expressed genes(DEGs),among them 99 genes are significantly up-regulated,and 53 genes that are significantly down-regulated.The DEGs concentrated in the pathway of flagella synthesis,chemotaxis,selenium compounds metabolism,sulfur metabolism,ABC transporters,and two-component system.In addition,we also found luxS gene involved in the negative regulation of plasmid conjugation tragene cluster,stress response genes,and in the positive regulation of organic phosphorus solubilization-related genes and maltose ABC transporter-related genes.This study aims to explore the regulation mechanisms of LuxS/AI-2 QS system in plant growth promotion and environment adaptation,and provide scientific basis and technical support for HX2 strain application in modern ecological agricultural production.