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Appl Environ Microbiol. Apr 2007; 73(7): 2037–2047.
Published online Feb 2, 2007. doi:  10.1128/AEM.02643-06
PMCID: PMC1855633

Characterization of an Escherichia coli O157:H7 Plasmid O157 Deletion Mutant and Its Survival and Persistence in Cattle[down-pointing small open triangle]

Abstract

Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic ΔpO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the ΔpO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the ΔpO157 mutant. The ΔpO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the ΔpO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the ΔpO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed ~50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 causes hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome (HUS) in humans worldwide (41). Infections have been associated with contaminated bovine food products, direct animal contact, and bovine manure contamination of vegetables, fruits, or water. Undercooked ground beef contaminated with E. coli O157:H7 causes more than half of the disease outbreaks in the United States (20). Cattle are considered to be the primary reservoir of E. coli O157:H7, and the rectoanal junction (RAJ) mucosa is the predominant colonization site in the bovine gastrointestinal tract (GIT) (22, 42). Healthy cattle carry E. coli O157:H7 transiently and sporadically and pass the bacteria in their feces (12, 57).

E. coli O157:H7 virulence factors include Shiga toxins (Stx) and the locus of enterocyte effacement (LEE) pathogenicity island. The LEE encodes a type III secretion system, an outer membrane protein called intimin (eae), the translocated intimin receptor called Tir (tir), and other secreted proteins (esp) (18). Vascular endothelial cell damage by Stx produces HUS, and several LEE-encoded genes mediate the adherence of E. coli O157:H7 to intestinal epithelial cells by a characteristic attaching-and-effacing mechanism (41). In addition to these elements, an F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other EHEC serotypes (36, 51). The sequence of pO157 reveals 100 open reading frames with many assignments of gene functions still lacking (8, 38). Putative virulence factors encoded by pO157 include enterohemolysin (ehxA) (2), the general secretory pathway (etpC to etpO) (8), serine protease (espP) (8), catalase-peroxidase (katP) (7), a potential adhesion (toxB) (55), a Cl esterase inhibitor (stcE) (34), and attaching and effacing gene-positive conserved fragments (ecf) (62). Nonetheless, the role of pO157 in bacterial virulence and survival is largely unknown. Several studies resulted in conflicting data as to the role of pO157 in bacterial adherence to epithelial cells in vitro and in vivo (19, 31, 41, 56). A limitation of this work, however, is that mice, gnotobiotic piglets, or rabbits have been used as animal models, none of which correlate perfectly with human HUS, so the contribution of pO157 to bacterial pathogenesis in vivo has not been demonstrated.

To study pO157 of E. coli O157:H7, it is important to analyze gene expression at the protein level. Proteome analysis by two-dimensional gel electrophoresis (2-DE) is a useful tool to determine genomic function. Coupled with the identification capabilities of mass spectrometry supported by the ionization of peptide fragments and the increasing availability of protein sequence data, 2-DE is the simplest and most powerful method for protein identification and characterization (4, 24, 43). This approach is even more efficient and reliable when the whole genome of an organism is fully sequenced and annotated, as has been done for E. coli O157:H7 ATCC 43895 (25, 61).

Recently, our laboratory showed that the presence of pO157 increased the level of E. coli O157:H7 adherence to bovine RAJ mucosa and showed its requirement for long-term colonization in cattle (52). These results indicate that pO157 may be required for optimal survival and persistence of E. coli O157:H7 in its bovine reservoir. In this study, we expanded our investigations of the role of pO157 of E. coli O157:H7 to include in vitro characterization and analysis of passage through the bovine gastrointestinal tract. To this end, wild-type (WT) E. coli O157:H7 and a whole pO157 deletion derivative were examined for (i) phenotypic characteristics including growth rates and antibiotic susceptibilities, (ii) survival in environments under various acidity, salt, or heat conditions, (iii) protein expression profiles and identifications using 2-DE and mass spectrometry, and (iv) survival and persistence in cattle following oral challenge.

MATERIALS AND METHODS

Bacteria and media.

Three E. coli O157:H7 strains were used in this study: strain ATCC 43894, a human stool isolate producing Shiga toxin types 1 and 2 (referred to as the WT); strain 277, an isogenic strain with the 92-kb plasmid pO157 deleted (referred to as the ΔpO157 mutant); and the complemented strain ΔpO157 mutant 277::pO157ΔehxA (referred to as the complement) (52). The ΔpO157 mutant was kindly provided by J. Shaw (Armstrong Laboratory, Brooks Air Force Base, TX) (52). All bacteria were grown and maintained in Luria-Bertani (LB) broth (pH 7.5), except for the complemented strain, which was grown in LB broth with kanamycin (50 μg/ml) (47). d-Sorbitol MacConkey agar supplemented with 4-methylumbelliferyl-β-d-glucuronide (MUG), cefixime, potassium tellurite, and vancomycin (SMAC-CTVM) was used to culture E. coli O157 strains from bovine samples (46). The reagents were used at the following concentrations: cefixime, 50 ng/ml; potassium tellurite, 2.5 μg/ml; and MUG, 0.1 mg/ml. Trypticase soy broth (TSB; BBL/Becton Dickinson, Detroit, MI) was used for enrichment cultures of bovine samples and survival assays as previously described (46).

Mutant verification.

Deletion of pO157 was verified by plasmid profile, PCR, pulsed-field gel electrophoresis (PFGE) of restricted genomic DNA, and Southern blot analysis. Plasmid DNA isolated from the WT and the ΔpO157 mutant was subjected to 0.6% agarose gel electrophoresis for pO157 visualization. PCR was performed with primers for the pO157-specific genes ecf1 (forward, 5′-GAGGTCGACGGTGTGGTTAATATCTACG-3′; reverse, 5′-TCACTGCAGTTTCCCGGACTTCAGACCAGC-3′) and hly (forward, 5′-GGTGCAGCAGAAAAAGTTGTAG-3′; reverse, 5′-TCTCGCCTGATAGTGTTTGGTA-3′) (49, 62). WT and ΔpO157 mutant PFGE patterns were compared. Briefly, DNA from whole-cell lysates in agarose plugs was digested with XbaI, and the gel was electrophoresed with a clamped homogeneous electric fielded (CHEF-DR-II; Bio-Rad, Richmond, CA). The total run time, switch time, and voltage for the run were 20 h, 2.2 to 54.2 s, and 6 V/cm, respectively. Following DNA separation, Southern blotting was performed with the ecf1 probe using a digoxigenin-based nonradioisotope system (Roche Diagnostics GmbH, Mannheim, Germany), and all procedures were based on the manufacturer's manual and standard protocols (47).

Bacterial phenotype analysis.

Growth of the WT, the ΔpO157 mutant, and its complement strain was analyzed using a Bio-Tek Power Wave XS reader and KCjunior software. Bacteria were grown overnight in 10 ml of LB broth or M9 minimal medium at 37°C. Ten wells of a 96-well plate were inoculated with one of these three strains after 1:100 dilutions with fresh LB broth. Empty wells were filled with fresh medium, and the plates were covered with breathable rayon sealing tape (Nunc, Rochester, NY) and mixed with high-intensity shaking prior to every spectrophotometric read. Optical densities at 600 nm were measured every 30 min for 24 h at 25°C, 37°C, or 42°C. API-20E test strips (bioMerieux, Basingstoke Hants, United Kingdom) for testing of 21 different biochemical reactions were used for metabolic profiles. Antibiotic susceptibility tests were performed by the Kirby-Bauer standard method using 35 different antibiotic disks (BBL/Becton Dickinson) in accordance with the manufacturer's instructions. The antibiotics tested were ampicillin-sulbactam, amoxicillin-clavulanic acid, azithromycin, carbenicillin, cefixime, cefotaxime, cefpodoxime, ceftriaxone, cephalothin, chloramphenicol, clindamycin, cloxacillin, colistin, erythromycin, gentamicin, imipenem, levofloxacin, lincomycin, lomefloxacin, minocyclin, nafcillin, nalidixic acid, nitrofurantoin, norfloxacin, penicillin, polymyxin B, sparfloxacin, spectinomycin, tetracycline, tilmicosin, tobramycin, trimethoprim, trimethoprim-sulfamethoxazole, trovafloxacin, and vancomycin.

Competition assay.

The WT and the ΔpO157 mutant were incubated together in LB broth at 37°C in WT/ΔpO157 mutant ratios of 1:1, 1:10, or 10:1. One hundred microliters of each strain was inoculated into 10 ml fresh LB broth. Each assay was done in triplicate. After incubation and aeration of these mixtures at 25°C for 48 h or at 37°C for 24 h, the numbers of cells in each condition were determined by plate counts, and the colonies were differentiated by PCR with pO157-specific ecf1 gene primers.

Heat and salt tolerance.

Cultures grown in TSB at 37°C for 18 h were used for tolerance assays. Cells were diluted with 0.9% saline as needed to obtain 108 CFU/ml, and 100 μl was transferred into 9.9 ml of fresh TSB prewarmed to 55°C or 9.9 ml of M9 minimal medium containing 2.5 M NaCl (15%). Cultures were exposed to 55°C for 2 h or to 2.5 M NaCl for 14 days, and 100 μl of the culture was withdrawn at 1 h or 2 h or 1 day, 7 days, or 14 days, respectively. The number of surviving CFU after exposure was enumerated by direct plating onto LB agar.

Acid resistance.

To determine strain resistance to acidic conditions that mimic the bovine gastrointestinal tract, synthetic bovine gastric fluid (SGF) was prepared with bovine bile (Sigma-Aldrich, St. Louis, MO) as previously described (10). Bacteria were grown at 37°C in TSB, inoculated into 20 ml of fresh SGF (108 CFU/ml) with various pHs from 1.5 to 7.5 (adjusted with 1 N HCl), and, after 1 h of incubation at 37°C without shaking, cultured by direct plating onto LB agar. For analysis of the specific components in SGF that affected bacterial death, TSB adjusted to pH 2.2 (TSB-2.2), TSB containing 0.15% bovine bile (final concentration) (TSB-bile), and TSB containing 0.15% bovine bile (final concentration) adjusted to pH 2.2 (TSB-bile 2.2) were prepared. Bacterial survival after exposure to these conditions was determined as described above for SGF plus bovine bile.

SDS-PAGE.

Each strain was grown overnight at 37°C with aeration in LB broth, and the cells were harvested by centrifugation. The pellets were washed in 1× phosphate-buffered saline and lysed by heating in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer (whole-cell lysate). The supernatants were filtered with a 0.2-μm syringe filter (Millipore Co., Bedford, MA) and concentrated to 10× using an Amicon Ultra-15 centrifugal filter (10 kDa) (Millipore Co.) and were considered to be representative of the secreted proteins. Membrane protein fractions were prepared from lyophilized cells using a ProteoPrep membrane extraction kit (Sigma-Aldrich) according to the manufacturer's protocol. All samples were diluted to 1:4 with 4× sample buffer, boiled for 10 min, and loaded onto SDS-polyacrylamide gels (33). Whole-cell lysates and secreted proteins were separated on 12.5% polyacrylamide gels, and proteins in membrane fractions were separated on 10% polyacrylamide gels. Proteins from whole-cell lysates and membrane preparations were visualized with Coomassie blue stain, and the secreted proteins were visualized with silver stain.

Sample preparation for 2-DE.

Each strain was grown overnight at 37°C with aeration in LB broth. Cellular proteins were prepared by centrifugation of the cultures, and the pellets were washed three times with low-salt washing sample buffer (28). The washed pellets were resuspended in deionized water and lysed by two passages through a French press at 18,000 lb/in2. The whole-cell lysate was separated by centrifugation into soluble cell proteins (the supernatant) and insoluble cell proteins (the pellet). Both protein fractions were treated with protein inhibitor and nuclease. The soluble protein fraction was lyophilized to reduce the volume. Membrane fractions were prepared as described above for SDS-PAGE samples. Each protein fraction (soluble cellular proteins, insoluble cellular proteins, and membrane proteins) was resuspended in a rehydration solution containing 7 M urea, 2 M thiol-urea, 2% ASB14, 0.5% Triton X-100, 20 mM dithiothreitol (DTT), and 0.1% bromophenol blue. These sample mixtures were used for 2-DE.

Isoelectric focusing and 2-DE.

All equipment and software used in 2-DE were obtained from Amersham Biotech (Uppsala, Sweden). Isoelectric focusing (IEF) was carried out on an 11-cm pH 3 to 11 IPG strip. The IPG strips, which have an immobilized pH gradient to improve reproducibility, were rehydrated overnight at room temperature in an Immobiline DryStrip Reswelling tray with prepared samples. After rehydration, IEF was run using the following conditions: (i) 300 V, 5 voltage hours (Vh); (ii) 3,500 V, 5,250 Vh; (iii) 3,500 V, 15,750 Vh. Voltage increases were made on a “gradient” basis. After IEF, gels were equilibrated in two steps by adding 1% DTT and 2.5% iodoacetamide with SDS-equilibration buffer stock solution according to the manufacturer's protocol. Two-dimensional SDS-PAGE was conducted using a 12.5%, 18- by 24-cm gel. Proteins were visualized using Coomassie blue stain and scanned on an Image Master Gel scanner. To analyze the protein patterns in the gels, Image Master 2D Platinum software was used for protein spot detection, matching, and correction, as previously described (28).

Peptide mass fingerprinting.

All procedures for peptide mass fingerprinting were performed according to the manufacturer's instructions. Selected spots were excised from the gel in ~1-mm cubes and placed into siliconized tubes. Proteins were destained in 50% acetonitrile-50 mM ammonium bicarbonate, reduced with 10 mM DTT, alkylated with 50 mM idoacetamide, and digested with sequencing-grade trypsin (Worthington Biochem Co., Lakewood, NJ) overnight at 37°C. Peptides were extracted from the gel by sonication in 5% acetronile-2% trifluoroacetic acid and centrifugation. The supernatant was used to determine peptide sequences with a nanoACQUITY UPLC system (Waters, Milford, MA) and quadrupole-time of flight (Q-TOF) Premier tandem mass spectrometry (Waters). The total membrane fraction was also analyzed with Q-TOF mass spectrometry using RapiGest SF (Waters). MassLynx V.4. 1. and ProteinLynx Global Sever V.2. 2. (Waters) were used for the identification of protein spots by matching sequences with the peptide fragment database.

Bovine oral challenge experiment.

All animal protocols were approved by the University of Idaho Animal Care and Use Committee. Twelve 4- to 6-month-old Holstein steers were used for this study, and steers were housed in a quarantined facility at the University of Idaho. E. coli O157:H7 cells were grown in LB broth at 37°C, and the total CFU were determined by measurements of the optical density at 600 nm and confirmed by plate counts on LB agar. Each animal received 1 × 1010 CFU directly into the oral cavity using a disposable syringe as described below.

(i) Individual-strain challenge.

Two groups of four steers were challenged orally with a single dose of 1.0 × 1010 CFU/cattle of the WT or the ΔpO157 mutant.

(ii) Two-strain cochallenge.

Four steers were challenged orally with a single dose of 1.0 × 1010 CFU containing both the WT and the ΔpO157 mutant.

Sample analysis.

Samples from steers were cultured as previously described (46). Briefly, fecal and rectoanal mucosal swab (RAMS) samples were collected from each steer twice a week into Whirl-Pak bags (Nasco, Fort Atkinson, WI) or into 3 ml fresh TSB (Difco Laboratories, Detroit, MI), respectively. Ten grams of feces diluted 1:5 in TSB or RAMS samples in TSB was cultured directly by plating onto SMAC-CTVM agar for quantitative data. The remainder of each sample was incubated at 37°C for 18 h. Sorbitol- and MUG-negative colonies on SMAC-CTVM were confirmed to be E. coli O157:H7 colonies by latex agglutination (Pro-Lab Diagnostics, Toronto, Canada). Samples that were negative by direct plating were cultured by enrichment procedures for qualitative data, as described previously (46).

Statistical analysis.

The Student's t test was used to determine differences among the WT, the ΔpO157 mutant, and the complement strains.

RESULTS

Confirmation of the pO157 deletion.

The ΔpO157 mutant was confirmed by plasmid profile, PCR, PFGE, and Southern blot hybridization. Plasmid profile analysis showed the presence of the plasmid in the WT and the absence of the plasmid in the ΔpO157 mutant. Results from PCR with primers for hly and ecf1, genes unique to pO157, resulted in products from the WT and not from the ΔpO157 mutant (data not shown). PFGE analysis of chromosomal DNA following digestion with XbaI showed that the ΔpO157 mutant differed from the WT by the loss of a single 92-kb band. This band was confirmed to be pO157 by Southern blot hybridization analysis with a probe for the pO157-specific ecf1 gene (Fig. (Fig.1).1). The complemented strain ΔpO157 mutant 277::pO157ΔehxA was similarly confirmed by plasmid profile, PCR, and PFGE for the acquisition of pO157 and the lack of ehxA on pO157 (data not shown).

FIG. 1.
Comparison of WT and ΔpO157 mutant genomic DNA. (A) PFGE of XbaI-digested genomic DNA from the WT or the ΔpO157 mutant. (B) Southern blot hybridization of A with a pO157-specific ecf1 probe. The size fragments shown at the left were estimated ...

Growth patterns, diagnostic metabolic reactions, and antibiotic resistance profiles of the ΔpO157 mutant, its complement, and the WT were indistinguishable.

The growth patterns of the ΔpO157 mutant, its complement, and the WT were indistinguishable during all growth phases in rich LB broth and M9 minimal medium at 24°C, 37°C, or 42°C (Fig. (Fig.22 and data not shown). Also, plate counts to determine CFU showed that the WT and the ΔpO157 mutant grew similarly on SMAC-CTVM agar at 24°C, 37°C, and 42°C (data not shown). Heat and salt tolerance assays using 55°C-prewarmed TSB and M9 minimal medium with 2.5 M NaCl (15%) showed no significant difference (P > 0.05) in survival between the WT and the ΔpO157 mutant as determined by plate counts (Fig. (Fig.2).2). Diagnostic metabolic profiles, as determined by API strip reactions and antibiotic susceptibilities determined by the Kirby-Bauer standard method with 35 different antibiotic disks, were identical for the ΔpO157 mutant and the WT (data not shown).

FIG. 2.
Comparison of the WT, the ΔpO157 mutant, and its complement for growth, competition, salt tolerance, and heat tolerance. Three strains were grown with aeration in LB medium or M9 minimal medium at 37°C (A). Growth was measured every 30 ...

The WT outcompetes the ΔpO157 mutant in an in vitro assay.

The ability to compete with other bacteria is often related to the ability to be pathogenic. We examined the abilities of the WT and the ΔpO157 mutant to compete with each other. Starting with synchronized cell numbers (2.0 × 108 CFU), the WT and the ΔpO157 mutant were incubated together for 24 h at 37°C or 48 h at 25°C. The number of cells following this competition were determined by plate counting, and the colonies were differentiated by PCR. In this situation, the WT was more abundant than the ΔpO157 mutant by 3- or 4.5-fold after incubation in LB broth at 37°C or 25°C, respectively (Fig. (Fig.2).2). We also tested competitive survivals using different ratios of the WT and the ΔpO157 mutant. For ratios of the WT/ΔpO157 mutant at 1:10 or 10:1, the WT outcompeted the ΔpO157 mutant, with the final numbers of bacteria after 24 h of growth at 37°C with the WT at 70 and 100%, respectively (data not shown).

The ΔpO157 mutant was more resistant to acidic SGF than the WT.

To mimic bovine gastrointestinal conditions, we compared the survival of WT and that of the ΔpO157 mutant in SGF at various pHs, from pH 1.5 to 5.5. Neither strain survived pH 1.5 (data not shown), but at pH 2.2, there were significant differences, with the ΔpO157 mutant surviving better than the WT (P < 0.001) (Fig. (Fig.3).3). In less acidic conditions (pH 3, 4, or 5.5), both the WT and the ΔpO157 mutant survived similarly (data not shown). To determine the SGF components responsible for the effects, the WT and the ΔpO157 mutant were exposed to TSB-2.2, TSB-bile, or TSB-bile 2.2. In TSB-bile, there is no difference between the survival of the WT and that of the ΔpO157 mutant. Also, about 75% of WT cells survived in TSB-2.2. However, in TSB-bile 2.2, the WT did not survive, similar to the effect of incubation in SGF at pH 2.2 (Fig. (Fig.3).3). This indicated that bile and low pH had a synergistic effect on the death of the WT. The ΔpO157 mutant survived in TSB-bile 2.2 better than in SGF (pH 2.2), and this suggested that other components of SGF, such as pepsin or lysozyme, may have affected ΔpO157 mutant survival. The survival of the complemented strain in all of these survival assays was similar to that of the WT (data not shown) and confirmed that the enhanced survival of the ΔpO157 mutant in acidic/bile conditions was due to the loss of pO157.

FIG. 3.
Effect of pO157 on survival of E. coli O157:H7 in synthetic bovine gastric fluid, bile, or low-pH medium. The WT or the ΔpO157 mutant was exposed to each medium for 1 h at 37°C. The number of E. coli cells was determined by direct plate ...

A 50-kDa protein was significantly increased in the ΔpO157 mutant.

Fig. Fig.44 shows protein profiles of whole-cell lysates, supernatants, and membrane fractions from the WT and the ΔpO157 mutant. There were many identical proteins among the strains; however, the ΔpO157 mutant had a unique band of ~50 kDa (Fig. (Fig.4).4). In the complemented ΔpO157 mutant, the missing ~50-kDa band confirmed that this specific band resulted when the pO157 was deleted.

FIG. 4.
Analysis of proteins from the WT and the ΔpO157 mutant. After overnight incubation at 37°C, either cells were collected by centrifugation, divided, washed in buffer, and lysed (whole-cell lysate) (A) or the membrane proteins (C) were extracted ...

Many proteins from whole-cell lysates were differentially expressed between the ΔpO157 mutant and the WT.

2-DE protein profiles from whole-cell lysates (soluble and insoluble fractions) were used to determine alterations in protein expression due to the loss of pO157. We compared 2-DE patterns of whole-cell lysates from the WT to those from the ΔpO157 mutant and the complemented strain. Equivalent amounts of protein from each sample were separated on 2-DE gels. The profiles were reproducible, and representative gels were scanned after Coomassie blue staining. The protein profiles of the WT and the complemented strain were similar except for the lack of one spot presumed to be the hemolysin protein (data not shown). After normalization and removal of low-confidence spot data, 387 spots from the WT and 392 spots from the ΔpO157 mutant were distinguishable from the insoluble factions (Fig. 5A and B). From the soluble factions, 576 proteins spots from the WT and 571 protein spots from the ΔpO157 mutant were detected (Fig. 5C and D). The 2-DE images were analyzed using image analysis software as described in Materials and Methods. The protein spots from the WT were used as a reference standard for spot matching. The relative changes in spot intensities (percent volume) between the WT and the ΔpO157 mutant were analyzed. Subsets of proteins (26 proteins in the insoluble fraction and 51 proteins in the soluble fraction) showed more-than-twofold differences in protein level relative to those of the WT. Among these, 11 and 19 proteins, respectively, exhibited more-than-threefold differences between the WT and the ΔpO157 mutant (Fig. (Fig.5).5). Most of the proteins from the ΔpO157 mutant with the greatest increase in expression compared to the WT were ~50 kDa, consistent with one-dimensional SDS-PAGE results (Fig. (Fig.44).

FIG. 5.
Whole-cell lysate protein profiles of WT (A and C) and the ΔpO157 mutant (B and D) using two-dimensional gel electrophoresis. Whole-cell lysates from the WT and the ΔpO157 mutant were prepared using a French press and divided into two ...

Many proteins from the membrane fraction were differentially expressed between the ΔpO157 mutant and the WT.

To investigate detailed differences in protein expression between the membrane fractions from the WT and the ΔpO157 mutant, we performed 2-DE analysis and mass spectrometry on selected proteins (Fig. (Fig.66 and Table Table1).1). A total of 241 (WT) and 239 (ΔpO157 mutant) protein spots were revealed on 2-DE images after normalization and removal of low-confidence spots. Analysis of the relative protein levels of each spot showed that 12 (spots 1 to 12) proteins exhibited a more-than-threefold difference between the WT and the ΔpO157 mutant. Only two protein spots in the WT showed increased expression levels.

FIG. 6.
Protein profiles of the WT (A) and the ΔpO157 mutant (B) in the membrane fraction using two-dimensional gel electrophoresis. Cell membrane fractions from the WT and the ΔpO157 mutant were extracted using the Sigma membrane extraction kit ...
TABLE 1.
Q-TOF mass spectrometry data for protein spots showing increased expression levels around 50 kDa on a 2-DE gel for the ΔpO157 mutant

To study the proteins around 50 kDa, we selected 11 protein spots (spots 7 to 17) with significant differences in expression between the WT and the ΔpO157 mutant. Protein spots 7 to 13, with more-than-threefold differences, and spots 13 to 17, with more-than-twofold differences, were selected for further analysis. Q-TOF mass spectrometry was used to analyze these 11 proteins. The results obtained by mass spectrometry are summarized in Table Table1.1. Two proteins at spots 9 and 11 were not identified by this procedure. As often occurs in membrane preparations, contaminating proteins were found. Sequences of 112 peptides from the membrane fraction, using Q-TOF mass spectrometry, showed that >60% of the proteins originated from the membrane, while the remaining <40% were cytoplasmic or of unknown subcellular origin (data not shown).

The ΔpO157 mutant survived passage through the bovine GIT better than the WT but did not colonize the bovine RAJ mucosa as well as the WT.

To compare the WT and the ΔpO157 mutant in vivo, we did two independent cattle experiments. First, four steers were inoculated orally with a single dose of both the WT and the ΔpO157 mutant and monitored for carriage of the bacteria by RAMS direct culture (Fig. (Fig.7).7). Over 90% of the isolates from all four steers were the ΔpO157 mutant on day 1 post-oral challenge (P < 0.001). Steers 3 and 4 were both culture positive for E. coli O157 for at least 3 weeks postdose, and both shed predominantly the WT after day 21. The predominant isolate from one steer (steer 1) oscillated between the ΔpO157 mutant and the WT. Fecal cultures from the four steers were similar to RAMS cultures (data not shown). The results from day 1 suggested that the ΔpO157 mutant survived passage through the bovine GIT better than the WT. Also, this result was consistent with the in vitro acid-resistant assay showing that the ΔpO157 mutant survived better in SGF at pH 2.2 (Fig. (Fig.33).

FIG. 7.
Effect of pO157 on survival of E. coli O157:H7 in cattle following oral administration of bacteria. Four steers (steers 1 to 4) were given a single oral dose of 1.0 ×1010 CFU containing both the WT and the ΔpO157 mutant. RAMS samples were ...

In the second experiment, eight steers were divided into two groups of four. Both groups were inoculated with a single oral dose of the WT or the ΔpO157 mutant, separately (Table (Table2).2). All steers in the WT group (WT1 to WT4) were culture positive for ≥7 weeks post-oral dose, and two steers (WT1 and WT3) were culture positive at ≥12 weeks. In contrast, among the animals in the ΔpO157 mutant group (MT1 to MT4), only one steer (MT3) remained culture positive until week 11, and the other three steers (MT1, MT2, and MT4) cleared the bacteria by week 1 or 2. Fecal culture results were similar to those of RAMS culture in both groups (data not shown). These results suggested that the WT persists in cattle better than the ΔpO157 mutant. However, culture results from steer MT3 indicated that in some animals, the ΔpO157 mutant had the ability to persist or colonize very well. The parameters that lead to this persistence may include the immune status of the animal, other competing microbial flora, and/or unknown factors.

TABLE 2.
E. coli O157:H7 carriage in cattle given a single oral dose of the WT or the ΔpO157 mutant

DISCUSSION

By comparing E. coli O157:H7 WT with the ΔpO157 mutant, this study suggested that pO157 influenced survival in the upper GIT, persistence at the RAJ mucosa, and expression of proteins encoded on the chromosome. These results expanded on our previous work that showed that pO157 is required for efficient E. coli O157:H7 colonization of cattle following rectal challenge (52) and that the ecf operon of pO157 affects E. coli O157:H7 survival in vivo and in the environment (62).

The oral administration of bacteria assesses both the ability of strains to survive passage through the GIT and the ability to colonize by attaching and surviving at the RAJ mucosa (52). The ΔpO157 mutant survived passage through the bovine GIT better than the WT. In vitro analysis of bovine synthetic gastric fluid showed that the ΔpO157 mutant survived better than the WT and was more resistant to both bile and acidic conditions. These characteristics should allow the bacteria to survive through the abomasum (bovine gastric stomach) and in the bile-laced upper intestines. Although we did not culture bacteria from these GIT sites, culture data at 1 day post-oral dose (Fig. (Fig.7)7) showed that larger numbers of the ΔpO157 mutant than the WT were in the feces and in RAMS. This situation presumably led to a better chance for the ΔpO157 mutant than the WT to attach to epithelial cells at RAJ mucosa. Surprisingly, this was not the outcome, and it was the WT that persisted best throughout the remaining days of the trial (Table (Table2).2). Bacterial colonization and persistence at the bovine RAJ mucosa are undoubtedly dynamic processes that depend on the ability of bacteria not only to attach to the epithelial cells but also to compete with other bacteria for potentially limited nutrients or a particular attachment site(s) in that niche (52). Although no differences between the WT and the ΔpO157 mutant were measurable in individual growth analyses, in vitro competition assays showed that the WT had an advantage over the ΔpO157 mutant in survival. A similar competitive edge may explain why the WT was able to persist in the RAJ mucosa better than the ΔpO157 mutant.

Several genes that likely impact bovine colonization have been identified on pO157. A myristoyl transferase gene in the ecf operon is involved in lipid A modification and represents one of two copies of this gene (the other is the chromosomal lpxM gene). Double mutants carrying deletions in both the ecf and lpxM genes lead to reduced survival in the bovine GIT and persistence in farm water troughs (62). The mucinase activity of StcE, a zinc metalloprotease, contributes to intimate adherence of E. coli O157:H7 to epithelial cells in culture and may be required for efficient colonization of cattle (34). Although Tatsuno et al. showed that the deletion of the toxB gene results in reduced adherence of E. coli O157:H7 to epithelial cells in culture (55), Stevens et al. showed that the toxB mutation did not affect E. coli O157:H7 colonization of calves or sheep (54). This suggests that increased adherence to cultured cells may not always predict increased colonization. In addition, Schmidt et al. found a type II secretion pathway, etpC to etpO, on pO157, and extracellular secretion of hydrolytic enzymes that may effect colonization are secreted via this pathway (48, 50). Using signature-tagged mutagenesis, Dziva et al. reported 59 E. coli O157:H7 genes that influence colonization of the gastrointestinal mucosa of preruminant calves but did not identify any pO157-encoded genes (16). This discrepancy with our results (52, 62) may be due to differences in pre- and postruminant animals.

Proteomic analysis is important for determining the complexities of gene expression and the protein interactions that affect functions, and comparing total gene expression of wild-type and mutant strains is a useful application. Functional proteomics play a key role in assigning biological roles for genes whose functions are unknown (23, 27). The whole genome of E. coli O157:H7 including pO157 has been sequenced, and most of the genes are annotated (8, 25), but not all functions have been identified. About 20% of the open reading frames on the 92-kb plasmid pO157 are characterized, but until the work presented here, there has been no analysis of total gene expression at the protein level after a mutation or deletion of pO157 genes. Here, both one-dimensional gel electrophoresis- and 2-DE-based total protein expression profiling were used to investigate the effect of deleting pO157. Unexpectedly, about 33% of proteins that showed significant differences in expression between the WT and the ΔpO157 mutant were found at increased levels in the ΔpO157 mutant. This suggests that some pO157 genes control or regulate chromosomal gene expression. There has been no direct study supporting this, but Lathem et al. showed that the expression of the pO157 stcE gene is up-regulated by the global regulator Ler, encoded in the chromosomal LEE elements (34). In enteropathogenic E. coli, the plasmid-encoded regulator Per is well known to activate chromosomal eaeA gene expression (21).

The fact that the pO157 is highly conserved among all clinical isolates suggests that pO157 encodes required virulence factors and/or elements required for efficient colonization and/or survival in the host. Identifying proteins that are expressed differently between the WT and the ΔpO157 mutant will be a key to understanding the mechanism(s) of the pO157 association with in vivo results. Nonetheless, there were too many protein spots and they were too close together in the whole-cell lysate gels to systematically and precisely excise individual proteins for analysis by mass spectrometry. One solution to this problem would be to separate proteins further using various IPG strips with more finely tuned pH ranges such as pH 3 to 5, pH 4 to 7, pH 6 to 9, and pH 7 to 11 rather than one gel with a pH from 3 to11. These experiments are ongoing in our laboratory.

It is well known that membrane alterations result in various changes in bacterial survival. Previous studies demonstrated that several genes on pO157 are involved in membrane structure and/or integrity (30, 32, 62), but no significant differences in bacterial survival were measured with single gene deletions. Our identification of changes in membrane fraction proteins showed that the deletion of pO157 affected proteins encoded on the chromosome. Two proteins from this fraction were identified as being membrane-associated proteins, and eight proteins were identified as being cytoplasmic proteins. These two membrane-associated proteins, GroEL and Agp, were not related to membrane structure. Chaperonin GroEL is an essential protein for bacterial growth, and GroEL interacts with SecA, a cytoplasmic membrane protein (6, 17). Periplasmic glucose-1-phosphatase encoded by agp has inositol phosphatase activity and is potentially involved in pathogenic inositol phosphate signal transduction pathways via type III secretion into the host cell (11, 14, 35). Contamination of membrane fractions with cytosolic proteins is not uncommon. Several studies reported that proteins spots other than membrane proteins were identified by 2-DE of membrane protein extracts (40, 45, 60). Rhomberg et al. identified 53 proteins from the sarcosin-insoluble outer membrane fraction, and among them, 37 were cytoplasmic or of unknown subcellular origin (45). There was also the possibility that some cytoplasmic proteins can be extracted in the membrane fraction due to their transient interaction with membrane-associated proteins.

Another interesting protein that was differentially expressed in this study is tryptophanase (TnaA), an enzyme that degrades tryptophan to indole, pyruvate, and ammonia (53). Previous studies showed that TnaA is induced to high levels in alkaline growth conditions (pH 9). The actions of this enzyme may allow E. coli to grow at extreme conditions by neutralizing excess alkali by metabolizing amino acids to produce acidic products (5). Di Martino et al. revealed that transposon insertion mutation of tnaA leads to a decrease in both epithelial cell adherence and biofilm formation on polystyrene by E. coli S17-1 (15). TnaA was up-regulated in the ΔpO157 mutant even without high-pH induction, suggesting that a certain gene(s) of pO157 may be suppressed under neutral conditions.

Similar to TnaA, GadAB was up-regulated in the ΔpO157 mutant without induction by acidic conditions. This may explain why the ΔpO157 mutant survived better in bovine SGF than the WT. The glutamate-dependent acid resistance system is one of the major acid resistance systems in E. coli and particularly protects cells exposed to extreme acid conditions below pH 3. This system requires glutamate decarboxylase, encoded by gadA and gadB, and a glutamate-γ-aminobutyric acid antiporter, encoded by gadC (3, 9, 26). Previous studies found several regulatory proteins, including RpoS, histone-like protein H-NS, and GadE, involved in the regulation of the glutamate-dependent acid resistance system, to be complicated and not clearly understood (13, 37, 39). Our work suggests that pO157 is involved in this complex system. Finally, the presumptive functions of other gene products were related to cell survival in salvage conditions or cell metabolism (Table (Table1)1) (1, 27, 29, 44, 58, 59).

In summary, genes encoded on pO157 influence E. coli O157:H7 metabolic activity, survival in acidic environments, survival in the bovine gastrointestinal tract, and colonization of the bovine terminal rectum. These effects likely occur both directly and through the regulation of chromosomal genes.

Acknowledgments

This work was supported in part by the Idaho Agriculture Experiment Station, the National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service, grant number 04-04562; Public Health Service grants U54-AI-57141, P20-RR16454, and P20-RR15587 from the National Institutes of Health; and grants from the Idaho Beef Council.

We thank Jang W. Yoon for helpful discussions and Lonie Austin for animal handling.

Footnotes

[down-pointing small open triangle]Published ahead of print on 2 February 2007.

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