APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2006, p. 4978–4986 Vol. 72, No. 7 0099-2240/06/$08.00ϩ0 doi:10.1128/AEM.02842-05 Copyright © 2006, American Society for Microbiology. All Rights Reserved. Functional Heterogeneity of RpoS in Stress Tolerance of Enterohemorrhagic Escherichia coli Strains Arvind A. Bhagwat,1* Jasmine Tan,1† Manan Sharma,2 Mahendra Kothary,3 Sharon Low,1† Ben D. Tall,3 and Medha Bhagwat4 Produce Quality and Safety Laboratory1 and Food Technology and Safety Laboratory,2 Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Bldg. 002, 10300 Baltimore Avenue, Beltsville, Maryland 20705-2350; Division of Virulence Assessment, Food and Drug Administration, Laurel, Maryland 207083; and National Center for Biotechnology Information, Bldg. 38A, National Institutes of Health, Bethesda, Maryland 208944 Received 2 December 2005/Accepted 25 April 2006 The stationary-phase sigma factor (RpoS) regulates many cellular responses to environmental stress con- ditions such as heat, acid, and alkali shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of the RpoS-mediated stress responses of enterohemorrhagic E. coli strains from food-borne outbreaks. E. coli strains belonging to serotypes O157:H7, O111:H11, and O26:H11 exhibited polymorphisms for two phenotypes widely used to monitor rpoS mutations, heat tolerance and glycogen synthesis, as well as for two others, alkali tolerance and adherence to Caco-2 cells. However, these strains synthesized the oxidative acid resistance system through an rpoS-dependent pathway. During the transition from mildly acidic growth conditions (pH 5.5) to alkaline stress (pH 10.2), cell survival was dependent on rpoS functionality. Some strains were able to overcome negative regulation by RpoS and induced higher ␤-galactosidase activity without compromising their acid resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the tested strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was also evident in the Caco-2 cell adherence assay. Wild-type O157:H7 strains with native rpoS were less adherent than rpoS-complemented counterpart strains, suggesting that rpoS functionality is needed. These results show that some pathogenic E. coli strains can maintain their acid tolerance capability while compromising other RpoS-dependent stress responses. Such adaptation processes may have significant impact on a pathogen’s survival in food processing environments, as well in the host’s stomach and intestine. Enteric food-borne pathogens have developed sophisticated Several recent studies have analyzed the occurrence of rpoS stress response mechanisms suited for a variety of ecological mutants under laboratory chemostatic growth conditions as conditions encountered in both animal hosts and the external well as from environmental and clinical isolates of enteric environment. The low infectious dose of enterohemorrhagic pathogens (27, 28, 38, 56). Under certain circumstances, vari- Escherichia coli, a food- and water-borne pathogen, is most ation at the rpoS locus has been thought to confer a selective likely due to a combination of several factors, such as its abil- advantage to cells within a bacterial population experiencing ities to produce Shiga-like toxin and to survive gastric acidity nutrient deprivation (17, 28, 57). Several of these studies uti- and acidic foods, as well as efficient stress resistance mecha- lized assays such as temperature tolerance, resistance to hy- nisms (19, 25, 36). Under adverse environmental conditions, drogen peroxide, and/or synthesis of glycogen to measure the such as nutrient limitation, osmotic shock, or low pH, cellular frequency of rpoS mutation (29, 38, 51). Mutations at the rpoS RpoS levels increase dramatically and RNA polymerase-con- locus were frequently observed under chemostatic growth taining RpoS transcribes over 70 genes involved in stress re- when E. coli K-12 cells were fed a suboptimal concentration of sistance and protection (39, 54). RpoS is a key element in the glucose (38). On the other hand, certain external stress condi- survival of several food-borne human pathogens, including Shi- tions, such as mildly acidic conditions, significantly attenuated gella flexneri, salmonellae, and diarrheagenic E. coli (24, 41, the frequency of rpoS mutations (14, 27). In spite of the diverse 47). In spite of having a central role in bacterial viability, data virulence characteristics of pathogenic E. coli, one common from genome sequence projects, as well as other comparative trait that is very evident is the ability of these strains to with- studies with pathogenic E. coli strains, have revealed that the stand gastric acidity (18, 30, 36, 53). In fact, acid tolerance rpoS locus is highly polymorphic (16, 33, 38). plays a vital role in the survival and virulence of diarrheagenic E. coli strains (9, 42, 46). In S. flexneri and diarrheagenic E. coli * Corresponding author. Mailing address: Produce Quality and strains, the induction of two of three acid resistance pathways Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research under aerobic growth conditions is positively regulated by Center, Agricultural Research Service, USDA, Bldg. 002, 10300 Bal- RpoS (3, 30, 47). The first acid resistance system (AR1) is timore Avenue, Beltsville, MD 20705-2350. Phone: (301) 504-5106. referred to as the glucose-repressible oxidative pathway, and it Fax: (301) 504-5107. E-mail: [email protected]. † Present address: School of Life Sciences and Chemical Technol- protects cells from acidic stress above pH 3.0 (30, 31, 46). The ogy, Ngee Ann Polytechnic, 535 Clement Road, Singapore 599489, structural components of this acid resistance system (other Singapore. than RpoS), as well as the mechanisms by which it protects the 4978 VOL. 72, 2006 HEAT AND pH TOLERANCE AND MICROBIAL FOOD SAFETY 4979 cells, are still unknown (2, 9). The second acid resistance sys- For heat shock assays, stationary-phase cells from LB-MES broth were diluted tem (AR2) is glutamate-dependent acid resistance (GDAR), directly (1:200) into PBS preequilibrated to 58°C. Viable counts were determined and it can protect cells from acidic stress below pH 3 (22, 46). at 10 s and at 1.5, 3, 5, and 7.5 min by dilution of the cells in PBS and immediate plating on LB agar. In spite of the central regulatory role of RpoS, clinical and For alkali shock assays, stationary-phase cells from LB, LB-MES, or LB- food-borne isolates of pathogenic E. coli with rpoS mutations MOPS broth were diluted directly (1:200) into 100 mM CAPS (3-cyclohexyla- have been reported (5, 53), suggesting that the pathogen may mine-1-propane sulfonic acid), pH 10.2 (adjusted with 100 mM NaOH), pre- rely on rpoS-independent induction of acid resistance systems. equilibrated to 37°C. Viable counts were determined at 10 s and at 1, 2, and 4 h by dilution of cells in PBS and immediate plating on LB agar. Intrigued by the frequent occurrence of mutations in rpoS Experiments involving stress tolerance, as well as ␤-galactosidase assays, were and by the fact that the general stress response is controlled by repeated three to five times, and the results were calculated with consideration this sigma factor, we undertook analyses of rpoS alleles from of all data points. outbreak and clinical isolates of pathogenic E. coli strains. E. Detection of glycogen synthesis. Glycogen synthesis was monitored as de- coli strains were subjected to an array of stress tests in which scribed previously (21) with minor modifications (55). Briefly, overnight cultures were plated onto Kornberg agar medium (55) and, after 24 h of incubation at 37°C, survival is primarily considered to be RpoS dependent (10, 39, plates were further incubated at 10°C for 24 h and then stained with an iodine 43). We observed several strains (26 out of 53) with phenotypic solution for 1 to 2 min. Dark brown colonies indicated the synthesis of glycogen, characteristics that were atypical of a functional RpoS allele while pale brown or white colonies indicated partial or no synthesis of glycogen. (i.e., heat sensitivity and/or lack of glycogen synthesis), even ␤-Galactosidase assays. Enzyme activity was determined from cells grown to the stationary growth phase in EG minimal medium for 22 to 24 h. Cells were though they had a functional rpoS according to their acid centrifuged, resuspended in Z buffer (60 mM Na2HPO4,40mMNaH2PO4,10 resistance phenotype (5). Temperature tolerance and ability to mM KCl, 1 mM MgSO4 [pH 7.0]), and then permeabilized by treatment with synthesize glycogen have been used previously to monitor the sodium dodecyl sulfate (SDS) and chloroform (37). For all experiments, station- frequency of rpoS mutations (27, 38). Isolates of Shiga toxin- ary-phase cultures at 24 h after inoculation with an OD600 of 2.8 or more were ␤ producing E. coli of serotypes O157:H7 (two strains), O111: used, and the assays were performed with Z buffer containing 50 mM -mer- captoethanol. In all experiments, ␤-galactosidase activity (change in OD per H11 (one strain), and O26:H11 (one strain) with a wide spec- 420 minute) was normalized to cell density (OD600) and was compared to appropri- trum of glycogen synthesis patterns were chosen for in-depth ate controls assayed at the same time. phenotypic analysis. The objective of the experiments de- Western blot analysis. Cultures were incubated for 18 to 22 h at 37°C to a scribed here was to evaluate rpoS heterogeneity and its effect stationary growth phase (OD600 of 3.5 or higher) in LB-MES medium. Cells of ϫ on stress responses among pathogenic E. coli strains. each strain were collected by centrifugation (12,500 g, 4 min), washed twice in saline, resuspended at 1 OD600 unit/ml in loading buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 2.5% ␤-mercaptoethanol, 0.01% Na-azide, 0.1% bromophenol blue), and placed in a boiling water bath for 5 min to make a MATERIALS AND METHODS whole-cell protein sample.