The General Stress Response Is Conserved in Long-Term Soil-Persistent Strains of Escherichia coli

Abstract

Although Escherichia coli is generally considered to be predominantly a commensal of the gastrointestinal tract, a number of recent studies suggest that it is also capable of long-term survival and growth in environments outside the host. As the extraintestinal physical and chemical conditions are often different from those within the host, it is possible that distinct genetic adaptations may be required to enable this transition. Several studies have shown a trade-off between growth and stress resistance in nutrient-poor environments, with lesions in the rpoS locus, which encodes the stress sigma factor RpoS (σ(S)). In this study, we investigated a unique collection of long-term soil-persistent E. coli isolates to determine whether the RpoS-controlled general stress response is altered during adaptation to a nutrient-poor extraintestinal environment. The sequence of the rpoS locus was found to be highly conserved in these isolates, and no nonsense or frameshift mutations were detected. Known RpoS-dependent phenotypes, including glycogen synthesis and γ-aminobutyrate production, were found to be conserved in all strains. All strains expressed the full-length RpoS protein, which was fully functional using the RpoS-dependent promoter reporter fusion PgadX::gfp RpoS was shown to be essential for long-term soil survival of E. coli, since mutants lacking rpoS lost viability rapidly in soil survival assays. Thus, despite some phenotypic heterogeneity, the soil-persistent strains all retained a fully functional RpoS-regulated general stress response, which we interpret to indicate that the stresses encountered in soil provide a strong selective pressure for maintaining stress resistance, despite limited nutrient availability.

Importance: Escherichia coli has been, and continues to be, used as an important indicator species reflecting potential fecal contamination events in the environment. However, recent studies have questioned the validity of this, since E. coli has been found to be capable of long-term colonization of soils. This study investigated whether long-term soil-persistent E. coli strains have evolved altered stress resistance characteristics. In particular, the study investigated whether the main regulator of genes involved in stress protection, the sigma factor RpoS, has been altered in the soil-persistent strains. The results show that RpoS stress protection is fully conserved in soil-persistent strains of E. coli They also show that loss of the rpoS gene dramatically reduces the ability of this organism to survive in a soil environment. Overall, the results indicate that soil represents a stressful environment for E. coli, and their survival in it requires that they deploy a full stress protection response.

FIG 1

Motility of E. coli strains on 0.25% (wt/vol) LB agar at 37°C for 16 h (A) and at 15°C for 40 h (B), and biofilm formation in LB broth and SMM (C). All soil-persistent and commensal strains were significantly (P < 0.0001) more motile than the laboratory strain BW25113 at 15°C and 37°C. The wild-type BW25113 strain was significantly (P < 0.0003) less motile than the BW25113ΔrpoS strain at 37°C but not at 15°C. Biofilm was produced in rich and minimal media, with more biofilm produced at 15°C than at 37°C. UTI, urinary tract infection-associated E. coli isolate. Error bars represent standard deviations from three independent replicates. Data with similar lowercase or uppercase letters are not significantly different (P > 0.05).

FIG 2

Survival of E. coli under various environmental conditions at 15°C. (A) All of the soil-persistent, commensal strains and E. coli BW25113 survived significantly (P < 0.0001) better in PBS than the BW25113ΔrpoS strain did, except SE11, after 35 h. (B) The E. coli BW25113 parental strain survived significantly better than the BW25113ΔrpoS strain in soil. Time is shown in days (d) on the x axis. (C) Long-term soil survival experiment showed that RpoS is required for survival in silty loam soil A, as there were no detectable BW25113ΔrpoS strains after 42 days in soil, whereas all other test strains survived long term. (D) The E. coli COB583 parental strain survived significantly better (P = 0.002) than the COB583ΔrpoS strain in silty loam soils A and B. Error bars represent standard deviations from three independent replicates. Dashed lines represent the detection limit of the soil survival assay.

FIG 3

(A) Phylogenetic tree of rpoS performed using the maximum likelihood method based on the Kimura 2-parameter model with bootstrap analysis (1,000 iterations) using MEGA6 showed that rpoS in soil-persistent strains is similar to previously known E. coli strains. (B) Gene outline of rpoS and its flanking genes in the soil-persistent and commensal strains shows four distinct patterns. PCR and sequencing of genes flanking rpoS revealed insertion of open reading frames (ORFs) between rpoS and inner membrane permease (ygbN). Pattern I (COB583), which is similar to the reference strain (BW25113), shows rpoS directly flanked by murein hydrolase activator (nlpD) and ygbN. Pattern II (COB584, COB586, COB587, and SE11) shows insertion of 4 ORFs between rpoS and ygbN. Pattern III (COB585) shows insertion of 5 ORFs after rpoS, with ygbN absent, while pattern IV (SE15) had insertion of 2 ORFs between rpoS and ygbN. The region between nlpD (which carried rpoS promoters) and rpoS was conserved in all the strains. Block arrows with similar patterns indicate the same ORF, while arrowheads represent rpoS promoter sites.

FIG 4

RpoS-dependent phenotypes show soil-persistent strains have functional RpoS. All E. coli strains survived significantly better than the BW25113ΔrpoS strain at pH 2.5 (A), produced significantly higher (P < 0.0001) amounts of gamma-aminobutyric acid (GABA) in response to acid stress (pH 4) compared to the BW25113ΔrpoS strain (B), and turned brown to dark brown upon iodine staining, showing the accumulation of glycogen compared to the BW25113ΔrpoS strain (C). Error bars represent standard deviations from three independent replicates. Data with similar lowercase or uppercase letters are not significantly different (P > 0.05).

FIG 5

A higher level of RpoS is expressed in the stationary phase at 15°C than at 37°C in E. coli. Protein preparations were obtained from E. coli strains grown in LB broth with agitation to the stationary phase at 15°C and 37°C. The same amount of protein (25 μg) from each strain at both temperatures was run on SDS-PAGE, and RpoS was detected by immunoblotting. Purified RpoS protein was used as the control. More RpoS was produced at 15°C than at 37°C in all of the strains, whereas RpoS was not detected in the BW25113ΔrpoS strain at either 15°C or 37°C. The image shown is representative of three independent replicates.

FIG 6

RpoS-dependent GFP expression of gadX::gfp promoter fusions in the stationary phase at 15°C and 37°C by fluorescence microscopy (A) and Western blotting using anti-GFP antibody (B). E. coli strains containing the reporter fusion were cultured in LB broth supplemented with kanamycin (50 μg/ml) with agitation. Samples were taken at stationary phase (17 h at 37°C and 36 h at 15°C), fixed with ethanol-methanol (1:1) solution, and resuspended in PBS, and 2 μl was placed on a slide and imaged with a Leica DMI3000 B microscope. RpoS activity indicated by fluorescence was higher at 37°C than at 15°C (A), and this correlated with GFP detection by immunoblotting (B). COB585 carrying the reporter fusion had the lowest RpoS activity among strains with intact RpoS. Fluorescence was not detected in the BW25113ΔrpoS strain either by microscopy or immunoblotting. Fluorescent images presented are representatives of 2 independent experiments with >3 field captures in each experiment. Western blot image is representative of 3 independent experiments.