Role of the ArcAB two-component system in the resistance of Escherichia colito reactive oxygen stress
© Loui et al; licensee BioMed Central Ltd. 2009
Received: 26 March 2009
Accepted: 28 August 2009
Published: 28 August 2009
The global regulatory system ArcAB controls the anaerobic growth of E. coli, however, its role in aerobic conditions is not well characterized. We have previously reported that ArcA was necessary for Salmonella to resist reactive oxygen species (ROS) in aerobic conditions.
To investigate the mechanism of ROS resistance mediated by ArcAB, we generated deletion mutants of ArcA and ArcB in E. coli. Our results demonstrated that both ArcA and ArcB were necessary for resistance to hydrogen peroxide (H2O2), a type of ROS, and their function in this resistance was independent from H2O2 scavenge. Mutagenesis analysis of ArcA indicated that ROS resistance was mediated through a distinct signaling pathway from that used in anaerobic conditions. An abundant protein flagellin was elevated at both the protein and mRNA levels in the ΔarcA mutant as compared to the wild type E. coli, and deletion of flagellin restored the resistance of the ΔarcA mutant to H2O2. The resistance of the ΔarcA mutant E. coli to H2O2 can also be restored by amino acid supplementation, suggesting that a deficiency in amino acid and/or protein synthesis in the mutant contributed to its susceptibility to H2O2, which is consistent with the notion that protein synthesis is necessary for ROS resistance.
Our results suggest that in addition to its role as a global regulator for anaerobic growth of bacteria, ArcAB system is also important for bacterial resistance to ROS in aerobic conditions, possibly through its influence on bacterial metabolism, especially amino acid and/or protein assimilation and synthesis.
Aerobic bacteria use oxygen as a terminal electron acceptor in oxygen-containing environments for their metabolism. Although aerobic growth has its obvious advantages (e. g. high energy efficiency, abundance of oxygen in the atmosphere, etc), bacteria must deal with the undesired consequences from exposure to oxygen and oxidative environments. Oxygen and its derivatives, such as superoxide and hydrogen peroxide, are often highly reactive and pose a threat to many macromolecules, such as enzymes with iron-sulfur centers, nucleic acids, and lipids. Therefore, bacteria undergoing aerobic growth must be able to sense, respond to, and detoxify reactive oxygen species (ROS), and maintain their structural and functional integrities.
The principle mechanism through which bacteria respond to environmental signals is through two-component and other regulatory systems [1, 2]. At least four global regulatory systems -OxyRS, SoxRS, Fnr and ArcAB – are identified to respond to oxygen and its derivatives [3, 4]. OxyRS and SoxRS systems control the response of bacteria to hydrogen peroxide and superoxide, respectively [3–12]. Fnr (fumarate and nitrate reduction) controls the transition from aerobic growth to anaerobic growth [13–17]. Fnr is believed to directly sense oxygen [18–20] and regulate at least 100 operons [21–23]. In addition to Fnr, the two-component regulatory system ArcAB also regulates the transition of bacteria from aerobic to anaerobic growth and is active under microaerobic conditions. It controls at least 100 operons that are involved in the TCA cycle and energy metabolism [16, 24–29]. The sensor kinase ArcB undergoes auto-phosphorylation at His292 under anaerobic conditions, and this activation is negatively regulated by the oxidized quinones under aerobic conditions . Activated ArcB undergoes a phosphorelay of His292 to Asp576 to His717, and subsequently activates its cognate transcriptional regulator ArcA by phosphorylating ArcA at Asp54 to repress genes contributing to aerobic metabolism (e.g. citrate synthase and isocitrate lyase) and activates genes necessary for anaerobic metabolism (e.g. pyruvate formate lyase and hydrogenase) [23, 25, 30–34].
Although the function of the ArcAB system in the anaerobic growth of E. coli has been well characterized, its function is unlikely to be limited to those required for the anaerobic growth of bacteria. For example, the ArcAB system has been reported to be involved in chromosomal replication, stress responses and aging of bacteria [35–37]. We have previously reported that ArcA of Salmonella enterica is necessary for its resistance to reactive oxygen and nitrogen species (ROS and RNS) . More recently, ArcA is implicated in the ROS stress response of Haemophilus influenzae . In this report, we analyzed the role of ArcAB in reactive oxygen resistance of E. coli and investigated the mechanism of ROS resistance mediated by the ArcAB two-component system.
ArcAB system is necessary for E. coli to resist hydrogen peroxide (H2O2)
Bacterial strains, plasmids and oligonucleotides used for mutagenesis.
Bacterial strains and plasmids
Source or reference
E. coli strains
Dr. Sydney Kustu, University of California
ΔarcA::kan derivative of K12
ΔarcB::cm derivative of K12
derivative of K12 in which Kanr was inserted adjacent to arcB while maintaining the function of arcB
kan derivative of ΔarcB with arcB::cm replaced by wild type arcB
fliC non-polar deletion mutant of K12
ΔarcA::kan/ΔfliC derivative of K12
Apr, low to medium copy number plasmid
derivative of pRB3-273C containing arcA
derivative of pRB3-arcA containing Asp54 → Ala mutation
mutagenesis of arcA
mutagenesis of arcA
mutagenesis of arcB
mutagenesis of arcB
generation of arcB::kan
generation of arcB::kan
mutagenesis of fliC
mutagenesis of fliC
We assayed the resistance of the ΔarcA mutant E. coli to hydrogen peroxide (H2O2). Overnight culture of the ΔarcA mutant E. coli was exposed to H2O2, and its survival was compared to that of the wild type E. coli. The ΔarcA mutant E. coli was more susceptible than the wild type E. coli (Figure 1A). Plasmid pRB3-arcA, which carries a wild type allele of arcA in plasmid pRB3-273C [38, 40], complemented the survival defects in H2O2. This indicates that the susceptible phenotype of the ΔarcA mutant E. coli was likely due to the deletion of the arcA allele (Figure 1A). Assays performed with log-phase culture of the ΔarcA mutant E. coli yielded similar results (data not shown). Similar results were obtained with LB broth and M9 minimal medium, results obtained with LB broth are shown (Figure 1).
The same analysis was carried out for ArcB, the cognate sensor-kinase of the ArcAB system. The ΔarcB mutant E. coli survived less than the wild type parental strain (Figure 1C). We had attempted to clone a wild type allele of arcB into plasmid pRB3-273C to complement the ΔarcB mutant E. coli. However, the cloning efficiency was unusually low as compared to similar cloning attempts we had conducted with the plasmid vector. Of a total of 7 recombinant plasmids we eventually obtained from several transformations, 5 contained mutations at the start codon of arcB and the remaining 2 had mutations that produced truncations early in the ORF (data not shown). This indicates that an over-expression of arcB from a plasmid is probably toxic to E. coli. As an alternative, we constructed a revertant of the ΔarcB mutant E. coli, in which a wild type arcB allele replaced the deleted arcB allele (see Materials and Methods). The revertant mutant of ΔarcB was shown to have the same resistance to H2O2 as the wild type E. coli (Figure 1C).
The ArcAB system is dispensable for H2O2scavenge
Phosphorylation at Asp54 is dispensable for H2O2resistance mediated by ArcA
Response of flagellin, OppA and GltI to H2O2 is altered in the ΔarcA mutant E. coli
Relative levels of differentially regulated proteins in the wild type and ΔarcA mutant of E. coli K12.
37.9 ± 16.7†
188.9 ± 29.8†
139.9 ± 57.8§
2555.5 ± 1343.1†
892.0 ± 555.8†
440.3 ± 202.2
717.5 ± 390.5†
205.2 ± 127.3
183.1 ± 67.9
Flagellin is the only one among the 10 most abundant proteins that responded to H2O2 treatment. In the wild type, un-treated E. coli flagellin was detected at a lower level than in the ΔarcA mutant E. coli, and H2O2 treatment further decreased the flagellin level (p < 0.05, Student's t-test, Table 2 and Figure 4). In the ΔarcA mutant E. coli H2O2 treatment also decreased flagellin level, however, the decrease was not statistically significant (Table 2). Therefore, compared to the wild type the E. coli, ΔarcA mutant displayed higher flagellin levels both constitutively and following H2O2 treatment, and its flagellin level did not respond to H2O2 treatment as that in the wild type E. coli.
The response of OppA and GltI expression was different from that of flagellin. In the untreated bacteria levels of both GltI and OppA appeared to be higher in the ΔarcA mutant than in the wild type E. coli (p < 0.05, Student's t-test for GltI, Table 2). Following H2O2 treatment the levels of OppA and GltI in the wild type E. coli became higher (p < 0.05, Student's t-test), while neither protein displayed a statistically significant change in the ΔarcA mutant E. coli (Table 2). This results in a lower GltI and OppA level in the H2O2 treated ΔarcA mutant than the wild type E. coli.
Flagellin messenger RNA is over-expressed in the ΔarcA mutant E. coli
Deletion of flagellin increased the survival of the ΔarcA mutant E. coli
In addition to flagellin, we have also attempted to delete other abundant proteins to determine if such deletions would improve the survival of the arcA mutant E. coli. Our efforts were not successful, however, because most abundant proteins such as elongation factors, 30 s ribosomal proteins, and chaperone proteins are either essential or important for E. coli, and such deletions would be detrimental to E. coli. We successfully deleted D-ribose periplasmic binding protein (RbsB) encoded by rbsB, a protein which is as abundant as or more abundant than flagellin. The ΔrbsB mutant itself was found to be susceptible to H2O2, therefore could not be used to test the effect of RbsB on the H2O2 resistance of the arcA mutant E. coli (data not shown).
Amino acid supplementation improved the survival of the ΔarcA mutant E. coli under H2O2stress
Antibiotic that inhibits protein synthesis increased susceptibility of E. coli to H2O2
Similar assays were carried out with the ΔarcA mutant E. coli and the results were consistent with those of the wild type E. coli. While incubation with H2O2 alone reduced the concentration of the ΔarcA mutant E. coli by over 5log10 after 6 hours of incubation (Figure 8, right half, diagonally-hatched bar), the addition of chloramphenicol to the assay eliminated all E. coli (Figure 8). The synergistic effect of the bactericidal activity of H2O2 and chloramphenicol on the ΔarcA mutant E. coli is not because it is more susceptible to chloramphenicol (Figure 8, vertically-hatched bars). Similarly to that observed with wild type E. coli, ampicillin reduced the bactericidal activity of H2O2, and the ΔarcA mutant E. coli survived better in the presence of both ampicillin and H2O2 than H2O2 alone (1.7 × 105 CFU/ml vs. 1.0 × 102 CFU/ml) (Figure 8).
Although the ArcAB system has been extensively investigated for its role as the global control system of E. coli in anaerobic growth, its role, if any, in aerobic growth is much less understood. We have previously reported that ArcA is necessary for the pathogenic bacterium Salmonella enterica to resist reactive oxygen and nitrogen species under aerobic conditions . In this report, we used E. coli as our model to further explore the role of both ArcA and ArcB in ROS resistance, and to investigate the mechanism of ROS resistance mediated by the ArcAB two-component system. Here we demonstrate that deletion mutants of ArcA and ArcB were more susceptible to H2O2, suggesting that both ArcA and ArcB were necessary for E. coli to resist the stress caused by H2O2 (Figure 1), and that their functions were not limited to anaerobic growth of bacteria. Interestingly, we have not detected any growth defects of ΔarcA or ΔarcB mutant E. coli under anaerobic conditions (data not shown) and to our knowledge no such defect has been reported in the literature. In addition, an ΔarcA mutant of Salmonella enterica grew normally in anaerobic medium . This further indicates that ArcAB has wider roles in the physiology and metabolism of enteric bacteria besides its well-characterized regulation of anaerobic growth of bacteria.
The signaling pathway of the ArcAB system under anaerobic conditions has been extensively characterized [25–28, 30–34, 42, 44]. The membrane-bound sensor-kinase ArcB is activated by reduced quinones under anaerobic conditions, and subsequently activates its cognate transcriptional regulator ArcA by phosphorylating ArcA at Asp54 [30, 42, 25]. Matsushika and Mizuno previously reported that ArcB can also phosphorylate ArcA directly through His292 under aerobic conditions , however, its physiological relevance to E. coli has not been reported. Our results on the role of ArcAB in ROS resistance suggest that ArcAB can be activated by novel signals other than reduced quinones and anaerobic conditions, and the activation is independent of phosphorylation at Asp54 of ArcA as demonstrated under anaerobic conditions [41, 42, 46], since phosphorylation-defective ArcA expressed from a plasmid fully complemented an ΔarcA mutant E. coli for its susceptibility to H2O2 (Figure 3). We would like to point out that our analysis was conducted using a phosphorylation-mutant ArcA (Asp54 → Ala) expressed from a plasmid. It is yet to be determined if a mutant carrying a corresponding mutation of arcA in the chromosome is susceptible to H2O2. (Our attempts to generate a mutant arcA encoding an Asp54 → Ala mutation in the chromosome were unsuccessful due to technical difficulties. Similar to what we observed for arcB, plasmids carrying arcA were prone to mutations during cloning.) We have also noticed that the wild type ArcA expressed from a plasmid confers a stronger H2O2 resistance phenotype than the phosphorylation-defective ArcA. The ΔarcA mutant E. coli complemented in trans with a wild type arcA allele demonstrated higher H2O2 resistance than the wild type E. coli (Figure 1 and 3), while the same mutant E. coli complemented with a phosphorylation-defective arcA allele has the same H2O2 resistance as the wild type E. coli (Figure 3).
In addition to novel signals and signaling pathways that may mediate the function of the ArcAB system in the ROS resistance, the ArcAB system may also regulate a distinct set of genes under aerobic conditions. Under anaerobic conditions ArcA mostly negatively regulates genes involved in the TCA cycle and electron transport [26–28]. Under aerobic conditions, a microarray study by Oshima et al. demonstrated that expression of a large number of genes in the ΔarcA or ΔarcB mutant E. coli was altered . Our results suggest that levels of at least three proteins (flagellin, GltI and OppA) were altered in the ΔarcA mutant E. coli both constitutively and in response to H2O2 treatment (Figure 4 and Table 2). Our further analysis on the messenger RNA level of fliC indicates that the RNA levels are higher in the ΔarcA mutant E. coli and corresponded to the protein levels, suggesting that the regulation is likely on the transcriptional or post-transcriptional level (Figure 5). Oshima et al. did not detect a significant alteration in the expression of fliC in their microarray analysis, although flagellar synthesis was identified as a system that was affected in the ΔarcA mutant but not the ΔarcB mutant E. coli . The discrepancy is possibly due to the differences in experimental conditions (shaking bacterial cultures at 120 rpm vs. 225 rpm) and detection methods (microarray vs. Real-Time Reverse Transcriptase PCR and 2-D gel electrophoresis). Since we detected an elevation of both mRNA and protein levels of flagellin in the ΔarcA mutant E. coli (Figures 4 and 5), we believe that our observation is valid. The regulation of ArcA on flagellin is likely to be indirect, as we did not detect specific binding of recombinant ArcA protein to the upstream sequence of fliC (data not shown).
Given that the ArcAB system regulates a large number of genes in E. coli, its role in the ROS resistance is likely to be complex. We have demonstrated that mutation of ArcA or ArcB did not alter the H2O2 scavenging ability of E. coli (Figure 2), however, the precise molecular mechanism on how ArcA regulates ROS resistance in E. coli is yet to be elucidated. ArcA was reported to be necessary for the ROS resistance of Haemophilus influenzae due to its regulation of Dps, a ferritin-like small protein that was previously reported to be involved in ROS resistance of Salmonella [39, 47]. The mechanism of the ROS resistance mediated by ArcA is likely to be different in E. coli, since dps is expressed close to the wild type level in the ΔarcA or ΔarcB mutant (84% and 99% respectively), and our preliminary microarray analysis with Salmonella ΔarcA mutant indicated that dps responded normally to H2O2 in the ΔarcA mutant (unpublished results). One possible clue on the mechanism of how ArcAB contributes to the ROS resistance of E. coli came from our proteomic analysis that showed altered expression of flagellin, GltI and OppA between the wild type and ΔarcA mutant E. coli (Table 2). The constitutive GltI and OppA levels are higher in the ΔarcA mutant than in the wild type E. coli, suggesting that the mutant may have a higher need for amino acid transport. In contrast to the GltI and OppA levels in the wild type E. coli that increased 6- and 24-fold respectively in response to H2O2 exposure (possibly due to a higher need for amino acid transport under ROS stress), the level of neither protein in the ΔarcA mutant increased under the same condition (Table 2). A higher level of flagellin in the ΔarcA mutant likely put further constraint on the protein synthesis, and as a result the ΔarcA mutant E. coli might have become less fit under H2O2 stress. Our genetic study demonstrating that deletion of fliC "rescued" the survival defect of the ΔarcA mutant E. coli under H2O2 stress (Figure 6) supports the hypothesis.
ROS stress conditions induce growth arrest in E. coli. Chang et al. has reported that in growth arrest induced by either glucose-lactose diauxie, entry into stationary phase, or H2O2 treatment, genes involved in amino acid biosynthesis pathways are down-regulated except those of histidine and arginine biosynthesis . Recently, Jang and Imlay have shown that H2O2 damages enzymes with iron-sulfur and impairs bacterial metabolism, especially the biosynthesis of leucine . This down regulation of amino acid synthesis may cause a strain on the protein synthesis of bacteria. Our results indicate that protein synthesis is important for E. coli to survive H2O2 treatment. Chloramphenicol, an antibiotic inhibiting protein synthesis, reduced the survival of both the wild type and ΔarcA mutant E. coli after H2O2 treatment, while ampicillin did not (Figure 8). Consistently, amino acid supplementation enhanced the survival of E. coli after H2O2 treatment (Figure 7). This is in agreement with the report by Calioz and Touati that amino acid supplementation facilitates the survival of superoxide dismutase-deficient E. coli under aerobic conditions .
Although our results and results from other investigators suggest that protein synthesis and amino acid availability are important for E. coli to survive ROS stress and the global regulatory system ArcAB plays a role this aspect of ROS stress resistance, protein synthesis and amino acid availability may be only one aspect of the pleiotropic effect of ArcAB system on E. coli, since chloramphenicol-treated ΔarcA mutant was still more susceptible than the similarly treated wild type E. coli. Further studies are necessary to elucidate more molecular mechanisms that control the ROS resistance mediated by the ArcAB global regulatory system.
The global regulatory system ArcAB of E. coli regulate many important functions of bacteria including anaerobic growth, motility, and cell division. Here we demonstrate that ArcAB regulates ROS resistance under aerobic condition, and the signalling pathway of this regulation is distinct from that under anaerobic conditions. The ArcAB system may regulate protein and amino acid synthesis and transport that influence the fitness of E. coli under ROS stress.
Growth media for bacteria were purchased from Becton Dickinson and Company (Franklin Lakes, NJ). Anaerobic peptone-yeast medium was obtained from Anaerobe Systems (Morgan Hills, CA). Chemicals and antibiotics were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) unless otherwise indicated. Restriction and modifying enzymes for manipulating DNA were purchased from the New England Biolabs (Beverly, MA). Custom oligonucleotides were purchased from Sigma Genosys (The Woodlands, TX).
Bacterial strains and plasmids
E. coli strain K12 isolate MG1655 (gift from Dr. Sydney Kustu, University of California) was used as the parental strain in all analyses described in this report. Mutagenesis was carried out using the one-step mutagenesis method by Datsenko and Wanner . Mutant bacterial strains and sequences of oligonucleotides used for mutagenesis are listed in Table 1. In the ΔarcA mutant, the wild type arcA allele was replaced by a kanamycin-resistance cassette (Kanr). In the ΔarcB mutant, the wild type arcB allele was replaced by a chloramphenicol-resistance cassette (Cmr). Each mutation was transduced into fresh E. coli by general transduction with phage P1 before further analysis. In the ΔfliC mutant, the wild type fliC allele was replaced by Cmr, which was subsequently removed to generate a non-polar mutant . The ΔarcA/ΔfliC mutant was prepared by transducing arcA::kan from the ΔarcA mutant into the ΔfliC non-polar mutant E. coli. A revertant of ΔarcB mutant E. coli was generated through a two-step process. First, a mutant, arcB(Kanr), was generated in which Kanr was inserted downstream to the arcB coding sequence without affecting the arcB open reading frame. Subsequently, phage P1 was prepared from arcB(Kanr) and used to transduce the ΔarcB mutant E. coli. Kanamycin-resistant and chloramphenicol-sensitive colonies were selected, in which the deletion mutant arcB allele in the ΔarcB mutant E. coli was replaced by a wild type allele from arcB(Kanr). The genome structure surrounding the arcB allele was determined to verify that wild type arcB allele was restored. The resultant bacterial strain was referred to as ΔarcB-rev.
Plasmid pRB3-arcA used to complement the ΔarcA mutant E. coli was described previously . Plasmid pRB3-arcD2A was constructed using megaprimer method as described . Briefly, a 260-bp section of the arcA gene that included the Asp54 was amplified using mutagenesis primer 5'-CAACCTGGTGATCATGGCGATCAATCTGCC-3' and an arcA primer 5'-CAACGCTACGACGCTCTTC-3'. Sequence in bold in the mutagenesis primer introduced an aspartate to alanine mutation (Asp → Ala) at amino acid 54 in ArcA. The PCR product was used as a megaprimer to amplify plasmid pRB3-arcA together with a vector primer 5'-GTTTTCCCAGTCACGAC-3'. The PCR product was subsequently digested with KpnI and cloned into KpnI-digested plasmid pRB3-arcA to replace the wild type arcA gene with the corresponding sequence that introduced an Asp54 → Ala mutation. The resulting plasmid pRB3-arcD2A contained the same sequence as the original plasmid pRB3-arcA except that GAT which codes for Asp54 of ArcA was mutated to GCG which codes for Ala.
Survival assays of bacteria after exposure to oxidative and other stresses
Survival of E. coli after H2O2 and other stress conditions was assayed as described previously [38, 52]. E. coli was cultured in 2 ml of Luria Bertani (LB) broth at 37°C overnight with shaking at 225 rpm. Antibiotics were added as appropriate. Twenty microliters of overnight cultures were added to 2 ml of LB containing one of the following chemicals: hydrogen peroxide, sodium chloride, or sodium dodecyl sulfate (SDS). Cultures in all assays were grown aerobically by shaking at 225 rpm. After exposure to H2O2 or other stresses, aliquots of cultures were diluted and plated in triplicates. Bacterial colonies were enumerated as colony-forming units (CFU) after overnight incubation to determine the bacterial concentration. Disc diffusion assay was carried out as described previously . Briefly, approximately 1 × 106 cfu bacteria were plated onto M9 minimal agar plates and paper discs of 1/4" diameter loaded with 10 μl of 30% H2O2 were placed in the center of plates onto the bacterial lawn. Plates were incubated overnight at 37°C, and the diameter of the inhibitory zone on each plate was measured.
Scavenging of H2O2 by E. coli
Wild type, the ΔarcA and the ΔarcB mutant E. coli were cultured overnight in LB broth at 37°C with shaking at 225 rpm. Twenty microliters of overnight bacterial culture was diluted in 1 mL of fresh LB broth containing 2 mM of H2O2 that had been pre-warmed to 37°C. An aliquot of 100 μL was taken as the 0 minute sample, and rest of the cultures were incubated at 37°C with shaking. Subsequently, aliquots were taken at 10' intervals. Aliquots of bacterial cultures were used for plating to determine the bacterial concentration, and the rest of the samples were used to determine the concentration of H2O2. A control sample of LB supplemented with H2O2 that contained no bacteria was included in all assays for spontaneous degradation of H2O2.
The concentration of H2O2 in bacterial cultures was determined as described . Briefly, bacterial cultures were spun down to remove bacteria and 40 μL of supernatant was diluted in 260 μL of 50 mM potassium phosphate (pH7.0). Diluted supernatant was mixed with 600 μL of a reaction mixture containing 500 nM H2O2, 2.5 mM phenol, 0.5 mM 4-aminoantipyrine, 40 μg horseradish peroxidase, and 1 mM potassium phosphate (pH 7.0) . The reactions were incubated at room temperature for approximately 10' till color stabilized, and OD505 nm was measured for each sample. The concentration of H2O2 was determined by a standard curve generated with known concentrations of H2O2 in LB broth. The H2O2 scavenging was determined as (initial H2O2 concentration – residual H2O2 concentration) (in mM)/bacterial concentration (in 107 cfu/mL).
Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis of gene expression
To analyze the expression of fliC messenger RNA, we cultured the wild type and ΔarcA mutant E. coli in LB broth to log phase and divided each culture into two aliquots. One of the aliquots was exposed to 5 mM H2O2 and samples were taken after different exposure periods. The other aliquot was used as an unexposed control. Total RNA was purified from E. coli using TRIzol reagent (Invitrogen, Carlsbad, CA) followed by digestion with DNase I (Qiagen, Valencia, CA) and purification by RNeasy kit (Qiagen, Valencia, CA). Subsequently, 1.5 μg RNA were reverse-transcribed using M-MLV reverse transcriptase (Promega, Madison, WI), and cDNA samples were used for Real-Time Reverse Transcriptase PCR analysis (RT-PCR). RT-PCR was performed using the iQ SYBR Green PCR supermix (Bio-Rad, Hercules, CA) in an iCycler (Bio-Rad, Hercules, CA). Primers 5'-GGCGGAACTAACCCAGCTTCA-3' and 5'-TGCTCCAGTCGCCATTGTCA-3' were used for the RT-PCR analysis of fliC expression. The 16S ribosomal RNA level was determined with primers 5'-GGGACCTTCGGGCCTCTTG-3' and 5'-ACCGTGTCTCAGTTCCAGTGTGG-3', and was used to normalize expression levels of fliC from different samples.
Q-Gene program and Relative Expression Software Tool (REST) were used for data analysis of threshold cycle numbers from the iCycler [54, 55]. Mean values of normalized expression and standard error measurements were determined as described . Comparisons of mean normalized expression were used to calculate expression ratios. REST was used to obtain statistical significance (p-value) as described .
Bacterial extracts and two-dimensional (2-D) gel electrophoresis
E. coli was cultured in LB broth overnight at 37°C with shaking. Overnight bacterial culture was diluted 1:100 in fresh LB and cultured for 4 hours at 37°C with shaking, and then split into two aliquots. Hydrogen peroxide was added to 5 mM to one of the aliquots, and both aliquots were further incubated for 2 hours at 37°C with shaking. Bacterial cultures were chilled on ice immediately and spun down. Bacterial pellets were then resuspended in 8 M urea and 4% CHAPS in 10 mM Tris 8.0 and sonicated. The insoluble fraction was removed by centrifugation, and soluble lysate was used for 2-D gel electrophoresis.
Two-dimensional gel electrophoresis of E. coli proteins was performed with the Zoom IPG Runner system following the manufacturer's instructions (Invitrogen, Carlsbad, CA). One hundred fifty micrograms of cellular proteins were diluted in rehydration buffer (8 M urea, 4% CHAPS and 0.5% pH 3–10 ampholytes) and loaded onto each pH 3–10 ZOOM strip (Invitrogen, Carlsbad, CA). The first dimension electrophoresis was carried out at 200 V for 20', 450 V for 15', 750 V for 15' and 2000 V for 60'. After isoelectric focusing, ZOOM strips were reduced and alkylated with 125 mM iodoacetamide and electrophoresed on NuPAGE Novex 4–12% Bis-Tris ZOOM gels (Invitrogen, Carlsbad, CA) at 100 V for 90'. Proteins were visualized by staining with ProteomIQ reagents (Proteome Systems, Woburn, MA), and then scanned with a HP Scanjet 5530 scanner (Hewlett-Packard, Palo Alto, CA). Individual proteins were quantified using ImageQuant (Amersham Biosciences, Piscataway, NJ) and normalized against the total protein content of the gel.
Mass spectrometry analysis of protein spots
Protein spots of interest were excised from gels and washed with 50% acetonitrile in 50 mM ammonium bicarbonate twice for 15' each. The gel spots were then dehydrated in acetonitrile for 30' and dried in a speed vac for 10'. Thirty microliters of 50 mM ammonium bicarbonate containing 0.3 μg of trypsin (Sigma-Aldrich, St Louis, MO) were added to each sample, and samples were incubated at 37°C for 16 hours. Digested peptides were extracted from gel spots by two washes of 50% acetonitrile/0.1% trifluoroacetic acid, and purified with Ziptips (Millipore, Billerica, MA). Purified peptides were eluted from Ziptips with 50% acetonitrile/0.05% trifluoroacetic acid with 10 mg/ml alpha-cyano-4-hydroxycinnamic acid, and spotted on a sample plate to obtain mass spectra using an Axima CFR Plus MALDI-ToF mass spectrometer (Shimadzu Biotech, Columbia, MD). Each spectrum was calibrated externally using the ProteoMass peptide MALDI-MS calibration kit (Sigma-Aldrich, St Louis, MO).
Peptide fingerprints obtained for each sample were used to search the databases at NCBI and SWISS-PROT using MASCOT search engine http://www.matrixscience.com. Search parameters used were variable carbamidomethyl and propionamide modifications of cysteines and oxidation of methionines. A peptide tolerance window of 0.5 daltons was used for all searches. Once an identification was made with a statistically significant score, data were accepted when the peptide coverage of the protein was at least 20%, and the molecular weight and isoelectric point of the protein matched those observed on the 2D gel electrophoresis.
We thank Drs. Stuart Linn and Hiroshi Nikaido for insightful discussions. This work was supported by USDA CALR-2005-01892 (to S. L.).
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