The Escherichia coli O157:H7 bovine rumen fluid proteome reflects adaptive bacterial responses

  • Indira T Kudva1Email author,

    Affiliated with

    • Thaddeus B Stanton1 and

      Affiliated with

      • John D Lippolis2

        Affiliated with

        BMC Microbiology201414:48

        DOI: 10.1186/1471-2180-14-48

        Received: 6 November 2013

        Accepted: 10 February 2014

        Published: 21 February 2014

        Abstract

        Background

        To obtain insights into Escherichia coli O157:H7 (O157) survival mechanisms in the bovine rumen, we defined the growth characteristics and proteome of O157 cultured in rumen fluid (RF; pH 6.0-7.2 and low volatile fatty acid content) obtained from rumen-fistulated cattle fed low protein content “maintenance diet” under diverse in vitro conditions.

        Results

        Bottom-up proteomics (LC-MS/MS) of whole cell-lysates of O157 cultured under anaerobic conditions in filter-sterilized RF (fRF; devoid of normal ruminal microbiota) and nutrient-depleted and filtered RF (dRF) resulted in an anaerobic O157 fRF-and dRF-proteome comprising 35 proteins functionally associated with cell structure, motility, transport, metabolism and regulation, but interestingly, not with O157 virulence. Shotgun proteomics-based analysis using isobaric tags for relative and absolute quantitation used to further study differential protein expression in unfiltered RF (uRF; RF containing normal rumen microbial flora) complemented these results.

        Conclusions

        Our results indicate that in the rumen, the first anatomical compartment encountered by this human pathogen within the cattle gastrointestinal tract (GIT), O157 initiates a program of specific gene expression that enables it to adapt to the in vivo environment, and successfully transit to its colonization sites in the bovine GIT. Further experiments in vitro using uRF from animals fed different diets and with additional O157 strains, and in vivo using rumen-fistulated cattle will provide a comprehensive understanding of the adaptive mechanisms involved, and help direct evolution of novel modalities for blocking O157 infection of cattle.

        Keywords

        O157 Rumen LC-MS/MS iTRAQ Proteome Growth-patterns

        Background

        Escherichia coli O157 (O157) have been implicated in several human outbreaks since their being established as foodborne pathogens in 1982; an estimated 63,153 illnesses, 2,138 hospitalizations and 20 deaths occur annually in the United States [14]. Human disease ranges from self-limiting watery diarrhea to debilitating bloody diarrhea that can advance into often fatal, extraintestinal, secondary sequelae in susceptible patients [3, 4]. Cattle are the primary reservoirs for O157, with their recto-anal junction (RAJ) serving as the colonization site at which these human foodborne pathogens persist [4, 5]. However, the first bovine gastrointestinal tract (GIT) compartment that O157 encounters is the rumen, where the dynamic environment of regurgitating food particles, bio-fermentation, changing pH, and production of varying amounts of volatile fatty acids (VFA) poses challenges for O157 survival [68]. Studies thus far, have concentrated on the recovery of O157 from the rumen, the in vitro O157 growth dynamics in modified rumen fluid or media with additives to mimic the rumen environment, expression of select O157 genes under controlled pH and VFA conditions, dietary effects on bacterial survival, and effects of select flora/metabolite on the growth/survival of O157 in the rumen or rumen fluid [611]. Despite this, however, a comprehensive study of the mechanisms used by O157 to survive the rumen environment is yet to be undertaken. Hence, as an initial step, we determined the repertoire of O157 proteins (proteome) as expressed in vitro in harvested, rumen fluid (RF). We included RF of varying compositions (with and without normal flora, or depleted of nutrients essential for bacterial growth), with no additives, and used diverse culture conditions, to identify bacterial factors that may enable O157 adaptation to the rumen.

        Methods

        Bacterial strain, inoculum preparation and animals

        Wild-type O157 strain 86–24 (Shiga toxin (Stx) 1-negative, Stx 2-positive; motile; clinical isolate) was used in this study [12]. Overnight culture of O157 in Luria-Bertani (LB) broth, grown at 39°C with aeration was used to prepare log-phase sub-cultures of the same in 50 ml LB broth, under the same growth conditions. Bacteria harvested from the log-phase cultures at an OD600 0.5-0.6, washed and re-suspended in sterile 0.9% saline, were used to inoculate various rumen fluid (RF) or LB aliquots as described under ‘Culture conditions and processing for proteomics’. All O157 cultures were confirmed serologically using latex agglutination kits (Remel Inc., Lenexa, KS). Two rumen-fistulated Holstein cows, routinely used as rumen fluid ‘donors’ at the National Animal Disease Center (NADC, Ames, IA) with approval from the NADC-Animal Care and Use Committee, were used in this study. Both animals, approximately 1 year of age, were fed the NADC Maintenance Diet (corn silage, grass hay, 520 pellets, protein supplements) at 25% fiber and 10% protein, with ad-lib access to water through out.

        Unfiltered (uRF), Filtered (fRF), and Depleted RF (dRF)

        Rumen fluid samples collected from the two animals (Samples A and B; Tables 1 and 2), on separate days, were used to prepare the RF-preparations for each experiment set (Experiment I and II). Two liters of RF was collected 2–3 hr post-feeding to allow for rumination to occur, at each sampling time [10, 13]. RF was strained through cheesecloth to remove large feed particles, and poured into collection flasks; pH was recorded on site and an aliquot frozen at –80°C for volatile fatty acid (VFA) analysis. Approximately 500 ml of the strained RF was stored as the unfiltered RF (uRF) at 4°C.
        Table 1

        Biochemical characteristics of rumen fluid used to analyze growth patterns of O157 strain 86–24 in Experiment I

        Sample analysis

        Depleted rumen fluid

        Filtered rumen fluid

         

        Sample A

        Sample B

        Sample A

        Sample B

        pH1

        7.9

        7.6

        7.6

        7.7

        Volatile Fatty Acids (μm/ml; VFA)

        Total VFA

        324

        207

        211

        157

        Acetic acid

        201

        142

        144

        112

        (62%)2

        (69%)

        (68%)

        (71%)

        Propionic acid

        41

        28

        31

        23

        (13%)

        (14%)

        (15%)

        (15%)

        Butyric acid

        43

        20

        16

        10

         

        (13%)

        (10%)

        (8%)

        (6%)

        1pH, post-depletion and/or post-filtration of the depleted and filtered rumen fluid samples, respectively.

        2Percent individual volatile fatty acid of the total is shown in parenthesis.

        Table 2

        Biochemical characteristics of rumen fluid used to analyze growth patterns of O157 strain 86–24 in Experiment II

        Sample analysis

        Depleted rumen fluid

        Filtered rumen fluid

        Unfiltered rumen fluid

         

        Sample A

        Sample B

        Sample A

        Sample B

        Sample A

        Sample B

        pH1

        7.6

        7.4

        7.7

        7.2

        6.4

        6.7

        Volatile Fatty Acids (μm/ml; VFA)

        Total

        203

        205

        144

        153

        210

        165

        Acetic acid

        139

        140

        103

        110

        141

        104

        (68%)2

        (68%)

        (72%)

        (72%)

        (67%)

        (63%)

        Propionic acid

        28

        28

        21

        23

        32

        30

        (14%)

        (14%)

        (13%)

        (15%)

        (15%)

        (18%)

        Butyric acid

        19

        19

        9

        10

        20

        17

         

        (9%)

        (9%)

        (6%)

        (7%)

        (10%)

        (10%)

        1pH, post-depletion and/or post-filtration of the depleted and filtered rumen fluid samples, respectively.

        2Percent individual volatile fatty acid of the total is shown in parenthesis.

        One half of the remaining strained RF was processed as follows to generate filtered RF (fRF). The strained RF was centrifuged at 27,000× g for 30 mins at 18°C, at least 3 times, to remove particulate matter and pressure filtered using a 0.5 μ pre-filter and a 0.2 μ filter in tandem (Pall Corporation, Port Washington, NY). The fRF was collected into sterile bottles and stored at 4°C after recording the pH and freezing an aliquot for VFA analysis.

        To prepare dRF, the other half of the remaining strained RF was first subjected to depletion, a process that involves exhaustion of residual nutrients in the RF by exploiting metabolic activities of the resident microflora, prior to the centrifugation-filtration steps. Specifically, the depletion process was initiated by adjusting the strained RF pH to 6.8-7.0, and incubating it under anaerobic conditions, at 39°C for four days. The strained RF was held in flasks fitted with stoppers bearing valves to release the fermentation gases throughout the incubation, following which the depleted RF was centrifuged and filtered as described above. This depletion protocol was adapted from previously described methods with no extraneous substrates added to the RF prior to depletion [11, 14]. The pH of the resultant filter-sterilized dRF was recorded and aliquots set aside for VFA analysis prior to storage at 4°C in sterile bottles.

        pH and volatile fatty acids (VFA) analysis

        Initial rumen fluid pH measurements were taken during collection by using a portable pH meter (Thermo Fisher Scientific Inc., Waltham, MA) [8, 11]. Subsequently, the pH meter or pH paper was used (pH range 5.0–8.0; Micro Essential Laboratory Inc., Brooklyn, NY), to record pH of the processed RF and media. VFA concentrations in rumen fluid and its preparations were determined by capillary gas chromatography of their butyl esters, as described previously [15, 16], on an Agilent 6890 N gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA).

        Culture conditions, and processing for proteomics

        RF preparations from Samples A and B were analyzed separately per experiment set, and each analysis in turn was conducted in duplicate. In Experiment I, 5 ml LB, dRF, or fRF media were aliquoted separately into 85, 16 × 150 mm tubes. Of these, five tubes per media were used as uninoculated controls. The remaining 80 tubes were inoculated with O157. To create anaerobic culture conditions, half of these tubes were transferred into the anaerobic Coy Chamber for 72 hrs, sealed and inoculated within the chamber and then removed. The log-phase O157 culture, re-suspended in 0.9% saline was inoculated to a starting OD600 0.05-0.06, into all the 80 tubes, which were then incubated at 39°C with shaking, along with the uninoculated control tubes. O157 was grown to an OD600 of 0.8-1.0, before harvesting cells from each tube by centrifugation at 7,000 rpm, 15 min at 4°C. Bacterial cells from like media, whether derived from RF-samples A or B, were pooled together and washed three times with an equal volume of ice-cold sterile phosphate buffered saline (PBS; pH 7.4), and processed to obtain cell lysate and pellet fractions for bottom-up proteomic analysis [17].

        In Experiment II, uRF was included to the media (LB, dRF, fRF) being evaluated and aliquoted as described above. However, the O157 inoculum diluted in saline to the starting OD600 0.05-0.06 was placed in sterile dialysis tubing (Spectra/Por Type F, PVDF: 80,000 kDa cut off; Serva Electrophoresis, Heidelberg, Germany) and suspended within the uRF containing tubes [18]. This was to ease the recovery of O157 from the complex uRF milieu and the colony counts recovered from the tubings matched those obtained by magnetic recovery of O157 from directly inoculated uRF (data not shown). O157-innoculated LB, dRF, fRF, and uRF were incubated for 48 h, anaerobically, before harvesting cells and processing for proteomic analysis [17] using iTRAQ. For this experiment, bacterial cells from like media were pooled together but kept separate between preparations derived from RF-samples A and B. The culture conditions used in Experiment II correlated with ruminal conditions and feed turnover rates [1921]. In both experiments, OD600 of each tube was recorded relative to uninoculated control tubes, centrifuged at 10,000 rpm for 10 min to remove any sediments or particulate matter which could interfere with the spectrophotometer reading. In addition, pH, and colony counts (on LB agar) were determined from the five uninoculated and ten inoculated tubes at different time points, for comparison.

        Bottom-up proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS)

        This proteomic analysis was done at the Proteomics Division, ICBR, University of Florida, Gainesville, Florida. O157 cell pellet and lysate fractions from Experiment I (LB, dRF, fRF) were concentrated using spin filters (MW cutoff 5000 Daltons), and digested with trypsin prior to tandem mass spectrometry (MS/MS) as described previously [17]. The enzymatically-digested samples were injected onto a capillary trap (LC Packings PepMap) and desalted for 5 min with a flow rate of 3 μl/min of 0.1% v/v acetic acid. The samples were loaded onto an LC Packing® C18 Pep Map nanoflow HPLC column. The elution gradient of the HPLC column started at 3% solvent B, 97% solvent A and finished at 60% solvent B, 40% solvent A for 95 min for protein identification.

        Solvent A consisted of 0.1% v/v acetic acid, 3% v/v acetonitrile (ACN), and 96.9% v/v H2O. Solvent B consisted of 0.1% v/v acetic acid, 96.9% v/v ACN, and 3% v/v H2O. LC-MS/MS analysis was carried out on a hybrid quadrupole-TOF mass spectrometer (QSTAR elite, Applied Biosystems, Framingham, MA). The focusing potential and ion spray voltage was set to 225 V and 2400 V, respectively. The information-dependent acquisition (IDA) mode of operation was employed in which a survey scan from m/z 400–1800 was acquired followed by collision-induced dissociation (CID) of the four most intense ions. Survey and MS/MS spectra for each IDA cycle were accumulated for 1 and 3 s, respectively.

        Tandem mass spectra were extracted by ABI Analyst version 2.0. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.2.2). Mascot was set up to search NCBI with taxonomy Bacteria database assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 0.50 Da. Iodoacetamide derivative of Cys, deamidation of Asn and Gln, oxidation of Met, were specified in Mascot as variable modifications. Scaffold (version Scaffold-03-3-2, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm [22]. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified unique peptides. Proteins with single peptide hits were included if they exhibited high confidence based on low false discovery rates [23]. Relative protein abundance was estimated using the normailized total spectral counts [24]. Protein probabilities were assigned using the Protein Prophet algorithm [25]. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.

        Quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ)

        O157 cell pellet and lysate fractions from Experiment II (LB, dRF, fRF, uRF; cultured 48 h, anaerobically) were analyzed by iTRAQ. Samples were processed, trypsin digested, and labeled with various iTRAQ reagents as described earlier [26], in accordance with the manufacture’s instructions for the iTRAQ 4-plex kit (Amine-Modifying Labeling Reagents for Multiplexed Relative and Absolute Protein Quantitation, Applied Biosystems, Foster City CA). Labeled peptides were combined, dried in one tube, and held at -80°C until use. A modification of the previously used protocol was used to analyze these labeled peptides that were resuspended in mobile phase A (72 mM triethlyamine in H2O, pH 10 with acetic acid) at a concentration of 200 μg/μl and incubated for 1 hour in a sonic-water bath at RT. 100 μg of sample was injected into a Waters 1525 μ Binary HPLC (Waters Corporation, Milford, MA) with a Waters XBridge C18, 3.5um, 1 × 100 mm column in mobile phase A and ran isocratically for 6 minutes. The gradient consisted of, 0-20% mobile phase B (72 mM triethlyamine in ACN, 52 mM acetic acid), over 34 minutes; 20-40% over 20 minutes; and finally 40-100% over 2 minutes, at a flow rate of 100 μl/minute throughout the entire gradient [27]. Two-minute fractions were collected, dried in a vacuum centrifuge, and resuspended in nano-HPLC buffer A (95% H2O: 5% ACN and 0.1% formic acid). Based on previous experience we combined, 3 fractions before and after, the fractions that contained the majority of the eluted peptides.

        Fractions from the first dimension chromatography were injected on a second dimension of chromatography using a Proxeon Easy-nLC (Thermo Fisher Scientific, West Palm Beach, FL) connected to the mass spectrometer. The second dimension chromatography used a trapping column (Proxeon Easy-Column, 2 cm, ID 100 μm, 5um, 120A, C18) and an analytical column (Proxeon Easy-Column, 10 cm, ID 75 μm, 3 μm, 120A, C18). The gradient using a mobile phase A (95% H2O: 5% acetonitrile and 0.1% formic acid) and mobile phase B (5% H2O: 95% acetonitrile and 0.1% formic acid). The gradient was, 0% B for 3 minutes, 0%-8% B from 3–5 minutes, 8-18% B from 5–85 minutes, 18-30% B from 85–100 minutes, 30-90% B from 100–105 minutes, and held at 90% B from 105–120 minutes at continuous flow rate throughout the gradient of 300 nl/min. The analytical column was connected to a PicoTip Emitter (New Objectives, Woburn, MA; FS360-75-15-N-20) and together attached to a LTQ OrbiTrap Velos Pro (Thermo Fisher Scientific, West Palm Beach, FL) mass spectrometer using the Proxeon Nanospray Flex Ion Source. The capillary temperature was set at 275°C and spray voltage was 2.9 kV. The mass spectrometer was used in a data dependent method. In MS mode, the instrument was set to scan 300–2000 m/z with a resolution of 30,000 FWHM. A minimal signal of 20,000 could trigger tandem MS and 10 consecutive MS/MS were possible. High-energy collision-induced dissociation (HCD) was used to resolve the iTRAQ reporter ions, 113–117. The normalized collision energy was set to 35 and repeat mass exclusion was set to 120 seconds.

        Tandem mass spectra were extracted and charge state deconvoluted by Proteome Discoverer version 1.4. Charge state deconvolution and deisotoping was not performed. All MS/MS samples were analyzed using Mascot, Sequest (XCorr Only; Thermo Fisher Scientific, San Jose, CA, USA; version 1.3.0.339) and X! Tandem (GPM.org; version CYCLONE (2010.12.01.1)) assuming digestion with trypsin. A custom E. coli database was generated by combining the fasta files from uniprot.org from the following E. coli strains: 12009/EHEC, 2009EL-2050, 2009EL-2071, 2011C-3493, 11128/EHEC, O157:H7, EC4115/EHEC, TW14359/EHEC, and 11368/EHEC. This E. coli fasta file consists of 47,819 entries and was generated in May 2013. Mascot, Sequest (XCorr Only) and X! Tandem were searched with a fragment ion mass tolerance of 0.100 Da and a parent ion tolerance of 10.0 PPM; carbamidomethyl of cysteine and iTRAQ4plex of lysine and the n-terminus were specified as fixed modifications while deamidation of asparagine and glutamine, oxidation of methionine and iTRAQ4plex of tyrosine were specified as variable modifications. Scaffold (version Scaffold_4.0.6) was used to validate MS/MS based peptide and protein identifications, as described above for ‘Bottom-up Proteomics’. The O157-proteome as expressed in LB was used as the reference against which all the other O157-proteomes were compared. Two biological replicate samples (Sample A and B), corresponding to the duplicate experiments described under ‘Culture conditions, and processing for proteomics’ above, were analyzed separately. In addition, each sample was analyzed twice (Run A and Run B; technical replicates) to cover the entire spectra of proteins in these samples. Only proteins that were consistently identified were selected for analysis.

        Statistics and bioinformatics

        The Student t-Test (two-tailed) was used to evaluate differences between the means of the O157 optical densities and viable counts recovered from the different cultures and a values of p < 0.05 was considered significant. Putative functions were determined by querying the Conserved Domain Database (CDD) at http://​www.​ncbi.​nlm.​nih.​gov/​Structure/​cdd/​wrpsb.​cgi, and associated metabolic pathways were determined using the KEGG pathway database at http://​www.​genome.​jp/​kegg/​pathway.​html. Cellular and sub-cellular locations of proteins were determined as described previously [17].

        Results

        pH and VFA content

        The pH and VFA concentrations were comparable amongst all rumen fluid samples, indicating consistency in maintenance diet being fed and the ruminal chemistry between the two animals enrolled in the study (Tables 1 and 2). The pH of the uRF ranged from 6.4-6.7 at collection [2831] but attained a more neutral pH after filtering, as seen with dRF (pH 7.4–7.9) and fRF (pH, 7.2–7.7) in both experiments (Tables 1 and 2). Concentrations of three VFAs, acetate, propionate and butyrate, were closely analyzed as these vary the most with changes in the forage versus starch compositions of the feed, and are of relevance to both host and bacterial growth. Consistent with the 25% forage and 10% protein diet that these cattle were being fed, the RF comprised a higher percentage of acetate [2831]. Acetate ranged from 72-62%, compared to the 13-18% propionate and 6-13% butyrate concentrations across the uRF, dRF and fRF samples in both experiments, irrespective of procedures used to prepare dRF and fRF (Tables 1 and 2). LB broth (pH 7.0-7.2) did not contain added VFAs.

        O157 growth characteristics

        Log phase O157 cultures, set up for the two experiments, were at 0.5-0.6 OD600, respectively, with viable counts around 1 × 108 cfu/ml. Hence, when each medium was inoculated to a starting 0.05-0.06 OD600, the corresponding O157 counts were at ~1-5 × 107 cfu/ml. In both experiments, O157 grew to an OD600 of 1.0 within 2 h in LB media, aerobically and anaerobically as anticipated, with an increase in viable count to 4 × 108 cfu/ml and the final culture pH at 6.0-6.2. However, significant differences were observed between aerobic and anaerobic growth patterns of O157 when cultured in dRF, fRF and uRF preparations.

        In Experiment I, O157 cultured in dRF and fRF achieved an average OD600 of 0.6-1.0 in 48 h aerobically, but remained at a low OD600 of ≤0.2 anaerobically, even after 14 days of incubation. Irrespective of the ODs, viable O157 was recovered from all cultures, but the viable counts at 106 (dRF)-2 × 107 (fRF) cfu/ml aerobically, and at 105 (dRF)-2 × 105 (fRF) cfu/ml anaerobically (data not shown) appeared to be static or decreasing. The pH for dRF and fRF cultures at the end of incubation was around 7.7 (aerobic)–7.3 (anaerobic). Similar O157 growth results were observed upon anaerobic culture for 48 h in dRF, fRF and uRF, in Experiment II (Figure 1), with the pH for uRF cultures being 6.8 at end of incubation. This was despite these media being prepared with RF from a separate animal and a shorter anaerobic incubation period than in the first experiment, thereby verifying the observations made initially. Here, the cultures reached an average OD600 of 0.97 (LB), ~0.03 (dRF), ~0.04 (fRF) and ~0.03 (uRF) in 48 h, with O157 viable counts of 2 × 108 cfu/ml (LB), 4 × 105 cfu/ml (dRF), 3 × 106 cfu/ml (fRF) and 1 × 106 cfu/ml (uRF), respectively.
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-14-48/MediaObjects/12866_2013_2209_Fig1_HTML.jpg
        Figure 1

        Growth characteristics of O157 in Experiment II, following anaerobic incubation for 48 h, in LB and RF-preparations. Optical densities (OD600) and viable counts (colony forming units [cfu]/ml), with the standard error of means, are shown in graph A and B, respectively. The p values shown on the graphs were calculated using the Student t-Test (significant, p < 0.05).

        Significant differences were observed among the optical densities and viable counts of LB cultures versus RF-preparation cultures, under all growth conditions. However, differences between the RF-preparations were not always significant (Figure 1). For instance, in Experiment II, the p values for the O157 viable counts were: LB: dRF, p = 0.0379; LB: fRF, p = 0.0385; LB: uRF, p = 0.0381, dRF: fRF, p = 0.0121 and fRF: uRF, p = 0.0655; dRF: uRF, p = 0.1077.

        Proteomics analysis

        (i) Bottom-up

        LC-MS/MS analysis of the O157 cell pellet and lysate fractions generated in Experiment I provided insights into the proteins being expressed by O157 in different media, under different growth conditions and at extended incubation time points. A total of 585 protein (2284 spectra) hits were identified by setting minimum characteristics for the identification confidence. However, of these only 218 O157 proteins matched a higher threshold cut off, with 90% protein-80% peptide probability in the Scaffold Viewer, and hence, were selected for analysis. The 218 O157 proteins were differentially expressed: 90 only under aerobic conditions, 37 only under anaerobic conditions and 91 under both conditions (data not shown), accounting for fewer proteins under anaerobic conditions. Interestingly, none of the O157 proteins expressed aerobically or anaerobically in either media were associated with direct virulence (e.g., the Locus of Enterocyte Effacement [LEE]-encoded proteins or Shiga toxins) but were primarily associated with sequences homologous to other E. coli genomes (Backbone) (Additional file 1: Table S1). Considering that the rumen is an anaerobic microbiome, the 128/218 O157 proteins expressed anaerobically were examined in greater detail. These proteins were either unique to growth in LB (93/128), dRF (2/128), fRF (10/128) or, expressed in more than one media (14/128 in LB/dRF/fRF, 9/128 in dRF/fRF) (Figure 2). Specifically, the 35 proteins expressed anaerobically in fRF and dRF (unique and shared combined), were functionally associated with the osmotic adaptation pathway (OsmE), anaerobic respiration and oxidative stress pathway (YggE, MoaB, DmsB, FdoH), heat stress response (HchA), carbon starvation response (Slp), energy metabolism and biosynthetic pathways (glycolytic/gluconeogenesis pathway, amino acid biosynthesis: AldoC, Crr, AnsB, PykF, Eno, GpmA, GadpH, CysK, Ttc, AhpC, YhcB), chaperones (DnaK, GroEL, HchA), transport (LamB, ManX, FadL, RbsB), outer membrane proteins/porins/channel (OmpC, TolC, YdeN, Slp, OmpA), tellurite resistance (TerD), lysozyme inhibitor (Ivy), chemotaxis (GgbP), and motility (FliC) (Table 3; Additional file 1: Table S1).
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-14-48/MediaObjects/12866_2013_2209_Fig2_HTML.jpg
        Figure 2

        Distribution of 128 anaerobically expressed O157 proteins, identified using bottom-up proteomics, amongst the media tested. LB, Luria-Bertani broth; dRF, depleted and filtered rumen fluid; fRF, filtered rumen fluid.

        Table 3

        O157-proteome expressed under anaerobic conditions in dRF and fRF in Experiment I

        Protein/Function/Pathway; Name

        Accession Number

        Molecular Weight (kDa)1

        Number of Peptides (Relative Abundance)2

           

        dRF

        fRF

        2,3-bisphosphoglycerate-dependent phosphoglycerate mutase; GpmA

        gi|157155502

        29 kDa

        1 (6)

        1 (4)

        3-isopropylmalate dehydrogenase/Amino acid Biosynthesis; TtC 3

        gi|170684236

        40 kDa

        1 (6)

        1 (4)

        Alkyl hydroperoxide reductase protein C/Energy; AhpC

        gi|15800320

        21 kDa

        0

        1 (4)

        Anaerobic dimethyl sulfoxide reductase/Anaerobic growth/Oxidative Stress; DmsB

        gi|145756

        23 kDa

        1 (6)

        1 (4)

        Chain A, Crystal Structure Of The Long-Chain Fatty Acid Transporter/Transport; Fadl

        gi|203282230

        47 kDa

        1 (6)

        1 (4)

        Chain A, Crystal Structure Of Ggbp. Glucose-galactose binding protein/Chemotaxis, Transport; Ggbp 3

        gi|126030485

        33 kDa

        0

        1 (4)

        Chain A, Structure Of Ivy/ Lysozyme inhibitor; Ivy

        gi|29726212

        15 kDa

        1 (6)

        1 (4)

        Chaperone protein, stabilizes proteins under heat stress/Heat Stress Related; HchA

        gi|15802400

        31 kDa

        0

        1 (4)

        Chaperonin, type 1 protein/Protein folding/Transport; GroEL 3

        gi|15834378

        57 kDa

        3 (18)

        3 (11)

        Cysteine synthase/Amino acid transport and Metabolism; CysK

        gi|145686

        35 kDa

        1 (6)

        1 (4)

        Cytochrome d ubiquinol oxidase subunit III/Oxidative phosphorylation/Energy; YhCB 3

        gi|157148804

        15 kDa

        0

        1 (4)

        D-ribose transporter subunit B/Transport; RbsB

        gi|110644091

        31 kDa

        1 (6)

        0

        DNA-binding transcriptional activator/Osmotically-inducible lipoprotein E; OsmE

        gi|15802150

        12 kDa

        2 (18)

        2 (7)

        DNA-directed RNA polymerase subunit alpha/Transcription; RpoA

        gi|123444073

        37 kDa

        0

        1 (4)

        Flagellin/Flagellar assembly/Motility; FliC

        gi|15802358

        60 kDa

        3 (24)

        4 (19)

        Formate dehydrogenase-O, iron-sulfur subunit, energy metabolism/Anaerobic Respiration, Glyoxylate & Dicarboxylate Metabolism; FdoH

        gi|15804482

        33 kDa

        0

        1 (4)

        Fructose-bisphosphate aldolase/Glycolysis, Gluconeogenesis, Amino acid Biosynthesis; AldoC

        gi|161984958

        38 kDa

        3 (24)

        4 (19)

        Glucose-specific PTS system component, phosphorylation/Transport; Crr

        gi|15802950

        18 kDa

        0

        1 (4)

        Glyceraldehyde 3-Phosphate Dehydrogenase; GadpH 3

        gi|1421424

        35 kDa

        2 (12)

        1 (4)

        Hypothetical protein CKO_00658/ Uncharacterized; DedA 3

        gi|157144929

        21 kDa

        0

        1 (4)

        Hypothetical protein EcE24377A_0553/Glyoxylate Utilization; GlxB

        gi|157157046

        29 kDa

        1 (6)

        0

        Hypothetical protein ECP_2911/Oxidative Stress; YggE

        gi|110643066

        25 kDa

        0

        2 (7)

        L-asparaginase II, induced by anaerobiosis/Nitrogen and Amino acid Metabolism; AnsB

        gi|157157301

        37 kDa

        0

        4 (15)

        Maltoporin/Receptor for lambda phage/Transport; LamB 3

        gi|110644375

        50 kDa

        1 (6)

        1 (4)

        Molecular chaperone/Protein folding/Transport; DnaK

        gi|157159481

        69 kDa

        7 (42)

        4 (15)

        Molybdopterin biosynthesis protein B/Cofactor Biosynthesis/ Oxidative Stress; MoaB

        gi|15800533

        19 kDa

        0

        1 (4)

        Outer membrane channel protein, efflux of hydrophobic molecules/Transport; TolC

        gi|110643281

        54 kDa

        0

        2 (7)

        Outer membrane porin protein C/Tranport of small molecules/Osmotic; OmpC 3

        gi|15802768

        41 kDa

        1 (6)

        5 (22)

        Outer membrane protein II, porin, receptor, integrity/Membrane Stability; OmpA

        gi|146983

        26 kDa

        2 (12)

        3 (11)

        Outer membrane protein induced after carbon starvation, stationary phase, environmental stress/ Membrane stability; Slp

        gi|110807343

        27 kDa

        0

        1 (4)

        Phosphopyruvate hydratase: enolase/Glycolysis, Gluconeogenesis; Eno 3

        gi|15832893

        46 kDa

        0

        1 (4)

        PTS system, mannose-specific IIAB component/phosphotransferase/Transport; ManX

        gi|110641934

        35 kDa

        0

        1 (4)

        Putative sulfatase/Inorganic ion transport and metabolism/Transport; YdeN 3

        gi|110641672

        63 kDa

        0

        1 (4)

        Pyruvate kinase/Glycolysis, Gluconeogenesis, Amino acid Biosynthesis; PykF

        gi|110805653

        59 kDa

        0

        1 (4)

        Tellurium resistance protein/Stress related; TerD

        gi|135596

        20 kDa

        0

        1 (4)

        1KDa, Kilodalton.

        2Relative abundance based on normalized total spectral counts.

        3Proteins not identified in Experiment II (see Table 4).

        (ii) iTRAQ

        To more closely examine and quantify O157 protein expression in the bovine rumen, especially in the uRF, the anaerobic O157-proteome expressed in LB, dRF, fRF and uRF after 48 h incubation was compared using iTRAQ, in Experiment II. Data generated in two runs for each biological replicate was condensed to create a single comprehensive file per sample, and the files for the two biological replicate samples compared (Additional file 2: Table S2) to identify unambiguous proteins. Using the anaerobic O157-proteome expressed in LB as the reference, a total of 394 O157 proteins that were either differentially or similarly expressed in dRF, fRF, and uRF were identified (Figure 3, Additional file 2: Table S2). Of the cumulative 35 O157 proteins expressed anaerobically in dRF and fRF, and identified via Bottom-up proteomics, 10 were not identified using iTRAQ in the second experiment (Table 3). Overall, only 134 proteins were common to the results of the two experiments, indicative of incubation-time related differences in the number and type of proteins expressed. Differentially expressed O157 proteins in the iTRAQ dataset distributed as 298/394 in dRF (169, up-regulated, 129, down-regulated), 241/394 in fRF (162, up-regulated, 79, down-regulated) and 237/394 in uRF (155, up-regulated, 82, down-regulated) (Table 4). Interestingly, similar expression patterns were observed between O157 proteins expressed in dRF and uRF; 90% of dRF-differentially regulated and 71% dRF-no change proteins were similarly expressed in uRF. This may have been due to shared growth conditions (nutrient limitation)/signals in these two media. The competing microflora in uRF may have decreased nutrients in that media.
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-14-48/MediaObjects/12866_2013_2209_Fig3_HTML.jpg
        Figure 3

        Log fold changes in the expression of O157 proteins, identified using iTRAQ, in media tested under anaerobic conditions. The O157-proteome expressed in LB was the reference against which the regulation of O157 proteins in other media was determined. The scatter plots represent O157 proteins expressed in the context of the 155 up-regulated in uRF (Panel A), 82 down-regulated in uRF (Panel B) and 157 with no change in expression levels in uRF (Panel C). LB, Luria-Bertani broth; dRF, depleted and filtered rumen fluid; fRF, filtered rumen fluid; uRF, unfiltered rumen fluid.

        Table 4

        O157 proteins up-regulated under anaerobic conditions in uRF, in Experiment II

        Protein/Function/Pathway; Name

        Accession Number

        Molecular Weight ( kDa)1

        2,3-bisphosphoglycerate-dependent phosphoglycerate mutase/Glycolysis/Energy; GpmA

        GPMA_ECO57

        29 kDa

        3-oxoacyl-[acyl-carrier-protein] reductase/Fatty acid biosynthesis; FabG

        Q8X8I5_ECO57

        26 kDa

        3,4-dihydroxy-2-butanone 4-phosphate synthase, Riboflavin (Vitamin B2) biosynthesis/ Cofactor Bisoynthesis; RibB

        RIBB_ECO57

        23 kDa

        30S ribosomal protein S6/Protein Translation; RpsF

        RS6_ECO57

        15 kDa

        4-hydroxy-tetrahydrodipicolinate reductase, Leucine synthesis/Amino acid Biosynthesis; DapB

        DAPB_ECO57

        29 kDa

        50S ribosomal protein L10/Protein Translation; RplJ

        RL10_ECO57

        18 kDa

        50S ribosomal protein L18/Protein Translation; RplR

        RL18_ECO57

        13 kDa

        Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta/Fatty acid Biosynthesis; AccD

        ACCD_ECO57

        33 kDa

        Acriflavine resistance protein A/Multidrug efflux system/Transport; AcrA

        B5Z3X3_ECO5E

        44 kDa

        ADP-L-glycero-D-manno-heptose-6-epimerase/LPS core Biosynthesis/Heat induced; HldD

        HLDD_ECO57

        35 kDa

        Agmatinase/Polyamine Biosynthesis/Acid Resistance; SpeB

        SPEB_ECO57 (+1)

        34 kDa

        AidA-I adhesin-like protein/Adherence; AidA

        K0AT24_ECO1C

        141 kDa

        Aminomethyltransferase/Nitrogen, Amino Acid Metabolism; GcvT

        GCST_ECO57

        40 kDa

        Argininosuccinate lyase, Arginine synthesis/ Amino Acid Biosynthesis; ArgH

        ARLY_ECO57

        50 kDa

        AsmA protein/LPS Biogenesis; AsmA

        B5YUC4_ECO5E (+5)

        69 kDa

        Aspartate carbamoyltransferase regulatory chain/Nucleotide Biosynthesis; PyrI

        PYRI_ECO57

        17 kDa

        Aspartate--tRNA ligase/Aminoacyl-tRNA Biosynthesis; AspS

        SYD_ECO57 (+1)

        66 kDa

        Aspartate-semialdehyde dehydrogenase/Amino acid Biosynthesis; AsD

        DHAS_ECO57

        40 kDa

        ATP synthase subunit b/Energy Production; AtpF

        ATPF_ECO57

        17 kDa

        ATP-dependent helicase/DNA Replication, Repair; HrpB

        Q8X904_ECO57 (+1)

        91 kDa

        ATP-dependent protease subunit/Proteolysis; HslV

        HSLV_ECO57

        19 kDa

        Bacterioferritin/Iron storage and Transport; BfR

        Q8X890_ECO57

        18 kDa

        Bacteriophage replication gene A protein/Predicted Phage replication; ECH74115_3058

        B5YUH3_ECO5E

        86 kDa

        Beta-D-glucoside glucohydrolase, degradation of small carbon compunds/Biosyntheis of secondary metabolites; BglX

        Q8X668_ECO57

        83 kDa

        Bifunctional N-acetylglucosamin-1-phosphate-uridyl transferase/Cell Wall Synthesis; GlmU

        GLMU_ECO57 (+2)

        49 kDa

        Bifunctional purine biosynthesis protein/Purine Biosynthesis; PurH

        PUR9_ECO57

        57 kDa

        Biofilm regulator/Biofilms, Adherence; BssR

        BSSR_ECO57

        15 kDa

        Biosynthetic arginine decarboxylase/Polyamine Biosynthesis, Acid Resistance; SpeA

        SPEA_ECO57

        74 kDa

        Biotin carboxyl carrier protein of acetyl-CoA carboxylase/Fatty Acid Biosynthesis; AccB

        BCCP_ECO57 (+8)

        17 kDa

        Branched-chain-amino-acid aminotransferase/Amino acid Transport and Metabolism; IlvE

        ILVE_ECO57

        34 kDa

        Catabolite repression sensor kinase for PhoB alternative sensor for pho regulon/Global Regulaor; CreC

        Q8XB07_ECO57

        52 kDa

        Catalase-peroxidase 1/Prevent Cell, DNA damage/Oxidative Stress; KatG1

        KATG1_ECO57

        80 kDa

        Cell Division protein; ZapB

        ZAPB_ECO5E (+8)

        9 kDa

        Cellulose synthase subunit/Biofilms, Adherence; BcsC

        C8TYF1_ECO10 (+2)

        126 kDa

        Chromosome partition protein/Cell Division; MukB

        MUKB_ECO57 (+2)

        170 kDa

        Curli production assembly/transport subunit/Biofilms, Adherence; CsgG

        B5YVQ8_ECO5E (+1)

        31 kDa

        Cyclic pyranopterin monophosphate synthase accessory protein/Cofactor Biosynthesis; MoaC

        MOAC_ECO57

        17 kDa

        Cytidylate kinase/Nucleotide Biosynthesis; Cmk

        KCY_ECO57

        25 kDa

        Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase

          

        Complex/Energy Metabolism; SucB

        ODO2_ECO57

        44 kDa

        Dihydroorotase/Nucleotide Biosynthesis; PyrC

        PYRC_ECO57

        39 kDa

        DNA helicase/DNA Replication, Transcription; RecB

        Q8X6M9_ECO57

        134 kDa

        DNA invertase from prophage CP-933H/DNA Replication; PinH

        Q8X7L1_ECO57

        22 kDa

        DNA topoisomerase IV subunit B/DNA Replication; ParE

        Q8XBQ4_ECO57

        70 kDa

        DNA-directed RNA polymerase subunit alpha/DNA Transcription; RpoA

        RPOA_ECO57

        37 kDa

        DNA-directed RNA polymerase subunit beta/DNA Transcription; RpoB

        RPOB_ECO57

        151 kDa

        DNA-directed RNA polymerase subunit omega/DNA Transcription; RpoZ

        RPOZ_ECO57

        10 kDa

        Elongation factor P-like protein/Translation, Protein synthesis; YeiP

        EFPL_ECO57

        22 kDa

        Evolved beta-D-galactosidase alpha subunit/Degradation of small carbon compounds; EbgA

        Q8XAM9_ECO57

        119 kDa

        Ferritin-1/Iron Uptake and Storage; FtnA

        FTNA_ECO57

        19 kDa

        Fimbrial usher family protein/Chaperone/Transport; ECH74115_2118

        B5Z1W3_ECO5E (+5)

        81 kDa

        Formate dehydrogenase-O major subunit/Energy Metabolism, Anaerobic Respiration; FdoH

        Q7A9A6_ECO57 (+1)

        113 kDa

        Fructose-bisphosphate aldolase, class 1/Glycolysis, Gluconeogensis; FbaB

        B5YV44_ECO5E (+1)

        38 kDa

        Galactose-1-phosphate uridylyltransferase/Galactose Metabolism; GalT

        Q8X943_ECO57

        40 kDa

        Glucans biosynthesis protein G/Osmotic Adaptation; MdoG

        OPGG_ECO5E

        59 kDa

        Glutamate--tRNA ligase/Amino acyl tRNA synthesis; GltX

        SYE_ECO57

        54 kDa

        Glutamine synthetase/Amino acid Biosynthesis; GlnA

        GLNA_ECO57

        52 kDa

        Glutathione synthetase/Cofactors, carriers Biosynthesis; GshB

        GSHB_ECO57

        35 kDa

        Glycerol kinase/Glycerolipid Metabolism; GlpK

        GLPK_ECO57

        56 kDa

        Glycine dehydrogenase [decarboxylating]/Amino acid Metabolism; GcvP

        GCSP_ECO57

        104 kDa

        GMP synthase [glutamine-hydrolyzing]/Nucleotide Biosynthesis; GuaA

        GUAA_ECO57

        59 kDa

        GTPase-activating protein/Transcriptional Activator; YihI

        YIHI_ECO57

        19 kDa

        Guanylate kinase/Nucleotide Biosynthesis; GmK

        KGUA_ECO57 (+2)

        24 kDa

        Hemin import ATP-binding protein/Transport; HmuV

        HMUV_ECO57

        29 kDa

        Histidine--tRNA ligase/Amino acyl tRNA synthesis; HisS

        SYH_ECO57

        47 kDa

        HTH-type transcriptional regulator/Maltooligosaccharide Uptake and Metabolism; MalT

        MALT_ECO57

        103 kDa

        Hydrogenase-2 large chain/Energy Metabolism/Anaerobic Respiration; HybC

        MBHM_ECO57

        62 kDa

        Hydroxyacylglutathione hydrolase/Pyruvate Metabolism; GloB

        GLO2_ECO57

        28 kDa

        Inosine-5′-monophosphate dehydrogenase/Nucleotide Biosynthesis; GuaB

        B5Z0X7_ECO5E

        54 kDa

        Iron-sulfur cluster insertion protein/Growth using alternate electron acceptors; ErpA

        ERPA_ECO57

        12 kDa

        Long-chain fatty acid transport protein/Transport; FadL

        FADL_ECO57

        49 kDa

        Low-affinity putrescine importer/Transport; PlaP

        PLAP_ECO57

        50 kDa

        LPS-assembly lipoprotein/LPS Biogenesis; LptE

        LPTE_ECO57

        21 kDa

        Macrolide export ATP-binding/permease protein/Transport; MacB

        MACB_ECO57

        71 kDa

        Major outer membrane lipoprotein/Membrane Integrity; Lpp

        LPP_ECO57

        8 kDa

        Mannitol-1-phosphate 5-dehydrogenase/Fructose, Mannose Metabolism; MtlD

        MTLD_ECO57

        41 kDa

        Membrane, TerC family/CBS/transporter associated protein/Transport/Tellurium Resistance; TerC

        B5YUC1_ECO5E (+6)

        59 kDa

        Methyl-accepting chemotaxis protein III, ribose and galactose sensor receptor/Chemotaxis; Trg

        C8U899_ECO10

        59 kDa

        Minor curlin protein/Biofilms, Adherence; CsgB

        B5YVR3_ECO5E (+1)

        17 kDa

        Multiple Resistance and pH adaptation protein, antiporter/Transport; Mrp

        B5YV63_ECO5E (+1)

        40 kDa

        Multidrug resistance efflux protein/Transport; MdtC

        C8TU07_ECO26 (+2)

        111 kDa

        Multiphosphoryl transfer protein/Fructose, Mannose Metabolism; FruB

        PTFAH_ECO57

        40 kDa

        N-acetylglucosamine-6-phosphate deacetylase/LPS Biosynthesis; NagA

        NAGA_ECO57

        41 kDa

        NADH-quinone oxidoreductase subunit C/D/Electron transfer/Energy Metabolism; NuoC

        NUOCD_ECO57 (+2)

        69 kDa

        NifU-like protein/Energy Production, Conversion; NifU

        NIFU_ECO57

        14 kDa

        Non-LEE-encoded type III secreted effector; EspX7

        C6URC5_ECO5T (+2)

        73 kDa

        Oligopeptide ABC transporter, periplasmic oligopeptide-binding protein/Transport; OppA

        B5YYE8_ECO5E

        63 kDa

        Osmotically-inducible lipoprotein E/Global Regulator; OsmE

        OSME_ECO57

        12 kDa

        Outer membrane lipoprotein/Cell Envelope Biogenesis; SlyB

        SLYB_ECO57

        16 kDa

        Outer membrane protein, porin, receptor, integrity/Membrane Stability; OmpA

        B5YT87_ECO5E

        38 kDa

        Outer membrane protein assembly factor/Membrane Assembly, Antibiotic Resistance; BamA/YaeT

        BAMA_ECO57

        91 kDa

        Outer membrane protein assembly factor/Membrane Assembly, Antibiotic Resistance; BamD/YfiO

        BAMD_ECO57

        28 kDa

        Outer membrane protein, efflux protein/Transport; TolC

        B5YR81_ECO5E (+2)

        54 kDa

        Outer membrane protein W/Outer Membrane Biogenesis; OmpW/YciD

        Q8XCB6_ECO57

        23 kDa

        Oxidoreductase subunit/Oxidative Stress; DmsA_YnfF

        C6UV29_ECO5T (+6)

        90 kDa

        Penicillin-binding protein activator/Lipid binding/Putative Adhesin; LpoB/YcfM

        LPOB_ECO57

        23 kDa

        Peptidase B/Protein Metabolism; PepB

        PEPB_ECO57

        46 kDa

        Peptide chain release factor 1/Protein Translation; PrfA

        RF1_ECO57

        41 kDa

        Peptidyl-prolyl cis-trans isomerase A/Post-translational Modification; PpiA

        PPIA_ECO57

        20 kDa

        Peptidyl-prolyl cis-trans isomerase/Post-translational Modification; FklB

        B5Z2L4_ECO5E (+2)

        22 kDa

        Periplasmic binding protein for nickel/Amino acid Transport and Metabolism; NikA

        Q8X5U3_ECO57 (+1)

        59 kDa

        Phenylalanine--tRNA ligase alpha subunit/Amino acyl tRNA synthesis; PheS

        SYFA_ECO57 (+2)

        37 kDa

        Phenylalanine--tRNA ligase beta subunit/Amino acyl tRNA synthesis; PheT

        SYFB_ECO57

        87 kDa

        Poly (A) polymerase I/DNA Transcription; PcnB

        PCNB_ECO57 (+3)

        54 kDa

        Proline--tRNA ligase/Amino acyl tRNA synthesis; ProS

        SYP_ECO57

        64 kDa

        Protease IgA1, Serine protease/Protection; EspP

        K0AWD8_ECO1C

        146 kDa

        Protein elaB/Uncharacterized; ElaB

        ELAB_ECO57

        11 kDa

        Protein grpE, prevents aggregation of denatured proteins/ Heat and Hyperosmotic Shock-Related; GrpE

        C8U980_ECO10 (+2)

        22 kDa

        Protein translocase subunit/Transport; SecD

        SECD_ECO57

        67 kDa

        PTS system, mannose-specific transporter subunit IID/Transport; ManZ

        B5YQW0_ECO5E (+1)

        31 kDa

        Putative anaerobic dimethyl sulfoxide reductase chain A; DmsA_YnfE

        Q7ABM3_ECO57 (+1)

        88 kDa

        Putative DNA replication factor encoded within cryptic prophage CP-933P/Hypothetical; Z6069

        Q8XAD9_ECO57

        28 kDa

        Putative endopeptidase of prophage CP-933X/Hypothetical; Z1877

        Q8X704_ECO57

        12 kDa

        Putative carboxypeptidase/Hypothetical; YagX

        Q8X6I4_ECO57 (+1)

        91 kDa

        Putative lipoprotein induced during stationary phase/Stress Response; YbjP

        Q8X6N7_ECO57 (+1)

        19 kDa

        Putative homeobox protein/Regulator; YbgS

        Q8X948_ECO57

        13 kDa

        Putative lipoprotein/Membrane protein; LppC

        B5YUN1_ECO5E

        20 kDa

        Putative membrane protein, peptidase/Uncharacterized; YibP

        Q8XDE2_ECO57

        47 kDa

        Putative multimodular enzyme/Energy Metabolism; Z3719

        Q8XBF4_ECO57

        82 kDa

        Putative pectinesterase, localizes to cellular poles/Membrane protein; YbhC

        Q8X891_ECO57

        46 kDa

        Putative replicase/DNA Replication; Z5187

        Q8XBZ7_ECO57

        37 kDa

        Pyridoxine 5′-phosphate synthase/Vitamin B6 (Pyridoxine) synthesis; PdxJ

        PDXJ_ECO57

        26 kDa

        Pyruvate dehydrogenase (Dihydrolipoyltransacetylase component)/Energy Metabolism; AceF

        Q8X966_ECO57

        66 kDa

        Pyruvate oxidase/Degradation of small carbon compunds; PoxB

        Q8X6L4_ECO57

        62 kDa

        RNase E/RNA Degradation; RnE

        Q8X8J5_ECO57

        118 kDa

        Serine protease/Protection; DegP

        B5Z0E1_ECO5E (+1)

        49 kDa

        Serine endoprotease/Protection; DegQ

        Q8X9F1_ECO57

        47 kDa

        Single-stranded DNA-binding protein/DNA Replication; SsB

        SSB_ECO57

        19 kDa

        Soluble cytochrome, electron transport/Energy Metabolism; CybC

        C562_ECO57 (+4)

        14 kDa

        Spermidine/putrescine import ATP-binding protein/Transport; PotA

        POTA_ECO57

        43 kDa

        Stringent starvation protein A, stationary phase induced acid tolerance/Global Regulator; SspA

        SSPA_ECO57

        24 kDa

        Stringent starvation protein B, ClpXP protease specificity enhancer/Global Regulator; SspB

        SSPB_ECO57

        18 kDa

        Succinate dehydrogenase flavoprotein subunit/Energy Metabolism; SdhA

        DHSA_ECO57

        64 kDa

        Tat-linked quality control protein/DNAse activity; TatD

        Q8X8J6_ECO57

        30 kDa

        Thiosulfate sulfurtransferase/Anaerobic Respiration, Energy Metabolism; GlpE

        C8TJL4_ECO26 (+2)

        12 kDa

        Threonine deaminase (Dehydratase)/Amino acid Biosynthesis; IlvA

        Q8X467_ECO57

        56 kDa

        Transcription termination/antitermination protein/Modulates DNA Transcription; NusG

        NUSG_ECO57

        21 kDa

        Transcriptional regulatory protein/Envelope Stress Response Protein/Downregulates LEE; CpxR

        CPXR_ECO57

        26 kDa

        Transketolase 1, thiamin-binding/Non-oxidative Metabloism; TktA

        C8TGU9_ECO26

        72 kDa

        Transketolase 2 isozyme, stationary phase induced/Non-oxidative Metabolism; TktB

        Q8XBF1_ECO57 (+6)

        73 kDa

        Translation initiation factor IF-2/Protein Translation; InfB

        IF2_ECO57

        97 kDa

        Trigger factor/Cell division; TiG

        TIG_ECO57 (+1)

        48 kDa

        Tryptophanase/Tryptophan Metabolism, Indole Production; TnaA

        TNAA_ECO57

        53 kDa

        Type II secretion pathway related protein/Transport; EtpE

        O82884_ECO57

        56 kDa

        Tyrosine-protein kinase/Downregulates colanic acid production; WzC

        WZC_ECO57

        79 kDa

        Uncharacterized protein/Hypothetical; ECs1547

        Q8X3G9_ECO57 (+1)

        17 kDa

        Uncharacterized protein/Hypothetical; ECs2891

        Q8X7H8_ECO57

        14 kDa

        Uncharacterized protein/Hypothetical; ECs2991

        Q8X2Z1_ECO57

        10 kDa

        Putative oxidative stress defense protein/ Oxidative Stress; YggE

        YGGE_ECO57

        27 kDa

        Uncharacterized protein/Membrane Protein; YqjD

        YQJD_ECO57

        11 kDa

        UPF0042 nucleotide-binding protein/putative ATPase; YhbJ

        YHBJ_ECO57

        32 kDa

        UPF0092 membrane protein, translocase/Transport; YajC

        YAJC_ECO57

        12 kDa

        UPF0337 protein, putative stress response protein/Osmotic Shock; YjbJ

        YJBJ_ECO57

        8 kDa

        UPF0352 protein/Uncharacterized; YejL

        YEJL_ECO57

        8 kDa

        Uridylate kinase/Nucleotide Interconversion; PyrH

        PYRH_ECO57

        26 kDa

        Valine--tRNA ligase/Amino acyl tRNA Biosynthesis; ValS

        SYV_ECO57

        108 kDa

        1kDa, Kilodalton.

        As observed with the Bottom-up proteomics results, none of the well-established O157 virulence factors were identified in either media after 48 h of anaerobic incubation (Additional file 2: Table S2). Specifically, the 155 up-regulated (Table 4), uRF-O157 proteins could be functionally associated with osmotic adaptation (MdoG, CreC, OsmE, YjbJ), oxidative stress pathway (KatG, DmsA_ynfE, DmsA_ynfF, YggE), heat shock response (HdlD, GrpE), carbon starvation response (SspA, SspB), anaerobic respiration (HybC, ErpA, GlpE), pH adaptation/acid resistance (SpeA, SpeB, Mrp), energy metabolism: degradation of carbon compounds (GalT, BglX, EbgA, MtlD), glycolysis/ gluconeogenesis (GpmA, SucB, FdhO, FbaB, GloB, NuoC, AceF, PoxB, SdhA), amino acid metabolism (GcvT, GcvP, HslV, IlvE, GlnA, TnaA), nitrogen and glycerolipid metabolism (GlpK), DNA degradation (RecB), biosynthetic pathways: fatty acid (FabG, AccD, AccB), amino acids (DapB, ArgH, AsD, IlvA), nucleotides (PyrI, PyrC, PurH, GlmU, CmK, GuaA, GuaB, GmK,), cellulose (BcsC), cofactors/carriers (MoaC, GshB), vitamins (RibB, PdxJ), chaperones (fimbrial usher protein,), transport (HmuV, FadL, PlaP, MacB, OppA, NikA, SecD, ManZ, PotA, YajC, EtpE), storage (BfR, FtnA), multi-drug efflux systems (AcrA, MdtC), tellurite resistance (TerC), serine proteases (DegP, DegQ, EspP), outer membrane proteins/porins/channel (AsmA, LptE, Lpp, NagA, SlyB, OmpA, BamA, BamD, TolC, OmpW, ElaB, YbjP, LppC, YqjD), chemotaxis (Trg), adherence (AidA-like, BssR, CsgG, CsgB, LpoB/YcfM, EspP), and cell division/DNA replication (HrpB, ZapB, MukB, ParE, Ssb, Tig) (Table 4; Additional file 2: Table S2).

        Discussion

        This study provides a snapshot of various proteins expressed by O157 in unfiltered, rumen fluid through a comparative analysis of the O157 proteomic-profile in different media, growth conditions and incubation times. Interestingly, none of the reported (LEE, Shiga toxins) O157 virulence proteins were detected, under all conditions, in any media tested. Overall, fewer O157 proteins were detected in more nutritionally complex RF-preparations versus LB and among these, differences were observed based on availability of oxygen, nutrients and incubation time. Also, the O157-proteome in the RF-preparations included more proteins with diverse functions at 48 h than after 14 days of incubation. In fact, proteins associated with adherence, cell division and growth were identified only at 48 h. However, under all conditions, a selective expression of proteins with a role in cell structure, transport, metabolism, chemotaxis, motility, resistance, stress and regulation was observed in RF-preparations , many of which were up-regulated in the unfiltered rumen fluid. The O157 growth patterns and proteome expressed in the rumen fluid is suggestive of an adapting O157, expending minimal energy, preparing for survival and downstream intestinal colonization.

        Since adult cattle are often fed a maintenance diet with less protein until ready for feedlots, we decided to analyze O157 growth dynamics in rumen fluid derived from animals on this diet. Rumen fluid from cattle fed a diet low in protein usually has a pH ranging from 6.2-6.8, and VFA concentrations at, 60-70% acetic acid, 15-20% propionic acid, 5-15% butyric acid [2831]. The rumen fluid VFA and pH values were within the limits described for this diet for both animals used in this study (Tables 1 and 2; 26–29). Irrespective of incubation times (14 days versus 48 h), O157 exhibited very distinctive growth patterns in RF-preparations compared to LB. O157 cultures in dRF, fRF and uRF were consistently at lower optical densities than LB, under both aerobic and anaerobic conditions. The anaerobic RF-preparation cultures never reached an OD600 ≅ 1.0 and the viable O157 recovered were at substantially lower counts when compared to LB. The low OD readings and viable counts recovered from RF-preparation grown cultures may have been due to inhibitory factors and /or limited nutrients in dRF, fRF, uRF, not seen in LB, having a bacteriostatic (aerobic) or bactericidal (anaerobic) effect on O157 and reflective of O157 growth in a stressful environment [11, 3236]. Using LB media for estimating viable counts may have helped recover the stressed bacteria [35]. Similar recovery of viable bacteria despite low OD reading has been reported among bacteria exposed to antimicrobial stress [36], and limited growth has been associated with bacteria entering into a stressed/starved state or stationary phase [3537].

        Overall, fewer O157 proteins were detected in RF-preparation cultures compared to LB, especially under anaerobic conditions. Irrespective of the media used to culture O157, its anaerobic proteome was functionally associated only with cell structure, transport, metabolism, chemotaxis, motility, resistance, stress-related and regulation, and not O157 virulence. Previous reports have demonstrated that O157 virulence genes, especially the Shiga toxin and LEE–encoded genes, are down-regulated in LB compared to minimal media [3840]. In addition, presence of trace amounts of glucose has also been shown to down-regulate LEE expression due to catabolite repression and/or acidic pH [3840]. Hence, the lack of virulence gene expression in LB in this study conforms to those findings. Experiments with acid-stressed, starved bacteria have shown that these are likely to be more virulent only on recovery, and over time [35]. Even in minimal media that usually supports O157 virulence gene expression, several of these are suppressed as cultures reach the stationary phase [41]. Butyrate, a key environmental cue in LEE gene expression was limited in the RF used in this study, which may have also caused the LEE suppression [9]. Conditioned media from unrelated cultures have been shown to suppress Shiga toxin gene expression while maintaining O157 growth or suppressing growth itself [33, 35, 42]. In fact, experimental studies have shown that it is easier to displace O157 in unfiltered rumen fluid versus autoclaved rumen fluid, by addition of “nonfermentable” sugars in the presence of the ruminal microflora [11]. Thus, the absence of O157 virulence gene expression in RF-preparations may be reflective of the stressful growth environment, suppression due to nutrient limitations, lack of inducers, oxygen deprivation, pH fluctuations and inhibitory metabolites released by resident microbiota.

        Previous studies have suggested development of acid resistance by Shiga-toxin producing E. coli (STEC) in the rumen as a means for better STEC survival through the ‘stomach-like’ acidic bovine abomasum [43, 44] and have prescribed a role for glutamate-dependent acid resistance system (Gad system) and the tryptophanase (tnaA) enzyme toward this end [45]. Hughes et al., recently demonstrated that O157 LEE expression is down-regulated while the Gad system is up-regulated in the rumen of cattle [46]. This observation made in animals being fed a grain diet, having a ruminal pH of 5.93, derived a role for the SdiA gene in sensing the acylhomoserine lactone (AHL) signals in the rumen fluid and affecting differential expression of these genes. AHLs formed by ruminal resident flora, are effective only under highly acidic pH and hydrolyze at neutral-alkaline pH [46, 47]. Similarly, the Gad system that relies on the decarboxylation (gadA/B) of glutamate via proton consumption to increase cytoplasmic alkalinity is active at pH 4–4.6 [48]. However, other degradative amino acid decarboxylase and acid-resistance systems are activated in response to low pH (5.2 to 6.9), fermentative-anaerobic growth and stationary phase growth [48, 49] and used more often than the Gad system to counter the deleterious effects of protons. We observed one such system, the arginine-dependent acid resistance system (Arg system) to be up-regulated in the RF-preparations after 48 h of anaerobic growth. Since the pH of the RF-preparations used in this study did not reach extreme acidic levels, the Gad system may not have been induced. In the Arg system, decarboxylation (speA) of arginine via proton consumption resulting in the formation of agmatine stabilizes the cytoplasmic pH. Agmatine is either exported via the arginine-agmatine antiporter (aidC) or converted (speB) to putresceine as part of the polyamine biosynthetic pathway.

        Considering that O157 is exposed to heat-shock, starvation and stationary-phase-like growth in the rumen, it is possible that these factors enhance acid-tolerance in the bacteria through other mechanisms such as outer membrane changes and synthesis of proton transport-related protective proteins, as well [49, 50]. Several stress (acid, low oxygen, osmolites, stationary phase)-responsive genes were expressed by O157 in this study, and included genes associated with the metabolism of arginine (speA, speB), lysine (lysU), formate (hyC), tryptophan (tnaA) and maltoporin (lamB), catalase (katG), DNA polymerase-1 (polA) and AidA-1 adhesin-like protein (aidA) [4951]. Flagellar genes are differentially expressed under varying acid-stress conditions [5153], and in our study, these genes were up-regulated in dRF and fRF but not uRF, suggesting less pH variation in the course of growth in uRF and limiting the role of flagella to motility alone. Stressed bacteria have been shown to be more adherent [35, 40, 53]; proteins associated with adherence (AidA-1 adhesin-like) and biofilm formation (BssR, CsgG, CsgB) were identified after 48 h incubation and not after longer incubation periods. Interestingly, several ‘resistance’ related proteins were up-regulated in RF-preparations, a subset of which (tellurite resistance, serine protease) have also been shown to contribute towards O157 adherence [54, 55]. This suggests that adherence may be critical during the initial phase of O157 colonization and although LEE is suppressed, the bacteria rely on other mechanisms to adhere or form biofilms in the rumen. It has been observed that bacteria and protozoa in the rumen tend to adhere to the fibrous mat layers comprising of plant material to remain in the rumen and assist in the digestion of insoluble feed materials [56]. While this may not be in the case of O157, initial adherence to or biofilm formation on available surfaces may give the bacteria time to adapt and survive the rumen environment [34]. It appears that much of the adaptive changes are initiated early in colonization as reflected in more stress-induced, structural integrity-related outer membrane proteins (AsmA, LptE, Lpp, NagA, SlyB, OmpA, BamA, BamD, TolC, OmpW, ElaB, YbjP, LppC, YqjD), and cell division and growth proteins, being expressed at 48 h. This supports the observation that O157 is maintaining slow growth in the RF-preparations as well.

        Conclusion

        Bottom-up proteomics provided a broad picture of differences in O157 protein expression after extended incubation in various media tested. Quantitative proteomics (iTRAQ)-based analysis of the O157 anaerobic proteome expressed in uRF with all normal rumen flora was performed to more closely determine O157 protein expression in the bovine rumen. The cumulative results of all RF-preparation analysis suggested that rumen specific protein expression enables O157 to adapt to this hostile environment and successfully transit to its colonization sites in the bovine GIT. To further verify our conclusions, we are evaluating the O157 proteomic-profile as expressed in vivo in a rumen-fistulated cow, and confirming the role of a subset of these ‘adaptive’ proteins in O157 survival.

        Declarations

        Acknowledgements

        Technical support provided by Bryan Wheeler, Deb Hinrichsen (NVSL) and Laurie Evans (NVSL) in collection & filtration of rumen fluid; Deb Lebo and Sam Humphrey in analyzing VFAs; Duane Zimmerman for assisting with iTRAQ labeling and Paul Amundson’s group of animal caretakers for assisting in rumen fluid collection is acknowledged with appreciation. Bottom-up proteomics was done at the Proteomics Division, ICBR, University of Florida, Gainesville, FL. We thank Dr. Manohar John, Dr. Thomas Casey and Dr. John Bannantine for their insightful review of this manuscript.

        Disclaimer

        Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

        Authors’ Affiliations

        (1)
        Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture
        (2)
        Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture

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