Immobilization Induces Alterations in the Outer ... - ACS Publications

Immobilization Induces Alterations in the Outer Membrane Protein Pattern of Yersinia ruckeri Laurent Coquet,†,| Pascal Cosette,†,| Emmanuelle De´ ,†,| Ludovic Galas,‡,| Hubert Vaudry,‡,| Christophe Rihouey,§,| Patrice Lerouge,§,| Guy-Alain Junter,†,| and Thierry Jouenne*,†,| IBBR Group, Laboratory “Polyme`res, Biopolyme`res, Membranes”, UMR 6522 CNRS, University of Rouen, Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA CNRS, University of Rouen, UMR 6037 CNRS, University of Rouen, and Proteomic Platform of the European Institute for Peptide Research (IFRMP23), France Received June 6, 2005

We compared the outer membrane protein (OMP) pattern of 2-day-old immobilized Yersinia ruckeri cells (IC) with that of early (FC24) and late (FC48) stationary-phase planktonic counterparts. Fifty-five OMPs were identified. Principal component analysis discriminated between the protein maps of FC and IC. Some OMPs involved in bacterial adaptation were accumulated by both FC48 and IC but the expression of other proteins was controlled by the sessile mode of growth. Keywords: biofilm • proteome • outer membrane • immobilized cells • pH

Introduction The innate property of bacteria to colonize accessible surfaces and to form biofilms is a well-established strategy for their survival. Biofilms are ubiquitous.1 Their occurrence is widely spread in natural environments as well as in industrial installations. Since they are involved in many infectious diseases and display a high resistance to antimicrobials,2 a lot of investigations have focused on the gene expression in biofilm bacteria.3-6 Proteomic analyses, based on the high separation efficiency of two-dimensional gel electrophoresis, represent a powerful approach to get detailed information on the physiology of bacteria,7 including biofilm organisms.8-12 While the cell envelope plays an important role in bacterial responses to environmental alterations,13-16 most proteomic analyses on biofilm cells consist in comparing the whole protein pattern of sessile and planktonic organisms, few being devoted to changes in the membranal proteome.17-18 We showed previously that Yersinia ruckeri, a common fish pathogen,19 displayed a high ability to form biofilms on solid supports.20,21 In the present work, we determined whether this species underwent changes in its outer membrane protein (OMP) pattern when it was artificially entrapped in a polysaccharide gel matrix, such immobilized-cell structure representing a good model of mature biofilms.22 The formation of biofilms involves in fact a coordinated series of molecular * To whom correspondence should be addressed. UMR 6522 CNRS, Faculty of Sciences, University of Rouen, 76821 Mont-Saint-Aignan Cedex, France. Fax: +33 2 35 14 67 13. E-mail: [email protected]. † IBBR Group, Laboratory “Polyme`res, Biopolyme`res, Membranes”. ‡ Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA CNRS, University of Rouen. § UMR 6037 CNRS, University of Rouen. | Proteomic Platform of the European Institute for Peptide Research (IFRMP23).

1988

Journal of Proteome Research 2005, 4, 1988-1998

Published on Web 11/08/2005

events.23 Appendages and envelope structures (e.g., flagella, fimbriae, curli, NlpE lipoprotein, ...) have been reported to have a substantial influence on attachment and may also play a role in biofilm development (see the review by Lejeune).24 By screening a library of Tn5 insertions in a nonpiliated Pseudomonas aeruginosa strain, Vallet et al.25 identified genes involved in bacterial adherence, i.e., a cluster of genes (cup genes) specifying the components of a chaperone/usher pathway involved in assembly of fimbrial subunits in microorganisms. Activation of the Cpx two-component signal transduction system by the adhesion of Escherichia coli cells to hydrophobic surfaces has also been reported.26 This two-component system has been implied in the modulation of curli expression.27 Recently, a new two-component system controlling the expression of P. aeruginosa fimbrial cup genes was described.28 Moreover, it is now recognized that multidrug resistance pumps29,30 and the decreased outer membrane permeability31 are implied in the resistance of Gram-negative bacteria to antibiotics. Some of the noteworthy changes in the outer membrane profiles of sessile cells pointed out the existence of environmental alterations within biofilms.18 It has been suggested that mature biofilm cells have stationary growth phase traits as reduced growth and metabolic activity.32-35 In a previous proteomic study,36 we confirmed this by showing a strong reduction of L-[35S]methionine incorporation in gelentrapped E. coli cells as compared to exponential- and stationary-phase planktonic organisms.. To investigate the stationary phase character of bacterial life within biofilms, we compared the OMP patterns to early and late stationary phase with that of immobilized cells.

Experimental Procedures Bacterial Strain and Free-Cell Growth Conditions. Y. ruckeri strain ATCC 29473 was used. The strain was maintained as 10.1021/pr050165c CCC: $30.25

 2005 American Chemical Society

OMP of Immobilized Y. ruckeri Cells

glycerol stocks and stored at -80 °C. Pre-cultures were performed in unbaffled Erlenmeyer flasks (50-mL volume) containing 15 mL of Columbia broth (Difco Laboratories, Detroit, MI). Flasks were incubated at 25 °C for 18 h in a gyratory water bath shaker (agitation speed, 115 rev/min). Bacteria were harvested by centrifugation and suspended again in sterile distilled water. The number of Colony Forming Units (CFU) was estimated by optical density measurement at 550 nm referred to a calibration curve. Free-cell cultures were performed in diluted (10% v/v) Columbia broth (initial cell concentration: 107 CFU/mL) for an incubation period of 24 or 48 h. Preparation and Incubation of Artificial Biofilms. A 2% w/v solution of agar (Diagnostics Pasteur, Marnes-la-Coquette, France) in sterile distilled water was cooled to 38 °C and inoculated with Y. ruckeri cells (final concentration, 107 CFU/ mL gel). After homogenization, a volume of 5 mL of the mixture was poured into a Petri dish (diameter, 5 cm) where it hardened at room temperature. A 5-mL volume of diluted (10% v/v) Columbia broth was then poured on the inoculated agar plate. The dishes (fifty per experiment) were incubated for 48 h at 25 °C. The medium was replaced after 24 h. Bacterial Growth Kinetics. Every 2 h during the incubation period (sampling was stopped during night), an immobilized cell structure was removed from its dish and disrupted in 15 mL of 0.1 M phosphate buffer, pH 7.0, using a blender. Appropriate dilutions of the resulting mixture were plated out on Tryptic Soy Agar (Difco). Colonies were enumerated after incubation of the plates for 48 h at 25 °C. All counts were performed in duplicate. The growth of free-cell cultures was also monitored by periodically plating out appropriate decimal dilutions of the cell suspension. pH Gradient Determination. To measure pH values within the immobilized-cell gel, we used confocal scanning laser microscopy with the Oregon Green 514 Dye from Molecular probes Europe BV (Leiden, Netherlands). Acidification progressively protonates this fluorinated analogue of fluorescein to different forms with specific spectral characteristics. The acidic pH range over which the probe can be used is that expected within the agar matrix. The staining protocol was the following. After incubation of the gel structure for 24 h, the probe was added into the growth medium (final concentration: 0.01 mM). Diffusion of the probe within the agar matrix was controlled by using a sterile gel. After 48 h of incubation (i.e., 24 h of contact with the probe), a thin portion of the gel (length, 5 mm; thickness, 0.1 mm; height, 3 mm) was cut using a microtome.22 The gel portion was then observed. Stained samples were examined by epifluorescence using a dual-channel confocal scanning laser microscope (CSLM, Leica, Heidelberg, Germany) equipped with a Diaplan optical system and an argon/krypton ion laser (excitation wavelength: 514 nm). Filter sets I2 (green fluorescence) and N2 (red fluorescence) were used. For pH calibration, the labeling was performed as described above by using a sterile agar matrix immersed in different buffer solutions (pH ranging from 3 to 8). The pH of free-cell cultures was monitored using an Ag/AgCl pH electrode (Fisher Bioblock Scientific, Illkirch, France). Extraction of Bacteria from Artificial Biofilms. After incubation for 48 h, each immobilized-cell structure was removed from its dish and crushed in 0.1 M phosphate buffer, pH 7, as described above. Gel particles were then eliminated by filtration through a glass-fiber membrane (GF/C from Whatman, Maid-

research articles stone, UK; pore size, 1,2 µm) under vacuum. Filtrates obtained from 50 dishes were pooled to obtain sufficient OMP contents. Bacteria remaining in the filtrate were collected by centrifugation (1300 × g for 15 min). Preparation and Analysis of Outer Membrane Extracts. Crude outer membrane extracts were prepared from bacterial pellets following the spheroplast procedure of Mizuno and Kageyama.37 Experiments were performed on free and immobilized cells. Briefly, bacterial pellets (free cells and microorganisms extracted from agar gel structures as described above) were washed twice with 20% (w/v) sucrose. Cells (ca. 1.5 g wet weight) were suspended in a digestion solution of the following composition: 27 mL of 1.25 M sucrose; 10 mL of 0.1 M Tris-HCl, pH 7.8; 0.8 mL of 1% (w/v) Na-EDTA, pH 7.0 and 1.8 mL of 0.5% lysozyme. The mixture was incubated for 1 h at 30 °C in the presence of DNase and RNase (3 µg/mL; Sigma, St Louis, MO) and MgCl2 (3µM). Spheroplasts were first collected by centrifugation (20 min at 10 000 × g). Outer membranes were next pelleted (150 000 × g for 1 h at 4 °C) and resuspended in 300 µL of IEF buffer composed of 5 M urea, 2 M thiourea, 33 mM 3-[3-chloamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 2 mM tributyl phosphine, 10 mM dithiothreitol and 2% (v/v) carrier ampholytes (pH 3.510; Sigma). The amount of proteins in the sample was measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Protein patterns were analyzed by two-dimensional gel electrophoresis (2-DE). One hundred micrograms of protein was added to IEF buffer containing 0.4% (w/v) Coomassie blue G 250 (Sigma) (final volume, 400 µL).38 The first-dimension gel separation was carried out with Immobiline Dry Strips NL (18 cm, pH 3-10NL, Amersham Pharmacia Biotech, Uppsala, Sweden). IEF was performed as follows: 150 V for 1 h, 350 V for 15 min, 750 V for 45 min, 1.5 kV for 1 h and 3.5 kV for 17 h (1 mA, constant) for a total of 61.8 kVh. The second dimension was obtained by SDS-PAGE using a 12.5% (w/v) polyacrylamide resolving gel (width, 16 cm; length, 20 cm; thickness, 0.75 mm). After migration, proteins were visualized by silver nitrate staining (developing duration: 15 min).39 Gel Analysis. All experiments were duplicated and the corresponding gels made in triplicate. Gels were scanned using the GS-800 Imaging densitometer (Bio-Rad) and analyzed using the PDQuest software (version 6.21, Bio-Rad). For each experimental condition, the six 2-D gels were matched together to form a standard image. The different standard gels were then matched together so that the same spot in different gels had the same number. All spot quantifications were performed on a Gaussian image as already detailed.40 The pH gradient was determined with an isoelectric focusing calibration kit (broad pI kit, pH 3-10, Pharmacia). Molecular masses were estimated on the basis of comigrating broad-range standards (Bio-Rad) in the second dimension. Data Processing and Statistical Analyses. Excel (Microsoft Corporation, Santa Clara, CA) and Sigmaplot 8.0 (Jandel Scientific, Corte Madera, CA) were used for data processing. Statgraphics Plus 4.0 (Manugistics, Rockville, MD) was used for multivariate analyses and descriptive statistics. Protein spots from sessile and planktonic bacteria were considered to display significant quantitative differences if they fulfilled the following criteria:12 p values of e 0.05 (t-test); detection threshold, average volume g 20 (n ) 6); differential tolerance, fold change g 2. N-Terminal Amino Acid Sequence Analysis. After 2-DE, proteins were electro-transferred onto 0,2-µm pore-size PVDF membranes (Millipore, Bedford, MA) using a semi-dry elecJournal of Proteome Research • Vol. 4, No. 6, 2005 1989

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Figure 1. Growth kinetics of planktonic ([0]) and immobilized (`) Y. ruckeri cells.

trophoretic transfer cell (Trans-BlotSD, Bio-Rad). Proteins were transferred for 1.5 h at 150 mA. Protein spots were visualized by staining with 0.1% (w/v) Coomassie brilliant blue G 250 and spots of interest were excised using an automatic spot cutter (ProXCISION, Perkin-Elmer, Boston, MA). The N-terminal sequences of proteins were determined by introducing the blots into an Applied Biosystems 492 automated protein sequencer. Runs of Edman degradation (13 cycles of pulsed-liquid chemistry) were carried out. The sequences obtained were matched to public protein sequence databases with PATTINPROT, a software developed at the PBIL in Lyon, France (http:\\ npsa-pbil.ibcp.fr), and with MS-PATTERN on the protein prospector web site (http://prospector.ucsf.edu). For inconclusive searches, sequences were matched against microbial genomes at the NCBI using the more general tBLASTn algorithm.41 Protein Identification by LC-MS/MS. Protein spots were visualized by G250 Coomassie blue, excised and tryptically digested with an automatic digester (MultiPROBE II, PerkinElmer). After lyophilization, the peptide extracts were resuspended in 10 µL of 0.2% formic acid/5% acetonitrile. The samples were analyzed by nanoLC/MS/MS. A 5-µL portion of the sample was injected onto an Ultimate nanoLC system (Dionex-LC Packings, Voisins Le Bretonneux, France). Peptides were enriched and desalted on an RP-C18 trap column, and separated on a 75 µm ID × 15 cm C18 column. A 45-min linear gradient (10% to 45% acetonitrile in 0.2% formic acid) at a flow rate of 200 nL/min was used. The eluent was analyzed on a Q-TRAP System (Applied Biosystems, Courtaboeuf, France) equipped with a nanospray source. For protein identification, MS/MS peak lists were extracted and compared to the NCBInr protein database restricted to bacteria, using the Mascot Software.

Results Bacterial Growth. The growth kinetics of planktonic cells (Figure 1) showed that Y. ruckeri grew exponentially (doubling time, 60 min) and reached a stationary growth phase in 1 day at a cell concentration of 109 CFU/mL. The stationary growth phase continued over 72 h with no decline (death) phase. After a latent phase of about 4 h, the cell population of immobilized cultures also reached 109 CFU/g gel at 1 day and then stabilized. Immobilized cells displayed the same generation time as planktonic counterparts. 1990

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Figure 2. Silver-stained experimental 2-DE gel obtained with OMP extract of 1-day-old planktonic bacteria. Protein loading, 100 µg of proteins.

Figure 3. Silver-stained experimental 2-DE gel obtained with OMP extract of 2-days-old planktonic bacteria. Protein loading, 100 µg of proteins.

Gel Analysis. Proteomics analyses were performed on planktonic bacteria in the early (FC24) and late (FC48) stationary phase of growth and on immobilized cells (IC). From computerassisted image analysis, a total of 213 spots was discriminated on 2-DE electropherograms (Figures 2-4). However, only 138 spots with average volume was g20, were considered for qualitative and quantitative analyses of protein pattern alter-

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Figure 4. Silver-stained experimental 2-DE gel obtained with OMP extract of immobilized bacteria. Protein loading, 100 µg of proteins.

ations. The corresponding proteins were expressed in at least one of the three tested incubation conditions. These spot data, i.e., a matrix of 138 rows (i.e., observations, consisting of the spot density values averaged over 6 gels) and 3 columns (variables), were first submitted to principal component analysis (PCA) to assess statistical analogies or differences between the three protein maps. PCA was performed after vertical standardization of the raw data (i.e., conversion to normal scores), implying that the same total amount of protein material was recovered on each 2-DE electropherogram. PCA (Figure 5) extracted a widely predominant component (PC1), accounting for 84.1% of the variability in the data (eigenvalue, 2.52) and clearly related to overall protein abundance. The 3 variables obviously displayed close coordinates along this first dimension, while observations were distributed along PC1 according to their intensity value averaged over the 3 incubation conditions (i.e., the variables). Variables were more discriminated along the second component (PC2) that reflected the protein content in a given incubation condition and opposed the two growth modes, i.e., free vs immobilized. This pattern remained unchanged when the most abundant protein spots (e.g., spots 11, 16, 25, and 20 in Figure 5) were excluded from PCA (not shown). To quantitatively assess the differences in protein levels according to the incubation conditions, spot intensities of proteins produced by FC24, FC48, and IC bacteria were compared spot by spot (Table 1). Increasing the incubation duration from 24 to 48 h doubled the number of expressed OMPs in planktonic cells. About 74% of the proteins expressed by IC were also present in FC48. The number of proteins whose level statistically varied between immobilized and planktonic organisms, depending on the incubation time, represented more than 50% of the detected spots. Protein Identification. Fifty-five proteins (whose amounts were changed and/or corresponding to the most intense spots)

Figure 5. PCA of the protein spot data matrix (138 rows × 3 columns) visualized by the score-loading biplot in PC1 × PC2. In PCA the original variables are transformed into new orthogonal ones, principal components (PCs). Each PC is a linear combination of the original variables. PC1 accounts for the largest amount of data variance, and so on. In this way, the original data are projected into a new coordinate system in which the observations are described by the scores and the variables by the loadings. The biplot shows scores (0) while loadings associated with each of the 3 variables (i.e., incubation conditions: FC24, FC48, bacteria in the early and late stationary phase of growth, respectively; IC, immobilized cells) are indicated as vectors. Numbers refer to identified: (see Table 2) majority spots (over the 3 incubation conditions). The biplot diagram provides a means of identifying variables which contribute strongly to each PC and facilitates an investigation of relationships between observations and variables. Table 1. Influence of the Growth Mode and Incubation Time on Membrane Protein Expression in Y. ruckeri Cells X/Ya no. of spots

FC24/FC48

FC24/IC

FC48/IC

total absent in X absent in Y overexpressed in X underexpressed in X unvarying

113 59 8 1 (1)b 67 (43)c 45 (39.8%)

103 49 15 6 (5)b 46(38)c 51 (49.5%)

129 24 41 32 (21)b 22 (19)c 75 (58.1%)

a Incubation conditions X and Y were compared. b In parentheses, absent in Y. c In parentheses, absent in X.

were identified by N-terminal microsequencing and LC-MS/ MS (Table 2). Most spots correspond to proteins that were found from the genome of Yersinia pestis. The other gene products were mainly identified from Escherichia coli or Salmonella typhimurium genomes. Two proteins were labeled as unknown since their N-terminal sequences did not match with any known sequence (spots 17 and 18). Some polypeptides involved in the bacterial stress response, e.g., GsrA, OsmY, DppA, and GroEL were accumulated in IC when FC24 were used as control. However, these polypeptides appeared unchanged in immobilized cells as compared with FC48, because they were accumulated by suspended cells during the last 24 h of incubation. On the other hand, the relative expression level of some proteins, e.g., FliC, OmpW, TolC, MalM, the malate dehydrogenase (Mdh), and the putative surface antigen, did not depend on the free-cell reference because they showed a very similar expression level in FC24 and FC48. Some of these spot alterations are shown in Figure 6 Isoform Detection. Four OMPs (spots 1-3 and 5) displayed the same apparent molecular mass (about 50 kDa) but differed in pI value (from 4.3 to 5.1). Three of these proteins had the Journal of Proteome Research • Vol. 4, No. 6, 2005 1991

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Table 2. Behavior of Identified Proteins in Immobilized Cells behaviorb in immobilized cells as compared with

function

motility

efflux pump Porin

spota

1 2 3 5 6 119 11 25 31 32 58

adaptation

113 12

16 21

metabolism

4 26 27 28 69

8

9 29

30 40

44

48

1992

N-terminal sequencing * and/or peptides MS/MS sequencing

FSQAVSGL* AVINTNSLSLLT* AVINTNSLSLLT* AVINTNSLSL* ENLLQVYKQA* QLDQTTQR LSQDLAR APKDNT-YTG* ATLKPEGQQALDQLYAQLSSIDPK APKDNT-YTGG* APKDNT* APKDNT* DGSVVVLGFADR AALIECLAPDR AQGVQLAAK DGSVVVLGFADR GEPLSFRLDGVIPGWTEGLK DQLIAGVQDAFANK LDGVIPGWTEGLK ITLVIPPELAYGK DSDTVVVNYK TDEEIEK YRDTFAK HEEGDYIFRA* AETASSSTQQ* RGELGIMGTELNSELAK NLTSQMVEFGQVK AEIGTLPVGSK IADSDQLR NLTAIK SKIVKVIG* GESTVTGGYA* EDTLLNKASS* KDVKFGNE* ITILPQEVESLAK LAIANSADSQALK AIADIAAQNR SGVGFHILK LPMAVAGQ* AFNIDAQR DDVIIAVNR DEQTDIALLQLTDAK LLGRDEQTDIALLQLTDAK IPNITLLATG* TIKVGINGFG* SYAGQEIVSNASCTTNCLAPLAK GVLGYTEDDVVSTDFNGEK VPTPNVSVVDLTAR STEIKTQVVV* ALAEHGIVFGEPK YDAVLVAIGR VNADYVEAFTKGEVK QSVDQPVQTGYK TALAIDAIINQR GYLADVELAK YAIALNLER ILEVPVGR TALAQYR IEEDLLGTR ISDVPEFVR AIENFYISNSK AFQVLLNEETK EVPAEAYYGVHTLR SNNIRIEEDLLGTR GEYQYLNPNDHLNK VNPVIPEVVNQVCFK AFQVLLNEETKNLQR TQLQDAVPMTLGQEFR TGLNEINLPELQAGSSIMPAK IEEDLLGTR ISDVPEFVR AIENFYISNSK AFQVLLNEETK


estimated

coverage (%)

microorganism

locus

1 day-old freecells

2 days-old free cells

2 3 3 3 2 3

Escherichia coli Y. pestis Y. pestis Y. pestis Y. pestis Y. pestis

b1076 YPO1842 YPO1842 YPO1842 YPO0663 YPO0663

----++

----++

10

Y. pestis

YPO1435

)

)

OmpA OmpA OmpA OmpA

3 2 2 9

Y. pestis Y. pestis Y. pestis Y. pestis

YPO1435 YPO1435 YPO1435 YPO1435

) ) ) )

) ) )

4.6 8.4

OmpA FkpA

4 32

Y. pestis Y. pestis

YPO1435 YPO0195

)

) )

17 57

5.4 8.5

OmpW GsrA

5 14

Y. pestis Y. pestis

YPO2201 YPO3382

+

)

55 16 18 62 53

5.2 6.1 8.5 4.6 7.3

R-enolase OmpX OsmY GroEL SurA

2 3 5 1 11

Y. pestis Y. pestis Y. pestis Y. pestis Y. pestis

YPO3376 YPO2506 YPO0431 YPO0351 YPO0494

) ) + + )

) ) ) ) )

52

8.9

DegQ

13

Y. pestis

YPO3566

)

)

37 36

7.8 8.3

AnsB GAPDH-A

3 20

Y. pestis Y. pestis

YPO1386 YPO2157

) )

)

64

6.9

LpdA

7

Y. pestis

YPO3417

)

)

62

5.9

AtpA

14

S. typhi

STY3911

+

)

55

5.1

AspA

31

Y. pestis

YPO0348

-

)

54

5.1

AspA

31

Y. pestis

YPO0348

-

)

Mr

pI

protein

51 50 51 50 56 57

4.3 4.8 5.1 5.6 6.1 7.5

FlgE FliC FliC FliC TolC TolC

39

8.0

OmpA

31 39 39 75

8.0 7.1 8.4 8.0

31 37

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OMP of Immobilized Y. ruckeri Cells Table 2 (Continued)

behaviorb in immobilized cells as compared with

function

metabolism

spota

EVPAEAYYGVHTLR SNNIRIEEDLLGTR GEYQYLNPNDHLNK VNPVIPEVVNQVCFK AFQVLLNEETKNLQR TQLQDAVPMTLGQEFR TGLNEINLPELQAGSSIMPAK 67 MKKTKIV-TI* 87 VILLDKVA*

120

124 transport


7 15 19

20 22 23 61 62

DIADAVTAAGVEVAK VANLGSLGDQVNVK NFLVPQGK LFGSIGTR MKVAVLGAAG* SDLFNVNAGIVR LFGITTLDTIR FFAQPILLGK GITFDSGGYSLK LPLAEFHR KIDGQGIK EVRIEITQGV* ADGTYQTIYK VTDADYFGTGLGIAVR DECQIMPYPNPADLAR TYTFHLR KPLDNVK LVEFK ILYK AVVPAGVQLA* AEQQLDKDSAIVPVYYYVNAR DTRIGVTIFK* KL-VATDT* IEGVPESNISR LLAEAGYTK AVVPA-VQ*

DSAIVPVYYYVNAR IEGVPESNISR 65 NKHINADTDYSIAEAAFNK HINADTDYSIAEAAFNK QTVEAALNDAATR TWEEIPALDK DKPLGAVALK GYNGLAEVGK SFQEQLAK VTIEHPDK 70 DECQIMPYPNPADLAR LVFSITPDASVR 73 ASTISPANVS* TTQLLDPAK LTPALGQK TFIGSVK 79 IPLLQNGTFDFECGSTTNNLER ESSVPFSYYDNQQK AVVVTSGTTSEVLLNK NNGVIVVGHR 84 GYHSVVVVKA EFLDKELNVKT TNLHNASDYSGVIQGILGGKI AFGFADPDSTSGFLIPNQSFK EINLGILGGQNQTQQIGDNMCVKE 135 MIEFKNVLKA* hypothetical 10 AILIIVL- -G*

estimated

2 days-old free cells

61 15

7.9 Pyruvate Kinase I 8.2 50S ribosomal protein L9

2 35

Y. pestis Y. pestis

YPO2393 YPO3536

+ +

+ )

34

7.2 Mdh

14

Y. pestis

YPO3516

++

++

57

6.4 Peptidase B

6

Y. pestis

YPO2889

++

++

54 29

7.0 TolB 6.3 ArtI

2 11

Y. pestis Y.pestis

YPO1124 YPO1351

) )

) )

58

7.1 DppA

7

E. carotovora

ECA4394

+

)

65

8.3 OppA

6

S. typhimurium STM1746

+

)

36 30 65

6.2 GBP 8.7 GlnH 8.1 OppA

3 3 4

S. typhimurium STM2190 S. typhimurium STM0830 Y. pestis YPO2182

+ + +

) ) )

65

8.4 OppA

6

Salmonella STM1746 typhimurium

+

+

46

8.6 MalE

24

Y. pestis

YPO3714

+

)

57

6.5 DppA

5

Erwinia carotovora

ECA4394

+

+

37

8.5 Mal M

11

Y. pestis

YPO3710

+

+

34

9.4 GlnH

23

Y. pestis

YPO2615

+

+

34

7.2 Putative substrate binding periplasmic transport protein 8.2 GlnQ 8.0 Putative membrane protein 8.6 Putative exported protein 8.7 Putative periplasmic binding protein 8.5 Putative OMP

28

Y. pestis

YPO1183

-

)

4 3

Y. pestis Y. pestis

YPO2514 YPO3058

++ -

++ -

2

Y. pestis

YPO2305

+

)

3

Y. pestis

YPO3633

+

)

4

S. typhimurium STM1131

+

)

13 ELAPEKA*

38

14 EKKHEIAVVA*

36

24 ATLDYRHEYA*

24

locus

1 day-old free cells

pI

33 36

protein

coverage (%) microorganism

Mr

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Table 2 (Continued) behaviorb in immobilized cells as compared with

function

hypothetical

spota


pI

33 FNIDSTQVSLTPDKK

79

5.1 Outer membrane protein

13

ERPTIASITFSGNK ALFATGNFEDVR GLEDFYYSVGK DIHFEGLQR DGNTLLVQVK LAGDLETLR QNLEASGVR YGYAYPR VNVDAGNR 74 LNELLDGALKDINAANLR

32

8.6 Putative exported protein 5.8 Hypothetical protein

7

VIVYPIGIGQLGR YVEVHQPLSR EGIVINLAELR 123 FNIDSTQVSLTPDKK ALFATGNFEDVR GLEDFYYSVGK SEQFQFNIGK DGNTLIVQVK LVFTEGVSAK LAGDLETLR GFFPTSGVK 17 ISITGEAGRH* 18 QKTAAP-QFP*

45

protein

coverage (%) microorganism

Mr

122 LIGENTTFTVPNDGRPLEAIAADYK

unknown

estimated

locus



Salmonella typhi

STY0247

)

-

E. carotovora

ECA3908

+

)

17

Y. pestis

y1940

++

++

90

5.2 Putative surface antigen

11

Y. pestis

YPO1052

++

++

15 16

7.2 8.3 -

-

-

-

) )

) -

a The spot numbers correlate with the numbers indicated in Figures 2, 3 and 4. b +, overexpressed; ++, appearing; ), unvariable; -, underexpressed; - -, disappearing.

same N-terminal amino acid sequence (A-V-I-N-T-N-S-LS-L-L-T). This sequence showed 100% identity with that of flagellin of Y. pestis. The sequence analysis of the fourth protein (spot 1) indicated that this protein had 100% identity with the flagellar hook protein of E. coli. The N-terminal sequence of spot 31 displayed 100% identity with the β-barrel membrane protein OmpA of Y. pestis. The same protein was identified from spots 11, 25, 32, 58, and 113. Protein TolC was also found in two spots (spots 6 and 119) showing an observed molecular mass of 56-57 KDa. The pI values of these spots differed considerably (1.4 pH units). The two spots displayed a different behavior: spot 6 intensity decreased in IC whereas protein corresponding to spot 119 was accumulated. Protein OppA was also recovered in 3 spots (spots 20, 61 and 62). pH Monitoring in Immobilized Structures. To evaluate the environmental conditions within the immobilized-cell structure, we measured the pH within the gel matrix. We also monitored changes of the pH values in the free-cell culture. While the pH remained stable in the free-cell culture over the whole incubation period (around 7.5, not shown), a sharp pH gradient was established within the polymer matrix after incubation for 2 days (Figure 7). Cell clusters with pH values of 4 were visible in the upper layers of the agar sheet, close to the liquid phase. Down to 1 mm depth, colonies at pH 5 were found whereas microcolonies exhibiting pH values between 6 and 7 were observed in the deeper gel areas. Not surprisingly, the biomass distribution inside the gel appeared heterogeneous. The cell concentration progressively decreased as the distance inside the gel increased. 1994


Discussion As confirmed by the biomass distribution within the gel matrix, very similar to that of biofilms,42 polysaccharide gelentrapped organisms represent a simple model structure of natural mature biofilms.22 This cellular concentration gradient is mainly due to mass transfer limitations in the polymer matrix.22 We previously showed that gel-entrapped cells displayed a low susceptibility to antibiotics.43,44 This high resistance of sessile bacteria to antimicrobials constitutes the main problem linked to biofilms in the medical field.2 The fact that Y. ruckeri was able to survive in fish farm water as biofilms that are resistant to antibiotics,21 emphasizes the risk of environmental and economical problems due to the establishment of Y. ruckeri biofilms in the environment. The biofilmlike structure was also used to compare the whole proteome of free and immobilized E. coli36,45 and Pseudomonas aeruginosa41 cells. In a recent paper,8 the validity of this model was indirectly proved in the results of a PCA that was used to interpret the variations in the protein maps of P. aeruginosa from two natural (attached) and one artificial (gel-entrapped) immobilized-cell systems together with their free counterparts. Indeed, the growth modes (immobilized vs planktonic) were fairly separated by the multivariate analysis. Protein identification showed the good efficacy of the outer membrane purification. Only 6 cytoplasmic proteins (the aspartate ammonia-lyase (AspA), the dihydrolipoamide dehydrogenase (LpdA), the glyceraldehyde-3-phosphate dehydrogenase (GADPH), the peptidase B, the pyruvate kinase I and the 50S ribosomal protein L9) were found among the 55 spots that were identified. Most polypeptides exhibited a high

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Figure 6. Comparison of the behavior of some proteins in planktonic and immobilized cells.

sequence identity with proteins of Y. pestis whose genome is probably the closest to that of Y. ruckeri. PCA of protein spot intensities allowed to discriminate between the two bacterial growth modes. This compares with previous data obtained on the whole proteome of agarentrapped P. aeruginosa cells,8,41 highlighting statistically significant differences in total protein expression by bacteria cultured in the free or immobilized state. The alteration level of the outer membrane proteome (around 50%) in IC (Table 1) is in accordance with those reported in the litterature17,18 and greater than most of those obtained on the whole proteome (see the review by Jouenne et al.46). In our previous study of the overall proteome of gel-immobilized P. aeruginosa, for instance, the proportion of spots affected by immobilization was only about 20% (Table 3). Immobilization was shown to induce essentially protein underexpression compared to free cell growth. Furthermore, increasing the incubation time of free

cells did not induce the drastic protein overexpression observed on Y. ruckeri subproteome. These differences probably point out the strategic role of the outer membrane in the cell adaptation.13-16 FC48 and IC accumulated some polypeptides involved in bacterial adaptation, e.g., GsrA,47 GroEL,48 and most bacterial periplasmic substrate-binding proteins (ArtI, DppA, GlnH, MalE, GBP, and OppA) that are implicated in protein folding, in addition to their transport function.49 Though predominantly cytoplasmicswe consequently cannot rule out the role of the cytoplasmic contamination (some cytoplasmique proteins were actually identified, see above)sGroEL has been localized in the membrane and periplasm50 and, more recently, in the extracellular medium.51 GroEL might be involved in the folding of membrane-associated proteins48 and in cell adherence.52 The overproduction of adaptation proteins is probably induced by alterations in the environmental conditions. It is in fact Journal of Proteome Research • Vol. 4, No. 6, 2005 1995

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Coquet et al.

Figure 7. CSLM images of a 2-day-old cell biofilm-like structure stained with Oregon Green 514 probe showing the pH gradient within the polymer matrix (magnification: ×32). Table 3. Influence of the Growth Mode and Incubation Time on Total Protein Expression in Pseudomonas aeruginosa Cellsd X/Ya no. of spots

FC18/FC48

FC18/IC

FC48/IC

total 777 824 816 absent in X 78 125 142 absent in Y 103 166 158 overexpressed in X 52 (35)b 131 (53)b 135 (51)b underexpressed in X 48 (25)c 33(25)c 37 (22)c unvarying 677 660 644 (87.1%) (80.1%) (78.9%) a Incubation conditions X and Y were compared. b In parentheses, absent in Y. c In parentheses, absent in X. P. aeruginosa was immobilized in agar gel layers and incubated for 48 h. Free cell cultures were incubated for 18 or 48 h. Adapted from Vilain et al.7

admitted that formation of a biofilm alters the microenvironment of its own inhabitants which leads to alterations in gene expression.53 This is due to diffusion limitations that occur within the biofilm, resulting in local variations in pH, nutrient and oxygen availability, and concentrations of bacterial metabolites. Confocal microscopy investigations confirmed the presence of local low pH in the highly colonized, peripheral areas of the polysaccharide matrix. The acidification of the culture medium could not be advanced to explain changes in the OMP pattern of planktonic cells during the last 24 h of incubation, however. Substrate depletions were more probably responsible for these alterations. In a recent paper, Beloin et al.34 showed that the biofilm lifestyle, although sharing similarities with the stationary growth phase, triggers the expression 1996


of specific sets of genes. The present study confirms these observations at the outer membrane level. Thus, the underexpression of OmpW (a little characterized protein), FliC or FlgE and the accumulation of GlnQ, Mdh, MalM or the putative surface antigen by immobilized cells, reflects the “biofilm phenotype” since these proteins exhibited the same quantities in 24-day- and 48-day-old planktonic bacteria. Another information brought by the study is the presence of isoforms for some OMPs. Thus, in accordance with our recent study performed on P. aeruginosa18 we found flagellin as 3 mass isoforms. The primary function of flagella seems to lie in initial cell-to-cell-interactions.54 The role of flagella in adherence and biofilm formation is still controversial and seems to be dependent on the carbon source and the experimental setup used.55 Once contact with the surface is made, bacteria use either flagella or pili to move along the surface until other bacteria are encountered and microcolonies are formed or enlarged.54 Both types A and B flagella can be phosphorylated56 but the precise role of phosphorylation on flagellin proteins remains unknown. It has been also shown that type A flagella of P. aeruginosa are glycosylated while type B are not.57 We reported previously that the high ability of Y. ruckeri to adhere on solid supports was correlated with a high flagella-mediated motility21 but genes involved in flagellum production were found to be down-regulated following initial adhesion.9 The disappearance of proteins FliC and FlgE in gelentrapped Y. ruckeri cells confirms this last observation. Recently, Patten et al.58 showed that flagellum expression in E. coli was repressed by RpoS, an alternative sigma factor that was described as essential in the biofilm growth mode.59 We also identified OmpA in six spots. Whereas spots 25 and 113, showing an apparent molecular mass of 31 kDa, corresponds to the native form of the protein60 and spot 58 (mass of 75 kDa) to the dimeric form, 3 spots of 39 kDa (spots 11, 31 and 32), corresponding to the denatured form of the protein, probably indicate posttranslation modifications whose exact nature remains to be determined (see Figure 2). The same mass for 3 spots rules out a protein degradation. The interpretation of the decrease in production of one of the isoforms (spot 31) is currently unclear. OmpA is able to form nonspecific diffusion channels with low permeability.60 The OmpA protein family is also required for structural integrity of the outer membrane and to maintain a normal cell shape.61 This porin has been involved in cell-to-cell interactions62 and binding to fibronectin.63 A decrease in OmpA amount has been reported in attached E. coli cells17 and might reflect a tendency of the microorganism to limit its exchanges with the extracellular medium, perhaps in response to stresses. A single gene codes for the TolC protein (gene YPO0663) in Y. pestis. However, we found TolC in two spots that showed different behaviors (one was accumulated by IC, the other under-expressed). The efflux system encoded by the acrABtolC operon expels a range of antibiotics64,65 but the resistance of biofilms to ciprofloxacin has been described as not mediated by the up-regulation of the acrAB operon. The different behaviors of the two isoforms call for further investigations. We detected GlnH in two spots (spots 23 and 79). Researches on databases showed that the sequence identity between these polypeptides was low (25%). N-terminal sequence of spot 23, which was accumulated by FC48 and IC, displayed 87% of identity with that of GlnH of S. typhimurium. In Y. pestis, two genes (YPO 2512 and YPO 2615) code for a GlnH. These proteins have a low sequence similarity (23%). Protein YPO


2512 displays a high similarity (87%) with GlnH of S. typhimurium whereas the sequence of YPO 2615 is very different. Peptide sequences of spot 79, which was accumulated in IC, were close to those of the protein coded by the gene YPO 2615. Consequently, Y. ruckeri expressed two GlnH proteins just like Y. pestis and only one of these transport systems was specifically mobilized by entrapped cells.

Concluding Remarks Analysis of outer membrane proteins remain a real challenge though most OMPs of gram-negative bacteria are mainly folded as β-sheets, which reduces their overall hydrophobicity as compared to integral helical membrane proteins. Together with the limited quantities of biological materials available from biofilm cultures, these difficulties explain why studies devoted to changes in the OMP patterns of sessile microorganisms are so scarce. Results show that although sharing similarities with that of late-stationary phase bacteria (suggesting these bacteria encounter similar environmental conditions), the OMP pattern of immobilized cells were markedly different from those of early- and late-stationary phase suspended bacteria. When the behavior of some OMPs (e.g., flagellins) in sessile organisms is understandable, the accumulation of some proteins (whose function is not sometimes clearly established) by entrapped cells demonstrates the specificity of this growth mode. This late observation questions about the impact of this protein landscape on bacterial physiology.

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