A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode

FEMS Microbiology Letters 215 (2002) 7^14

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A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in bio¢lm or in planktonic growth mode Fre¤de¤ric Tre¤moulet a , Ophe¤lie Duche¤ a , Abdelkader Namane b , Brigitte Martinie c , Jean-Claude Labadie a;
a

c

Station de Recherches sur la Viande, Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Theix, 63122 Saint Gene's Champanelle, France b Institut Pasteur, Genopole, Plate-forme Prote¤omique, 28 Rue du Docteur Roux, 75724 Paris Cedex 15, France Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Theix, 63122 Saint Gene's Champanelle, France Received 18 March 2002; received in revised form 24 June 2002; accepted 2 July 2002 First published online 28 August 2002

Abstract Escherichia coli 0157:H7 biofilms were studied by a new method of cultivation in order to identify some of the proteins involved in the biofilm phenotype. A proteomic analysis of sessile or planktonic bacteria of the same age was carried out by two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. Comparison of two-dimensional gels showed clear differences between protein patterns of sessile and planktonic cells. Fourteen proteins increased in biofilms, whereas three decreased. From these 17 proteins, 10 were identified by MALDI-TOF-MS and could be classified into four categories according to their function: (1) general metabolism proteins (malate dehydrogenase, thiamine-phosphate pyrophosphorylase), (2) sugar and amino acid transporters (D-ribose-binding periplasmic protein, D-galactose-binding protein, YBEJ), (3) regulator proteins (DNA starvation protein and H-NS) and (4) three proteins with unknown function. The results of this study showed that E. coli O157:H7 modified the expression of several proteins involved in biofilm growth mode. < 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Bio¢lm; Proteome ; Two-dimensionnal electrophoresis; Matrix-assisted laser desorption ionization-time of £ight; Escherichia coli O157 H7

1. Introduction It has long been recognized that in natural environments, bacteria are found predominantly as bio¢lms. These bio¢lms generally are de¢ned as microbial cells attached to a surface and encased in an extracellular polysaccharide matrix [1]. It is also widely accepted that bio¢lms exhibit a speci¢c phenotype which is characterized by a greater resistance to antimicrobial agents and biocides [2]. This property is important notably in the food industry because established bio¢lms on food plant facilities are very often responsible for contamination during the product processing. Escherichia coli O157:H7 is a Gram-negative pathogenic bacterium which is responsible for hemorrhagic colitis and

* Corresponding author. Tel : +33 (4) 73 62 41 57; Fax : +33 (4) 73 62 42 68. E-mail address : [email protected] (J.-C. Labadie).

the hemolytic^uremic syndrome. This organism is recognized as an important cause of food-borne diseases reported in the USA and other developed countries[3]. The highly virulent nature of this bacterium demands a rigorous control of its dissemination in the environment to ensure food safety. Several studies reported that E. coli O157:H7 are able to form bio¢lms on various abiotic surfaces [4] in food plants and to display a marked ability to survive harsh conditions applied in the food industry [5]. In order to ¢nd out the solutions limiting the impact of bio¢lms in food-processing environments, it is necessary to understand the physiology of these bacterial communities. One of the most powerful methods which gives global information on the physiology of bacteria is the proteomic analysis of cell-free ultrasonic extracts including two-dimensional (2-D) electrophoresis of soluble proteins and mass spectrometry analysis. The aim of the present work was to study the modi¢cations in protein expression of mature bio¢lms of E. coli O157:H7 NCTC 12900, with

0378-1097 / 02 / $22.00 < 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 7 9 - 0

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the combined use of 2-D electrophoresis, matrix-assisted laser desorption ionization-time of £ight mass spectrometry (MALDI-TOF-MS) and database searching.

2. Materials and methods 2.1. Bacterial strains and growth conditions E. coli O157:H7 NCTC 12900 is a non-pathogenic strain, phenotypically similar to the toxigenic strain of E. coli O157:H7 but devoid of the ability to produce verotoxins. E. coli O157:H7 NCTC 12900 was grown in modi¢ed M9 medium (Na2 HPO4 H2 O, 6 g; KH2 PO4 , 3 g; NH4 Cl, 1 g; NaCl, 0.5 g) supplemented with 0.05% (w/v) yeast nitrogen base (YNB ; Difco), 0.1% (w/v) MgSO4 and 0.3% (w/v) glucose. Prior to each experiment, E. coli O157:H7 was streaked on a nutrient agar (BHI, Difco, Detroit, MI, USA), incubated overnight at 37‡C and transferred to a 50-ml £ask containing 20 ml of M9 medium. The £ask was agitated in an orbital shaker (150 rpm) for 24 h at 20‡C and then transferred to 100 ml of the same medium which was cultivated in the same conditions. The bacteria were then harvested by centrifugation at 8000Ug for 10 min at 20‡C and washed twice in saline water (8.5 g of NaCl per liter). Dilutions with saline water were carried out to obtain accurate bacterial concentrations. 2.2. Planktonic and bio¢lm cultures For planktonic culture, 100 ml of M9 medium were adjusted to an optical density at 600 nm of 0.1. Cultures were grown in a rotary shaker (150 rpm) at 20‡C for 7 days. The glass ¢ber ¢lter method was used to produce bio¢lms[6]. Brie£y, bio¢lms were grown on a pre¢ltration membrane disk Glass Fiber Filter Extra Thick (GFF, Pall Bio Pharma, Ann Arbor, MI, USA) with 1 Wm pore size, 25 mm diameter and 1270 Wm thickness. Sterile GFF were inoculated with 500 Wl of the bacterial suspension. The adhesion of the bacterial cells to the glass ¢bers was completed in 5 min. After washing with saline water, the inoculated GFF were placed on a sterile GFF deposited in the center of a Petri plate (90 mm diameter) containing M9 medium with agar. The bio¢lms were incubated at 20‡C for 7 days. Mature bio¢lms or planktonic bacteria of the same age were diluted (1/10, w/v) in saline and stomached during 5 min. After serial decimal dilution in saline, bacteria were streaked on tryptic soy agar (TSA, Difco) and incubated for 24 h at 37‡C before counting. 2.3. Protein sample preparation Bacteria were centrifuged (20‡C, 8000Ug, 10 min) three times, washed and resuspended in 1 ml of 20 mM Tris bu¡er (pH 7.5) containing 5 mM EDTA and 5 mM

MgCl2 . The cells were sonicated (Vibra cell, Bioblock, Illkirch, France) three times for 2 min at 4‡C using a microtip setting at power 5 and 50% pulse duration. Protein concentration was determined according to the method of Bradford [7] with bovine serum albumin as standard. Samples were centrifuged at 14 000Ug for 10 min at 4‡C. The supernatant proteins were precipitated with ice-cold acetone for 2 h at 320‡C and centrifuged at 15 000Ug for 20 min at 4‡C. The pelleted proteins were solubilized in isoelectric focusing bu¡er containing 8 M urea, 2% (w/v) CHAPS and traces of bromophenol blue. The protein samples were stored at 320‡C until analysis. 2.4. 2-D electrophoresis 2-D electrophoresis was performed essentially according to the method described by O’Farrell [8]. Precast Immobiline DryStrip with non-linear gradient pH 3^10 (Pharmacia-Biotech, Orsay, France) was rehydrated overnight with 60 Wg protein sample in 8 M urea, 2 mM tributylphosphine (TBP), 2% (v/v) ampholytes pH 3^10, 2% (w/v) CHAPS and traces of bromophenol blue. TBP was used to improve protein solubility according to Herbert [9]. The ¢rst dimension was carried out with a Multiphor II system (Pharmacia Biotech) for a total of 63.7 kVh according to the manufacturer’s instructions. After isoelectric focusing, the strips were ¢rst equilibrated for 15 min in a 50 mM Tris^HCl bu¡er (pH 6.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), and 2 mM TBP. The same solution without TBP but with 2.5% (w/v) iodoacetamide and traces of bromophenol blue was used for another 15-min equilibration period. The second-dimensional separation was a vertical SDS^ polyacrylamide gel electrophoresis (PAGE) with 12.5% acrylamide-resolving gels (13.5U15 cm) and 1 cm stacking gel (4%). Separation was performed in two steps. 12.5 mA per gel were applied during the stacking period and 25 mA were used for the separation period. Gels were stained either with Coomassie brilliant blue (Brilliant blue R 250, Sigma) or with a silver-staining method [10]. Gels were scanned using a GS-700 Imaging densitometer (BioRad, Ivry sur Seine, France). 2.5. Statistical and image analysis Gels were analyzed with Melanie III software (release 3.03, Bio-Rad) for qualitative and quantitative analysis of protein spots visualized on 2-D gels. Calibration of gels with isoelectric point (pI) and molecular mass was performed with internal protein standards (2-D SDS^PAGE standards, Bio-Rad). Six gels per condition resulting of three independent protein extractions were analyzed and compared. Statistical analysis was performed with Student’s t-test (95% con¢dence interval) to ensure that only signi¢cant changes in spot intensity between the two conditions tested were taken into account.

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2.6. Protein identi¢cation by MALDI-TOF-MS After excision from 2-D gels staining with Coomassie blue, proteins were in-gel digested by trypsin (Promega). After extraction, peptides were mixed with a 10 mg l31 solution of K-cyano-4-hydroxycinnamic acid and deposited on the sample plate. Samples were allowed to dry and analyzed by MALDI-TOF (Voyager DE-STR, Perkin-Elmer, USA). Protein identi¢cations were realized by the method of peptide mass ¢ngerprinting and database searches in SWISS-PROT with minimal restrictions. Protein identi¢cation was con¢rmed by the comparison of the apparent pI and molecular mass obtained experimentally by 2-D electrophoresis and the theoretical value available in the SWISS-PROT database. 2.7. Database search for protein identi¢cation Monoisotopic peptide masses obtained from mass spectra were used to identify proteins using the MS-¢t program and searching in the SWISS-PROT database. The following parameters were used in the searches : E. coli species, protein molecular mass range from 1000 to 100 000 Da, trypsin digest with one missing cleavage, fragment ion mass tolerance of S 50 ppm and possible oxidation of methionine. 2.8. Scanning electron microscopy Bio¢lm bacteria were ¢xed with 6% glutaraldehyde, 0.2 M cacodylate bu¡er (pH 7.4) for 1 h at 4‡C. GFF were rinsed three times in 0.2 M cacodylate bu¡er for 10 min and post-¢xed for 1 h with osmic vapors. GFF samples were dehydrated in a graded ethanol series of 70, 95 and 100% (three times) for 10 min, then in a graded acetone series of 30, 50 and 100% (three times) for 10 min. Samples were mounted on aluminum stubs then coated with gold (Emscope SC500) and observed with a Philips SEM 505 scanning electron microscope.

3. Results

Fig. 1. Growth kinetics at 20‡C of (A) planktonic and (B) bio¢lm E. coli O157:H7 NCTC 12900 in modi¢ed synthetic M9 medium at 20‡C. Initial inoculation at Log10 = 6 (8) and at Log10 = 8 for bio¢lm growth (a). Results are averages of at least three independent repetitions.

during the ¢rst 2 or 3 days at 20‡C, reaching 109 CFU per GFF. Observation of bio¢lm formation was performed by scanning electron microscopy. One- or 7-day-old bio¢lms are shown in the micrographs of Fig. 2B and C, respectively. After overnight incubation at 20‡C, the E. coli O157:H7 cells adhered to the glass ¢bers presented in Fig. 2A and appeared encapsulated in a polymeric matrix (Fig. 2B). Seven-day-old bio¢lms showed numerous embedded adherent bacteria colonizing the ¢lters (Fig. 2C).

3.1. Bio¢lm growth and scanning electron microscopy 3.2. 2-D gel electrophoresis analysis The growth conditions of E. coli O157:H7 NCTC 12900 were ¢rst investigated on planktonic bacteria and bio¢lms. Bacteria were grown at 20‡C in synthetic M9 medium modi¢ed by supplementation with YNB, MgCl2 and glucose as previously described. This medium allowed a good growth of the free-living bacteria and bio¢lms, as shown in Fig. 1. In liquid medium at 20‡C, the stationary phase was reached in 1 or 2 days (Fig. 1A) and the cells numbers (109 CFU ml31 ) remained stable for up to 7 days. For the bio¢lms, the initial population was per ¢lter either 106 or 108 CFU (Fig. 1B). The E. coli O157:H7 bio¢lms grew

Total soluble proteins of E. coli O157:H7 cultured either in liquid medium or with GFF for 7 days at 20‡C were separated by 2-D gel electrophoresis. A protein was considered to be culture mode dependent if the spot density was signi¢cantly di¡erent after Student’s t-test performed with Melanie III software. Computer analysis of the protein pattern showed that more than 600 spots were visualized by silver staining (Fig. 3). Among the 600 spots detected, at least 17 proteins were signi¢cantly (P 6 0.05) up- or down-regulated by the growth of E. coli O157:H7

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MHþ matching in the E. coli database were also reported with the precision of the results given in v mass. The maximal mass tolerance accepted for protein identi¢cation was 50 ppm. The percentage of protein coverage is also indicated in Table 2 and is included between 22% and 42%. Finally, the peptide sequence of matched fragments with their position is noted. As shown in Table 2, protein spots 1, 2, 5, 6, 8, 10, 14, 15 and 17 which were expressed at a higher level in sessile E. coli O157:H7 were tentatively identi¢ed as YBFU_ECOLI protein (YBFU), DNA-binding protection during starvation (DPS), hypothetical oxidoreductase in METC-SUFI intergenic region, D-ribosebinding periplasmic protein (RBP), D-galactose-binding protein (GBP), malate dehydrogenase, amino acid ABC transporter-binding protein, thiamine-phosphate pyrophosphorylase (TPP-PPASE) and DNA-binding protein (H-NS), respectively. Only one protein (spot 4) expressed at a higher level in planktonic bacteria was identi¢ed by MALDI-TOF-MS and corresponded to a hypothetical 24.8-kDa protein. Comparison of the theoretical pI and molecular mass of the identi¢ed protein with experimental results obtained by 2-D gel electrophoresis permitted the con¢rmation of a correct identi¢cation.

4. Discussion

Fig. 2. Electron micrographs of (A) sterile glass ¢ber ¢lter, (B) 24-h-old bio¢lms and (C) 7-day-old bio¢lms of E. coli O157:H7 NCTC 12900 grown in modi¢ed synthetic M9 medium at 20‡C. Black bars = 10 Wm.

in bio¢lms. Table 1 shows these proteins, with their experimentally estimated pI, and molecular masses. Fourteen proteins showed increased synthesis when E. coli O157:H7 was cultured in bio¢lm. Only three showed a decreased synthesis in similar conditions and no protein was totally repressed. 3.3. Protein identi¢cation by MALDI-TOF-MS Ten proteins were identi¢ed by MALDI-TOF-MS as shown in Table 2. The name of the protein with the accession number given by the SWISS-PROT database is indicated in Fig. 3. The masses of peptide fragments

The bio¢lms studied in this work were grown on GFF, according to a new and convenient approach [6] successfully used to identify proteins after 2-D electrophoresis combined with MALDI-TOF-MS and database searching. The growth of E. coli O157:H7 on GFF at 20‡C lasted between 24 and 48 h. After that time, the E. coli O157:H7 bio¢lms remained stable during 7 days and microscopical observations of the ¢lters showed microcolonies in the 24h-old bio¢lms and numerous bacteria embedded in exopolymers in those of 7 days (Fig. 2C). Although the EPS produced in such conditions was not identi¢ed, the E. coli 0157:H7 bio¢lms observed on GFF resembled those already observed on other types of surface with other bacterial species or strains [1]. As the time necessary for a bio¢lm phenotype to be fully expressed is not known, protein extractions were made on mature bio¢lms (7-day-old) in order to be sure that the characters observed are related to true phenotypic properties. The comparisons realized between proteins extracted from bio¢lms and planktonic cells of the same age revealed numerous di¡erences in 2-D electrophoresis patterns. Several studies have already shown that protein synthesis is modi¢ed in bio¢lm versus planktonic grown bacteria [1], but only a limited number was dedicated to a global 2-D electrophoresis study allowing identi¢cation of proteins involved in the bio¢lm phenotype. In the present work, 17 proteins were identi¢ed and most of them were up-expressed in bio¢lms. From 17 varying proteins, 10 were tentatively identi¢ed by MALDI-TOF-MS

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Fig. 3. Silver-stained 2-D gel analysis of total soluble proteins of 7-day-old bio¢lms of E. coli O157:H7 growing at 20‡C for 7 days. Proteins were ¢rst subjected to isoelectric focusing (pH range 3^10) and resolved in the second dimension by SDS^PAGE (12.5%). Planktonic and bio¢lm protein patterns were compared with Melanie III software (release 3.03, Bio-Rad). Spots indicated by a black arrowhead are proteins with a signi¢cantly increased level in bio¢lms versus planktonic cells. Black circles indicate decreased spots in bio¢lms.

Table 1 Proteins induced or repressed in E. coli O157:H7 by bio¢lm development compared to planktonic mode Identi¢cation No.a

pIb

Molecular mass (kDa)b

Protein spots induced (+) or repressed (3) by bio¢lm growthc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

5.69 5.68 5.41 5.65 5.70 5.83 5.13 4.94 4.92 5.59 5.13 5.23 5.86 7.05 5.57 5.82 5.29

16.5 20.4 26.1 27.1 31.1 31.2 33.8 34.8 29.5 36.5 22.1 36.7 44.5 33.9 27.4 24.9 16.3

+ + + 3 + + 3 + + + + + + + + 3 +

a

Number of protein spots indicated on the gel of Fig. 3. pI and molecular mass are experimentally determined with known protein standards. c +, protein spot signi¢cantly (P 6 0.05) more important in E. coli O157:H7 grown in bio¢lm mode; 3, protein spot signi¢cantly (P 6 0.05) less important in E. coli O157:H7 grown in bio¢lm mode. b

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Table 2 Summary of the E. coli O157:H7 proteins identi¢ed by MALDI-TOF-MS showing di¡erent expression levels in bio¢lms Spot No. 1

2

4

5

6

8

10

14

Name; SWISS-PROT accession No. YBFU; P76492

Peptide MHþ (Da)a

770.4 824.4 951.5 1235.6 1397.7 1553.8 DNA protection during 951.5 starvation protein; P27430 1020.5 1485.7 1676.8 1678.8 1692.8 Hypothetical 24.8-kDa 923.3 protein in DCM-SHIA 1414.7 intergenic region ; P76344 1459.8 1490.7 2265.1 2281.1 Hypothetical oxidoreductase 807.4 in METC-SUFI intergenic 897.5 region; Q46857 1411.6 1621.8 2644.4 2660.4 D-Ribose-binding periplasmic 1242.6 protein ; P02925 1257.7 1385.8 1479.7 1509.9 1527.8 1555.8 1571.8 D-Galactose-binding protein ; 1135.5 P02927 1160.6 1243.7 1245.6 1248.6 1261.5 1439.7 1643.8 1717.9 1766.0 1780.9 1926.1 Malate dehydrogenase; 703.3 Q60150 703.3 791.5 1149.6 1247.7 1405.7 2402.2 Amino acid ABC transporter- 833.3 binding protein; P37902 950.5 1041.6 1511.8 1625.8 1641.7 1691.7

v in mass (ppm)b 38.5 320.1 18.1 3.5 311.6 9.9 336.2 23.5 12.1 13.5 35.1 8.5 324.0 37.6 9.8 3.3 3.9 0.5 32.6 39.2 3.5 3.3 312.6 310.2 34.7 5.9 14.7 15.3 6.0 10.6 14.0 5.9 5.9 38.3 36.7 10.11 319.3 31.4 4.1 312.6 2.0 37.6 39.4 311.9 317.1 9.4 7.8 36.8 26.2 20 0.9 5.7 8.0 10.9 1.5 31.3 35.1 0.9

Protein coverage (%)c

Start^end positiond

Peptide sequence of matched fragmente

27

103^108 39^45 109^116 46^55 90^102 89^102 10^17 124^132 70^82 55^69 105^118 55^69 128^134 89^99 161^172 40^53 100^119 100^119 37^43 212^219 238^248 63^76 148^170 148^170 167^178 71^81 151^164 179^191 254^268 165^178 102^115 102^115 171^181 161^170 137^148 35^44 105^115 35^44 149^160 270^286 105^119 85^102 316^331 251^269 82^87 273^278 234^240 263^272 143^153 218^233 57^81 186^192 39^47 90^98 123^136 199^213 199^213 48^61

(R)YLGYVR(F) (R)GYGLQMR(E) (R)FMVNVEGR(Y) (R)ELDREFGELK(E) (R)VTFLGFDAATEAR(Y) (R)RVTFLGFDAATEAR(Y) (K)ATNLLYTR(N) (R)YAIVANDVR(K) (R)TALIDHLDTMAER(A) (R)GANFIAVHEMLDGFR(T) (K)SYPLDIHNVQDHLK(E) (R)GANFIAVHEMLDGFR(T) 1 Met-ox (K)YDYDGYK(I) (K)TFAEIKDYYHK(G) (K)YIQFSDHIIAPR(K) (K)AANGVFDDANVQNR(T) (K)GYATDIEMIGIEDGIVEFHR(N) (K)GYATDIEMIGIEDGIVEFHR(N) 1 Met-ox (K)ALEVGYR(S) (K)TPAQIVIR(W) (R)IAENFDVWDFR(L) (K)NASVNREELFITTK(L) (R)LIDETGVTPVINQIELHPLMQQR(Q) (R)LIDETGVTPVINQIELHPLMQQR(Q) 1 Met-ox (R)GEGFQQAVAAHK(F) (K)ELANVQDDLTVR(G) (K)VIELQGIAGTSAAR(E) (K)FNVLASQPADFDR(I) (K)LAATIAQLPDQIGAK(G) (R)ERGEGFQQAVAAHK(F) (K)MANQANIPVITLDR(Q) (K)MANQANIPVITLDR(Q) 1 Met-ox (K)GEPGHPDAEAR(T) (K)DGQIQFVLLK(G) (K)ESGIIQGDLIAK(H) (K)YDDNFMSVVR(K) (R)GQNVPVVFFNK(E) (K)YDDNFMSVVR(K) 1 Met-ox (K)HWAANQGWDLNK(D) (K)SGALAGTVLNDANNQAK(A) (R)GQNVPVVFFNKEPSR(K) (K)ALAINLVDPAAAGTVIEK(A) (R)VPYVGVDKDNLAEFSK(K) (K)SSIPVFGVDALPEALALVK(S) (R)KPGMDR(S) (K)NGVEER(K) (R)FGLSLVR(A) (R)FFSQPLLLGK(N) (K)LFGVTTLDIIR(S) (K)AGGGSATLSMGQAAAR(F) (K)GFSGEDATPALEGADVVLISAGVAR(K) (K)DHGDSFR(T) (K)NGVIVVGHR(E) (K)LIPITSQNR(I) (K)QAAFSDTIFVVGTR(L) (R)AVAFMMDDALLAGER(A) 1 Met-ox (R)AVAFMMDDALLAGER(A) 2 Met-ox (R)ESSVPFSYYDNQQK(V)

35

29

22

27

42

25

22

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Table 2 (Continued). Spot No.

Name; SWISS-PROT accession No.

Peptide MHþ (Da)a

15

Thiamine-phosphate pyrophosphorylase ; P30137

17

DNA-binding protein H-NS ; P08936

743.4 1493.7 1656.8 2449.2 742.4 835.4 957.5 1575.7

v in mass (ppm)b 37.9 12.8 31.2 35.0 328.3 336.6 15.4 6.1

Protein coverage (%)c 27

25

Start^end positiond 28^34 1^12 45^60 78^99 6^11 56^61 32^39 40^53

Peptide sequence of matched fragmente

(R)LLDAGVR(T) MYQPDFPPVPFR(S) (R)DEEVEADVVAAIALGR(R) (K)HQAYGVHLGQEDLQATDLNAIR(A) (K)ILNNIR(T) (R)KLQQYR(E) (K)LEVVVNER(R) (R)REEESAAAAEVEER(T)

a

Values indicate monoisotopic masses. Mass di¡erences between the peptides from the unknown protein and a known peptide from a known E. coli protein. c Coverage of the identi¢ed peptides on the protein. d Position of the peptide in the protein. e Peptide sequence of the matching peptide from the identi¢ed protein. b

and database searches. The proteins identi¢ed could be classi¢ed into four categories based on their function: two general metabolism proteins (spots 10 and 15), three transporters (spots 6, 8 and 14), two DNA-binding protein regulators (spots 2 and 17) and three unknown proteins (spots 1, 4 and 5). Among general protein metabolism, malate dehydrogenase is the principal enzyme of the tricarboxylic acid cycle (TCA) ; it catalyzes the reversible conversion of L-malate and oxaloacetate and is NAD-dependent. It is well known that changing the conditions of cell growth such as oxygenation (aerobic or anaerobic growth) and the nature of carbon substrates highly in£uences the synthesis of this enzyme [11]. It was found in this study that this important enzyme of the TCA cycle was up-regulated when E. coli O157:H7 was grown in bio¢lm. This result revealed undoubtedly that the central metabolism of E. coli O157:H7 was a¡ected by the culture mode. The TPP-PPASE proteins, which also increased in bio¢lm, strengthened this observation. Indeed, this protein was involved in the phosphorylation of thiamine [12]. Thiamine pyrophosphate is an important cofactor of enzymes involved in carbon metabolism and particularly the pentose pathway. The increase in TPP-PPASE, which participates in the transketolase reaction of this pathway in bio¢lms, could favor the degradation of the ribose transported via the spot 6 protein identi¢ed as an RBP. Spot 8, which is a GBP, also indicated that lactose metabolism could be essential in mature bio¢lms devoid of glucose, particularly for bio¢lms of E. coli, which is a well known lactose-degrading bacterium. Spot 14, which also increased in bio¢lm-grown cultures, was an amino acid ABC transporter-binding protein (YBEJ). This protein belongs to the ABC superfamily transporters containing both uptake and e¥ux transport systems. The protein identi¢ed in this study is a solute periplasmic binding protein of E. coli O157:H7 with high substrate-speci¢c a⁄nity. YBEJ is probably speci¢c for two amino acids, aspartate and glutamate. This observation is interesting when aiming to propose characters

particularly involved in the bio¢lm phenotype. Indeed, it has been shown that glutamate as carbon source in liquid medium restores the ability of Pseudomonas £uorescens to form bio¢lms [13]. Spots 2 and 17 were identi¢ed as two DNA-binding protein regulators, DPS and H-NS, respectively. The DPS protein, which is overexpressed in bio¢lm, is also known to increase during the stationary phase and prolonged starvation [14]. DPS is a protein which binds to DNAs without apparent speci¢city and protects DNAs from oxidative damage [15]. As it also contributes to the acid tolerance of E. coli O157:H7 [16], it could be necessary for an overall protection of the bacterial chromosome of bacterial cells surviving in bio¢lms. In this work, H-NS was also found as overexpressed in bio¢lms of E. coli O157:H7. As this protein is a global gene regulator and in£uences the synthesis of many bacterial proteins [17], it seems a priori normal to observe that some of those which are necessary for bio¢lm growth or survival, are regulated by H-NS. Overexpression of DNA-binding proteins could also be related to the protection and/or regulation of some genes, before DNA exportation outside the cells in the bio¢lms. As a matter of fact it was recently shown by Whitchurch et al. [18] in Pseudomonas aeruginosa that extracellular DNA is required for bio¢lm formation. Although extracellular DNA was not yet observed in E. coli bio¢lms, it is possible that certain virulent strains do export their DNA in bio¢lms in order to protect them from speci¢c damage in human tissues. Finally, three other proteins (spot 1, YFBU, spot 4, hypothetical 24.8 kDa, and spot 5) were also identi¢ed, but no function was attributed to them except for spot 5, which corresponds to a hypothetical oxidoreductase. In conclusion, this study showed that 17 proteins were di¡erentially synthesized in bio¢lms of E. coli O157:H7 NCTC 12900. The combination of the methods used for bio¢lm formation, i.e., cultures on GFF with 2-D gel electrophoresis and MALDI-TOF-MS, has proven to be a convenient approach for studying the phenotype. Among

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