The low-salt stimulon in Vibrio parahaemolyticus

International Journal of Food Microbiology 137 (2010) 49–54

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o

The low-salt stimulon in Vibrio parahaemolyticus Lei Yang a,c,1, Lingjun Zhan a,c,1, Haihong Han b, He Gao a, Zhaobiao Guo a, Chuan Qin c, Ruifu Yang a, Xiumei Liu b,⁎, Dongsheng Zhou a,⁎ a b c

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing, 100050, China Institute of Laboratory Animal Sciences, Chinese Academy of Medical Peking Union Medical College, Beijing, 100021, China

a r t i c l e

i n f o

Article history: Received 8 July 2009 Received in revised form 15 October 2009 Accepted 11 November 2009 Keywords: Vibrio parahaemolyticus Osmotic stress Osmoadaptation Microarray

a b s t r a c t Vibrio parahaemolyticus is the leading cause of seafood-associated bacterial gastroenteritis and is a moderately halophilic, salt-requiring bacterium. Global gene expression profiles of V. parahaemolyticus grown under 2% and 0.66% NaCl were compared to define the low-salt stimulon. The ectABC-lysC operon for synthesis of the compatible solute ectoine, as well as three compatible solute transport systems, namely ProU (glycine betaine), OpuD1 (glycine betaine) and Pot2 (spermidine), was up-regulated under 2% NaCl relative to 0.66% NaCl. The 2% NaCl condition favored the inducible expression of OmpW, OmpN and OmpA2, while repressed the expression of OmpA1, OmpU and VP1008. These results indicated that, to master the hyperosmotic stress of saline environments, V. parahaemolyticus might not only accumulate osmoprotectants through uptake or endogenous synthesis of compatible solutes, but also remodel its profiles of outer membrane protein to restore its cell membrane. The above differentially regulated genes will provide novel candidates for the further investigation of the molecular mechanisms of osmoadaptation in V. parahaemolyticus. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Vibrio parahaemolyticus is a natural inhabitant in estuarine and coastal environments. It is the leading cause of seafood-associated bacterial gastroenteritis in North American and Southeast Asian countries (Mead et al., 1999; Su and Liu, 2007). Relevant infections are primarily due to the consumption of undercooked or raw seafood especially including oyster. Its characterized virulence determinants include thermostable direct haemolysin (TDH), TDH-related haemolysin (TRH), two different type III secretion systems, and the ability of adhesion and invasion of enterocytes (Park et al., 2004; Yeung and Boor, 2004; Zhang and Austin, 2005). Pandemic outbreaks of V. parahaemolyticus infections occurred since 1996 were initially linked to a clonal pandemic complex [GS-PCR+ (a positive detection of toxRS/new sequence), tdh+ and trh−] consisting of serovars O3:K6, O4:K68, O1:K25 and O1:KUT (Matsumoto et al., 2000; Okura et al., 2003; Nair et al., 2007). V. parahaemolyticus is a moderately halophilic, salt-requiring organism, and able to grow in NaCl concentrations of up to 8%. Its

⁎ Corresponding authors. E-mail addresses: [email protected] (X. Liu), [email protected] (D. Zhou). 1 L. Y. and L. Z. contributed equally to this work. 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.11.006

growth rate in the medium is enhanced by moderate increases in salt concentration, and the optimum growth is observed at 2 to 3% NaCl. In addition, a Na+ motive force (an electrochemical potential of Na+ across the cytoplasmic membrane) drives not only various transport systems for carbohydrates and amino acids, but also the flagellar rotation that is important for cell motility (Kuroda et al., 2005). At least three different Na+ / H+ antiporters contribute to the cytoplasmic Na+ circulation in V. parahaemolyticus (Kuroda et al., 2005). A high NaCl concentration in the growth medium denotes elevated extracellular osmolarity. Bacterial cells are required to maintain an intracellular osmotic pressure that is greater than that of growth medium in order to generate cell turgor, which is generally considered to be the driving force for cell extension, growth and division, and hence, bacteria have evolved a number of osmoadaptive strategies (Sleator and Hill, 2002). The ability to adapt to the elevated extracellular osmolarity is of fundamental importance for growth and survival of V. parahaemolyticus in estuarine and coastal environments. Mechanism of osmoadaptation is yet poorly known in this bacterium. In the present work, cDNA microarray in combination with real-time reverse transcription polymerase chain reaction (RT-PCR) and primer extension assay was used to compare the global mRNA profiles of V. parahaemolyticus grown in 2% and 0.66% NaCl, respectively. This analysis disclosed various potential osmoadaptive functions whose gene transcription was greatly up-regulated by 2% NaCl, especially those responsible for remodeling of porins and transport/synthesis of compatible osmolytes.

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2. Materials and methods 2.1. Bacterial growth V. parahaemolyticus strain LAMVP1 (GS-PCR+, tdh+ and trh−) used in this study is a pandemic O3:K6 strain isolated from a diarrhoeal patient in Thailand in 1996. According to the DNA microarray-based comparative genomic hybridization (data not shown), its genomic content is almost identical to that of another pandemic O3:K6 strain RIMD2210633 whose genomic sequence has been determined (Makino et al., 2003). A modified V-5 (MV-5) minimal medium (Sarker et al., 1994), consisting of 2% or 0.66% NaCl, 40 mM D-Mannitol, 10 mM (NH4)2SO4, 100 mM K2HPO4, 0.75 mM MgSO4, 0.05 mM CaCl2, 0.025 mM ZnCl2, and 0.025 mM FeSO4 (pH 8.0), was used for bacterial cultivation. Strain LAMVP1 was grown at 37 °C to an OD600 of 1.0, and then diluted 50-fold into fresh MV-5 medium. For the ‘continuous growth’ (CTG) design, bacteria were grown in MV-5 containing 2% (reference condition) or 0.66% (test condition) NaCl, and cell cultures with an OD600 of 1.2 at the middle exponential phase were harvested for RNA isolation. For the ‘shift growth’ (STG) design, bacteria were grown in MV-5 containing 2% NaCl to an OD600 of 1.2, and cells were then harvested by centrifugation at 3000 ×g for 5 min and then the culture supernatant was removed; the remaining cell pellets were resuspended in fresh MV-5 containing 2% (reference condition) or 0.66% (test condition) NaCl, and cells were allowed to grow at 37 °C for 1 h and then harvested for RNA isolation. Before bacterial harvest, double-volume of RNAprotect Bacteria Reagent (Qiagen) was added immediately to each cell culture.

genes. The transcriptional variation of mRNA levels was then calculated for each gene. A mean ratio of two was taken as the cutoff of statistical significance. 2.5. Primer extension An oligonucleotide primer (Supplementary Table 1) complementary to a portion of the RNA transcript of each gene was employed to synthesize cDNAs from the RNA templates by using the Primer Extension System-AMV Reverse Transcriptase kit (Promega) (Gao et al., 2008). Electrophoresis of primer extension products was performed with a 6% polyacrylamide/8 M urea gel. The gel was analyzed by autoradiography (Kodak film). To serve as sequence ladders, sequencing reactions were also performed with the same primers used for primer extension, using the fmol® DNA Cycle Sequencing System (Promega). 3. Results and discussion 3.1. Bacterial growth under 2% or 0.66% NaCl The bacterium was cultivated in a chemically defined minimal medium MV-5 with 0.66 or 2% NaCl, and its growth curves were measured. As shown in Fig. 1, the growth rate under 2% NaCl was significantly higher than that under 0.6 NaCl at the exponential phase, but the bacterial cultures under the two different conditions gave almost the same cell density (a OD600 of about 2.4) when entered the stationary phase. 3.2. Overview of microarray analysis

2.2. RNA isolation Total RNA extraction was performed by using MasterPure™ RNA Purification kits (Epicenter) as mentioned in our previous report (Yang et al., 2009). RNA quality was monitored by agarose gel electrophoresis and RNA quantity was measured by spectrophotometer. 2.3. Microarray expression analysis

The mRNA profiles of bacterial cells grown under 0.66 (test) and 2% (reference) NaCl were compared by cDNA microarray. Fold change of gene expression (test/reference) was obtained for each gene, and the significant change was determined by the SAM software (Tusher et al., 2001) in combination of a cutoff value of two fold change. The CTG design disclosed 151 up-regulated genes and 147 down-regulated ones (0.66% vs. 2% NaCl), while STG gave 143 and 62, respectively.

A cDNA microarray imprinted with 4669 genes representing about 97% of V. parahaemolyticus genome was used for transcriptome analysis (Han et al., 2007; Yang et al., 2009). For a two-sample (reference vs. test) microarray hybridization, four independent bacterial cultures from each condition were prepared as biological replicates for RNA isolation (Yang et al., 2009). Accordingly, for each time point, four dual-fluorescence-labeled cDNA probes were prepared to hybridize with four slides, respectively. Pairwise comparisons were made using dye swaps to avoid labeling bias. A ratio of mRNA levels (test/reference) was calculated for each gene. Significant changes of gene expression were identified with the SAM software (Tusher et al., 2001). After the SAM analysis, only genes with at least 2-fold changes in expression were collected for further analysis. 2.4. Real-time quantitative RT-PCR Gene-specific primers (Supplementary Table 1) were designed to produce a 150 to 200 bp amplicon for each gene tested in RT-PCR. Contaminated DNA in RNA samples was further removed by using the Amibion's DNA-free™ Kit. cDNAs were generated by using 5 µg of RNA and 3 µg of random hexamer primers. Using three independent cultures and RNA preparations, real-time PCR was performed in triplicate as described previously (Yang et al., 2009), through the LightCycler system (Roche) together with the SYBR Green master mix. Based on the standard curve of 16 S rRNA expression for each RNA preparation, the relative mRNA level was determined by the classic ΔCt method. 16S rRNA gene was used to normalize that of all the other

Fig. 1. Growth curves of V. parahaemolyticus. Strain LAMVP1 was grown in the MV-5 medium at 37 °C to an OD600 of 1.0, and then 50-fold diluted into 50 ml of fresh MV-5 medium containing 0.66 or 2% NaCl. Bacterial cells were cultivated at 37 °C with shaking at 200 rpm. The OD600 values were monitored for each culture with a 1 h interval until the cultures reached the stationary phase. Experiments were done in triplicate. The arrow indicated the time point for cell harvest at the middle exponential phase (an OD600 of 1.2).

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Table 1 The low-salt stimulon in V. parahaemolyticus.

Gene ID

Gene name

Fold change (test/reference)

Product

CTG

CTG

Remodeling of major OMPs VPA0096 ompW ompN VPA0166 ompA2 VPA1186 ompA1 VP0764 ompU VP2467 VP1008

-4.06 (-3.23) -6.44 (-8.89) -3.57 (-4.43) 5.21 (1.15) 7.67 (3.23) 2.87 (2.61)

-10.19 (-3.59) -6.91 (-8.92) -4.36 (-4.72) 5.40 (6.79) 3.20 (3.71) 7.44 (7.2)

Outer membrane protein W Outer membrane protein N Outer membrane protein A2 Outer membrane protein A1 Outer membrane protein U Putative outer membrane porin protein

Synthesis of ectoine VP1722 VP1721 VP1720 VP1719

-17.46 (-8.19) -21.47 -27.50 -11.12

-22.96 (-9.63) -10.00 -10.62 -7.35

L-2,4-diaminobutyric acid acetyltransferase Diaminobutyrate-2-oxoglutarate aminotransferase L-ectoine synthase Aspartate kinase

Transport of glycine betaine proV VP1726 proW VP1727 proX VP1728 opuD1 VP1456

-20.36 (-46.44) -37.65 -51.58 -4.95 (-8.61)

-15.2 (-20.4) -16.31 -22.54 -4.16 (-1.85)

Glycine betaine-binding periplasmic protein Glycine betaine transport system permease Glycine betaine transporter periplasmic subunit Betaine-choline-carnitine (BCCT) family transporter

Transport of spermidine potA2 VP1336 potB2 VP1337 potC2 VP1338

-2.32 (-4.93) -2.03 -2.35

-4.27 (-24.19) -2.98 -4.68

Spermidine transport system ATP-binding protein Spermidine transport system permease protein Spermidine transport system permease protein

Transduction of energy for active transport ttpC VP0166 exbB2 VP0165 exbD2 VP0164 tonB2 VP0163

-2.93 -2.78 -2.48 -2.27 (-4.67)

-3.88 -4.44 -4.41 -4.21 (-2.13)

TonB2 complex-associated transport protein C TonB system transport protein ExbB2 TonB system transport protein ExbD2 TonB2 protein

ectA ectB ectC lysC/aspK

Chaperonins VPA0286 VPA0287

groES2 groEL2

8.84 (3.64) 3.77 (2.16)

7.69 (7.93) 7.62 (2.19)

Chaperonin GroELS Chaperonin GroELS

Iron uptake VP0857 VP0858

feoA feoB

2.26 (2.3) 2.22

5.56 (5.3) 6.53

Ferrous iron transport protein A Ferrous iron transport protein B

-2.74 (-4.47) -3.06 -2.71 -3.99 -2.71 (-1.81)

-2.81 (-3.09) -2.46 -2.81 -4.07 -3.04 (-2.71)

Flagellins VP0790 VP2256 VP2257 VP2258 VP2259 Regulators VP0350 VPA0593 VPA0961

fliD FlaG

leuO

Small molecule Metabolism sdhC VP0843 VP1331 ccoO VP1543 glnA VP0121 VP1188 hemH VP1263 VP1941 argD VP2797 VPA0127 VPA0170 VPA0962

speA fdhD

Various/unknown functions metI VP0705 VP0706 metN VPA0173 VPA1209 secD VPA0851 VPA0973 VP0525 VP0634 VPA0040 VPA0172 VPA0310

2.73 (12.78) 11.22 (26.02) 2.10 (1.86)

2.32 (23.75) 4.80 (11.57) 5.08 (3.21)

-2.09 -2.08 -2.56 3.02 3.10 2.42 2.47 2.06

-3.24 -3.79 -2.00 2.37 2.55 3.40 2.48 3.37

2.01 2.69 2.15

5.11 4.88 2.68

2.98 2.85 2.21 2.91 5.49 3.08 3.21 5.35 2.79 2.05 2.97

3.97 3.82 3.14 5.57 22.01 2.07 2.47 3.69 4.41 3.12 4.89

Flagellin Flagellar capping protein Flagellar protein FlaG Flagellin Flagellin

Leucine transcriptional activator Putative transcriptional regulator Putative transcriptional regulator Succinate dehydrogenase cytochrome b556 large membrane subunit D-amino acid dehydrogenase, small subunit Cytochrome c oxidase, subunit CcoO Glutamine synthetase Putative ferredoxin oxidoreductase protein Phosphoribosylaminoimidazole-succinocarboxamide synthase Putative carboxynorspermidine dehydrogenase Bifunctional N-succinyldiaminopimelate-aminotransferase/ acetylornithine transaminase protein Cytochrome c-type protein YecK Arginine decarboxylase Putative formate dehydrogenase oxidoreductase protein

Methionine transport system permease protein Methionine transporter ATP-binding subunit Ribosomal protein S6 modification protein Preprotein translocase subunit SecD Putative formate transporter MFS family transport protein (permease) Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein (continued on next page)

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Table 1 (continued)

The data were presented as the mean change of mRNA level for each gene. The positive number stood for fold increased, while minus decreased. Those in the brackets represented the RT-PCR data for the 19 genes in Fig. 2. Putative operons were boxed.

Only genes (62 in total) each giving the same differential regulation under both CTG and STG designs were subjective to further analysis (Table 1), so as to exclude the genes with altered expression due to systemic errors (cell handing in STG) or subordinary effects (different growth rates in CTG). Thus, these 62 genes were considered as the members of the low-salt stimulon in V. parahaemolyticus, and they could be further divided into 11 functional categories as listed in Table 1. The microarray data (GSE16530) had been deposited in Gene Expression Omnibus (GEO).

NaCl, as disclosed by both microarray and RT-PCR. A single primer extension product was detected (Fig. 3) for each of the two genes, and thus a single inducible promoter was transcribed for each gene under the stimulating condition (0.66% NaCl). The nucleotide number of the transcription start site was taken as ‘+1’, and accordingly the promoter − 10 and − 35 elements for RNA polymerase (RNAP) recognition were predicted (Fig. 3).

3.3. Validation of microarray data by real-time RT-PCR

Elevated external osmolarity will trigger efflux of water from cells that causes the decrease of cytoplasm volume and thus the increase of ion concentration in the cytosol, which will trigger uptake or synthesis of water-soluble organic osmolytes (so-called compatible solutes) that can be accumulated to high intracellular concentrations without adversely affecting cellular processes (da Costa et al., 1998; Welsh, 2000; Sleator and Hill, 2002). Compatible solutes not only play their role as osmotic balancers, but also function as effective stabilizers of enzyme function, providing protection against salinity, high temperature, freeze–-thaw treatment and even drying (da Costa et al., 1998; Welsh, 2000; Sleator and Hill, 2002). Ectoine, betaine, glycine betaine, and spermidine have been recognized as effective compatible solutes in bacterial osmoadaptation (da Costa et al., 1998; Welsh, 2000; Sleator and Hill, 2002; Makino et al., 2003). Relevant genes for synthesis or transport of the above compatible solutes (Table 2) can be found in the genome of V. parahaemolyticus RIMD2210633 (Makino et al., 2003). V. parahaemolyticus harbors two putative compatible solute synthesis systems (ectoine and betaine) that are encoded by ectABC and betABI, respectively (Table 2). In the present work, ectABC and lysC was greatly up-regulated under 2% NaCl relative to 0.66% NaCl, for both CTG and STG designs (Table 1). Co-expression of ectoine biosynthesis genes ectABC and lysC-encoding aspartate kinase leads to markedly increased production of ectoine in E. coli, and thus aspartate kinase acts as the main limiting factor for ectoine production. Interestingly, ectABC and lysC constitutes a single putative operon in strain RIMD2210633 (Makino et al., 2003). Indeed, V. parahaemolyticus is able to use ectoine as a compatible solute, and the ΔectB mutant has a growth defect at high NaCl concentrations (Naughton et al., 2009). The ectABC-lysC operon appears to encode a major endogenous compatible solute synthesis system in V. parahaemolyticus. Beside endogenous synthesis, bacteria have evolved sophisticated mechanisms for uptaking compatible solutes from extracellular environments (Sleator and Hill, 2002). Strain RIMD2210633 harbors eight putative compatible solute transporters that are encoded by choVWX, proVWX, four opuD paralogues (opuD1, opuD2, opuD3 and opuD4), and two pot paralogues (potA1B1C1D1E1 and potA2B2C2), respectively (Table 2). ChoXWV is an ABC (ATP-binding cassette) transporter highly specific for choline uptake, and gives high homology scores with components of ProU (ProVWX) (Dupont et al., 2004). Both ProU and OpuD are the high-affinity glycine betaine transport system (Lucht and Bremer, 1994; Kapfhammer et al., 2005),

Real-time quantitative RT-PCR was employed to confirm the microarray data. Nineteen genes were chosen to compare data from the two techniques (Table 1). The resulting transcriptional ratio from real-time RT-PCR analysis was logarithm-transformed and then plotted against the average log ratio values obtained by microarray analysis (Fig. 2). As shown in Fig. 2, there was a strong positive correlation (R2 = 0.8141) between the two techniques. In addition, the two kinds of data gave the same tendency of transcriptional alteration for each of 34 (89%) of the 38 data points (19 genes × 2 growth designs) tested, confirming the reliability of microarray data. 3.4. Primer extension assays Primer extension experiments were performed for two arbitrarily selected genes, VP0857 (feoA) and VP1008, with total cellular RNA of the CTG growth design. The yield of primer extension product would indicate the mRNA expression level of the corresponding gene in each strain, and further could be employed to map the 5′ terminus of RNA transcript for each gene. This assay confirmed the up-regulation of these two genes under 0.66% NaCl relative to 2%

Fig. 2. Comparison of transcription measurements by microarray and RT-PCR. The relative transcriptional levels for 19 genes for both CTG and STG designs (Table 1) were determined by microarray and real-time RT-PCR. The real-time RT-PCR log2 values were plotted against the microarray data log2 values.

3.5. Transport or synthesis of compatible osmolytes

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Fig. 3. Primer extension assay. Lanes 1 and 2 represented 0.66 and 2% NaCl in MV-5, respectively. Lanes C, T, A and G represented the Sanger sequencing reactions. The yield and length of each primer extension product can be used to map the 5′ terminus of the RNA transcript, and thus the transcription start site. The transcription start sites were underlined in the upper electrophoresis images, and shown with asterisk-underlined bent arrows in the lower sequence images. The predicted promoter −10 and/or −35 elements were boxed.

Table 2 Putative osmoadaptation systems in V. parahaemolyticus RIMD2210633.

while Pot is competent for spermidine uptake (Arai et al., 2005). This study disclosed that at least three compatible-solute transport systems, namely ProU (ProVWX), OpuD1 and PotA2B2C2, were upregulated under 2% NaCl for both CTG and STG designs (Table 1). 3.6. Remodeling of major outer membrane proteins (OMPs) Differential expression of OMPs, especially including the porinforming OmpF and OmpC in E coli, upon changes in environmental conditions has been widely characterized. The OmpC channel appears to be slightly smaller than the OmpF channel on the basis of diffusion rates of organic molecules (Nikaido, 2003; Matsuyama et al., 2008). High osmotic strength favors the production of OmpC and represses the production of OmpF, which is important for minimizing their influx (Nikaido, 2003; Matsuyama et al., 2008). On the other hand, the increased production of OmpF under low-osmolarity conditions will benefit the bacterium by facilitating the influx of scarce nutrients (Nikaido, 2003; Matsuyama et al., 2008). In the present work, ompW, ompN and ompA2 were up-regulated under 2% NaCl for both CTG and STG designs, while ompA1, ompU and VP1008 were downregulated (Table 1), indicating the remodeling of major OMPs upon osmotic stress. Whether these six proteins form porin channels in outer membrane and whether/how they account for the alteration of bacterial outer membrane permeability during osmotolerance need to be elucidated. A previous proteomic study disclosed that OmpW was preferentially expressed under high osmolarity, while OmpV expression increased under low one (Xu et al., 2004). 3.7. Other salt-responsive genes

+, Detected to be the member of the low-salt stimulon in the present work. Putative operons were boxed.

Active transport across the outer membrane in gram-negative bacteria requires the energy that is generated by the proton motive force in the inner membrane. This energy is transferred to the outer membrane by the TonB protein in complex with the proteins ExbB and ExbD. Vibrios have two functional exbDB-tonB systems (TonB1 and TonB2) (Seliger et al., 2001), and herein ttpC, exbB2, exbD2 and tonB2 were up-regulated under 2% NaCl for both CTG and STG designs (Table 1). The ttpC, exbB2, exbD2 and tonB2 genes constitute a putative

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operon and TtpC plays a role in the TonB2-mediated transport, which appears to be a general feature in Vibrio TonB2 system (Stork et al., 2007). In bacteria, GroESL is one of the major molecular chaperones that protect newly synthesized or stress-denatured polypeptides from misfolding and aggregation in the highly crowded cellular environments (Frisk et al., 2002). Strain RIMD2210633 harbors two groESL systems, namely GroE1 (VP2851–2852) and GroE2 (VPA0286–0287). Herein, groES2L2 and feoAB were up-regulated under 0.66% NaCl for both CTG and STG designs (Table 1). The FeoAB system is specific for ferrous iron transport (Hantke, 2003). Five flagellin genes were upregulated under 2% NaCl for both CTG and STG designs, which confirmed the previous observation (Xu et al., 2004). The inducible expression of flagellin genes at a higher salt concentration suggested that increased polar flagellin may make the bacterium move faster. Three regulatory genes (VP0350, VPA0593 and VPA0961) were upregulated under 0.66% NaCl for both CTG and STG designs. Transcriptional changes in these genes may have pleiotropic effects, and hence may significantly influence osmotic regulation. 4. Conclusions Compared to 2% NaCl (high salt), 0.66% NaCl (low salt) in the synthetic medium lead to a restricted growth of the moderately halophilic V. parahaemolyticus. As determined by cDNA microarray, a total of 62 genes differentially expressed under 2% and 0.66% NaCl were collected to be the members of low-salt stimulon in V. parahaemolyticus, especially including those encoding major OMPs and compatible osmolyte transport/synthesis systems. To master high salt environments, V. parahaemolyticus might accumulate osmoprotectants through either exogenous uptake or endogenous synthesis of compatible solutes. A remodeling of OMPs was detected under high salt conditions, which appeared to play a role in the osmoadaptation of V. parahaemolyticus. These results provide a list of genes as novel candidates for probing the molecular physiology to shed light on the osmoadaptation of V. parahaemolyticus. Acknowledgements Financial supports came from the National Natural Science Foundation of China (30871370), the National Key Technology R&D program (2006BAK02A15), and the National Key Program for Infectious Disease of China (2009ZX10004-103 and 2008ZX10004-009). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijfoodmicro.2009.11.006. References Arai, H., Hayashi, M., Kuroi, A., Ishii, M., Igarashi, Y., 2005. Transcriptional regulation of the flavohemoglobin gene for aerobic nitric oxide detoxification by the second nitric oxide-responsive regulator of Pseudomonas aeruginosa. J. Bacteriol. 187, 3960–3968. da Costa, M.S., Santos, H., Galinski, E.A., 1998. An overview of the role and diversity of compatible solutes in bacteria and archaea. Adv. Biochem. Eng. Biotechnol. 61, 117–153. Dupont, L., Garcia, I., Poggi, M.C., Alloing, G., Mandon, K., Le Rudulier, D., 2004. The Sinorhizobium meliloti ABC transporter Cho is highly specific for choline and expressed in bacteroids from Medicago sativa nodules. J. Bacteriol. 186, 5988–5996.

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