OmpW and OmpV Are Required for NaCl Regulation in Photobacterium damsela Lina Wu,† Xiangmin Lin,† Fengping Wang,‡ Dezan Ye,‡ Xiang Xiao,‡ Sanying Wang,§ and Xuanxian Peng*,†,§ State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China, Third Institute of Oceanography, State Oceanic Administration, Xiamen, People’s Republic of China, and Center for Proteomics, Department of Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, People’s Republic of China Received February 11, 2006
Photobacterium damsela is a marine pathogen to both fish and human beings. The bacterium can shift between the ambient seawater and hosts, suggesting the existence of proteins rapidly responding to salt concentration. In the current study, proteomic methodologies were applied to screen the outer membrane proteins (OMPs) related to salt stress. OmpW and OmpV were determined in the response in this bacterium as OmpC and OmpF did in E. coli. Furthermore, the two genes were overexpressed in E. coli Top10F and complemented in V. paraheamolyticus mutants. The ability in salt-tolerance was elevated in the E. coli overexpressed OmpW and reduced in the cells overexpressed OmpV. These V. paraheamolyticus mutants could recover their response to environmental salt concentration when they were complemented by P. damsela OmpW and OmpV. These findings indicate that OmpW and OmpV are required for environmental salt regulation in P. damsela, in which OmpW and OmpV, respectively, elevate and reduce the ability in salinity-tolerance. Keywords: P. damsela • OmpW • OmpV • salt regulation
Introduction The ability of osmoregulation is crucial to marine pathogens that always face a change in osmotic pressure when they shift between the ambient seawater and hosts, which refer to high and low osmolarity, respectively. The environment of high osmolality causes rapid loss of water (plasmolysis), loss of turgor, and shrinkage of the cells, while the converse is true at the low osmolarity. However, bacteria can sense and respond to salt changes in their external environment for survival. Accumulating data have indicated that the cell surface structure of gram-negative bacteria is essential for bacterial physiology as well as communication with the external environment,1 in which outer membrane proteins (OMPs) show their great ability in regulation because they are on the outmost of a bacterium and face the osmotic changing environment. The regulation has been documented by the fact that bacteria can regulate the synthesis of OMPs when they are transferred between different salinities of environments by a pattern of osmolarity regulation with two proteins. The pattern has been determined in E. coli, Klebsiella pneumoniae, and Vibrio cholerae as OmpF and OmpC,2 OmpK35 and OmpK36,3 and OmpT and OmpU,4 * To whom correspondence should be addressed. E-mail: wangpeng@ xmu.edu.cn. Tel: (86)-592-218-7987. Fax: (86)-592-218-1015. Address: School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, People’s Republic of China. † Sun Yat-Sen University. ‡ State Oceanic Administration. § Xiamen University.
2250
Journal of Proteome Research 2006, 5, 2250-2257
Published on Web 08/11/2006
respectively. OmpF and OmpC, two abundant OMPs of E. coli in the most classical model, are reciprocally regulated by the osmolarity of the growth medium, showing that intensive OmpC expression and less abundant OmpF are at high osmolarity, and the opposite is true at low osmolarity.5,6 Recently, we have characterized OmpW and OmpV as regulating OMPs in response to different NaCl concentrations in Vibrio parahaemolyticus and Vibrio alginolyticus with the use of differential proteomic methodologies.7,8 The two OMPs may be related to high salt resistance because the ability in salt-resistance is stronger in V. alginolyticus and V. parahaemolyticus than E. coli, K. pneumoniae, and V. cholerae. The OmpW is a major protein of 22 kD and localizes on the bacterial outer membrane (OM). Its function is still not clear. Reports indicated that there was a great homology of OmpW between V. cholerae and E. coli. In E. coli, it was an S4 colicin receptor9 and elicited proinflammatory response that was independent of TLR4 and Ca2+ signaling.10 Its expression increased when strains were challenged with acid condition.11 OmpW was still considered as a putative function category of iron acquisition and utilization, showing 3- to 5-fold increases in transcription in Shewanella oneidensis and a 1.7-fold elevated level in response to transferrin in Pasteurella multocida, when the ferric uptake regulator (Fur) gene was knockout.12,13 The OmpW might be linked to the adaptive response of the organism under stress conditions with it being highly immunogenic.14-16 More recently, the protein of E. coli has been crystallized, indicating that it functions in the transport of small 10.1021/pr060046c CCC: $33.50
2006 American Chemical Society
OmpW and OmpV for Salt-Regulation in P. damsela
hydrophobic molecules across the bacterial OM.17,18 OmpV of 25 kD is a heat-induced, highly immunogenic protein associated with peptidoglycan in Vibrio cholerae19 and its homology is MipA in E. coli. Vollmer W et al. assumed that overproduction of MipA affected the membrane integrity.20 However, information regarding the ability of salt regulation for the two proteins is not available except for our two primary reports in Vibrios.7,8 Photobacterium damsela (P. damsela) was initially isolated by Love et al in 1981 from skin ulcers in damsel fish swimming in the temperate-water of California,21 being a halophilic, facultatively anaerobic gram-negative rod.22 It could cause not only a significant bacterial fish disease in the Mediterranean affecting marine fish species, but also necrotizing fascitis in human beings just as Vibrios did. The bacterium was first classified as Vibrio, terming Vibrio damsela, and then reassigned to the genus Photobacterium on the basis of phenotypic data in 1991. Therefore, the investigation of salt-regulation proteins will be helpful in an understanding of mechanism surviving between a host and seawater for this bacterium. The results reported here were obtained with the use of proteomic methodologies for the characterization of altered OmpW and OmpV of P. damsela cultured in LB medium with different NaCl concentrations. Furthermore, the ability of functioning as salt-regulating OMPs was evaluated and determined by overexpression and complementation of the two genes. Our results suggest that OmpW and OmpV are two proteins responding to salt regulation not only in Vibrios but also in a rod, showing intensive OmpW expression at high osmolarity and abundant OmpV at low osmolarity.
Materials and Methods Bacterial Strains and Culture Conditions. P. damsela was obtained from the Third Institute of Oceanography, Xiamen, P. R. China. V. parahaemolyticus and E. coli strains were from the bacterial collection of our laboratory. P. damsela, V. parahaemolyticus, and E. coli strains were grown in LB medium at 28 °C and 37 °C, respectively. When needed, the growth medium was supplemented with antibiotics at the following concentration: E. coli strains with plasmids AmpR were provided with 100 µg of ampciline per mL; to E. coli strains with plasmids CmR were added 34 µg of chloramphenicol per mL. P. damsela was separately grown in LB medium supplemented with 0.5, 1, or 4% concentrations of NaCl, 28 °C in a shaker bath at 200 rpm. These cultures were harvested in 18 h. E. coli cells were grown in LB medium or TSB medium containing appropriate antibiotics. The Physiological Characteristics of P. damsela. Measurement of Growth. P. damsela was grown in 50 mL of LB medium with 1% NaCl overnight. The initial optical density (OD600) of the culture was 0.10-0.12, which corresponds to approximately 107 cells per mL. Fresh overnight cultures were diluted into 1:100 in the same medium, and growth was separately continued in 200 mL of medium with 1 or 4% NaCl. Samples were obtained from the two cultures at 0, 2, 4, 6, 8, 10, 12, 14, 24, 32, 36, and 48 h. The cell density was determined by measurement at 600 nm. For every result, the value of growth was determined over a minimum of three independent measurements. Motility Testing of Isolates Which Responded to the Different Salinities. Motility testing was carried out according to a procedure described previously.23 Precultures of P. damsela separately realized in LB medium with 1 or 4% NaCl, which had reached the early stationary phase, were layered on the corresponding salinity plates with LB medium containing 1.5%
research articles agar. The plates were incubated until the colonies developed. Swimming mobility was evaluated using plates realized with LB medium supplemented with 0.4% agar. A single colony from 1 or 4% NaCl was inoculated by puncture in the middle of the plate with 1 or 4% NaCl. The diameter of the halo was measured at 4, 8, 12, 14, 24, and 28 h after inoculation. For every result, the value of mobility was determined over a minimum of three independent measurements. Isolation and Analysis of OMPs. Extraction of OMPs. OMPs of P. damsela were prepared according to a procedure described previously.24 Briefly, the bacterial cells were harvested by centrifugation at 4000g for 15 min at 4 °C. The cells were then washed in 40 mL of sterile saline (0.15 M NaCl) for three times and then resuspended in 5 mL of sterile saline. Cells were disrupted by intermittent sonic oscillation. Unbroken cells and cellular debris were removed by centrifugation at 5000g for 20 min, and the supernatant was collected and was further centrifuged at 100000g for 40 min at 4 °C. The pellet was resuspended in 10 mL of 2% w/v sodium lauryl sarcosinate (Sigma) and incubated at room temperature for 1 h. The solution was centrifuged at 100000g for 40 min at 4 °C. The resulting pellet was resuspended in 20 µL of sterile saline and stored at -20 °C. The concentration of the OMPs in the final preparation was determined using the Bradford method. 2-DE and Mass Spectrometric Analysis. The 2-DE was performed according to a procedure described previously.24,25 Briefly, OMP extracts containing 15 µg proteins were dissolved in a solution (8 M urea, 2 M thiourea, 4% CHAPS, and 80 mM DTT). IEF was carried out using a pH 3-9.5 carrier ampholyte for 8000 Vh. After being equilibrated for 15 min, the IEF gels were transferred to the second dimension electrophoresis using 12% acrylamide gel. The 2-DE gels were stained with Coomassie brilliant blue (CBB). Subsequently, gels were scanned in an AGFA white-light scanner at a resolution of 400 mm × 200 mm, and the raw images were processed using the 2-D software Melanie 4.0 (Swiss Institute of Bioinformatics, Geneva, Switzerland). Following background subtraction and spot detection, the gel patterns were matched to each other by visual comparison. Altered spots of OmpW and OmpV were compared on the basis of their volume percentages in the total spot volume over the whole gel image. MALDI-TOF/MS analysis was carried out according to a procedure described previously.24,25 Peptide masses were searched using the program Protein Prospector MS-Fit (http:// prospector.ucsf.edu), in which the V. parahaemolyticus database was defined as a matching species. Partial sequences of peptides were determined by ESI-MS/MS, which was performed on Q-Star by the Biological Department of the National University of Singapore. Homology searching was performed by the program provided at the NCBI server: http://www. ncbi.nlm.nih.gov/BLAST/. Cloning and Sequencing of the Full-Length ompW and ompV. Two pairs of primers were designed using a primer computer program according to the complete sequence of V. paraheamolyticus ompW and ompV. The following oligonucleotides with restriction sites were generated: ompW-forward primer, (5′-GGGGATCCATGAAAAAAACAATCTG-3′) with BamHI site; ompW-reverse primer, (5′-CCGAATTCTTAGAACTTGTAACCGC-3′) with EcoRI site; ompV-forward primer, (5′-GGGGATCCATGAACAAGACACT-3′) with BamHI site; ompV-reverse primer, (5′-CCGCTAGCTTAGAAGTTGTAAGACAC-3′) with HindIII site. Standard PCR and molecular biology protocols were used to amplify the two genes of P. damsela. The PCR fragments were Journal of Proteome Research • Vol. 5, No. 9, 2006 2251
research articles directionally cloned into plasmid pET-His digested with the same enzyme, and then expressed in E. coli BL21. Recombinant plasmids were checked by restriction enzyme digestion and direct sequencing. The sequencing was carried out by Shanghai Bioasia Biotechnology Co. Ltd. The nucleotide sequence data for the two genes of P. damsela reported in this study have been assigned in GenBank (accession no. DQ251175 for ompW and DQ251176 for ompV). Raising of Antiserum and Western Blotting. Purification of Recombinant ompW and ompV. E. coli BL21 harboring pET-His-OmpW or pET-His-OmpV was cultured in 100 mL of LB and the expression of recombinant proteins was induced by 0.25 mM IPTG. After 3 h induction at 37 °C, cells were harvested, washed, resuspended in buffer A (10 mM Tris-HCl, 200 mM NaCl, pH 7.5), and lysed by sonication. Following centrifugation at 5000g for 10 min, the pellet containing inclusion bodies of recombinant proteins was washed, and then 2 mL of buffer A containing 8 M urea was added. The recombinant protein was subsequently purified by affinity chromatography using Ni-NTA resin (Qiagen) and elution buffer A containing 8 M urea and 500 mM imidazole. The purified recombinant proteins (His6-OmpW and His6-OmpV) were renatured by stepwise dialysis against decreasing concentrations of urea (6 M to nil), and then 200 mL of buffer A containing 1% (v/v) Tween 20 or 1% (v/v) Triton X-100 was added. Finally, the protein solution was dialyzed further against buffer A containing decreasing concentrations of either of the detergents. The purified protein solution was stored at 4 °C at a concentration of about 600 µg mL-1 in buffer A containing 0.1% detergent. Raising of Antiserum. Antisera to the purified recombinant protein His6-OmpW or His6-OmpV were separately raised by immunizing a mouse with 500 µg of purified protein emulsified with Freund’s complete adjuvant. The first injection was followed by three similar injections of the purified protein with Freund’s incomplete adjuvant at weekly intervals. Serum was collected and stored at -20 °C for use. SDS-PAGE Analysis and Immunoblotting. SDS-PAGE analysis of samples was performed in 12% (w/v) polyacrylamide gels. The samples were separately solubilized at 100 °C for 5 min in sample buffer containing 2% (w/v) SDS and 5% (v/v) β-mercaptoethanol. Following electrophoresis, resolved bands were visualized by CBB staining. For immunoblotting, proteins in gels were transferred to nitrocellulose membranes by electrophoreses, followed by immunodetection of OmpW and OmpV using corresponding mouse antisera as the first antibodies and peroxidase-conjugated goat antimouse IgG as the secondary antibody. Effect of ompW and ompV Overexpression of P. damsela on Salinity-Tolerance of E. coli. Cloning ompW and ompV to Vector PLLP-ompA. Standard molecular cloning techniques were used to clone ompW and ompV to vector PLLP-ompA. This vector is accompanied by an ompA signal which can secrete the expression protein to the periplasmic space. Verification of the Overexpression Proteins Locating in Periplasmic Space. E. coli Top10F harboring PLLP-ompAOmpW or PLLP-OmpA-OmpV were separately cultured in 1000 mL of LB + glucose + Amp until the optical density (OD600) of the cultures was 0.5. Induction of the expression of OmpW and OmpV was obtained by adding IPTG to a final concentration of 100 µg/mL for 3 h at 30 °C. Cells were harvested by centrifugation and suspended with 30 mM TrisHCl, 20% sucrose, and 1 mM EDTA, pH 8.0. The cells were put 2252
Journal of Proteome Research • Vol. 5, No. 9, 2006
Wu et al.
in the ice, surged lightly for 10 min and centrifuged at 8000g. The pellet was suspended in 20 mL of 5 mM MgSO4, put in the ice, and surged lightly for 10 min again. Suspension was obtained for the cold osmotic shock fluid at 12000g centrifugation for 15 min. The verification of the secretion overexpression was performed by analysis of sarcosine-insoluble OMPs with the use of SDS-PAGE and Western blotting methods. Salinity-Tolerance Analysis. The single colony of TOP10F, TOP10F-PLLP-ompA, TOP10F-PLLP-ompA-ompW, TOP10F-PLLP-ompA-ompV was grown in 5 mL of LB medium at 37 °C until the optical density (OD600) of the cultures was 0.5. Induction of the expression of OmpW and OmpV was obtained by adding IPTG to a final 100 µg/mL concentration for 3 h at 30 °C. These bacterial cultures were separately streaked to LB plates with 1, 2, 3, 4, 5, 6, and 7% concentrations of NaCl and incubated at 37 °C for 24 h. The highest NaCl concentration was recorded as bacterial livability when the bacterium could survive. For every result, the value was determined over a minimum of three independent measurements. Effect of Complementation of P. damsela ompW and ompV on Salinity-Tolerance of V. paraheamolyticus Mutants. Construction of the Deletion Mutants of ompW and ompV in V. paraheamolyticus. We engineered unmarked nonpolar internal deletions of the coding sequences for ompW and ompV by allelic exchange as described previously.26 A forced directional ligation of the target suicide plasmid pRE112 was constructed with two chromosomal DNA fragments that each comprised at least 1 Kb of DNA flanking the deleted ORF, which was replaced by a kpnI site. (i) ompW deletion mutant oligonucleotides ∆ompW-01F (kpnI restriction site) and ∆ompW02R (kpnI restriction site) were used to amplify ompW with chromosomal DNA from V. paraheamolyticus. PCR products were confirmed by sequencing. The amplification product was cloned into pGEM T-Vector (Promega), generating pCR-ompW. Primers ∆ompW-03R and ∆ompW-04F were used to create an in-frame deletion of the ompW in pCR-ompW by using inverse PCR amplification. Both oligonucleotides ∆ompW-03R and ∆ompW-04F introduced a BamHI restriction site. The 2049-bp kpnI ompW deletion fragment was cloned into the positive-selection suicide vector pRE112, which had been digested with kpnI and dephosphrylated. The resulting plasmid, pRE112_∆ompW, was used to construct the ompW deletion mutant in V. paraheamolyticus (amp resistant) by allelic exchange as described previously,26 generating the V. paraheamolyticus _∆ompW strain. Primers ∆ompW-05F and ∆ompW-06R were used to identify the target mutant. (ii) The V. paraheamolyticus _ ∆ompV strain was constructed as described above by using primers ∆ompV-07F, ∆ompV-08R, and ∆ompV-09R, and ∆ompV-10F, ∆ompV-11 F, and ∆ompV-12R, which functions were equal to ∆ompW-01F, ∆ompW-02R, and ∆ompW-03R, and ∆ompW-4F, ∆ompW-5F, and ∆ompW-6R, respectively (see Table 1). Complementation of V. paraheamolyticusW- and V. paraheamolyticuV- by the ompW and ompV of P. damsela, Respectively. By using the appropriate primer pairs, ompW and ompV were amplified from P. damsela, introducing BamHI restriction sites. The amplified products were cloned into the BamHI sites in pACYC184 (New England Biolabs), leaving the genes under the control of the tetracycline resistance gene promoter, thus generating pompW and pompV. pompW and pompV were electrotransformed into V. paraheamolyticus _∆ompW and V. paraheamolyticus_∆ompV electrocompetents,
research articles
OmpW and OmpV for Salt-Regulation in P. damsela Table 1. Oligonucleotides Used in This Mutant Study
a
designation
DNA sequence (5′-3′)a
restriction enzyme sites
∆ompW-01F ∆ompW-02R ∆ompW-03R ∆ompW-04F ∆ompW-05F ∆ompW-06R ∆ompV-07F ∆ompV-08R ∆ompV-09R ∆ompV-10F ∆ompV-11F ∆ompV-12R
5′-AAGGTACCAAGGTTCCCCATTGTTC-3′ 5′-TTGGTACCATGTGAGTAAGATCGGTTTC-3′ 5′-CCGGATCCTTATTTCCTTTTGTAGGG-3′ 5′-AAGGATCCAAGCGGTAGTTACACACATTA-3′ 5′-GATCGGTATGGCGTTTCTCTAT-3′ 5′-TATGTGACCAGTGTAGGACTTTTCG-3′ 5′-AAGGTACCCTGTTTTCACACCACAACG-3′ 5′-AAGGTACCATGTTAGCCACGCCACA-3′ 5′-TTGGATCCTATTTCCTGTACCGTTTATT-3′ 5′-CGGGATCCGAAATCTAAAAAAGCCA-′3 5′-ACTCTGACCTGATAGATGTTGAAAA-3′ 5′-TATTTGCCTTTGTTCATTGGCTACG-3′
kpnI kpnI BamHI BamHI
kpnI kpnI BamHI BamHI
Restriction enzyme sites used for cloning of PCR products are underlined.
Figure 1. Growth cycle of P. damsela in different salinities: 1%, 1% NaCl; 4%, 4% NaCl.
thus generating V. paraheamolyticus-+ompW and V. paraheamolyticus-+ompV, respectively. Salinity-Tolerance Analysis. The single colony of wild type V. paraheamolyticus, V. paraheamolyticusW-, V. paraheamolyticuV-, V. paraheamolyticus-+ompW and V. paraheamolyticus+ompV was separately grown in 5 mL of LB medium at 28 °C overnight. The cultures were separately streaked to LB plates with 1-10% concentrations of NaCl and incubated at 28 °C for 24 h. The highest NaCl concentration was recorded as bacterial livability when the bacterium could survive. For every result, the value was determined over a minimum of three independent measurements.
Results The Physiological Characteristics of P. damsela. To probe into the effect of salinity on its growth, the bacterium was separately cultured in LB medium with 1 or 4% NaCl, representing the osmotic conditions in host bodies and in various marine water bodies, respectively. Figure 1 showed the growth curves of P. damsela in the two salinities. As shown in Figure 1, there was no difference in bacterial amounts between the two salinities in 4 h of incubation. Then the bacterium grew faster in 1% NaCl than in 4% NaCl. The OD600 value indicated a distinction of approximately 0.1 between the two cultures from 4 to 24 h. Moreover, the OD distinction separately increased to 0.2 at 36 h and 0.5 at 48 h. These results suggest that 1% salinity is more suitable for the growth of the bacterium. During the experiment, we interestingly found that the colony of P. damsela was quite different in size among different salinities of plates, showing its diameter to be about 4.5 mm in a plate with 1% NaCl and about 1 mm in one with 4% NaCl (Figure 2). It suggests the effect of different salinities on the colony morphology of this bacterium.
Swimming performances of P. damsela grown at different salinities were evaluated in four groups. Group 1 and group 2 showed that the bacterium from the medium with 1% NaCl separately migrated in the plates with 1% and 4% NaCl, respectively. Group 3 and group 4 indicated that the bacterium from the medium with 4% NaCl separately migrated in the plates with 1% and 4% NaCl, respectively. The results showed that the bacterium moved faster in group 1 and group 3 than in group 2 and group 4 (Figure 3), suggesting that the bacterium showed stronger ability in motility in the 1% NaCl plate. Combining with the data in Figure 2, we conclude that salinity could obviously affect the movement of P. damsela. Therefore, salinity is a significantly environmental regulation factor for the motility of P. damsela. Proteomic Approach to Altered OMPs of P. damsela. SDSPAGE showed that the band patterns were significantly different between these cultures (Figure 4A). With the increase of NaCl concentrations, two major bands corresponding to molecular weights (Mr) of 25 and 22 kD were distinctly altered, showing that the band of 22 kD appeared and the band of 25 kD disappeared step by step. Moreover, 2-DE was used to analyze the expressional differentiation of sarcosine-insoluble OMPs from P. damsela cultured in the three different salinities. Figure 4C shows the micropreparatively loaded (15 µg) 2-DE gels. The gels were stained with CBB R-250, and approximately 40 protein spots could be detected. Of the approximately 40 spots observed, two distinctly altered spots, namely spot W and spot V, were excised. Spot w markedly increased in cells cultured at the concentration of 4% NaCl, whereas spot v only expressed in cells cultured at the concentrations of 0.5 and 1% NaCl. Changes in spots W and V corresponded to the finding in SDSPAGE combining with analysis of Mr. Therefore, the two proteins must play a significant role in the salinity adaptation of P. damsela. The two altered proteins and bands were identified as OmpW and OmpV by MALDI-TOF/MS. Table 2 shows the parameters in detail. Meanwhile, one of them, OmpV, was randomly selected to further analysis, and their identities were determined by partial sequence by ESI-MS/MS. Furthermore, Western blotting was applied for the confirmation of the two proteins using mouse antiserum against V. paheamolyticus OmpW or OmpV as the first antibody and HRP-antimouse IgG as the secondary antibody. The result indicated that the band of 22 kD was recognized by mouse anti-OmpW and the band of 25 kD by mouse anti-OmpV (Figure 4B). Therefore, they were successfully identified as OmpW and OmpV of P. damsela. In addition, besides OmpW and OmpV, several other protein spots Journal of Proteome Research • Vol. 5, No. 9, 2006 2253
research articles
Wu et al.
Figure 2. Effect of salinity on the colony of P. damsela in size: (A) 4%: 4% NaCl; 1%: 1% NaCl; (B) histogram displays showing the change in colony diameter.
Figure 3. Effect of salinity on the swimming mobility of P. damsela: group 1, bacterium from the medium with 1% NaCl migrated in the plate with 1% NaCl; group 2, bacterium from the medium with 1% NaCl migrated in the plate with 4% NaCl; group 3, bacterium from the medium with 4% NaCl migrated in the plate with 1% NaCl; group 4, bacterium from the medium with 4% NaCl migrated in the plate with 4% NaCl.
were altered with NaCl concentration and were not identified because OmpW and OmpV were the focus of the current study. Gene Analysis of ompW and ompV. In searching by MS data above, P. damsela OmpW and OmpV were identified against the V. paraheamolyticus database. Because the complete genome of P. damsela is not available, we try to use the primers designed for amplification of V. paraheamolyticus ompW and ompV to perform PCR of P. damsela ompW and ompV in a pretest. Luckily, a single band was obtained from both PCR for amplification of ompW and ompV, showing about 642 and 777 bp, respectively. The two DNA fragments in size were the same to those of V. paraheamolyticus ompW and ompV (data not shown). Then the two genes were cloned using standard molecular cloning techniques. Recombinant plasmids, pET-His-ompW and pET-His-ompV, were checked by restriction enzyme digestion and direct sequencing. After the IPTG induction of the pET-His-ompW and pET-His-ompV in E. coli BL21, two enlarged bands were separately obtained in SDS-PAGE (data not shown). They could match the Mr of the two proteins. Therefore, the ompW and ompV of P. damsela were really ligated and transformed into E. coli BL21 The nucleotide sequences of ompW and ompV were obtained from recombinant pET-ompW and pET-ompV by forward and reverse sequencing, and then analyzed by DNAsis 2254
Journal of Proteome Research • Vol. 5, No. 9, 2006
system. Their integral open reading frames (ORF) were 642 bp for ompW and 777 bp for ompV. Homology searching revealed that the nucleotide sequences of the ORF of the ompW and ompW-like genes shared the following identities: V. parahaemolyticus ompW, 96%; V. alginolyticus ompW, 91%; V. cholerae ompW, 42%; E. coli YciD, no homology. The ompV shared the identities with other ompV or ompV-like genes as following: V. parahaemolyticus ompW, 99%; V. cholerae ompV, 25%; E. coli mipA, no homology. The Function Analysis of OmpW and OmpV. Four bacteria were cultured on LB plates with NaCl concentrations from 1 to 12, for their survival. The expression of OmpW and OmpV, respectively, was up-regulated and down-regulated with an increase of NaCl concentration in the four bacteria (data not shown). Table 3 showed the results at 18 h-cultures, indicating that V. alginolyticus, V. paraheamolyticus, P. damsela, and V. vulnificus could survive at 12%, 8%, 8%, and 3% NaCl plates as the highest salt concentration (V. alginolyticus may survive at more than 12% concentration of NaCl), respectively. Therefore, of the four bacteria, V. alginolyticus is the most saltresistant bacterium, and V. vulnificus is the lowest one. For the observation of ability in salt-regulation of ompW and ompV, the two genes were separately overexpressed using the PLLP-ompA expression vector in the host E. coli strain TOP10F as two experimental groups and using the pET-His expression vector in the host E. coli strain BL21 as two corresponding controls. Meanwhile, original hosts only with a vector were used as two background controls. These six transformed strains were cultured on plates containing 1-8% concentrations of NaCl for survival evaluation. The results were shown in Table 4. We can see from Table 4 that the host E. coli strain TOP10F harboring ompW could survive in the medium with 6% NaCl, whereas the cells harboring ompV only survived in the medium with 4% NaCl. The two transformed controls and the two original hosts survived at 5% NaCl. These results indicated that overexpressed ompW could elevate the ability of host cells in salttolerance and overexpressed ompV could reduce the ability. To further examine the salt-regulated functions of ompW and ompV, we constructed their mutants, because the complete genome of P. dalema is not available now, and the complete genome of V. paraheamolyticus was accomplished in 2003.27 We constructed the two mutants by separately knocking out the ompW and ompV of V. paraheamolyticus with the use of the allelic exchange method as described in the experimental procedures to get ∆ompW and ∆ompV. Then the complementation of the ∆ompW and ∆ompV by the P.
research articles
OmpW and OmpV for Salt-Regulation in P. damsela
Figure 4. (A) CBB stained SDS-PAGE of OMPs of P. damsela grown in 0.5% (line 1), 1% (line 2), and 4% (line 3) NaCl concentrations; (B) immunoblot detection of OmpW and OmpV using antiserum to OmpW or OmpV as the primary antibody, respectively; (C) 2-DE profile of OMPs expressed in 0.5, 1, and 4% NaCl concentrations (C1, C2, and C3, respectively). Line M is the molecular weight standard. Target spots are shown by arrows. Panel D shows the enlarged partial 2-DE gels showing altered spots W and V among 0.5, 1, and 4% NaCl concentrations (D1) and the histogram displays, besides showing the changes in spot intensity, the differential expression of selected proteins between different NaCl concentrations (D2). From left to right in each group, the bars represent the spot intensity obtained in 0.5, 1, and 4% NaCl concentrations. Vol (%) represents the relative volume (according to Melanie 4.0 software description) divided by the total volume over the whole image. Table 2. Spot W and V Identified by PMF Searching against V. Parahaemolyticus Genomic Database spot no.
W V
identified protein
sequence coverage
accession
amino acid sequence (partial)
MOWSE score
protein Mr (Da)/pI
OmpW OmpV
25% 30%
28899951 28900173
LVMPINDNWQINQTTQYTR
1287 3.088 × 104
23468/5.0 28148/5.1
Table 4. Effect of ompW or ompV Overexpression on Bacterial Salinity-Tolerance
Table 3. Salinity-Tolerance Analysis of Four Species of Bacteria salinity (%):
1
2
3
4
5
6
7
8
9
10
11
12
salinity (%):
1
2
3
4
5
6
7
V. vulnificus V. parahaemolyticus P. damsela V. alginolyticus
+ + + +
+ + + +
+ + + +
+ + +
+ + +
+ + +
+ + +
+ + +
+
+
+
+
E. coli BL21 + PET E. coli BL21 + PET-ompW E. coli BL21 + PET-ompV E.coli Top10 + PLLP-ompA E.coli Top10 + PLLP-ompA-ompV E.coli Top10 + PLLP-ompA-ompW
+ + + + + +
+ + + + + +
+ + + + + +
+ + + + + +
+ + + + +
+
-
dalema ompW and ompV, respectively, obtained +ompW and +ompV. The highest surviving NaCl concentration of ∆ompW, ∆ompV, and V. paraheamolyticus was 7%, 10%, and 8%, respectively, whereas both the +ompW and +ompV could survive at 8% NaCl, suggesting that the ability in salt-tolerance was recovered in the complemented V. paraheamolyticus (Table 5). These results indicated that ompW could elevate the bacterial ability in salt-tolerance and ompV could reduce the ability. Therefore, ompW and ompV are really salt-regulating genes.
Discussion P. damsela causes the death of infected fish and is of major importance to the fish farming industry. More importantly, it has also been isolated from human beings, usually from wounds inflicted by fish in seawater or brackish water,28 where it results in necrotizing fascitis.29-31 P. damsela-associated necrotizing fascitis in particular tended to demonstrate more serious complications and a higher mortality rate than that Journal of Proteome Research • Vol. 5, No. 9, 2006 2255
research articles
Wu et al.
Table 5. Effect of Complementation by P. Damsela ompW and ompV on the Salinity-Tolerance of V. Paraheamolyticus Mutants salinity (%):
7
8
9
10
11
V. paraheamolyticus ∆ompW ∆ompV +ompW +ompV
+ + + + +
+ + + +
+ -
+ -
-
observed in cases attributable to V. vulnificus, suggesting the importance of further investigation of P. damsela. Osmolarity is distinctly different between the human body and seawater. In the current study, 1 and 4% concentrations of NaCl were used to mimic the osmotic conditions in host bodies and in various marine water bodies, respectively.7,8 The observation on the growth indicated that the bacterium could survive very well in both low and high salinities, although the low salinity is more suitable for it, suggesting that there is a powerful NaCl-regulation system in this bacterium. In this regard, proteomic methodologies were used to analyze altered OMPs involved in different concentrations of NaCl in the current study. Two proteins markedly altered were determined in both of the 2-DE subproteome and the SDS-PAGE map, showing a disappeared spot (band) and an increased one with increased NaCl concentration, which was the same behavior as OmpW and OmpV of V. paraheamolyticus and V. alginolytixus reported by our laboratory.8 The two proteins were indeed identified as homologies of the OmpW and OmpV of V. paraheamolyticus by MALDI-TOF/MS and ESI-MS/MS analysis and Western blotting identification. Further sequence alignment indicated that their genes showed high homologies with Vibrios rather than with rod. Therefore, the two proteins were named as OmpW and OmpV of P. damsela. Interestingly, the behavior of OmpW and OmpV in P. damsela was the same as that of OmpC and OmpF in E. coli in response to salinity stress. The influence of osmolarity on the regulation of OmpC and OmpF expressions has been extensively studied in E. coli, indicating that OmpF is preferentially expressed in the media of low osmolarity, whereas OmpC expression increases in the media of high osmolarity.32 In the model, a larger-channel porin is always highly expressed in normal conditions, whereas a smaller-channel porin is upregulated in hostile circumstances including a medium containing toxic agents, detergents, or antibiotics to decrease membrane permeability. With the long-term goal of identifying the molecular basis for this difference in salinity sensitivity between the two OMPs, we have undertaken a detailed gene clone and orientation overexpression and are constructing a nonpolar deletion mutant for the two genes encoding OmpW and OmpV and a complementation of these genes to give the function of these two OMPs a further investigation. The bacteria harboring PLLP-ompA-ompW could elevate the ability in salttolerance, and the ones having PLLP-ompA-ompV could reduce the ability compared to the controls only with a vector. The bacteria harboring pET-His-ompW and pET-His-ompV did not have the effect on salt-tolerance. This is because the two OMPs could function in OM as they do in natural conditions when the vector PLLP-ompA was used, whereas they might only work in cytoplasm when the vector pET-His was applied, indicating the elevation or reduction of bacterial salt-tolerance was closely related to whether overexpressed recombinant proteins were located in the OM. These results 2256
Journal of Proteome Research • Vol. 5, No. 9, 2006
strongly suggest that the function in salt-tolerance is elevated by OmpW and reduced by OmpV. Moreover, the deletion and complementation mutants of the two genes were separately constructed for the further evaluation in response to salt concentration. These experiments were performed in a V. paraheamolyticus strain because genome information was not available for P. damsela. Our results indicated that the complementation by P. damsela ompW could recover the ability in 8% NaCl tolerance of the V. paraheamolyticus mutant that survived at the medium with 7% NaCl because of the losing of ompW, whereas the complementation of the V. paraheamolyticus mutant without ompV by P. damsela ompV could reduce the ability of the mutant in salt-tolerance from 10% to 8% NaCl. The deletion and complementation mutants give us corroboration on the salt-regulation function of OmpW and OmpV. Undoubtedly, OmpW keeps the host to survive at high salinity and OmpV does at low salinity in P. damsela. In summary, OmpW and OmpV of P. damsela were determined to be osmotic stress responsive proteins at both levels of genome and protein. To the best of our knowledge this is the first report that OmpW and OmpV of P. damsela may act as OmpF and OmpC of E. coli in response to salinity stress, in which OmpW elevates the ability in salt-tolerance and OmpV reduces it.
Acknowledgment. This work was sponsored by grants from NSFC Project 30530610, Comra fund Grant (D.Y.105-0402-7), and China Hi-Tech Development Project “863” (No. 2005AA626013). References (1) Nikaido, H. Microdermatology cell surface in the interaction of microbes with the external world. J. Bacteriol. 1999, 181, 4-8. (2) Cai, S. J.; Inouye, M. EnvZ-OmpR interaction and osmoregulation in Escherichia coli. J. Biol. Chem. 2002, 277, 24155-24161. (3) Hernandez-Alles, S.; Albert, S.; Alvarez, D.; Domenech-Sanchez, A.; Martinez-Martinez, L.; Gil, J.; Tomas, M.; Benedi, V. J. Porin expression in clinical isolates of Klebsiella pneumoniae. Microbiol. 1999, 145, 673-679. (4) Wibbenmeyer, J. A.; Provenzano, D.; Landry, C. F.; Klose, K. E.; Delcour, A. H. Vibrio cholerae OmpU and OmpT porins are differentially affected by bile. Infect. Immun. 2002, 70, 121-126. (5) Hall, M. N.; Silhavy, T. J. The OmpB locus and the regulation of the major outer membrane porin proteins of Escherichia coli K12. J. Mol. Biol. 1981, 146, 1-15. (6) Jovanovich, S. B.; Martinell, M.; Record, M. T., Jr.; Burgess, R. R. Rapid response to osmotic upshift by osmoregulated genes in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 1988, 170, 534-539. (7) Xu, C. C.; Ren, H. X.; Wang, S. Y.; Peng, X. X. Proteomic analysis of salt-sensitive outer membrane proteins of Vibrio parahaemolyticus. Res. Microbiol. 2004, 155, 835-842. (8) Xu, C. X.; Wang, S. Y.; Ren, H. X.; Lin, X. M.; Wu, L. N.; Peng, X. X. Proteomic analysis on the expression of outer membrane proteins of Vibrio alginolyticus at different sodium concentrations. Proteomics 2005, 5, 3142-3152. (9) Pilsl, H.; Smajs, D.; Braun, V. Characterization of colicin S4 and its receptor, OmpW, a minor protein of Escherichia coli outer membrane. J. Bacteriol. 1999, 181, 3578-3581. (10) So¨derblom, T.; Oxhamre, C.; Wai, S N.; Uhle´n, P.; Aperia, A.; Uhlin, B. E.; Richter-Dahlfors, A. Effects of the Escherichia coli toxin cytolysin A on mucosal immunostimulation via epithelial Ca2+ signaling and Toll-like receptor 4. Cell. Microbiol. 2005, 7, 779788. (11) Sainz, T.; Perez, J.; Villaseca, J.; Hernandez, U.; Eslava, C.; Mendoza, D. G.; Wacher, C. Survival to different acid challenges and outer membrane protein profiles of pathogenic Escherichia coli strains isolated from pozol, a Mexican typical maize fermented food. Int. J. Food Microbiol. 2005, 105, 357-67.
research articles
OmpW and OmpV for Salt-Regulation in P. damsela (12) Thompson, D. K.; Beliaev, A.; Giometti, C. S.; Lies, D. P.; Nealson, K. H.; Lim, H.; Yates, J.; Tiedje, J. M.; Zhou, J. Transcriptional and proteomic analysis of a ferric uptake regulator (fur) mutant of Shewanella oneidensis: possible involvement of fur in energy metabolism, transcriptional regulation, and oxidative stress. Appl. Environ. Microbiol. 2002, 68, 881-92. (13) Paustian, M. L.; May, B. J.; Cao, D.; Boley, D.; Kapur, V. Transcriptional response of Pasteurella multocida to defined iron sources. J. Bacteriol. 2002, 184, 6714-6720. (14) Nandi, B.; Nandy, R. K.; Sarkar, A.; Ghose, A. C. Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae. Microbiol.-SGM 2005, 151, 29752986. (15) Iltanen, S.; Tervo, L.; Halttunen, T.; Wei, B.; Braun, J.; Rantala, I.; Honkanen, T.; Kronenberg, M.; Cheroutre, H.; Turovskaya, O.; Autio, V.; Ashorn, M. Elevated serum anti-12 and anti-OmpW antibody levels in children with IBD. Inflammatory Bowel Dis. 2006, 12, 389-394. (16) Kurupati, P.; The, B. K.; Kumarasinghe, G.; Poh, C. L. Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis. Proteomics 2006, 6, 836-844. (17) Hong, H.; Patel, D. R.; Tamm, L. K.; van den Berg, B. The outer membrane protein OmpW forms an eight-stranded beta-barrel with a hydrophobic channel. J. Biol. Chem. 2006, 281, 7568-7577. (18) Albrecht, R.; Zeth, K.; So¨ding, J.; Lupas, A.; Linke, D. Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli. Acta Crystallogr., Sect. F 2006, 62, 415-418. (19) Stevenson, G.; Leavesley, D.; Lagnado, C. A.; Heuzenroeder, M. W.; Manning, P. A. Purification of the 25-kDa Vibrio cholerae major outer-membrane protein and the molecular cloning of its gene: OmpV. Eur. J. Biochem. 1985, 148, 385-390. (20) Vollmer, W.; von Rechenberg, M.; Ho¨ltje J-V. Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli. J. Biol. Chem. 1999, 274, 6726-6734. (21) Love, M.; Teebken-Fisher, D.; Hose, J. E.; Farmer, J. J.; Hickman, F. W. Fanning, G. R. Vibrio damsela, a marine bacterium, causes skin ulcers on the damselfish Chromis punctipinnis. Science 1981, 214, 1139-1140. (22) Yamane, K.; Asato, J.; Kawade, N.; Takahashi, H.; Kimura, B.; Arakawa, Y. Two cases of fatal necrotizing fascitis caused by
(23)
(24) (25)
(26) (27)
(28) (29) (30)
(31) (32)
Photobacterium damsela in Japan. J. Clin. Microbiol. 2004, 42, 1370-1372. Picot, L.; Mezghani-Abdelmoula, S.; Chevalier, S.; Merieau, A.; Lesouhaitier, O.; Guerillon, J.; Cazin, L.; Orange, N.; Feuilloley, M. G. Regulation of the cytotoxic effects of Pseudomonas fluorescens by growth temperature. J. Res. Microbiol. 2004, 155, 39-46. Chen, Z. J.; Peng, B.; Wang, S. Y.; Peng, X. X. Rapid screen of highly efficient vaccine candidate by immunoproteomics. Proteomics 2004, 4, 3203-3213. Peng, X. X.; Xu, C. X.; Ren, H. X.; Lin, X. M.; Wu, L. N.; Wang, S. Y. Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Pseudomonas aeruginosa responding to ampicilin, kanamycin and tetracycline resistance. J. Proteome Res. 2005, 4, 2257-2265. Edwards, R. A.; Keller, L. H.; Schifferli, D. M. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 1998, 207, 149-157. Makino, K.; Oshima, K.; Kurokawa, K.; Yokoyama, K.; Uda, T.; Tagomori, K.; Iijima, Y.; Najima, M.; Nakano, M.; Yamashita, A.; Kubota, Y.; Kimura, S.; Yasunaga, T.; Honda, T.; Shinagawa, H.; Hattori, M.; Iida, T. Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae. Lancet 2003, 361, 743-749. Coffey, J. A. J.; Harris, R. L.; Rutledge, M. L.; Bradshaw, M. W.; Williams, T. W. J. Vibrio damsela: another potentially virulent marine vibrio. J. Infect. Dis. 1986, 153, 800-802. Knight-Madden, J. F. M.; Barton, M.; Gandretti, N.; Nicholson, A. M. Photobacterium damsela Bacteremia in a child with sicklecell disease. Pediatr. Infect. Dis. J. 2005, 24, 654-655. Goodell, K. H.; Jordan, M. R.; Graham, R.; Cassidy, C.; Nasraway, S. A. Rapidly advancing necrotizing fasciitis caused by Photobacterium (Vibrio) damsela: A hyperaggressive variant. Crit. Care Med. 2004, 32, 278-281. Asato, J.; Kanaya, F. Fatal infection of the hand due to Photobacterium damsela: A case report. Clin. Infect. Dis. 2004, 38, E100E101. Leonardo, M. R.; Forst, S. Reexamination of the vole of the periplasmic domain of EnvZ in sensing of osmolarity signals in Escherichia coli. Mol. Microbiol. 1996, 22, 405-413.
PR060046C
Journal of Proteome Research • Vol. 5, No. 9, 2006 2257
