Downregulation of Tsx and OmpW and Upregulation of OmpX Are

Notably, three novel proteins, Tsx, OmpW, and OmpX, related to iron homeostasis were identified; Tsx and OmpW were downregulated, and OmpX was ...
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Downregulation of Tsx and OmpW and Upregulation of OmpX Are Required for Iron Homeostasis in Escherichia coli Xiang-min Lin,† Li-na Wu,† Hui Li,†,‡ San-ying Wang,§ and Xuan-xian Peng*,† Center for Proteomics, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China, School of Life Sciences, Guangxi University, Nanning 530004, People’s Republic of China, and Center for Proteomics, School of Life Sciences, Xiamen University, Xiamen 361005, People’s Republic of China Received September 11, 2007

Upregulation of outer membrane (OM) proteins was systematically investigated in response to poor iron availability in the host and natural environments, but downregulation of OM proteins was illdefined in this response. We utilized proteomic methodologies to characterize altered OM proteins in the sarcosine-insoluble fraction of Escherichia coli K12 cultured in LB medium with iron limitation. Notably, three novel proteins, Tsx, OmpW, and OmpX, related to iron homeostasis were identified; Tsx and OmpW were downregulated, and OmpX was upregulated. These alterations were functionally validated with the use of gene overexpression and deletion methods. Of the two downregulated proteins, Tsx was more sensitive to an iron-deficient environment than OmpW. In addition, the significantly negative correlation between Tsx with OmpW was achieved when overexpressed strains were used. These findings strongly indicate that the downregulation of Tsx and OmpW and the upregulation of OmpX are required for iron homeostasis in E. coli. Keywords: Proteomic • outer membrane proteins • Escherichia coli K-12 • iron limit

Iron is an essential element in most organisms, playing a significant role in many biological processes involved in metabolic electron transport chains as a cofactor, such as photosynthesis, N2 fixation, methanogenesis, respiration, the trichloroacetic acid (TCA) cycle, and DNA biosynthesis.1 Unfortunately, the availability of iron in natural environments is usually very low because of scarce iron content in many places in the world and the low solubility of ferric iron. The content of free iron is much lower than what the bacteria essentially need so it is unavailable for them.2,3 To maintain iron homeostasis, bacteria have developed several tactics to scavenge iron from natural environments over their endless evolution. These tactics include elevating the capability of iron transport, iron stores, and redox stress resistance systems, downregulating the expression of iron-containing proteins under iron-restricted conditions, and an overarching iron-responsive regulatory system.1 A line of evidence has indicated that OM proteins show key ability in the regulation of small molecules between cells and their environments, including the ability in response to iron limitation,4 drug resistance,5 osmotic stress,6 antibiotics,7,8 and acid stress.9 OM proteins played a critical role in iron homeo-

stasis by expressing high-affinity OM protein receptors for siderophores which were secreted by endogenesis and chelated with ferric iron or host iron binding proteins such as lactoferrin, transferrin, and hemoglobin.10 Six OM proteins (CirA, FecA, FepA, FhuA, FhuE, and YbiL) were identified in iron acquisition as Fe3+ siderophore receptors in Escherichia coli K12, a model organism for understanding iron homeostasis maintenance.1 Furthermore, IroN was reported to be a siderophore receptor of enteropathogenic E. coli.11 Recently, high-throughput of proteomic and microarray methodologies have been utilized for the screening of OM proteins related to iron regulation. Molloy et al. identified five upregulated siderophore receptors (CirA, FepA, FhuE, YbiL, and FhuA) with the use of proteomic methodologies.12 McHugh et al. characterized iron- and Furdependent gene expression in E. coli K12 using a microarray, in which six siderophore receptors (CirA, FecA, FepA, FhuA, FhuE, and YbiL) were included.13 Some of these siderophore receptors (FepA, FecA, and FhuA) were well-elucidated in crystal structures to explain their iron uptake mechanism.14–16 These findings indicate the importance of OM proteins in bacterial iron homeostasis. However, downregulated OM proteins in response to iron limitation are ill-defined, since both positive and negative regulators should be components of a biological network.

* To whom correspondence should be addressed: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China. Telephone: +86-20-3145-2846. Fax: +8620-8403-6215. E-mail: [email protected]. † Sun Yat-sen University. ‡ Guangxi University. § Xiamen University.

Sodium carbonate and sodium lauryl sarcosinate (sarcosine) enrichments of bacterial OM proteins are conventionally used for subproteome analysis of the OM fractions (the sodium carbonate method may enrich all membrane proteins, including OM proteins and inner membrane proteins). The significant

Introduction

10.1021/pr7005928 CCC: $40.75

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The Journal of Proteome Research 2008, 7, 1235–1243 1235 Published on Web 01/26/2008

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Table 1. Strains and Plasmids Used in This Study strain or plasmid

strain E. coli K12 K99+

E. coli BL21(DE3) E. coli Top10F′

E. coli K12 BW25113

∆ompW JW0401 (∆tsx) JW0799 (∆ompX) plasmid pET-32a(+) pLLP-ompA

pompW pompX ptsx

relevant characteristic(s)

wild type

F– ompT hsdSB (rB– mB-) gal dcm (DE3) mcrA∆(mrr-hsdrms-mcrBC) Φ80∆lac DM15∆lacX74 deoR recA1 endA1 lacIq rrnBT14∆lacZWJ16 hsdR514 ∆araBADAH33 ∆rhaBADLD78 derivative of BW25113 that lacks ompW derivative of BW25113 that lacks tsx derivative of BW25113 that lacks ompX cloning vector; Ampr expression vector, containing an ompA signal peptide Ampr pLLP-ompW pLLP-ompX pLLP-tsx

difference in OM protein resolution has been appreciated between the two extractions.17 As mentioned above, Molloy et al. identified siderophore receptors (CirA, FepA, FhuE, YbiL, and FhuA) from the fraction enriched by a carbonate extraction.12 However, information regarding subproteomics on the sarcosine-insoluble fraction is not available. We supposed novel OM proteins related to iron regulation may be achieved in the lauryl sarcosinate fraction due to the difference between the two fractions in OM resolution. In this regard, we utilized proteomic methodologies to characterize altered protein spots in the sarcosine-insoluble fraction of E. coli K12 cultured in Luria-Bertani (LB) medium with iron chelator 2,2′-dipyridyl (DIP). This reagent could bind ferrous iron to form an iron-DIP complex that was unavailable for bacteria and was widely used to investigate the effect of iron limitation on bacterial physiology.18 Eleven altered spots representing eight proteins were characterized, of which YbiL, FecA, FepA, FhuE, and CirA were known iron regulation proteins and Tsx, OmpW, and OmpX were novel proteins related to iron homeostasis. Furthermore, the ability in iron homeostasis maintenance of the three novel proteins was further investigated by using genetically modified E. coli strains. Our results provide negative regulation data for the construction of a biological network of iron homeostasis maintenance in E. coli.

Materials and Methods Bacterial Strains and Culture. The bacterial strains and plasmids used in this work are described in Table 1, in which E. coli K12 BW25113 and its two derivatives, ∆ompX and ∆tsx, were kindly provided by the Nara Institute of Science and Technology.19 These strains were routinely grown in LB medium. Iron limitation was conducted by inclusion of the ferrous iron chelator DIP (Sinopharm Chemical Reagent Co., Ltd.) at 200, 400, or 500 µM in LB medium. A single colony was propagated in 200 mL of LB medium at 37 °C while being 1236

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source or reference

Institute of Microbiology, Chinese Academy of Sciences, Beijing, China laboratory collection laboratory collection

Nara Institute of Science and Technology laboratory collection Nara Institute of Science and Technology Nara Institute of Science and Technology laboratory collection laboratory collection

laboratory collection laboratory collection laboratory collection

shaken overnight at 200 rpm. The overnight culture was diluted 1:100 to fresh LB medium with DIP added at 37 °C with shaking at 200 rpm to late exponential phase. Ampicillin (100 µg/mL) and IPTG (68 µg/mL) were appropriately added to the medium. Measurement of Bacterial Growth and Survival. Bacteria were grown overnight in 50 mL of LB medium at 37 °C with shaking. The overnight culture was separately diluted 1:100 in the 200 mL of LB medium without or with DIP and then incubated at 37 °C in the same medium with shaking at 200 rpm. Ampicillin (100 µg/mL), IPTG (68 µg/mL), and FeCl3 (50, 100, and 200 µM) were added following shaking for 2 and 4 h when necessary. Cultures were separately collected at intervals. The cell density was determined via measurement at 600 nm. Analysis of Metal Accumulation in Bacterial Cells. Metal concentrations were measured by inductively coupled plasma optical emission spectroscopy (ICP-OES). Briefly, E. coli K12 BW25113 cells were cultured in LB medium without and with 200 µM DIP for 4 h. The cultures were harvested and cell pellets washed with sterile saline three times. The pellets were heated in 80 °C to a constant dry weight before addition of concentrated H2SO4 (800 µL) and then 4 mL of concentrated HNO3 until the solutions were clear at 150–200 °C. The cell digests were transferred to 5 mL volumetric flasks and diluted to the final volume with distilled water. Then the solutions and washing were analyzed by ICP-OES. All of the samples were repeated in triplicate. The glassware in this work was washed with 1 M HCl and then rinsed extensively with distilled water. Isolation of OM Proteins. OM proteins were prepared according to a procedure described previously.20 Briefly, bacterial cells were harvested by centrifugation at 4000g for 15 min at 4 °C. The cells were then washed in 40 mL of sterile saline (0.85% NaCl) several times. The pellet was resuspended in 10 mL of sonication buffer [50 mM Tris-HCl (pH 7.4)] and disrupted by intermittent sonic oscillation for a total of 15 min at 50% power with intervals of 9 s on ice. Unbroken cells and

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Tsx, OmpW, and Tsx for Iron Homeostasis

Figure 1. Effect of DIP on iron content, growth, and OM protein expression of E. coli. (A) Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis of iron, copper, and magnesium contents in bacterial cells cultured in LB medium without and with 200 µM DIP. Two asterisks indicate P < 0.01. (B and C) Growth curve of E. coli K12 K99+ in LB medium without and with DIP and an iron supplement. Cells were cultured with 200 or 400 µM DIP for 4 h before the addition of different concentrations of FeCl3 and assessed at 6 and 10 h: (B) 200 µM DIP and (C) 400 µM DIP. (D) Histogram analysis of bacterial growth from panels B and C at 10 h. (E) 1-DE proteomic analysis. CBB-stained SDS-PAGE of OM proteins of E. coli K12 K99+ grown in LB (lane 1) and LB with DIP (lane 2); lane M contained molecular mass standards. Four altered protein bands were found, excised, and identified by MALDI-TOF MS. The results are shown on the right side. Table 2. Identification of Altered Bands by PMF Searching in LB Medium with an Iron Limit spot

accession name

a

FEPA_ECOLI FECA_ECOLI

b c d

CIRA_ECOLI OmpW_ECOLI OmpX_ECOLI

character description

ferric enterobactin receptor iron(III) dicitrate transport system outer membrane receptor precursor colicin I receptor colicin S4 receptor OmpX

subcellular location

molecular mass (kDa)/pI

no. of peptides recovered

sequence coverage (%)

score

integral OMP integral OMP

75413/5.32 85269/5.59

8 8

22 17

68 62

integral OMP integral OMP integral OMP

73850/5.11 25861/5.93 16350/5.04

10 6 6

25 35 52

90 57 65

cellular debris were removed by centrifugation at 4000g for 20 min before supernatant was collected, and the supernatant was further centrifuged at 100000g for 40 min at 4 °C. The pellet was resuspended in 2% (w/v) sodium lauryl sarcosinate (Simga) in 50 mM Tris (pH 7.6) and incubated at room temperature

for 20 min, followed by centrifugation at 100000g for 40 min at 4 °C and resuspension in sonication buffer. The concentration of the OM proteins in the final preparation was determined using the Bradford method, and the samples were frozen at -80 °C until they were required. The Journal of Proteome Research • Vol. 7, No. 3, 2008 1237

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Figure 2. Analysis of 2-DE proteomics. (A) Comparison of OM protein maps of E. coli K-12 K99+ cultured in LB medium without (A-1) and with DIP (A-2). The histograms (A-3 and A-4) display the changes in spot intensity of altered proteins between LB medium without (black) and with DIP (white). Bars represent spot intensity with relative volume divided by the total volume over the whole image, according to the description for Melanie 4.0. (B) Enlarged partial two-dimensional gels showing the altered expression of Tsx (spot 7), OmpW (spots 8-10), and OmpX (spot 11). (C) Confirmation of these altered proteins (Tsx, OmpW, and OmpX) related to iron limitation by Western blotting analysis. The anti-rabbit sera against Tsx or OmpX and OmpW were used as the primary antibodies.

One-Dimensional Electrophoresis Proteomics. The discontinuous buffer system of Laemmli with 12% resolving gels and 5% stack gels was used to resolve OM proteins. All samples were heated for 5 min in boiling water after being mixed with the same volume loading buffer and electrophoresed with a constant voltage of 120 V until the dye front reached the bottom of the gels. The protein bands were visualized by staining with CBB R-250 (0.15% Coomassie brilliant R-250, 1% acetic acid, and 45% methanol). Altered bands were excised and used for mass spectrometric analysis. Two-Dimensional Electrophoresis Proteomics. 2-DE was performed according to a procedure described previously.20 Briefly, OM protein extracts containing 20 µg of proteins were dissolved in a solution (8 M urea, 2 M thiourea, 4% CHAPS, and 80 mM DTT). IEF rod gels were carried out using pH 3–9.5 carrier ampholyte. Protein samples were added to the anodic side of the gel and focused at 7200 Vh. After being equilibrated for 15 min, the IEF gels were subjected to two-dimensional 1238

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electrophoresis using a 12% acrylamide gel. The preparative gels were stained with CBB R-250 and scanned in an AGFA white-light scanner at a resolution of 600 bpi. The raw images were analyzed by the 2-DE software Melanie 4.0 (Swiss Institute of Bioinformatics, Geneva, Switzerland). Following background subtraction and spot detection, the gel patterns were matched to each other by a visual comparison. Mass spectrometric analysis was carried out according to a procedure described previously.17 In brief, protein spots separated by 2-DE were excised and dehydrated several times with ACN as a purified sample. The sample solution (30–100 ppm) with an equivalent matrix solution was applied to the MALDITOF target and prepared for MALDI-TOF MS analysis. CHCA was used as the matrix. MALDI-TOF spectra were calibrated using trypsin autodigestion peptide signals and matrix ion signals. All MALDI analysis was performed by a fuzzy logic feedback control system (Reflex III MALDI-TOF system, Bruker) equipped with delayed ion extraction. Peptide masses were

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Tsx, OmpW, and Tsx for Iron Homeostasis Table 3. Identification of Altered Proteins by PMF Searching in LB Medium with Iron Limitation spot accession name

1 2

YBIL_ECOLI FECA_ECOLI

3 4 5 6 7

FEPA_ECOLI FHUE_ECOLI CIRA_ECOLI FEPA_ECOLI TSX_ECOLI

8 9 10 11

OmpW_ECOLI OmpW_ECOLI OmpW_ECOLI OmpX_ECOLI

character description

tonB-dependent receptor iron(III) dicitrate transport system outer membrane receptor ferric enterobactin receptor ferric-coprogen receptor colicin I receptor ferric enterobactin receptor nucleosides and deoxynucleosides, specific channel colicin S4 receptor colicin S4 receptor colicin S4 receptor OmpX

subcellular location

molecular no. of peptides sequence mass (Da)/pI recovered coverage (%) score

volume difference (iron limit vs control)

integral Omp 81876/5.55 integral Omp 85269/5.59

7 16

15 37

71 1.5 ( 0.36:0 165 1.14 ( 0.99:0

integral integral integral integral integral

Omp Omp Omp Omp Omp

82056/5.39 81182/4.75 73850/5.11 75413/5.32 33568/5.07

9 12 10 7 7

21 25 25 18 44

69 133 95 64 98

4.89 ( 2.54:0 2.47 ( 2.83:0 6.74 ( 2.07: 0.47 ( 0.66 0.51 ( 0.30: 0.03 ( 0.04 0.39 ( 0.22: 2.26 ( 1.56

integral integral integral integral

Omp Omp Omp Omp

258615.93 25861/5.93 25861/5.93 16350/5.04

5 5 4 5

28 28 71 43

73 62 62 68

0.93 ( 0.46: 4.28 ( 1.08 0.43 ( 0.61: 8.35 ( 0.66 0.51 ( 0.50: 4.09 ( 0.73 2.40 ( 0.50: 1.13 ( 0.69

searched using MASCOT (http://www.matrixscience.com), in which the E. coli protein database was defined as a matching species, and the mass tolerance was 150 ppm. Gene Cloning and Overexpression. Full-length DNAs encoding Tsx, OmpW, and OmpX were separately isolated by PCR with E. coli K-12 DNA as the template: forward primer 5′-GCG GAA TTC ATG AAA AAA ACA TTA CTG GC-3′ and reverse primer 5′-GAC AAG CTT TCA GAA GTT GTA ACC TAC-3′ for tsx, forward primer 5′-CCC GAA TTC ATG AAA AAG TTA ACA G-3′ and reverse primer 5′-GGG AAG CTT TTA AAA ACG ATA TCC TGC-3′ for ompW, and forward primer 5′-GCG GAA TTC ATG AAA AAA ATT GCA TGT CT-3′ and reverse primer 5′-GAC AAG CTT TTA GAA GCG GTA ACC AAC-3′ for ompX. The PCR products were digested with EcoRI and HindIII and ligated into pET-32a with the same sites to allow expression of these proteins in E. coli BL21. Meanwhile, the PCR fragments were cloned into vector pLLP-ompA, namely, ptsx, pompW, and pompX, and overexpressed in E. coli Top10F′. This vector is accompanied with an ompA signal which can secrete the expression protein into the periplasmic space. These plasmids were identified by sequencing. These transformed strains were grown in the presence of ampicillin (100 µg/mL) and induced by IPTG (68 µg/mL) or/and DIP when necessary. Antibody Preparation and Western Blotting. The preparation of antibodies was performed essentially as described by Peng et al.21 Each 0.5 mL of immunogen consisted of the recombinant OmpX, OmpW, or Tsx (one-half) and Freund’s complete adjuvant (one-half) for the first injection and Freund’s incomplete adjuvant (one-half) for the following injections. Rabbits received three subcutaneous injections. We drew blood on day 5 after the last injection and collected sera for use. Western blotting on a 1-DE gel was performed as previously described.20 Briefly, equal amounts of OM proteins were loaded for separation by SDS-PAGE on a 12% gel. Gels of OM proteins were transferred to a NC membrane for 3 h at 200 mA in transfer buffer (48 mM Tris, 39 mM glycine, and 20% methanol) at 4 °C. Rabbit anti-OmpX, -Tsx, and -OmpW were separately used as the primary antibodies, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody was used as the secondary one.

Results Effect of Iron Limitation on Bacterial Growth and OM Protein Expression. We first investigated the effect of DIP on the iron, copper, and magnesium content of bacterial cells and

found that the level of iron was sharply decreased but those of copper and magnesium did not when DIP was added (Figure 1A). Then, the effect of iron limitation on bacterial growth was observed as shown in Figure 1B,C, indicating that the growth was dramatically inhibited when both 200 and 400 µM DIP were used, but this inhibition was completely and partly recovered via addition of FeCl3. Figure 1D shows these significant differences in a 10 h culture. These results indicate that DIP may chelate iron, which results in the inhibition of bacterial growth. Approximately 20 bands were detected in the preparations of the OM proteins by SDS-PAGE (Figure 1E). Altered band patterns were appreciated between the two cultures without and with DIP. Four protein bands, namely, bands a-d, altered in the sample with iron limitation at approximately 80 kDa (seen only in the experimental group), 76 kDa (increased), 16 kDa (increased), and 25 kDa (disappeared). The four bands were identified by MALDI-TOF MS. The results indicate that there was a mixture of proteins FepA and FecA in band a, whereas a single protein (CirA, OmpW, or OmpX) was located at bands c and d, in which OmpW was the OM protein that had disappeared. These results are summarized in Table 2. Indeed, care should be used in judging the result of each protein band which is actually a mixture of proteins if using MALDI-TOF analysis only because the proteins are overlapped and crowded in the 1-DE gel. Thus, we utilized 2-DE-based proteomic methodologies for further investigation of these samples. Analysis of OM Proteins using 2-DE Proteomic Methodologies. OM proteins were isolated by the use of the sarcosin-insoluble extraction method and analyzed by 2-DE. Figure 2A shows the two maps of OM proteins of E. coli K12 K99+ grown under normal and iron-deficient conditions. Approximately 80 protein spots were well-visualized on each of the two gels. Their molecular weights ranged from 20–90 kDa. Comparative analysis of the two gels with Melanie 4.0 indicated that 11 altered expression spots, namely, spots 1–11, were identified in the cells cultured in iron-deficient medium compare to the control. Of the 11 spots, spots 1–6 and 11 were upregulated and spots 7–10 were downregulated. The enlarged partial two-dimensional gels showing altered expression spots and a detailed comparative histogram view are shown in panels B and panels A-3 and A-4 of Figure 2, respectively. The 11 altered spots were excised from gels and digested with trypsin after being destained. The resulting digestion The Journal of Proteome Research • Vol. 7, No. 3, 2008 1239

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Figure 3. Effect of overexpression of OmpW, OmpX, and Tsx on the growth of E. coli Top10F′. (A) Growth curve of E. coli Top10F′ separately harboring pLLP-ompW, -ompX, and -tsx plasmids in LB medium without (A) and with (B) 500 µM DIP following IPTG induction. (C) Western blotting analysis of OM fractions from E. coli Top10F′ separately harboring pLLP-ompW, -ompX, and -tsx plasmids after incubation for 6 h with respect to no IPTG induction. (D) Survival rates from bacterial OD without divided by that with DIP for a 10 h culture. One asterisk indicates P < 0.05 vs Top10F′-pLLP; two asterisks indicate P < 0.01 vs Top10F′pLLP.

mixture was analyzed by MALDI-TOF MS to assign putative functions to these proteins. Table 3 shows the identification of the 11 altered spots using MALDI-TOF MS. Peptide masses 1240

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Lin et al. were searched using MASCOT, and the E. coli protein database was selected as a matching species with a mass tolerance of 150 ppm. The 11 spots were identified as eight uniquely OM proteins: YbiL (up spot 1), FecA (up spot 2), FepA (up spots 3 and 6), FhuE (up spot 4), CirA (up spot 5), Tsx (down spot 7), OmpW (down spots 8-10 or disappeared spots 9–10), and OmpX (up spot 11). Of the eight altered proteins, YbiL, FhuE, and Tsx were not identified with the use of 1-DE proteomic methodologies. Furthermore, protein isoforms can be profiled by using 2-DE proteomics. Two spots and three spots representing FepA and OmpW, respectively, are characterized in this study. These results indicate the advantages of 2-DE over 1-DE analysis. Tsx, OmpW, and OmpX were reported here to be novel proteins related to bacterial iron homeostasis (downregulated ompW was seen with the use of the DNA array method,13 and altered OmpW was noted in our latest report17). We cloned the three genes and prepared their antibodies to confirm the alteration in 1-DE and 2-DE by Western blotting analysis as shown in Figure 2C. The results show that Tsx and OmpW were downregulated in iron-deficient medium, whereas OmpX was upregulated in the same medium, consistent with the findings of 1-DE and 2-DE. Functional Analysis of Tsx, OmpW, and OmpX in Response to Iron Limitation by Using Gene-Overexpressed Stains. Moreover, the characterization of Tsx, OmpW, and OmpX in response to iron limitation was further investigated using gene-overexpressed strains. In this regard, tsx, ompW, and ompX were separately cloned into a pLLP-ompA vector and expressed in E. coli Top10F′, which can make these target proteins work in OM as natural OM proteins do. These bacteria harboring tsx, ompW, and ompX using a pLLP-ompA vector, ptsx, pompW, and pompX were separately cultured in LB medium for 2 h at 37 °C and then divided into two groups at dilution 1:100 without and with 500 µM DIP following IPTG induction for 2 h. The OD values of the two cultures were measured in intervals of 2 h (Figure 3A, B), and OM proteins were isolated after being cultured for 6 h and were used as Western blotting samples to confirm whether these overexpressed target proteins were located in OM. The results indicate that the levels of these three proteins were significantly elevated in the OM fraction when bacteria were induced with IPTG (Figure 3C). Meanwhile, the survival percentages of these bacteria separately harboring ptsx, pompW, and pompX were obtained by comparison of 10 h cultures without and with DIP (Figure 3D). We normalized the survival percentages of these bacterial strains by defining as a control the Top10F′-pLLP strain as 100% to limit the effect of DIP on bacterial growth. Our results show that the growth of bacteria harboring ptsx was distinctly disturbed (37.8 ( 2.9%; P < 0.01), and that with pompW was also significantly inhibited (83.2 ( 2.1%; P < 0.01); on the other hand, significantly greater growth was observed in the cells harboring pompX (106 ( 3.4%; P < 0.05). These results were consistent with the alteration of these proteins in 2-DE, suggesting that upregulated Tsx and OmpW affected bacterial growth whereas upregulated OmpX improved their growth. Functional Analysis of Tsx, OmpW, and OmpX in Response to Iron Limitation by Using Gene Deletion Mutants. We next investigated the function of tsx, ompW, and ompX in response to iron limitation using their gene deletion mutants. The growth curve of ∆tsx, ∆ompW, and ∆ompX was similar to that of their parental strain, E. coli K12 BW25113,

Tsx, OmpW, and Tsx for Iron Homeostasis

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Figure 4. Effect of ompW, ompX, and tsx deletion on the growth of E. coli K12 BW25113. Growth curves of E. coli K12 BW25113 and its derivatives, strains ∆ompW, ∆ompX, and ∆tsx, in LB medium without DIP (A) and with 500 µM DIP (B) and survival rates from bacterial OD without divided by that with DIP for a 10 h culture. Two asterisks indicate P < 0.01 vs E. coli K12 BW 25113 (C). (D) Effect of DIP on growth curves of ∆tsx. (E) Effect of DIP on growth curves of ∆ompW.

except for somewhat less growth of the ∆tsx stain at an early stage (Figure 4A), but the three mutants grew slower in LB medium with 500 µM DIP (Figure 4B). This analysis was further focused on a 10 h culture. On the basis of normalization of the control as 100%, the survival percentages of ∆tsx, ∆ompW, and ∆ompX were 86.8 ( 2.6, 84.8 ( 0.8, and 93.0 ( 1.3%, respectively (all P < 0.01) (Figure 4C). Of the three strains, ∆tsx and ∆ompW were more sensitive to DIP than ∆ompX. Thus, the effect of DIP on the growth of ∆tsx and ∆ompW was further investigated. The bacteria were separately cultured in LB medium with 0, 25, 50, and 200 µM DIP. Significant changes were characterized when the bacteria were cultured in LB medium with 200 µM DIP but not with 50 and 25 µM DIP, in which the growth was significantly inhibited in ∆tsx as opposed

to ∆ompW (Figure 4D, E). In summary, we failed to observe faster growth of ∆tsx and ∆ompW compared to their parent strain, but higher and lower survival percentages were found in ∆tsx and ∆ompX, respectively, than in their corresponding overexpressed strains. Interrelation of Tsx and OmpW. For an understanding of the interrelation of the two downregulated OM proteins, we separately extracted OM proteins of these bacteria harboring ptsx and pompW cultured without and with IPTG induction. These OM proteins were analyzed by SDS-PAGE, and the abundances of the bands were normalized with relative volume divided by the total volume over the whole line image. As a result, the expression of either of the two target proteins was upregulated by IPTG induction when another was downreguThe Journal of Proteome Research • Vol. 7, No. 3, 2008 1241

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Figure 5. Interactions between OmpW and Tsx. (A) CBB-stained SDS-PAGE of OM proteins of E. coli Top10F′ strains harboring ompW or tsx plasmids, grown in LB without or with IPTG induction. (B) Histogram comparative analysis of the altered abundance of the interrelation between OmpW and Tsx. (C) Western blotting of samples obtained from panel A and to confirm the alteration of the interrelation between OmpW and Tsx.

lated as shown in Figure 5A. The histogram comparative analysis of volume percentage shows that Tsx was downregulated from 6.68 to 3.47% when OmpW was upregulated from 13.52 to 22.30% while OmpW was downregulated sharply from 9.59 to 1.17% by overexpression of Tsx from 10.75 to 20.22% (Figure 5B). The interrelation was further validated by Western blotting as shown in Figure 5C. These results suggest the significantly negative regulation between Tsx with OmpW.

Discussion Reports have indicated that OM proteins play a critical role in iron uptake as siderophore receptors, of which CirA, FecA, FepA, FhuA, FhuE, and YbiL are widely known.22 Our current study highlights the identification of the two downregulated OM proteins, Tsx and OmpW. Tsx possesses such a relatively small size porin that it can be blocked by nucleosides.23 It functions as a substrate-specific channel for nucleosides and 1242

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deoxynucleosides and constitutes the receptor for colicin K and phage T6.25 OmpW representing three downregulation spots belongs to the ompW/alkL family. This protein functions as an ion channel in planar lipid bilayers26,27 and a receptor for colicin S4.28 Downregulated ompW was characterized in iron limitation medium with the use of a microarray in Pasteurella multocida.29 Our latest finding indicates that the OmpW-Frd complex is dissociated in medium with 200 µM DIP when downregulated OmpW was detected.17 Besides, OmpW of Shewanella oneidensis and P. multocida, being 52% and 45% identical to E. coli, respectively, experienced decreases in their levels under iron limitation conditions.29,30 Reports have indicated that OmpW is a salt-regulated and antibioticresistant OM protein in Vibrio parahaamolyticus, Vibrio alginolyticus, and Photobacterium damsela,6,31 and in E. coli.28 However, information regarding iron homeostasis for Tsx is not available. In this study, the functions of Tsx and OmpW in response to iron limitation were reversed with respect to that of siderophore receptors, indicating that they may function as iron exporters or users. Our results further indicate that Tsx may be a more important OM protein than OmpW in this response. Meanwhile, our results provide a key evidence that the two OM proteins may regulate each other. Therefore, Tsx and OmpW should be annotated as new iron regulation proteins. The identification of downregulated OM proteins shed light on a pathway for clarifying the network of OM proteins used in response to iron limitation. In addition, upregulated OmpX was first reported here to be an iron homeostasis protein in E. coli besides siderophore receptors CirA, FecA, FepA, FhuA, FhuE, and YbiL. OmpX belongs to a group of integral OM proteins that play important roles in adhesion32 and serum resistance.33 This protein was induced under acid or base conditions compared to pH 7.034 and involved in resistance to β-lactams. However, whether OmpX is a siderophore receptor or a porin for iron awaits investigation. In conclusion, we have clearly demonstrated that downregulated Tsx and OmpW in companion with upregulated OmpX form a characteristic feature in E. coli surviving in irondeficient medium, which highlights the pathway of iron homeostasis by regulation of OM proteins. Therefore, the significance of downregulated OM proteins in iron homeostasis should be great.

Acknowledgment. This work was sponsored by grants from China Hi-Tech Development Project “863” (2006AA09Z432), NSFC Project 30530610, the National Basic Research Program of China (2006CB101807), and the Foundation of Guangdong for Natural Sciences (7117645). References (1) Andrews, S. C.; Robinson, A. K.; Rodriguez-Quinones, F. Bacterial iron homeostasis. FEMS Microbiol. Rev. 2003, 27, 215–237. (2) Lin, J.; Huang, S.; Zhang, Q. Outer membrane proteins: Key players for bacterial adaptation in host niches. Microbes Infect. 2002, 4, 325–331. (3) Otto, B. R.; Verweij-van Vught, A. M.; MacLaren, D. M. Transferrins and heme-compounds as iron sources for pathogenic bacteria. Crit. Rev. Microbiol. 1992, 18, 217–233. (4) Faraldo-Gomez, J. D.; Sansom, M. S. Acquisition of siderophores in gram-negative bacteria. Nat. Rev. Mol. Cell Biol. 2003, 4, 105– 116. (5) Trias, J.; Nikaido, H. Outer membrane protein d2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 1990, 34, 52–57.

research articles

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