An Osmoregulatory Mechanism Operating Through OmpR-LrhA Signal Cascade Reversely Regulates Biofilm And Motility In Pantoea Alhagi

Bacteria use a variety of adaptive strategies or means to cope with the changing environment in naure.It is worth-noting that most bacteria grow in two different ways in response to environmental changes: planktonic model and biofilm model.The formation of biofilm is controlled by various factors,which are important participants in the transition of bacteria from planktonic status to biofilm status.Various regulators and molecular switches have been found so far.It has been found that some moving molecular switches in bacteria combined with complex regulatory networks can respond to various environmental signals to control the transition of their own lifestyle,and further affect the growth of plants or other hosts.However,the internal potential regulatory mechanisms remain unclear.Osmotic pressure stability is essential for bacterial growth and survival.Although many osmotic stress response-related regulatory genes have been identified in various bacterial species,how do the permeability changes affect bacterial movement,biofilm formation and colonization of plants is still unknown.In this study,we found an OmpR-LrhA osmotic response signal cascade system in Pantoea alhagi,which can reversely regulate the biofilm and motility of Pantoea alhagi and affects the colonization of bacteria on the plants.These results bring new insight on the regulatory mechanism in this bacteria.LrhA is a transcriptional regulator of the LysR family,which is highly conserved among bacteria and has diverse functions in different bacteria(Proteus,Escherichia coli,Yersinia,etc.).HexA,PecT and RovM,the homologous protein of LrhA,play important roles in necessary growth and metabolism processes like bacterial motility,biofilm formation,toxicity,drug resistance.In this paper,the LrhA functions of Pantoea alhagi LTYR-11 Z strain in regulating bacterial biofilm,migration,and plant colonization were explored by using molecular biological methods.The research in this paper confirms:(1)In P.alhagi LTYR-11 Z,LrhA can positively regulate the formation of biofilm and negatively regulate the ability of bacterial migration.Through wheat adsorption and colonization experiments,it detected the deletion of lrhA gene at different time reduced the bacterial attachment and colonization on wheat.It was more intuitively observed by confocal microscopy that the deletion of lrhA gene significantly reduced the ability of bacteria to compete with the host.This indicates that lrhA gene may act as an important plant colonization regulator,affecting the bacterial attachment and colonization on plants.(2)Analysis based on transcriptome data and qRT-PCR results showed LrhA plays a positive role in regulating eps,while in reverse regulation of flhDC.Further quantitative detection of LrhA by Congo red and extracellular polysaccharides showed LrhA can positively regulate the expression of EPS.In addition,the lacZ reporter gene was used to detect the difference in expression of eps in wild-type strains and lrhA mutants,further confirming the positive regulation on the transcriptional level of LrhA against eps.EMSA experiments confirmed that LrhA can directly bind to an upstream gene sequence of the eps promoter,and positively regulate the expression of EPS,which in turn affects the formation of biofilm.Similarly,it found that lrhA can reverse-regulate the activity of the flhDC gene cluster promoter and directly regulate the expression of flhDC at the transcriptional level negatively.EMSA assay detected specific binding sequences between LrhA and flhDC promoters.Bacterial migration assay further confirmed that LrhA affects bacterial motility by negative regulation of flhDC.(3)A fusion strain of the opgGH promoter with lacZ gene was separately constructed to detect the activity of the opgGH promoter.The effect between the LrhA and opgGH promoters was still tested using the EMSA assay.It found a specific binding between the LrhA and opgGH promoters,which positively regulates the transcriptional activity of the opgGH promoter.Moreover,it confirmed that opgGH plays an important role in the positive regulation of biofilm and the negative regulation of bacterial motility.Finally,we examined the content of OPGs in four different strains and found that the ΔlrhA significantly decreased the content of OPGs compared to the wild-type strain(WT).It indicated that LrhA can positively regulate the expression of opgGH promoter,which in turn affects the concentration of OPGs in the strain.(4)Western blotting analysis revealed that phosphorylated RcsB protein levels were significantly reduced after knocking out the genes opgH and lrhA,respectively.However,after replenishing opgGH in the strain ΔlrhA,the phosphorylated RcsB protein expression level increased to a higher level.It confirmed that an increase in the concentration of OPGs can activate the RCS phosphorylation pathway inside the bacteria.Finally,it was verified that OPGs/OpgGH regulates the transcriptional activity of eps and flhDC by controlling the phosphorylation level of RcsB in P.alhagi,thereby regulating the formation of biofilm and the motility of bacteria.Furthermore,the expression of LrhA is simultaneously suppressed by RCS phosphoric System.(5)Real-time PCR analysis showed that the mRNA levels of lrhA,opgG and opgH increased with the increasing of NaCl and sorbitol.The activity of promoters of eps and opgGH were highly increased,the activity of the flhDC was lowed.Interestingly,once the strain lacks the gene lrhA,the strain does not respond to changes in external osmotic pressure.These results suggest that lrhA is affected by different osmotic pressures in the surrounding environment during the regulation of eps,flhDC and opgGH.Similarly,when the NaCl and sorbitol concentration increased,the biofilm production of wild-type strains increased,the migrated ability of bacteria decreased,and the colonization ability of plants increased.However,after the deletion of lrhA,no significant change was detected in the biofilm formation,swimming experiments,root colonization,etc.(6)The possible binding sites of OmpR to lrhA promoter region was analyzed by bioinformatics,and the difference of lrhA expression between wild-type strain and ΔompR was detected using lacZ reporter gene,which further clarified the positive regulation of OmpR on lrhA.EMSA experiment proved that OmpR can directly bind to an upstream sequence of the lrhA promoter.In addition,it was verified that OmpR further regulates the activity of lrhA in bacteria by responding to changes in environmental osmotic pressure.Based on these results,this paper proposes a corresponding dynamic model: OmpRLrhA regulates the expression of eps,flhDC and opgGH promoters in response to changes in osmotic pressure of the surrounding environment,thereby controlling the shift of planktonic or biofilm mode.