PMID:18519731

Citation	Hansen, S , Lewis, K and Vulić, M (2008) Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli. Antimicrob. Agents Chemother. 52:2718-26
Abstract	Bacterial populations produce a small number of persister cells that exhibit multidrug tolerance. Persister cells are largely responsible for the antibiotic recalcitrance of biofilm infections. The mechanism of persister cell formation largely remains unknown due to the challenges in identifying persister genes. We screened an ordered comprehensive library of 3,985 Escherichia coli knockout strains to identify mutants with altered antibiotic tolerance. Stationary-state cultures in 96-well plates were exposed to ofloxacin at a concentration which allows only tolerant persister cells to survive. The persister cell level of each culture was determined. A total of 150 mutants with decreased persistence were identified in the initial screen, and subsequent validation confirmed that neither the growth rate nor the ofloxacin MIC was affected for 10 of them. The genes affected in these strains were dnaJ and dnaK (chaperones), apaH (diadenosine tetraphosphatase), surA (peptidyl-prolyl cis-trans isomerase), fis and hns (global regulators), hnr (response regulator of RpoS), dksA (transcriptional regulator of rRNA transcription), ygfA (5-formyl-tetrahydrofolate cyclo-ligase), and yigB (flavin mononucleotide [FMN] phosphatase). The prominent presence of global regulators among these strains pointed to the likely redundancy of persister cell formation mechanisms: the elimination of a regulator controlling several redundant persister genes would be expected to produce a phenotype. This observation is consistent with previous findings for a possible role of redundant genes such as toxin/antitoxin modules in persister cell formation. ygfA and yigB were of special interest. The mammalian homolog of YgfA (methenyltetrahydrofolate synthetase) catalyzes the conversion of 5-formyl-tetrahydrofolate (THF) into the rapidly degraded 5,10-methenyl-THF, depleting the folate pool. The YigB protein is a phosphatase of FMN which would deplete the pool of this cofactor. Stochastic overexpression of these genes could lead to dormancy and, hence, tolerance by depleting the folate and FMN pools, respectively. Consistent with this scenario, the overexpression of both genes produced increased tolerance to ofloxacin.
Links	PubMed PMC2493092 Online version:10.1128/AAC.00144-08
Keywords	Anti-Bacterial Agents/pharmacology; Drug Resistance, Multiple, Bacterial; Escherichia coli/drug effects; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Expression Regulation, Bacterial/drug effects; Microbial Sensitivity Tests; Mutation; Nucleotides/metabolism; Ofloxacin/pharmacology
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Materials and Methods Used

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Phenotype Annotations

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Phenotype of	Taxon Information	Genotype Information (if known)	Condition Information	OMP ID	OMP Term Name	ECO ID	ECO Term Name	Notes	Status
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: fis+ Genotype of Experimental Strain : BW25113 fis(del)::FRT-Kan-FRT	Reference Condition:					The deletion of fis gene and insertion of Kanamycin resistant cassette caused a slight level of persistence toward Ofloxacin. See Figure 2.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: hns+ Genotype of Experimental Strain : BW25113 hns(del)::FRT-Kan-FRT	Reference Condition:					The deletion of hns gene and insertion of Kanamycin resistant cassette caused a slight level of persistence toward Ofloxacin. See Figure 2.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: hnr+ Genotype of Experimental Strain : BW25113 hnr(del)::FRT-Kan-FRT	Reference Condition:					The deletion of hnr gene and insertion of Kanamycin resistant cassette caused a slight level of persistence toward Ofloxacin. See Figure 2.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: dksA+ Genotype of Experimental Strain : BW25113 dksA(del)::FRT-Kan-FRT	Reference Condition:					The deletion of dksA gene and insertion of Kanamycin resistant cassette caused a slight level of persistence toward Ofloxacin. See Figure 2.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: apaH+ Genotype of Experimental Strain : BW25113 apaH(del)::FRT-Kan-FRT	Reference Condition:					The deletion of apaH gene and insertion of Kanamycin resistant cassette caused a slight level of persistence toward Ofloxacin. See Figure 2.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: surA+ Genotype of Experimental Strain : BW25113 surA(del)::FRT-Kan-FRT	Reference Condition:					The deletion of surA gene and insertion of Kanamycin resistant cassette caused a slight level of persistence toward Ofloxacin. See Figure 2.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: dnaJ+ Genotype of Experimental Strain : BW25113 dnaJ(del)::FRT-Kan-FRT	Reference Condition:					The deletion of dnaJ gene and insertion of Kanamycin resistant cassette caused a slight level of persistence toward Ofloxacin. See Figure 2.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: dnaK+ Genotype of Experimental Strain : BW25113 dnaK(del)::FRT-Kan-FRT	Reference Condition:					The deletion of dnaK gene and insertion of Kanamycin resistant cassette caused a slight level of persistence toward Ofloxacin. See Figure 2.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: yigB+ Genotype of Experimental Strain : BW25113 yigB(del)::FRT-Kan-FRT	Reference Condition:					The deletion of yigB gene and insertion of Kanamycin resistant cassette caused a slight level of persistence toward Ofloxacin. See Figure 2.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: ygfA+ Genotype of Experimental Strain : BW25113 ygfA(del)::FRT-Kan-FRT	Reference Condition:					The deletion of ygfA gene and insertion of Kanamycin resistant cassette caused a slight level of persistence toward Ofloxacin. See Figure 2.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: recA+ Genotype of Experimental Strain : BW25113 recA(del)::FRT-Kan-FRT	Reference Condition:					The deletion of the recA gene and replacement with the Kanamycin cassette conferred increased sensitivity toward ofloxacin.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: recB+ Genotype of Experimental Strain : BW25113 recB(del)::FRT-Kan-FRT	Reference Condition:					The deletion of the recB gene and replacement with the Kanamycin cassette conferred increased sensitivity toward ofloxacin.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: recG+ Genotype of Experimental Strain : BW25113 recG(del)::FRT-Kan-FRT	Reference Condition:					The deletion of the recG gene and replacement with the Kanamycin cassette conferred increased sensitivity toward ofloxacin.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: recJ+ Genotype of Experimental Strain : BW25113 recJ(del)::FRT-Kan-FRT	Reference Condition:					The deletion of the recJ gene and replacement with the Kanamycin cassette conferred increased sensitivity toward ofloxacin.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: recN+ Genotype of Experimental Strain : BW25113 recN(del)::FRT-Kan-FRT	Reference Condition:					The deletion of the recN gene and replacement with the Kanamycin cassette conferred increased sensitivity toward ofloxacin.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: recQ+ Genotype of Experimental Strain : BW25113 recQ(del)::FRT-Kan-FRT	Reference Condition:					The deletion of the recQ gene and replacement with the Kanamycin cassette conferred increased sensitivity toward ofloxacin.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: ruvA+ Genotype of Experimental Strain : BW25113 ruvA(del)::FRT-Kan-FRT	Reference Condition:					The deletion of the ruvA gene and replacement with the Kanamycin cassette conferred increased sensitivity toward ofloxacin.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: ruvB+ Genotype of Experimental Strain : BW25113 ruvB(del)::FRT-Kan-FRT	Reference Condition:					The deletion of the ruvB gene and replacement with the Kanamycin cassette conferred increased sensitivity toward ofloxacin.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: xseA+ Genotype of Experimental Strain : BW25113 xseA(del)::FRT-Kan-FRT	Reference Condition:					The deletion of the xseA gene and replacement with the Kanamycin cassette conferred increased sensitivity toward ofloxacin.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113 NCBI Taxon ID: 83333	Genotype of Reference Strain: xseB+ Genotype of Experimental Strain : BW25113 xseB(del)::FRT-Kan-FRT	Reference Condition:					The deletion of the xseB gene and replacement with the Kanamycin cassette conferred increased sensitivity toward ofloxacin.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113	Genotype of Reference Strain: ygfA+ Genotype of Experimental Strain : BW25113 ygfA(del):: FRT-Kan-FRT	Reference Condition:					The deletion of the ygfA gene caused a significant decrease of persistence cells grown to stationary phase with ofloxacin. See figure 4 for experimental results.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: KLE910	Genotype of Reference Strain: ssrS+ Genotype of Experimental Strain : KLE910 ssrS(del)	Reference Condition:					The deletion of the ssrS gene caused minimal decrease in persister cells compared to the deletion of the ygfA gene with ofloxin. See figure 4 for experimental results.
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: BW25113	Genotype of Reference Strain: ygfA+ ssrS+ Genotype of Experimental Strain : KLE911 ygfA-ssrS(del)	Reference Condition:					The double deletion of the ygfA and ssrS genes caused a significant decrease in persister cells. See figure 4 for full experimental results.
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