The Effect Of Density On Formation Of Persister In Escherichia Coli And Its Molecular Mechanisms

Objective:To investigate the impact of bacterial density in vitro on formation of persister in Escherichia coil and its underlying molecular mechanisms.Methods:1.E.coil MG1655 strain cultured in Luria-Bertani(LB)broth for 3 hours was used as the exponential phase low-density bacterial suspension(EEP-3O),and its 100-fold concentrated culture was used as the exponential phase high-density bacterial suspension(EEP-3C).Additionally,the culture incubated for 24 hours was used as the stationary phase high-density bacterial suspension(EEP-24O),and its 1000-fold diluted cultue was used as the stationary phase low-density bacterial suspension(EEP-24D).The formation of persisters of these samples were evaluated by ampicillin(256 μg/m L),norfloxacin(40 μg/m L),and gentamicin(10 μg/m L)exposure assays,respectively.2.The samples of EEP-3O,EEP-3C,EEP-24 O,and EEP-24 D were collected and the differentially expressed genes(DEGs)and their associated pathways were explored by transcriptome sequencing(RNA-seq)and bioinformatics analysis,and were validated by RT-q PCR.3.Plasmids p KD3,p KD46,and p CP20 were used to construct single-gene knockout strains(Δssn A,Δyge W,Δtdc B,Δgrc A,Δdpa L,Δnar U,Δpfl B,Δglt B)of E.coil strain MG1655 by λ-red homologous recombination technology,and the mutants were validated by PCR and DNA sequencing.4.Growth curve and MIC were determined on the parent strain and the singlegene knockout strains(Δssn A,Δyge W,Δtdc B,Δgrc A,Δdpa L,Δnar U,Δpfl B,Δglt B).The formation of persisters under different density conditions during the stationary phase was evaluated by exposure to levofloxacin(40 μg/m L)and gentamicin(10 μg/m L)in both parent and mutant strains.5.The recombinant plasmid p Trc99a-tdc B was constructed and transformed into the Δtdc B mutant strain to obtain the complemented strain Δtdc B::p Trc99a-tdc B.The empty plasmid p Trc99 a was transformed into the MG1655 and Δtdc B to obtain WT::p Trc99 a and Δtdc B::p Trc99 a strains,respectively.6.Growth curve of WT::p Trc99 a,Δtdc B::p Trc99 a,and the complemented strainΔtdc B::p Trc99a-tdc B under IPTG induction was performed.The formation of persisters of the above strains under different density conditions in stationary phase was evaluated by levofloxacin(40 μg/m L)and gentamicin(10 μg/m L)exposure assays,respectively.Results:1.Under the action of ampicillin,the bacteria in the low-density bacterial suspensions(EEP-3O and EEP-24D)were completely killed after 5 or 4 days exposure,respectively,while the bacteria in high-density bacterial suspension(EEP-3C)of exponential phase was completely killed after 9 days of exposure.When the highdensity bacterial suspension(EEP-24O)of stationary phase bacteria exposed to ampicillin,a large number of bacteria still survived after 10 days of exposure.When exposed to norfloxacin,the bacteria in the low-density bacterial suspensions(EEP-3O and EEP-24D)were completely killed after 4 days,while the bacteria in the highdensity bacterial suspensions(EEP-3C and EEP-24O)still had surviving bacteria after10 days.When exposed to gentamicin,the bacteria in the low-density bacterial suspensions(EEP-3O and EEP-24D)were completely killed after 2 days,while the bacteria in the high-density bacterial suspensions(EEP-3C and EEP-24O)were completely killed after 5 and 9 days,respectively.2.Results of RNA-seq showed that under Log2(Fold Change)> 2 or <-2,compared with the bacteria in high and low density bacterial suspension,103 genes were up-regulated differentially in exponential phase and 129 in stationary phase.Among them,43 genes(AW869＿RS05950,dpa L,ssn A,ygf M,xdh D,ygf K,dms B,glp T,grc A,yge W,gua D,xdh A,xan Q,hyp D,bss R,AW869＿RS05955,AW869＿RS06010,hyb A,hyb O,omp F,frd D,tdc A,tdc B,tdc C,tdc D,ibp B,AW869＿RS05965,ftn A,yqe C,gld A,AW869＿RS05585,AW869＿RS17575,hyp B,usp F,asp A,AW869＿RS12540,kat G,bio D,frd A,omp W,glp F,ydf Z,arc C)were upregulated collectively.There were 124 differentially down-regulated genes in exponential phase and 140 in stationary phase,among which 6 were co-down-regulated genes(AW869＿RS07960,AW869＿RS20560,spy,AW869＿RS21530,nfu A,AW869＿RS02005).The main functions of common differential genes were distributed in two component system,purine and pyrimidine metabolism,selenium compound metabolism,sulfur metabolism,carbon metabolism,pyruvate metabolism,amino acid metabolism,secondary metabolites biosynthesis and other metabolic pathways.3.Using λ-red homologous recombination technique,the single-gene knockout strains including Δssn A,Δyge W,Δtdc B,Δgrc A,Δdpa L,Δnar U,Δpfl B,and Δglt B were constructed successfully.4.Compared with the parent strain,the MIC of the Δssn A,Δyge W,Δtdc B,Δgrc A,Δdpa L,Δnar U,Δpfl B,and Δglt B did not show significant changes.In low density bacterial suspension,the mutant of Δssn A and Δtdc B had a reduced ability to form persisters in response to gentamicin exposure,while the Δgrc A showed no significant difference compared with the parent strain.The mutant of Δyge W,Δdpa L,Δnar U,Δpfl B,and Δglt B showed an increased ability to form persisters in response to gentamicin exposure compared with the parent strain.The mutant of Δssn A,Δtdc B,and Δdpa L showed a reduced ability to form persisters in response to levofloxacin exposure,whileΔyge W,Δgrc A,Δnar U,Δpfl B,and Δglt B showed no significant difference compared with the partent strain.Under high-density conditions,the mutant of Δtdc B,Δnar U,Δpfl B,and Δglt B showed a weakened ability to form persisters in response to gentamicin exposure,while the Δssn A,Δyge W,and Δgrc A showed no significant difference compared with the parent strain,and Δdpa L showed an increased ability to form persisters in response to gentamicin exposure.The mutant of Δtdc B,Δpfl B,andΔglt B showed a weakened ability to form persisters in response to levofloxacin exposure,while the Δssn A and Δnar U showed no significant difference compared with the parent strain,and the Δyge W,Δgrc A,and Δdpa L showed an increased ability to form persisters in response to levofloxacin exposure compared with the parent strain.5.The bacteria of WT::p Trc99 a,Δtdc B::p Trc99 a,and complemented strainΔtdc B::p Trc99a-tdc B were constructed successfully.Further gentamicin and levofloxacin exposure experiments showed that the persister formation ability of the complemented strain was partially restored compared with the knockout strain,indicating that the gene tdc B plays an important role on formation of persister in highdensity of E.coli.Conclusions:1.The density of bacterial suspension affects significantly on the formation of persister in E.coli.Several pathways,including two component system,purine and pyrimidine metabolism,selenium compound metabolism,sulfur metabolism,carbon metabolism,pyruvate metabolism,amino acid metabolism,secondary metabolites biosynthesis,are involved in the influence of density on the formation of persister in E.coli.2.tdc B is involved in the formation of persister in E.coli under high-density conditions.