AHL-SdiA Inhibits Conjunction By Suppression Of Tral

ObjectiveBacteria communicate with each other with quorum sensing(QS)system.QS system helps bacteria sense the quantity dynamics of their own.Therefore,bacteria can make a strategic decision according to population.QS system can impacts gene regulation by a kind of signal molecule called Acyl-homoserine lactones(AHL).Bacteria secrete AHL for excretion until environmental concentration reach threshold.AHL bond its receptor to regulate genes.When bacteria encounters threat,low amount of bacteria activates the component to survive.In some situation,bacteria defend themselves by group behaviors like forming biofilm.Biofilm also creates an enabling environment for conjugation.Bacteria can transfer plasmids by pilus which is a connection between bacteria.The process is called conjunction and is the main route of transfering plasmids.Because plasmids can carry resistance genes,conjugation is considered as an access to antibiotic resistance.Antibiotics inhibit biofilm formation by suppressing QS system.In other words,antibiotics enhance plasmids transfer indirectly.However,some antibiotics promote conjugation with low concentration In this paper,we put forward a hypothesis.Antibiotics may reduce number of bacteria or degrade AHL,QS system therefore become paralysed.If QS system dominate and repress conjunction,then antibiotics would play as a disinhibition.As a result,antibiotics upregulate plasmids transfer.The issue is to illuminate that AHL repress plasmids transfer.MethodsWe use Escherichia coli SM10? ? as a donor of plasmid,Pseudomonas aeruginosa PA01 as a receptor.SM10? ? is unable to synthesis AHL but has SdiA as receptor protein.PA01 produce AHL which can combine SdiA from SM10? ?.Conjugational transfer gene traI dominate plasmids transfer in SM10???.By constructing the model of SM10? ?-PA01,both bacteria were shake cultured for enrichment.When bacterial cell density increased to 0.5 MCF,dilute it with LB broth to 1.0×107/mL.100?L of each bacteria were mixed and placed at 37? for 6h static cultured.Conjugants were then selected by using antibiotic plate.Bacteria solution was collected by centrifugation.Then total RNA was isolated for reverse transcription.Using qPCR to detect gene expression.Plasmid pQF50 was used as a reporter.Promoter of tral was cloned in the upstream of ?-galactosidase gene in pQF50.Then pQF50-PtraI was transformed into BW25113 for ONPG test.Results? To knockout of AHL gene las?rhlI of A01 enhanced SM10? ?-PA01 conjugation(P<0.001),and tral of SM10? ? was upregulated.Added by AHL,the decline of conjugation was observed(P<0.05).? Conjugation of SM10? ?-PA01 also was improved(P<0.001)if sdiA of SM10? ? was removed.Correspondly,expression of gene traI was increased(P<0.001).? Conjugation of SM10? ?-EC600 was not reinforced if sdiA of SM10? ?was deleted until AHL was added(P<0.001).? In the ONPG test,BW25113which contained pQF50-PtraI promoter reporter had the same enzyme activity when sdiA was erased.But the deletion of sdiA decreased enzyme activity in the presence of AHL(P<0.05).ConclusionTo mutate ether lasI?rhlI of PAO1 or sdiA of SM10? ? could enhanced SM10? ?-PA01 conjugation.Meanwhile,it induced the upregulation tral of SM10? ? accordingly.In other words,it illustrates AHL-SdiA inhibited plasmids transfer by depressing tral.The ONPG test result indicated AHL-SdiA played a role as tral promoter suppression.They were the supporting points for our assumption.Bacteria would not like to accept plasmids because it would be a heavy burden for bacteria to support plasmids replication.Therefore,QS system prevent bacteria from plasmids.When QS system collapses,bacteria will accept plasmids and attempt to survive.