Regulatory Mode Of AI-2 Quorum Sensing System In Escherichia Coli And Staphylococcus Aureus

In most environmental niches,bacteria exist in complex communities of single or multiple species that develop on abiotic(for example,rocks) or biotic(for example, host mucosal tissues) surfaces rather than as single,planktonic cells suspended in liquid.As such niches are diverse,microorganisms depend on their capacity to sense the local environmental conditions and adapt by regulating the expression of specific genes.This is particularly vital for pathogenic bacteria,which encounter a range of habitats in a host.Consequently,bacteria have developed mechanisms for quorum sensing(QS),by which bacteria communicate with one another using chemical signal molecules.QS bacteria secrete small,diffusible signalling molecules called autoinducers that accumulate in the external environment.When the concentration of the autoinducers reaches a threshold,an alteration of gene expression is induced, allowing the bacteria to adopt behaviours which are only productive when the bacteria are working together as a group.Among the many different mechanisms for QS that have evolved,the most-characterized quorum-sensing systems involve the production of N-acyl-l-homoserine lactones(AHLs) by Gram-negative bacteria.AHLs are usually produced by a protein homologous to LuxI,but can be generated by other enzymes. Signaling through the production of post-translationally modified peptides is confined to Gram-positive bacteria.The only quorum-sensing mechanism shared by both Gram-positive and Gram-negative bacteria involves the production of autoinducer 2 (AI-2) by the enzyme LuxS.In contrast to other autoinducers that are specific for a narrow range of organisms,the widely conserved AI-2 has been hypothesized to be a universal language for interspecies communication.However,LuxS has an alternative role in the cell,in which it functions as an integral component of the activated methyl cycle.The LuxS/AI-2 quorum sensing system has been suggested to be involved in the regulation of bioluminescence in Vibrio harveyi,biofilm formation in Escherichia coli,virulence-associated traits in Vibrio cholerae,and pathogenesis in Pseudomonas aeruginosa.However,the precise role of the LuxS/AI-2 system in some Gram-positive bacteria remains debating such as Staphylococcus aureus,an important human pathogen that constitutes the principal causes of a variety of diseases.Although the pathway for AI-2 production is widely conserved among bacteria, the mechanisms of AI-2 detection and signal transduction are specific and have only been established in a few species such as V.harveyi,V.cholerae,Salmonella typhimurium and E.coli.In E.coli and S.typhimurium,AI-2 is required to be imported into the cell for gene expression control.In E.coli and S.typhimurium, extracellular AI-2 accumulates in the exponential phase,but decreases drastically upon entry into the stationary phase.The rapid disappearance of AI-2 is due to its import by the Lsr transporter.Upstream of the lsr operon,there is a divergently transcribed gene encoding LsrR,which was reported previously to repress the transcription of the lsr operon and itself.Since LsrR contains a helix-turn-helix (HTH) DNA-binding domain,it was hypothesized that LsrR represses the expression of lsr operon and itself by binding to their promoter regions.However,no evidence on their direct binding has been shown yet.Here we have demonstrated for the first time that LsrR represses the transcription of the lsr operon and itself by directly binding to their promoters by gel shift assays and DNase I footprinting assays.Theβ-galactosidase assays further confirm that two motifs in both the lsrR and lsrA promoter regions are crucial for the LsrR binding.Furthermore,in agreement with the conclusion that pbosphorylated AI-2 can relieve the repression of LsrR in previous studies,our data shows that phospho-AI-2 makes LsrR unable to bind to its own promoter in vitro.Besides,we applied experiments to investigate the role of the AI-2/LuxS system in S.aureus.We identified genes regulated by LuxS using microarray analysis and demonstrated that AI-2,as an autoinducer,is involved in capsular polysaccharide synthesis,biofilm formation,and autolysis in S.aureus.Inactivation of luxS in S. aureus strain NCTC8325 resulted in decreased susceptibility to cell-wall-synthesis inhibitor antibiotics,enhanced biofilm formation,decreased autolysis and up-regulated vancomycin-resistant associated VraSR two-component regulatory system.These alterations can be restored by either genetic complementation or addition of exogenous AI-2,suggesting that the LuxS/AI-2 system in S.aureus is associated with these physiological functions mainly by quorum sensing.Our findings demonstrate the functional role of LuxS/AI-2 system in cellular processes in S.aureus, and provide novel clues for understanding of bacteria AI-2 quorum sensing regulation in adaptation to environmental stress.