Outer membrane Modifications of Pseudomonas fluorescens MF37 in Response to Hyperosmolarity Muriel Guyard-Nicodème,† Alexis Bazire,‡ Gaëlle Hémery,†,‡ Thierry Meylheuc,§ Daniel Mollé,4 Nicole Orange,† Laurène Fito-Boncompte,†,‡ Marc Feuilloley,† Dominique Haras,‡ Alain Dufour,‡,# and Sylvie Chevalier*,†,# Laboratoire de Microbiologie du Froid, UPRES EA 2123, Université de Rouen, Evreux, France, Laboratoire de Biotechnologie et Chimie Marines, EA 3884, Université de Bretagne-Sud. Lorient, France, Laboratoire Bioadhésion et Hygiène des Matériaux, UMR/INRA-ENSIA, Massy, France, and INRA-Agrocampus, UMR 1253, Science et Technologie du Lait et de l’Oeuf, Rennes, France Received August 17, 2007
The effect of hyperosmotic condition on the outer membrane protein (Omp) composition of Pseudomonas fluorescens was investigated by proteomic analyses. The abundances of 12 proteins, including porins, lipoproteins, and the flagella subunit FliC, were modified. This was at least partly explained by altered gene expression, as shown by mRNA level study. In agreement with Omp changes, hyperosmotic condition resulted in vesicle formation and modifications of mobility and antibiotic susceptibility. Keywords: Pseudomonas fluorescens • outer membrane proteome • osmotic stress • porin • lipoprotein • FliC • swimming • vesicles
Introduction Pseudomonads are well-known for their striking ability to adapt to various ecological niches, including environments that are submitted to major osmotic modifications, such as seawater, marine hosts, soils, or even human hosts during chronic lung infection of cystic fibrosis patients by Pseudomonas aeruginosa.1,2 To colonize such environments, bacteria must be able to respond immediately to dramatic changes in extracellular osmolarity and sustain growth under lasting conditions of osmotic stress.3 Bacteria generally cope with hyperosmotic conditions by accumulating low molecular mass molecules that are compatible with cellular processes at high internal concentrations.4 These molecules, termed compatible solutes or osmoprotectants, are either synthesized by the bacteria or imported from the environment. Osmotically stressed P. aeruginosa PAO1 was shown to synthesize and accumulate glutamate, trehalose and N-acetylglutaminylglutamine amide (NAGGN) in the absence of exogenous osmoprotectant, but preferentially accumulated glycine betaine when the latter was added to the culture medium.5,6 To gain insight into other osmoadaptative processes than osmoprotectant accumulation, the transcriptional response of P. aeruginosa PA14 to both osmotic up-shock and growth under steady-state osmotic stress conditions was recently investigated using microarray analyses.7 This work * Corresponding author: Laboratoire de Microbiologie du Froid “Signaux et Microenvironnement”, 55 rue St. Germain-27000 Evreux, France. Phone: (33) 2.32.29.15.60. Fax: (33) 2 0.32.29.15.50. E-mail: sylvie.chevalier@ univ-rouen.fr. † Université de Rouen. ‡ Université de Bretagne-Sud. Lorient. § UMR/INRA-ENSIA. 4 INRA-Agrocampus. # These authors contributed equally to this work.
1218 Journal of Proteome Research 2008, 7, 1218–1225 Published on Web 01/25/2008
suggested the involvement of putative hydrophilins and revealed that the expression of 26 genes encoding proteins of known or probable regulatory function responds to osmotic shock. Except the genes for a type three secretion system, very few genes encoding membrane proteins were identified as osmotically induced by Aspedon et al.7 However, because of their location at the outmost area of the cell, outer membrane proteins (Omps) and lipoproteins are likely to play a key role in the bacterial adaptation to changes of environmental conditions, including osmotic stress. Indeed, the following Omp examples are well-documented. In Escherichia coli, the genes encoding porins OmpF and OmpC (proteins forming channels through the outer membrane), are regulated by osmotic stress: ompF and ompC expressions are, respectively, reduced and enhanced, at elevated osmolarity.4 Similar regulations were observed for porins MOMP and Omp50 in Campylobacter jejuni,8 OmpK35 and OmpK36 in Klebsiella pneumoniae,9 and OmpW and OmpV in Vibrio species.10,11 To our knowledge, no such example of osmotically regulated porins was reported in Pseudomonas species. In the latter, proteomic studies revealed large modifications of Omp patterns in response to various modifications of growth conditions,10–15 but hyperosmotic stress was not investigated. In the present study, we describe the effect of hyperosmolarity on the proteic composition of the outer membrane (OM) of Pseudomonas fluorescens strain MF37, a raw milk contaminant, using complementary and multidisciplinary approaches including OM proteome mapping, peptide mass fingerprinting, quantitative RT-PCR (qRT-PCR), and phenotype screening.
Experimental Section Bacterial Strains and Culture Conditions. P. fluorescens MF37 is a spontaneous rifampycin-resistant mutant of the 10.1021/pr070539x CCC: $40.75
2008 American Chemical Society
Outer Membrane Modifications of P. fluorescens MF37 16
strain MF0 which was isolated from raw milk, and P. fluorescens MF373 is an OprF-deficient mutant of strain MF37.17,18 These strains were grown at 28 °C on a rotary shaker (180 rpm) in minimal medium containing citrate as carbon source (MMC16). Except where otherwise indicated, hyperosmotic conditions were obtained by including 0.5 M NaCl in MMC. Growth was monitored spectrophotometrically at 580 nm. All cultures were inoculated at an initial OD580 of 0.05. Omp Extraction and SDS-PAGE Procedure. P. fluorescens MF37 cells were harvested at the entry into stationary phase, and proteins were specifically extracted from the OM as described by Jaouen et al.19 Protein concentrations were determined according to the Bio-Rad protein assay (Bio-Rad Laboratories, Marnes la Coquette, France) using bovine serum albumin as standard. The discontinuous buffer system of Laemmli with 17% polyacrylamide resolving and 7% polyacrylamide stacking gels at 8 mA overnight was used to separate Omps (40 µg). The proteins were visualized after Coomassie Brilliant Blue (CBB) R-250 staining (Sigma-Aldrich, SaintQuentin Fallavier, France). Two-Dimensional Gel Electrophoresis (2-DE). Immobilized linear pH gradients (IPGs) (11 cm, pH 4–7, Bio-Rad Laboratories) were used for the first dimension. A total of 200 µg of proteins was resolved in a solubilization/rehydratation solution containing 7 M urea, 2 M thiourea, 4% (w/v) Chaps, 65 mM dithiothreitol (DTT), and 1% (v/v) ampholytes pH 4–7. IPG strips were rehydrated for 12 h at 30 V. Proteins were focused at 8000 V until a total of 27 000 V was reached. The IPG strips were then reduced and alkylated in equilibration buffer made of 6 M urea, 0.375 M Tris-HCl, pH 8.8, 2% (w/v) SDS, 20% (v/ v) glycerol, and 2% (w/v) DTT for reduction or 2.5% (w/v) iodoacetamide for alkylation. Second dimension SDS-PAGE was accomplished on 17% polyacrylamide gels as described by Jaouen et al.19 The proteins were then visualized by CBB R-250 staining. Gel images were captured using a GS-800 densitometer (Bio-Rad Laboratories). Image analyses were performed using PDQuest 2-DE analysis software (Bio-Rad Laboratories). For each condition, three independent samples were analyzed on nine gels (each sample was loaded onto three gels). Spots which showed reproducible NaCl-dependent variations of intensities were selected for further analyses by trypsin digestion and peptide mass fingerprinting. In-Gel Trypsin Digestion. A slightly modified procedure of that developed by Schevchenko et al.20 was used for in-gel digestion. Briefly, bands or spots of interest were excised from SDS-PAGE or 2-DE gels. The gel pieces were washed in acetonitrile/50 mM ammonium bicarbonate (1:1) and dried in a Speed Vac concentrator (Bioblock, Illkirch, France). SDSPAGE bands were previously reduced with DTT (10 mM) at 60 °C for 40 min and alkylated by iodoacetamide (55 mM) in dark for 30 min. Each sample was then digested for 18 h at 37 °C with 0.5 µg of sequencing-grade modified porcine trypsin (Promega, Charbonnières, France) in 25 µL of 50 mM ammonium bicarbonate pH 8.0. The reactions were stopped by adding 2 µL of 5% trifluoroacetic acid (Pierce, Touzart et Matignon, Vitry sur Seine, France). Peptide Mass Fingerprinting. In-gel trypsin digestion products were analyzed by Matrix Assisted Laser Desorption Ionization-Quadrupole/Time Of Flight-Mass Spectrometry (MALDIQ/TOF-MS). In these experiments, 1 µL of peptide mixture was spotted onto the MALDI target plate, and 1 µL of R-cyano-4hydroxycinnamic acid matrix (2 mg · mL-1 in a solution of 0.1% trifluoroacetic acid and 70% acetonitrile) was added onto each
research articles sample. The spots were dried on the target plate and the latter was introduced into the hybrid Quadrupole Time Of Flight (Q/ TOF) mass spectrometer Qstar XL (MDS Sciex, Toronto, Canada). The samples were ionized with a laser beam (λ ) 337 nm). Each spectrum was established over an average of 250-500 laser shots. The more representative monocharged ions were automatically submitted to fragmentation with energy of collision near 0.05 eV/Da.21 Typically, oMALDI Xpert 2.0 software (MS and MS/MS) treated each sample well individually and generated an MS peak list which was submitted for a peptide mass fingerprinting search and used as a “survey scan” to determine peptide precursors for MS/MS acquisition. All data (MS and MS/MS) were analyzed using MASCOT software (v.2.1) for search into several databases (Swiss-Prot, NCBI) to identify the proteins present into each gel band or spot. PCR Amplification and Sequencing of Genes Coding for the Identified Proteins. Bacteria from a single colony of P. fluorescens MF37 were suspended into 100 µL of sterile water and the mixture was boiled for 10 min. After centrifugation at 13 000g for 2 min, 1 µL of the supernatant was subjected to PCR amplification. Primers (Supporting Information Table S1) were designed from P. fluorescens SBW25 gene sequences using Primer Express Software Version 3.0 (Applied Biosystems, Courtaboeuf, France). The PCR products were sequenced on both strands (Genome Express, Grenoble, France) and the sequences were deposited into the EMBL database. Accession numbers are given in Table 1. Quantitative Reverse Transcription-PCR (qRT-PCR). Bacteria were grown until midexponential growth phase or beginning of stationary phase was reached, and 2 vol of RNAprotect bacteria reagent (Qiagen, Hilden, Germany) was added to 5 × 109 cells. RNAs were extracted with the RNeasy Midi Kit and RNase-Free DNase Set (Qiagen). Residual DNAs were eliminated by acid phenol treatment. The absence of DNA was confirmed by verifying that PCR reactions failed without prior cDNA synthesis. RNAs were nonspecifically converted to singlestranded cDNAs using the High Capacity cDNA Archive Kit (Applied Biosystems). mRNAs of interest were quantified by real-time PCR amplification of their cDNAs. Primers (Supporting Information Table S1) were designed with Primer Express 3 software in order to have a Tm between 58 and 60 °C. Each primer pair was validated by verifying that the PCR efficiency E was above 0.95, and that a single PCR product with the expected Tm was obtained. PCR reactions were performed in triplicate with the 7300 Real Time PCR System apparatus (Applied Biosystems). The 25 µL reactions contained 12.5 µL of SYBR Green PCR Master Mix (including AmpliTaq Gold DNA Polymerase, Applied Biosystems), 900 µM of each primer, and cDNAs generated from 0.01 ng of total RNA. The conditions were 95 °C for 10 min for polymerase activation, and 40 cycles at 95 and 60 °C for 60 and 30 s, respectively. ROX dye was used as passive reference to normalize for non-PCR related fluorescence variations. The relative quantification of the mRNAs of interest was obtained by the comparative CT (2–∆∆CT) method,22 using 16S rRNA as endogenous control23 (Supporting Information Table S1). ∆CT values were calculated by subtracting the 16S CT value of a sample from the CT value of an mRNA of interest of the same sample. ∆∆CT values were then obtained by calculating the difference between: (i) the ∆CT value of a given mRNA resulting from cells grown to a specific stage (midexponential growth phase or beginning of stationary phase) in the presence of 0.5 M NaCl, and (ii) the ∆CT value of Journal of Proteome Research • Vol. 7, No. 3, 2008 1219
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Guyard-Nicodème et al.
Table 1. Putative Functions of the Identified Proteins and Effect of the Presence of NaCl on These Proteins and Their mRNA NaCl effect on band or spot nba
P. fluorescens protein nb of matched coverage MF37 EMBL ID name peptides (%) MW (Da)
2–2′
AAA85840
OprF
12
63
32104
7 10–11 13–14–16 15
AM494063 AJ888456 AJ866544 AJ866545
OprG OprD OprQ OprE
7 16 19 13
53 49 53 36
17740 47230 46224 48846
5
AM494064
OprL
5
29
6
AM494067
OprI
4
1 8–9
AM494068 AM494069
FliC GroEL
3 4–4′ 12
AM494065 AM494066 AM494070
P28 P27 P45
pI
putative functions
b
Porins 5.22 Major general porin, Growth in low-osmolarity medium, Cell shape, Environment sensing 5.77 General porin 5.94 Basic amino acid uptake 7.73 Specific porin, unknown substrate 8.65 Specific porin (arginine or proline uptake?), Anaerobic growth
proteinc
mRNAd
+
none
-e -e -
-(E,S) ND -(E) -(E)
-
-(E)
49
Lipoproteins 17698 5.68 Tol-OprL system, OM integrity, Cell morphology, OM biogenesis 8798 7.87 Cell shape Membrane fluidity
+
-(E)
10 5
35 17
Membrane-Associated Proteins 49241 5.08 Swimming, Cell-surface interaction 57064 4.99 Protein folding
+
-(E) -(E), +(S)
12 7 8
47 34 34
+ + -
+(E) +(E) +(S)
28560 27256 45629
Omp-like 9.21 OmpA-like protein, Peptidoglycan-bound 5.45 Unknown function 6.11 Long chain fatty acid transporter, Aromatic hydrocarbon degradation
a Numbering is according to Figure 2. b The functions are proposed on the bases of functions of similar proteins. c +, up-regulation; -, down-regulation. +, mRNA level increased at least 1.5-fold; -, mRNA level divided by at least 1.5 (relative level
