Global Protein Expression Profile Response of Escherichia coli ATCC 25922 Exposed to Enrofloxacin

Abstract

Although enrofloxacin (ENR) is widely used for therapy of bacterial infections in the veterinary clinical, bacterial resistance to ENR is becoming an increasing worldwide problem. The primary global response of Escherichia coli to ENR exposure before resistance is largely unknown on the proteomic level. The purpose of this study was to understand the physiological response of E. coli to a subinhibitory concentration of ENR using proteomic methods. Differentially expressed proteins of the whole-cell extracts were visualized by two-dimensional gel electrophoresis, and the selected proteins were purified and identified by MALDI-TOF/mass spectrometry analysis. The result showed that the number of proteins (mean±standard deviation) detected in the ENR-treated strains was significantly (p<0.05) reduced from 1115±25 to 732±19. In total, 42 differentially expressed proteins with more than twofold difference were identified, including 13 upregulated proteins (p<0.05) and 17 downregulated proteins (p<0.05), as well as the specific proteins expressed in the group with or without ENR-treated cells. The results show that the differentially expressed proteins identified in E. coli exposed to ENR included proteins involved with a classic resistance mechanism, such as bacterial cell membrane permeability mediated by OmpX and OmpW, and other adaptive changes that appear to represent the physiological basis and background of resistance to ENR.

Introduction

E scherichia coli is probably the best understood of the simple model organisms, and the availability of its complete genome sequence⁵ facilitates its proteomic analysis by using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The 2-DE technology/MALDI MS has become a useful proteomic tool for comparative analysis of protein expression.⁴⁰ Enrofloxacin (ENR) is currently used in veterinary practice as an FDA-approved fluoroquinolone antibiotic for treatment of pets and domestic animals. Fluoroquinolones are potent antimicrobial agents that target two key enzymes related to DNA replication and transcription, DNA topoisomerase II (DNA gyrase) and DNA topoisomerase IV.¹³ However, fluoroquinolone resistance is a growing problem after its overuse worldwide.

In E. coli, DNA damage induces the SOS response for DNA repair, which allows a higher mutation rate of gyrA, gyrB, parC, and parE coding the target enzymes, particularly in their quinolone resistance-determining regions (QRDRs), and induction of the SOS response after exposure to quinolones is also responsible for extensive bacterial cell filamentation.³⁰ The profound morphological alterations in the presence of ENR and the effect of ENR on cell growth were described in our previous study.² Most studies on fluoroquinolone resistance mechanisms focus on the mutations of the topoisomerase genes, the expression of efflux pumps, and decreased permeability of the outer membrane proteins.^24,28,38 In our previous studies, we demonstrated that––regardless of the ENR concentration that bacteria were exposed to––their efflux pumps were triggered, that the expression of the SOS regulatory genes recA and umuC and the ENR target gene parC were significantly upregulated, and that the expression of the porin gene ompW was significantly downregulated. No mutations were detected in the QRDRs of E. coli treated with the subinhibitory concentration of ENR.¹ Further microarray analysis of E. coli ATCC 25922 exposed to a subinhibitory concentration of ENR for 5 hours showed that the expression of 519 genes was altered by the treatment, of which 239 were up- and 280 were downregulated. The functions of these differentially expressed genes included the SOS response, cell division, the stress response, biosynthesis, metabolism, transport, and transcription regulation.² However, on the protein expression profile, only limited information was available concerning the early global physiological response of susceptible bacteria to external drug stresses such as ENR.

In the present study, 2-D gel-based proteomic methods were used to identify the altered proteins of the whole-E. coli-cell lysates after ENR treatment, to explore the global physiological changes and the early protein expression profiles of E. coli exposed to ENR.

Materials and Methods

Bacterial strains, culture, and antibiotics

E. coli ATCC 25922 was purchased from the Institute of Microbiology, Chinese Academy of Sciences. Bacteria were routinely grown in a Luria-Bertani (LB) broth at 37°C. To measure bacterial growth, 1 ml of an overnight culture in LB was inoculated in 100 ml of LB, with or without a supplement of 1/2 minimum inhibitory concentration (MIC) of ENR, and the cultures were incubated at 37°C with shaking (160 rpm). Cultures treated with ENR were considered the experimental group, and cultures without treatment were used as the control. ENR was purchased from a commercial source (Amresco, Inc.) and of 99.5% purity.

Preparation of the total proteins of E. coli

Total proteins of ENR treated and untreated E. coli cells were extracted as described previously with a few modifications.¹⁹ Briefly, the bacterial cells were harvested at the late exponential growth phase (5 hours of growth) by centrifugation at 6,000 g for 10 minutes at 4°C. The supernatant was discarded, and the cells were washed three times with ice-cold 40 mM Tris–HCl (pH 8.0) buffer and centrifuged at 6,000 g for 10 minutes each time. Then, the pellets were resuspended in 600 μl 40 mM Tris–HCl (pH 8.0) buffer containing 1 mM phenylmethylsulfonyl fluoride and 65 mM dithiothreitol (DTT). The suspension was mixed well and disrupted by ultrasonication in 3–5-second bursts for a total of 5 minutes with a cell disrupter (MODEL: VC-750; Ultrasonics) on an ice bath. The benzonase nuclease (Merck) at a final concentration of 1 mM was added to degrade nucleic acids at room temperature for 1 hour. The supernatant was lysed with lysis buffer [8 M urea, 40 mM Tris, 4% 3-[(3-Cholamidopropyl) dimethylammonio] propane sulfonate (CHAPS), pH 8.5] at a volume ratio of 1:4, mixed well, and sonicated with the ultrasound probe as above for 5 minutes again for a better dissolution. The suspension was collected and centrifuged at 35,000 g for 15 minutes at 4°C. The supernatants were filtered through a 0.45-μm membrane to remove residual bacteria or debris, divided into single-use aliquots, and stored at −70°C. The concentration of the total proteins in the final preparation was determined by a microplate modification of the Bradford assay,⁸ and a calibration curve was made with 0.5 mg/ml of bovine serum albumin as standard. Three replicates of parallel preparation of total proteins for each sample were performed in our study.

2-DE proteomics and image analysis

The proteins were resuspended in an immobilized pH-gradient (IPG) rehydration buffer (8 M urea, 4% CHAPS, 50 mM DTT, 0.2% Bio-Lyte 4/7 ampholyte, 0.001% bromophenol blue; GE healthcare) and centrifuged at 10,000 g for 15 minutes at room temperature to remove undissolved materials. Samples equivalent to 200 μg total proteins were adsorbed onto an 18-cm Ready Strip IPG strips (linear, pI 4–7; Bio-Rad), and isoelectric focusing (IEF) was performed in the Bio-Rad Protein IEF Cell according to the manufacturer's instructions. The IPG strips were first rehydrated passively at 20°C for 8 hours and were then automatically focused using the following parameters: 50 V, 4 hours; 500 V, 1 hour; 1,000 V, 1 hour; 8,000 V, linear, 1 hour; 8,000 V, rapid; and 72,000 V-hour. The isoelectric focused strips were incubated for 15 minutes in an equilibration buffer (6 M urea, 30% glycerol, 2% sodium dodecyl sulfate [SDS], and 0.375 M Tris, pH 8.8) containing 1% DTT and then incubated for 15 minutes in an equilibration buffer containing 2.5% iodoacetamide. The equilibrated IPG strips and molecular weight standards (14–97 kDa; Bio-Rad) were subjected to 12% SDS–polyacrylamide gel electrophoresis at 80 V for 45 minutes and then 200 V until the dye front reached the bottom of the gels. Three replicate gels for each sample were performed in our study.

The gels were stained by the modified silver staining method compatible with MS³⁹ and scanned at a resolution of 500 dots/inch, using the Uniscan D3000 scanner (Tsinghua). Spot detection, spot matching, and quantitative intensity analysis were performed using PDQuest 2-D analysis software (Bio-Rad). Altered spots were compared based on their volume percentages in the total spot volume over the whole gel image. Significantly changed spots were selected if their intensity had changed (increased/decreased) by at least twofold.¹⁵

Identification of differentially expressed proteins by MALDI-TOF/MS and database searches

For MALDI-TOF/MS analysis, the selected protein spots were manually excised from the silver-stained gels and transferred into V-bottom 96-well microplates containing 100 μl of 25 mM NH₄HCO₃ (pH 8.0) in 50% acetonitrile (ACN) per well. After destaining for 1 hour, gel plugs were dehydrated with 100 μl of 100% ACN for 20 minutes and then thoroughly dried in a SpeedVac concentrator (Thermo Savant) for 30 minutes. The dried gel particles were rehydrated at 4°C for 45 minutes with 2 μl per well 0.1 μg/μl sequencing-grade-modified trypsin (Promega) in 25 mM NH₄HCO₃ (pH 8.0), and then incubated at 37°C for 12 hours.

The in-gel-digested peptide fragments were extracted from the gel segments using 100 μl of 5% trifluoroacetic acid (TFA) in 50% ACN aqueous mixture, followed by vortex mixing for 1 hour. After three repeated washing, the solute materials, including the peptide fragments, were dried by vacuum centrifugation. A zip-tip column (Millipore), in which C₁₈ resin was fixed at the tip end, was used to eliminate the sample impurities. The peptide solution was prepared using an equal volume of saturated R-cyano-4-hydroxycinnamic acid solution in 50% ACN and 0.1% TFA on a sample plate of the MALDI-TOF/MS. The MS analysis was obtained on an Ultraflex MALDI-TOF (Bruker). The annotation of peptide mass fingerprinting (PMF) was performed by the MASCOT search engine (Matrix Science). The search was done against the E. coli database.³⁶ The parameters used were Taxonomy: Escherichia coli; Type of search: Peptide Mass Fingerprint; Enzyme: Trypsin; Fixed modifications: Carbamidomethyl(C); Variable modifications: Gln->pyro-Glu (N-term Q), Oxidation (M); Mass values: Monoisotopic; Protein Mass: unrestricted; Peptide Mass Tolerance:±100 ppm; Peptide Charge State: 1+; Max Missed Cleavages: 1. The identification of a protein with respect to theoretical parameters (pI, molecular mass, etc.) was accepted if the peptide mass matched within a mass tolerance of 100 ppm.

Bioinformatic analysis for subcellular location and statistical analysis

The subcellular location of proteins identified by MALDI-TOF/MS was determined by the Program PSORTb Version 2.0 (http://psort.org/psortb/).

Two-tailed Student's test was used for statistically analyzing the data extracted from the comparison window of Image Master Software that normalized volumes for each protein spot. All the statistical analyses were carried out with Statistical Package for the Social Sciences 13.0 for Windows software. p-Value<0.05 was considered a significant difference, and p-value<0.01 was considered as very significant.

Results

2-DE profiles of the total proteins of E. coli cultures treated with ENR

We analyzed nine pairs of samples, including nine gels for the experimental group and nine gels for the control group to evaluate the differentially expressed proteins. The percentage of common spots detected from two replicate gels using the identical sample was more than 90%. The gel-to-gel reproducibility was credible, and the matching rate of the same protein spot interclass reached more than 90%. Figure 1 shows the staining spots separated by 2-DE with sharp focusing as well as wide distribution along pI 4–7. The number of proteins (mean±SD) derived from the ENR-treated strain was significantly (p<0.05) reduced from 1115±25 to 732±19, and a total of 101 proteins showed more than twofold difference in relative spot volumes when the gel carrying ENR-treated samples (Gel A) was matched with the reference gel (Gel B) carrying control samples.

FIG. 1.

2-DE profiles of the total proteins from Escherichia coli ATCC 25922 with or without ENR treatment. Equal amounts (200 μg) of cell lysates were separated on the 17-cm pI 4–7 linear-gradient IPG strips and by 12% denaturing (SDS) PAGE. (A, B) Representative 2-DE gel images of E. coli ATCC 25922 control cultures or ENR-treated cultures, respectively. The differentially expressed protein spots are shown with their unique sample spot protein (SSP)-identifying numbers. Circles indicate the successfully identified differentially expressed proteins spots with their unique SSP numbers. The upregulated (gel B) and downregulated (gel A) protein spots are indicated with black or red circles, respectively, and the spot numbers are shown in Table 1. Protein spots specifically seen in controls (gel A) or after ENR treatment (gel B) are indicated with yellow or blue circles, respectively, and the spot numbers are shown in Table 2. 2-DE, two-dimensional gel electrophoresis; ENR, enrofloxacin; IPG, immobilized pH gradient; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. Color images available online at www.liebertpub.com/mdr

Identification and subcellular location of differentially expressed proteins

To identify the differentially expressed proteins on 2-DE images, a total of the matched 101 protein spots were excised from these 2-DE gels and subjected to in-gel trypsin digestion and subsequent MALDI-TOF identification. Of these, 42 protein spots were of reliably high quality that allowed matching with the SWISSPROT protein database. These proteins included 13 significantly upregulated protein spots (>2-fold) and 14 significantly downregulated protein spots (>2-fold), two spots specific to the ENR treatment group and 13 spots specific to the control group (Tables 1 and 2). Based on the annotations from the UniProt Knowledgebase (SwissProt/TrEMBL) and Gene Ontology Database, these proteins were involved in bacterial resistance, protein biosynthesis, amino acid metabolism, transport systems, or cell division. Figure 2 shows a comparative analysis of the differential protein expression in the ENR-treated cultures and control cultures according to their biological functions. According to the Program PSORTb Version 2.0 (http://psort.org/psortb/), 26 of the proteins were cytoplasmic; four were periplasmic, two were outer membrane proteins; one was an inner membrane protein; and nine were unknown proteins (Tables 1 and 2).

FIG. 2.

Comparative 2-DE profiles of the differentially expressed proteins in E. coli ATCC 25922 after ENR treatment. Circles indicate the differentially expressed protein spots. E and C indicate the proteins from cultures treated with ENR (0.025 μg/ml), and those from untreated control cultures, respectively. Representative proteins are classified into groups A, B, C, D, E, and F according to their biological functions.

Table 1.

Proteins Differentially Expressed During Growth in an Enrofloxacin-Supplemented Medium

Fold change	Spot no.	Protein description	Subcellular location	Accession no.	No. of peptides matched	MW (Da)/pI theoretical	MW (Da)/pI experimental	Score
Significantly upregulated proteins
6.6±0.1	112	Chain A of outer membrane protein, OmpX	Outer membrane	gi\|6435772	10	16,350/5.04	16,749/5.48	161
5.9±0.2	498	F0F1 ATP synthase subunit alpha	Unknown	gi\|15804334	20	55,416/5.80	32,480/6.57	187
5.7±0.2	947	Queuine tRNA-ribosyl transferase	Cytoplasmic	gi\|15800133	19	43,022/5.97	46,847/6.55	135
5.4±0.2	490	Chain A of elongation factor complex Ef-TuEF-Ts	Cytoplasmic	gi\|1942721	13	42,321/5.22	31,048/5.22	106
5.3±0.3	103	30S ribosomal protein S6	Cytoplasmic	gi\|15804789	15	15,177/5.26	16,029/5.56	163
4.3±0.3	375	Guanylate kinase	Unknown	gi\|91213166	12	26,011/7.14	28,763/6.72	106
3.8±0.2	839	4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase	Cytoplasmic	gi\|15803038	15	40,943/5.87	41,781/6.41	173
3.7±0.3	1009	Bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase	Cytoplasmic	gi\|26250473	17	49,372/6.09	51,192/6.78	176
3.1±0.3	656	Methionyl-tRNA formyltransferase	Unknown	gi\|26249872	12	34,409/6.01	36,953/6.57	128
3.1±0.2	563	Chain A of elongation factor complex Ef-TuEF-Ts	Cytoplasmic	gi\|1942721	12	42,321/5.22	32,861/5.06	100
2.1±0.2	442	Chain B of trypsin-modified elongation factor Tu in complex with tetracycline	Cytoplasmic	gi\|118137902	8	37,020/4.98	30,626/5.43	103
2.1±0.2	265	Chain A of elongation factor complex Ef-TuEF-Ts	Cytoplasmic	gi\|1942721	8	42,321/5.22	25,837/5.48	112
2.1±0.2	1187	30S ribosomal protein S1	Cytoplasmic	gi\|15800772	22	61,235/4.89	66,799/5.00	143
Significantly downregulated proteins
11.8±0.2	224	Putative bacteriophage protein	Unknown	gi\|188493182	6	14,876/5.74	23,779/5.73	65
10.8±0.3	791	Asparagine synthetase A	Cytoplasmic	gi\|16131612	14	36,742/5.45	40,157/6.04	115
5.5±0.4	634	Chain A, high-resolution glucose-bound crystal structure of Ggbp	Periplasmic	gi\|126030485	21	33,347/5.25	35,324/5.35	258
4.9±0.4	1020	Tryptophanase	Cytoplasmic	gi\|188494319	12	53,138/6.00	50,202/5.81	96
4.9±0.4	1148	Fumarate hydratase (fumarase A), aerobic Class I	Cytoplasmic	gi\|16129570	28	60,774/6.11	62,055/6.71	279
4.9±0.3	1145	Phosphoenolpyruvate carboxykinase	Cytoplasmic	gi\|16131280	17	59,891/5.46	59,766/5.87	124
4.1±0.2	86	Hypothetical protein Z2335	Unknown	gi\|15801753	6	18,491/5.96	15,313/6.33	66
3.8±0.1	871	Succinyl-CoA synthetase subunit beta	Cytoplasmic	gi\|15800432	14	41,652/5.37	41,879/5.70	206
3.6±0.2	1185	Succinate dehydrogenase flavoprotein subunit	Unknown	gi\|91209757	32	65,456/5.85	69,463/6.30	236
3.3±0.4	156	Chain A, outer membrane protein OmpW	Outer membrane	gi\|88192831	6	21,661/6.15	19,176/5.72	70
3.3±0.4	330	Molybdate transporter periplasmic protein	Periplasmic	gi\|26246730	19	27,397/6.85	28,244/6.65	191
3.0±0.3	1070	Glycerol kinase	Cytoplasmic	gi\|33516898	19	56,525/5.37	53,393/5.71	230
2.4±0.3	869	Aromatic amino acid aminotransferase	Cytoplasmic	gi\|91210030	15	43,827/5.55	41,814/6.04	151
2.1±0.2	694	Malate dehydrogenase	Cytoplasmic	gi\|27526134	15	28,194/6.22	37,473/6.10	178

A threshold of ≥2-fold was fixed, and differentially expressed protein spots were identified on two-dimensional gels. Altogether, 27 protein spots were identified, of which 13 were upregulated, and 14 were downregulated. In terms of subcellular location, two were outer membrane proteins, 17 cytoplasmic proteins, two periplasmic proteins, and six unknown proteins.

Ggbp, glucose-/galactose-binding protein.

Table 2.

Proteins Detected Exclusively in the Enrofloxacin-Treated or Control Cultures

Spot no.	Protein description	Subcellular location	Accession no.	No. of peptides matched	MW (Da)/pI experimental	MW (Da)/pI theoretical	Score
Exclusively detected in ENR-treated cultures
138	30S ribosomal protein S6	Cytoplasmic	gi\|15804789	9	15,177/5.26	23,731/5.45	75
313	Gnd	Unknown	gi\|14517811	7	47,821/4.92	24,167/5.27	63
Exclusively detected in untreated control cultures
68	Cell division topological specificity factor MinE	Cytoplasmic	gi\|15801396	5	10,286/5.15	14,582/5.37	81
88	Primase	Cytoplasmic	gi\|52697340	6	18,919/5.50	16,002/5.78	62
136	Periplasmic protein	Unknown	gi\|89110540	6	19,832/5.09	18,403/4.59	71
163	Leucine/isoleucine/valine transporter ATP-binding subunit	Cytoplasmic membrane	gi\|26250067	6	28,527/6.67	20,238/5.31	69
451	Tryptophanase	Cytoplasmic	gi\|54401993	12	31,824/6.02	31,602/6.29	103
509	Tryptophanase	Cytoplasmic	gi\|54401993	8	31,824/6.02	32,948/6.30	76
512	Tryptophanase	Cytoplasmic	gi\|41018359	13	53,167/5.88	33,081/6.60	108
740	Tryptophanase	Cytoplasmic	gi\|41018359	7	53,167/5.88	39,974/5.69	68
1021	Tryptophanase	Cytoplasmic	gi\|170681687	31	53,107/5.88	52,343/6.44	36
1032	Tryptophanase	Cytoplasmic	gi\|170681687	27	53,107/5.88	52,497/6.26	277
1090	Periplasmic nickel-binding protein NikA	Periplasmic	gi\|194435944	17	58,750/5.94	57,076/6.03	131
1133	Oligopeptide transport periplasmic binding protein	Periplasmic	gi\|15830997	11	61,052/5.95	60,261/6.22	109
1157	Fumarate hydratase class I	Unknown	gi\|26250935	15	60,592/5.80	62,604/6.34	183

There were two protein spots seen only in the samples from ENR treatment, and 13 were seen only in the control samples. Subcellular analysis showed that one was an inner membrane protein, nine cytoplasmic proteins, two periplasmic proteins, and three unknown proteins.

ENR, enrofloxacin.

Discussion

In our study, differentially expressed proteins were identified using 2D gel analysis and MALDI-TOF/MS. Functional analyses of the differentially expressed proteins of E. coli exposed to ENR were related to antibiotic susceptibility, growth, and morphology of the bacteria, transport system, protein biosynthesis, the tricarboxylic acid (TCA) cycles, gluconeogenesis, and amino acid metabolism.

Proteins related to antibiotic susceptibility

Adjustments in the expression levels of the different outer membrane proteins are considered to be an adaptive response to antibiotic exposure as exemplified by a recent report.²⁵ For example, OmpX can regulate the uptake of antibiotics such as β-lactams and fluoroquinolones because of the role in regulating membrane permeability.¹⁴ An increased synthesis of OmpX accompanied by a reduction in the major porins was linked to multidrug resistance, including resistance to fluoroquinolones, in two clinical strains of Enterobacter aerogenes.¹⁸ In our study, OmpX was upregulated 6.6±0.1-fold, whereas OmpW was downregulated 3.3±0.4-fold after exposure to ENR for 5 hours. The results suggest that the early regulation of OmpX may represent a preliminary response to ENR and the initial line of defense of bacteria against antibiotic therapy.

Previous studies suggested that members of the OmpW family were involved in the transport of small hydrophobic molecules across the bacterial outer membrane and in the protection of bacteria against various forms of environmental stress.²⁰ Downregulation of Tsx and OmpW and upregulation of OmpX are known to be required for iron homeostasis in E. coli,²⁴ a characteristic feature in E. coli surviving in an iron-deficient medium. Lin et al. reported that expression of the ompW gene was downregulated by exposure to chlortetracycline, and its deletion resulted in a decrease in survival capabilities.²⁵ The downregulated expression of OmpW in the study presented here is consistent with the downregulated gene expression of ompW, demonstrated in our previous study.² In contrast to these reports, OmpW was said to be upregulated in E. coli after exposure to nalidixic acid, another gyrase inhibitor like ENR.²³ It will be interesting to study the molecular interactions between ENR and OmpX and OmpW, especially in view of the changes in the size and shape of ENR-treated cells, which may influence the structure of these proteins.

The reasons why OmpF and OmpC,³¹ the major outer membrane proteins in E. coli, have not been detected in our study is not clear. Possibly, OmpX and OmpW may play a dominant role in the early response to environmental stress such as ENR treatment. Moreover, the use of CHAPS, a less-denaturing zwitterionic detergent, might have been a hindrance to the solubilization of the missing major outer membrane proteins.

Another protein upregulated in the ENR-treated cells was the guanylate kinase encoded by the gmk gene is a target of an antitumor drug, 6-thioguanine, and is required for activation of some of the antiviral drugs such as acyclovir and ganciclovir.^6,33 An earlier study showed that gmk was also transcriptionally regulated by heat stress during nutrient starvation and growth phase in the gram-positive bacterium, Staphylococcus epidermidis, such that the transcript levels of gmk declined after the mid-exponential phase.³⁵ In the present study, sampling was done in the late-exponential phase when the gmk transcriptional activity would be expected to be low, but we found a higher level (4.3±0.3-fold upregulated) of protein expression in the presence of ENR. It will be interesting to investigate if this enzyme has a role to play in the mode of action of this fluoroquinolone drug.

Proteins related to growth and morphology of the bacteria

Primase is one of the essential enzymes of chromosomal and plasmid DNA replication that initiates leading-strand synthesis once and lagging-strand synthesis multiple times during the course of replication.⁷ Primase-catalyzed reactions are known to be inhibited under conditions of starvation or stress, which leads to an arrest of DNA replication and retardation in growth rate.²⁶ MinE is a cell division topological specificity factor in gram-negative and gram-positive bacteria. Disruption of the minE gene in Sinorhizobium meliloti resulted in large, swollen, and branched cells that could not enter into a symbiotic association with the host plant.¹⁰ In our study, spots corresponding to primase and MinE were found only in untreated control culture samples.

Yet, another protein induced by ENR treatment was the bifunctional N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate acetyltransferase, the product of the glmU gene. This enzyme catalyzes a two-step reaction yielding UDP-N-acetylglucosamine, an important precursor in cell wall (peptidoglycan) biosynthesis, and hence is considered an excellent drug target.²⁹ In a recent long-term evolution study in populations of E. coli, a single base change in the glmUS operon has led to reduced expression of these genes and conferred an improved fitness on the population.³² Hence, it is intriguing to see that the amount of this protein increased 3.7±0.3-fold after ENR treatment, although the growth rate was reduced. Whether this effect is an adaptive response to ENR or is related to the dramatic changes in the cell morphology because of the role of this enzyme in cell wall synthesis remains to be investigated.

Tryptophanase, encoded by the tnaA gene, degrades l-tryptophan to indole, pyruvate, and ammonia, allowing microorganisms to utilize tryptophan as a source of carbon, nitrogen, and energy.²¹ The product of tryptophan metabolism, indole, has been proposed as an extracellular signaling molecule that plays an important role during transition to stationary phase and biofilm formation in E. coli.²⁷ A recent study proposed that in a culture treated with an antibiotic, the altruistic extracellular secretion of indole by a few, highly resistant, mutant cells can result in an increased antibiotic resistance in the entire population.²² In our study, one tryptophanase protein (spot no. 1020) was repressed 4.9±0.4-fold after treatment with ENR, whereas several tryptophanase proteins (spot nos. 451, 509, 512, 740, 1021, and 1032) were only detected in the control samples. This suggests that the indole concentration presumably decreased, thereby compromising the fitness of the cells to survive the antibiotic stress.

Proteins related to transport systems

NikA is a periplasmic binding protein involved in nickel uptake and transport in E. coli. It plays a dual role as the primary binding protein for Ni in the uptake process and also in negative chemotaxis in conditions of excess of Ni.¹⁶ The oligopeptide-binding protein, OppA, binds to oligopeptide substrates and facilitates their uptake via the membrane-associated oligopeptide permease (Opp).³ Here we found up to three- to fivefold reduction in the expression of proteins involved in active transport, for example, the NikA and OppA discussed above, as well as the glucose-/galactose-binding protein and molybdate transporter periplasmic protein, in extracts from cells treated with ENR. It is believed that importing of amino acid and other carbon sources via periplasmic binding proteins is essential for E. coli cells during energy crisis.³⁷

Proteins related to protein biosynthesis

Alterations in the expression of proteins related to protein biosynthesis, in a particular elongation factor complex EF-Tu, EF-Ts, and 30S ribosomal protein, were also demonstrated in ENR-treated cells. A two- to fivefold upregulation of the elongation factor complex (protein spot nos. 490, 563, 442, and 265) was recorded (Table 1). In prokaryotes, three elongation factors are required for translation: EF-Tu, EF-Ts, and EF-G. Apart from their essential role in protein synthesis, the elongation factors also play role in stress response. For instance, EF-Tu acts as a chaperone to assist in folding of proteins damaged due to stress.⁹ Other proteins such as the 30S ribosomal proteins were seen to be upregulated: S6 (spot no. 103) and S1 (spot no.1187) showed increases in expression of about fivefold and twofold, respectively, after treatment with ENR, whereas one spot, no. 138 that also corresponded to S6, was detected only in the ENR-treated cells. In some reports, the ribosomal S1 protein was shown to be downregulated under oxidative stress, while its expression was unaltered under other environmental stresses such as heat or acidic pH.³⁴ However, in the present study, this protein was clearly upregulated during growth in the presence of ENR. Similarly, the ribosomal protein S6 is known to be induced under various environmental stress conditions such as heat shock¹² and was upregulated 5.3±0.3-fold in ENR-treated cells.

Proteins related with the TCA cycles, gluconeogenesis, and amino acid metabolism

The introduction of ENR into the growth medium appeared to influence the carbon flux by repressing the expression of related enzymes for the TCA cycle, gluconeogenesis, and fatty acid synthesis. For example, malate dehydrogenase and succinate dehydrogenase are important enzymes in the TCA cycle and were repressed after ENR treatment (Table 1; Fig. 1). Fumarate hydratase was downregulated with ENR treatment or expressed only in the control cultures. In addition, phosphoenol pyruvate carboxykinase involved in gluconeogenesis was repressed in the ENR-treated cultures (Table 1; Fig. 1). 6-Phosphogluconate dehydrogenase involved in the hexose monophosphate shunt was detected only in the extracts from ENR-treated cultures. The phosphotransferase enzyme glycerol kinase showed downregulation with ENR treatment. Another important enzyme, asparagine synthetase A (AsnA), was downregulated 10.8±0.3-fold, which was the largest fold change seen for proteins of known functions in this study.

Other proteins

There were a number of other proteins showing significantly higher expression (between 3.1±0.3 and 5.9±0.2-fold) in the group with ENR treatment, for example, F0F1-ATP synthase subunit-alpha (a response to fluoroquinolone¹¹), queuine tRNA-ribosyl transferase, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, and methionyl-tRNA formyltransferase, which have been mentioned in other studies as stress- or growth-regulated proteins.^4,17 Two of the downregulated proteins, spot nos. 224 and 86, were putative proteins with unknown functions, of which the former showed the highest level of change (11.8±0.2-fold).

Presumably, some of the differentially expressed proteins may be related to the reduced growth rate or to the changes in cell morphology. Some other proteins may be specifically regulated by ENR, for example, due to its effect on DNA gyrase and DNA topoisomerase IV. We did not detect the proteins associated with the SOS response that can be induced by fluoroquinolone drugs as was demonstrated in our microarray result.² The reasons for this discrepancy are not clear at the present time. Further studies on individual proteins will be necessary to identify those that may be involved in development of resistance to ENR.

Conclusion

This study adopted a gel-based comparative proteomic approach to analyze and identify the differentially expressed proteins of E. coli ATCC 25922 during growth in a medium containing ENR at a concentration of 1/2 MIC (0.025 μg/ml). The proteomic analysis revealed the early protein expression profiles in E. coli on exposure to ENR and identified 42 differentially expressed proteins. An upregulated expression (p<0.05) of 13 proteins was detected, including OmpX, F0F1-ATP synthase subunit-alpha, and elongation factor complex proteins, Ef-Tu and EF-Ts. Downregulated expression (p<0.05) of 17 proteins was detected, including OmpW, AsnA, and tryptophanase. MinE and primase, which are involved in cell shape and division, were absent from the proteome of ENR-treated cells.

Our results demonstrate on the level of protein expression––the global response of E. coli to ENR exposure. The proteins involved included such classic components of resistance mechanisms as OmpX and OmpW. The other adaptive changes may represent the physiological basis and background of resistance to ENR. The data generated in this study will serve a base to launch future studies for detailed analysis of the role of each of the potentially interesting proteins in bacterial adaptive responses to and development of resistance to fluoroquinolone drugs.

Footnotes

Acknowledgments

This project was supported by the National Natural Science Foundation of China (No. 30770044). The authors would like to thank Mr. Tang Chong (Beijing Genomics Institute), Mr. Wang Guibin (Beijing Genomics Institute), and Ms. Zheng Xiaojuan (Zhejiang University) for technical assistance and experimental help.

Disclosure Statement

No competing financial interests exist.

References

Bai

, Du

J.F.

, Hu

, Qi

, Cai

Y.N.

, Niu

W.W.

, Liu

Y.Q.

2011. Analysis of mechanisms of resistance and tolerance of Escherichia coli to enrofloxacin. Ann. Microbiol., 62:293–298.

Bai

, Su

W.Z.

, Zhu

X.L.

, Hu

, Liu

Y.Q.

2010. Effect of enrofloxacin on gene expression profiles of Escherichia coli. Ann. Microbiol., 60:653–660.

Balan

, Ferreira

R.C.

, Ferreira

L.C.

2008. Production of the refolded oligopeptide-binding protein (OppA) encoded by the citrus pathogen Xanthomonas axonopodis pv. Citri. Genet. Mol. Res., 7:117–126.

Barriuso-Iglesias

, Schluesener

, Barreiro

, Poetsch

, Martín

J.F.

2008. Response of the cytoplasmic and membrane proteome of Corynebacterium glutamicum ATCC 13032 to pH changes. BMC. Microbiol., 8:225–241.

Blattner

F.R.

, Plunkett

3rd , Bloch

C.A.

, Perna

N.T.

, Burland

, Riley

, Collado-Vides

, Glasner

J.D.

, Rode

C.K.

, Mayhew

G.F.

, Gregor

, Davis

N.W.

, Kirkpatrick

H.A.

, Goeden

M.A.

, Rose

D.J.

, Mau

, Shao

1997. The complete genome sequence of Escherichia coli K-12. Science, 277:1453–1462.

Boehme

R.E.

1984. Phosphorylation of the antiviral precursor 9-(1,3-dihydroxy-2-propoxymethyl) guanine monophosphate by guanylate kinase isozymes. J. Biol. Chem., 259:12346–12349.

Bouché

J.P.

, Rowen

, Kornberg

1978. The RNA primer synthesized by primase to initiate phage G4 DNA replication. J. Biol. Chem., 253:765–769.

Bradford

M.M.

1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72:248–254.

Caldas

T.D.

, El. Yaagoubi

, Richarme

1998. Chaperone properties of bacterial elongation factor EF-Tu. J. Biol. Chem., 273:11478–11482.

10.

Cheng

, Sibley

C.D.

, Zaheer

, Finan

T.M.

2007. A Sinorhizobium meliloti minE mutant has an altered morphology and exhibits defects in legume symbiosis. Microbiology, 153:375–387.

11.

Coldham

N.G.

, Webber

, Woodward

M.J.

, Piddock

L.J.

2010. A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli. J. Antimicrob. Chemother., 65:1655–1663.

12.

De Angelis

, Di Cagno

, Huet

, Crecchio

, Fox

P.F.

, Gobbetti

2004. Heat shock response in Lactobacillus plantarum. Appl. Environ. Microbiol., 70:1336–1346.

13.

Drlica

, Zhao

1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev., 61:377–392.

14.

Dupont

, James

C.E.

, Chevalier

, Pagès

J.M.

2007. An early response to environmental stress involves regulation of OmpX and OmpF, two enterobacterial outer membrane pore-forming proteins. Antimicrob. Agents Chemother., 51:3190–3198.

15.

Egnaczyk

G.F.

, Pomonis

J.D.

, Schmidt

J.A.

, Rogers

S.D.

, Peters

, Ghilardi

J.R.

, Mantyh

P.W.

, Maggio

J.E.

2003. Proteomic analysis of the reactive phenotype of astrocytes following endothelin-1 exposure. Proteomics, 3:689–698.

16.

Eitinger

, Mandrand-Berthelot

M.A.

2000. Nickel transport systems in microorganisms. Arch. Microbiol., 173:1–9.

17.

Eleaume

, Jabbouri

2004. Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth. J. Microbiol. Methods, 59:363–370.

18.

Gayet

, Chollet

, Molle

, Pagès

J.M.

, Chevalier

2003. Modification of outer membrane protein profile and evidence suggesting an active drug pump in Enterobacter aerogenes clinical strains. Antimicrob. Agents Chemother., 47:1555–1559.

19.

Han

K.Y.

, Park

J.S.

, Seo

H.S.

, Ahn

K.Y.

, Lee

2008. Multiple stressor-induced proteome responses of Escherichia coli BL21 (DE3) J. Proteome Res., 7:1891–1903.

20.

Hong

, Patel

D.R.

, Tamm

L.K.

, Van den Berg

2006. The outer membrane protein OmpW forms an eight-stranded beta-barrel with a hydrophobic channel. J. Biol. Chem., 281:7568–7577.

21.

Hopkins

F.G.

, Cole

S.W.

1903. A contribution to the chemistry of proteids: Part II. The constitution of tryptophane, and the action of bacteria upon it. J. Physiol., 29:451–466.

22.

Lee

H.H.

, Molla

M.N.

, Cantor

C.R.

, Collins

J.J.

2010. Bacterial charity work leads to population-wide resistance. Nature, 467:82–85.

23.

Lin

X.M.

, Li

, Wang

, Peng

X.X.

2008b. Proteomic analysis of nalidixic acid resistance in Escherichia coli: identification and functional characterization of OM proteins. J. Proteome Res., 7:2399–2405.

24.

Lin

X.M.

, Wu

L.N.

, Li

, Wang

S.Y.

, Peng

X.X.

2008a. Downregulation of Tsx and OmpW and upregulation of OmpX are required for iron homeostasis in Escherichia coli. J. Proteome Res., 7:1235–1243.

25.

Lin

X.M.

, Yang

J.N.

, Peng

X.X.

, Li

2010. A novel negative regulation mechanism of bacterial outer membrane proteins in response to antibiotic resistance. J. Proteome Res., 9:5952–5959.

26.

Maciag

, Kochanowska

, Lyzeń

, Wegrzyn

, Szalewska-Pałasz

2010. ppGpp inhibits the activity of Escherichia coli DnaG primase. Plasmid, 63:61–67.

27.

Martino

P.D.

, Fursy

, Bret

, Sundararaju

, Phillips

R.S.

2003. Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria. Can. J. Microbiol., 49:443–449.

28.

Peng

, Xu

, Ren

, Lin

, Wu

, Wang

2005. Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Pseudomonas aeruginosa responding to ampicilin, kanamycin, and tetracycline resistance. J. Proteome Res., 4:2257–2265.

29.

Pereira

M.P.

, Blanchard

J.E.

, Murphy

, Roderick

S.L.

, Brown

E.D.

2009. High-throughput screening identifies novel inhibitors of the acetyltransferase activity of Escherichia coli GlmU. Antimicrob. Agents Chemother., 53:2306–2311.

30.

Piddock

L.J.

, Walters

R.N.

, Diver

J.M.

1990. Correlation of quinolone MIC and inhibition of DNA, RNA, and protein synthesis and induction of the SOS response in Escherichia coli. Antimicrob. Agents Chemother., 34:2331–2336.

31.

Pratt

L.A.

, Hsing

, Gibson

K.E.

, Silhavy

T.J.

1996. From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli. Mol. Microbiol., 20:911–917.

32.

Stanek

M.T.

, Cooper

T.F.

, Lenski

R.E.

2009. Identification and dynamics of a beneficial mutation in a long-term evolution experiment with Escherichia coli. BMC Evol. Biol., 9:302.

33.

Stolworthy

T.S.

, Krabbenhoft

, Black

M.E.

2003. A novel Escherichia coli strain allows functional analysis of guanylate kinase drug resistance and sensitivity. Anal. Biochem., 322:40–47.

34.

Teixeira-Gomes

A.P.

, Cloeckaert

, Zygmunt

M.S.

2000. Characterization of heat, oxidative, and acid stress responses in Brucella melitensis. Infect. Immun., 68:2954–2961.

35.

Vandecasteele

S.J.

, Peetermans

W.E.

, Merckx

, Van Eldere

2001. Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. J. Bacteriol., 183:7094–7101.

36.

Vijayendran

, Burgemeister

, Friehs

, Niehaus

, Fla schel

2007. 2DBase:2D-PAGE database of Escherichia coli. Biochem. Biophys. Res. Commun., 363:822–827.

37.

Wang

, Wu

S.L.

, Hancock

W.S.

, Trala

, Kessler

, Taylor

A.H.

, Patel

P.S.

, Aon

J.C.

2005. Proteomic profiling of Escherichia coli proteins under high cell density fed-batch cultivation with overexpression of phosphogluconolactonase. Biotechnol. Prog., 21:1401–1411.

38.

, Lin

, Ren

, Zhang

, Wang

, Peng

2006. Analysis of outer membrane proteome of Escherichia coli related to resistance to ampicillin and tetracycline. Proteomics, 6:462–473.

39.

Yan

J.X.

, Wait

, Berkelman

, Harry

R.A.

, Westbrook

J.A.

, Wheeler

C.H.

, Dunn

M.J.

2000. A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis, 21:3666–3672.

40.

Zimny-Arndt

, Schmid

, Ackermann

, Jungblut

P.R.

2009. Classical proteomics: two-dimensional electrophoresis/MALDI mass spectrometry. Methods Mol. Biol., 492:65–91.