A Switch In The Poly(dC)/RmlB Complex Regulates Pseudomonas Aeruginosa PAO1 Persister Formation

The abuse of antibiotics has led to the serious problem of microbial resistance in the environment.In addition to the antibiotic resistance genes(ARGs),the broad-spectrum resistance of microorganisms mediated by persisters has attracted global attention.The regulation mechanism of persisters has become a hot topic in international research.Bacterial persisters are dormant cells that form stochastically within isogenic bacterial populations.These transient and non-hereditary phenotypic cells are highly tolerant to lethal doses of antibiotics.Unlike normal vegetative cells(majority of cells in a population),persisters(minority of cells in a population)remain viable in the presence of antibiotics.Furthermore,following antibiotic withdrawal the dormant cells resume growth.At the same time,the researchers also found that the founctional bacteria becomes into viable but non-culturable state(VBNC)in the long-term application of ecological environment,these aforementioned VBNC is initially identified as persisters.The transient nature of persistence makes this phenomenon inherently difficult to study;therefore,many of the molecular mechanisms that underlie persistence remain to be elucidated.Most of what we know about mechanisms of persister formation,namely toxin/antitoxin(TA)modules,environmental stimuli,and several global regulators,comes from the studies of common bacteria,such as Escherichia coli,Mycobacterium tuberculosis,Salmonella Typhimurium,and Staphylococcus aureus.Pseudomonas aeruginosa is a common bacteria in the ecological environment.Pseudomonas aeruginosa is an important antibiotic resistant bacteria that causes severe pollution in the ecological environment.As a functional microorganism,Pseudomonas aeruginosa is widely used in short-term ecological restoration.But the long-term ecological application of Pseudomonas aeruginosa has no yet been realized.With the deepening of the research on the mechanism of persisters and the cooperation of various disciplines,ecologists speculate that persisters lead to the antibiotic resistence of Pseudomonsa aeruginosa and the defects in the long-term ecological appilication of functional microorganisms.Although P.aeruginosa persistence formation mechanisms is still remain poorly understood compared to the above strains.Two previously reported mechanisms,namely type II TA module HigBA and quorum sensing signaling molecules pyocyanin and acyl-homoserine lactone,do not reveal the charm of persister formation mechanisms in P.aeruginosa.However,deletion of the TA modules and pyocyanin biosynthetic pathway has no effect on persistence,further raising the possibility that additional signaling systems control the bacterial persistence phenotype.In this study,we report a persistence-associated complex that directly controls persister formation in response to environmental stimuli in P.aeruginosa PAO1.The complex consists of two components,an extracellular 5'-dephospho-polydeoxycytidylic acid(poly(dC))and its membrane-associated binding protein RmlB.Mechanism underlying Poly(dC)/RmlB-mediated bacterial persistence as following:1.Previous studies have demonstrated that environmental insults can induce persister formation.We found that a routine procedure for washing cells increased persister levels 102 to 103-fold in exponential and stationary P.aeruginosa PAO1,suggesting the existence of an independent extracellular effector that modulates persister formation.2.P.aeruginosa PAO1 has an independent extracellular effector that modulates persister formation in the whole growth stage.Poly(dC)as the extracellular persistence negative regulator in the exponential and stationary phase,functional antagonism between Poly(dC)and P-poly(dC)that exists in the death phase.Poly(dC)and P-poly(dC)are potentially length-independent effectors and highly dependent on its base composition.RmlB is a bona fide common binding site for both Poly(dC)and P-poly(dC).RmlB has a high affinity for P-poly(dC)and a low affinity for Poly(dC),thereby resulting in one-sided competition in empty site;Poly(dC)occupying the site in advance resulted in P-poly(dC)unable to bind the same site;P-poly(dC)occupying the site in advance resulted in Poly(dC)unable to bind the same site.3.Removal of the DNA component,or switching of the complex to P-poly(dC)/RmlB by in-situ DNA phosphorylation or tailoring(from the 5' terminus)reduces proton motive force(PMF)across the membrane with a concomitant reduction in intracellular ATP levels and dissolved oxygen concentrations.These occurrences ultimately lead to persistence.4.RmlB has previously been assigned as a putative dTDP-L-glucose 4,6-dehydratase.The dTDP-L-rhamnose pathway is essential for PAO1 viability.It is likely that shutdown of this pathway inhibits cell wall synthesis since rhamnose is a fundamental component of the PAO1 cell wall.We also reveal that the switch-triggered shutdown of the dTDP-L-rhamnose pathway is necessary for P-poly(dC)/RmlB or RmlB-mediated persistence.Furthermore,P-poly(dC)/RmlB or RmlB-triggered persistence is also an adaptive response to the shutdown of the dTDP-L-rhamnose pathway.The complex switch-triggered persistence might be considered as an adaptive response to the adverse conditions.5.RmlB is a ubiquitous protein in microorganisms.RmlB in PAO1 shares high identity(>70%)with its homonlogues in other Pseudomonas spp.RmlB-based regulatory strategies regulate bacterial persister formation is ubiquitous nature in the Pseudomonas genus.