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Characteristics of Na+-dependent respiratory chain in Vibrio anguillarum, a fish pathogen, in comparison with other marine Vibrios
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Characteristics of Naþ-dependent respiratory chain in Vibrio anguillarum, a ¢sh pathogen, in comparison with other marine Vibrios Erina Fujiwara-Nagata a , Kazuhiro Kogure b , Kumiko Kita-Tsukamoto b , Minoru Wada b , Mitsuru Eguchi a; b
a Department of Fisheries, Kinki University, Nara 631-8505, Japan Ocean Research Institute, The University of Tokyo, Tokyo 164-8639, Japan
Received 27 September 2002; received in revised form 13 December 2002; accepted 29 December 2002 First published online 12 February 2003
Abstract The activity of membrane-bound NADH oxidase of Vibrio anguillarum M93 (serotype J-O-1), which causes vibriosis in freshwater area was activated by Naþ in the same manner as other marine Vibrios. However, in addition to Naþ , Kþ was also found to positively enhance the NADH oxidase activity of strain M93. This tendency has not been recognized in other marine Vibrios. Furthermore, the Naþ dependent NADH oxidase of strain M93 required less Naþ (0.1 M) for its maximum activity than those of other Vibrios such as Vibrio alginolyticus and ‘Vibrio angustum’ S14, which were in the range of 0.4 M NaCl, similar as seawater. Destruction of Hþ motive force by a proton conductor carbonylcyanide m-chlorophenylhydrazone (CCCP) revealed high dependency of V. anguillarum on the primary Hþ pump. Even at pH 8.5, V. anguillarum strains other than serotype O-4 could not grow well with the addition of CCCP. In contrast, marine-type Vibrios such as V. alginolyticus and V. angustum S14 can grow well at pH 8.5 even with the addition of CCCP. The lower requirement for Naþ in V. anguillarum probably reflects the salinity of their original habitats. > 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Naþ -dependent NADH oxidase; Marine bacteria ; Primary Naþ pump; Fish pathogen; Vibrio anguillarum
1. Introduction Why do marine bacteria require Naþ for growth? This has long been a major question for marine microbiologists, and studies have uncovered a few Naþ functions in marine bacteria, such as Naþ stabilization of the cell membrane, Naþ -dependent active transport, and £agellar motility [1^5]. Besides these physiological Naþ functions, it was reported that marine bacteria possessed a Naþ -dependent respiratory chain (Naþ -NQR, Naþ -NADH :quinone oxidoreductase; primary Naþ pump) in addition to the Hþ -dependent respiratory chain [2,6^12,23]. From a bioenergetic point of view, the Naþ -rich and alkaline environment of the sea must make it advantageous for marine
* Corresponding author. Tel. : +81 (742) 431511; Fax : +81 (742) 431316. E-mail address : [email protected] (M. Eguchi).
bacteria to possess a primary Naþ pump in addition to the primary Hþ pump [12]. Vibrio anguillarum is a well-known ¢sh pathogen that requires around 2% NaCl for optimal growth [13,14]. This pathogen has caused serious damage to aquaculture systems worldwide, especially salmon cultures in brackish and coastal water regions. An unusual case is a vibriosis by V. anguillarum that occurs to landlocked salmonoid in freshwater areas in Japan [15,16]. Although this pathogen was detected from bottom sediments of freshwater areas by an indirect immuno£uorescence method with a speci¢c monoclonal antibody [15,17], it has never been detected by conventional culturing methods. The infectious process and ecology of the pathogen in freshwater regions remain unclear. When V. anguillarum cells were exposed to sterilized freshwater directly, their ability to form colonies on agar plates and their pathogenicity to ¢sh disappeared rapidly [18^20]. The main reason for this rapid loss of culturability seems to be the lack of Naþ in freshwater [18].
0168-6496 / 03 / $22.00 > 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-6496(03)00031-X
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In this report, we demonstrate the presence and describe the characteristics of a Naþ -dependent respiratory chain in V. anguillarum isolated from freshwater ¢sh killed by vibriosis. The relationship between the characteristics of the primary Naþ pump and the salinity of the original habitat is also discussed.
2. Materials and methods 2.1. Bacterial strains In the bacterial growth experiments under the presence of a proton conductor, carbonylcyanide m-chlorophenylhydrazone (CCCP, Sigma), 28 strains of Vibrio species were used as shown in Table 1. V. anguillarum M93 was originally isolated from diseased freshwater ¢sh, Plecoglossus altivelis (Salmoniforms) in Lake Biwa, Japan. It causes vibriosis in freshwater and seawater ¢sh [20,21]. Its Japanese serotype is J-O-1, which probably corresponds to the Danish serotype O-2 [17]. Naþ -NADH oxidase activities and inhibitory e¡ects on the activities by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO, Sigma) were measured with V. anguillarum M93, and V. anguillarum serotype O-4. V. anguillarum serotype O-4 is more common in the marine environment [22]. ‘Vibrio angustum’ S14 (Vibrio sp. CCUG15956) and Vibrio alginolyticus were used as representatives of marine Vibrio. V. alginolyticus was known to possess a marinetype Naþ -NQR [23]. 2.2. Growth with CCCP Strains were pre-incubated in a VNSS medium, a complex medium for marine bacteria [24,25]. For pH control, 40 mM of MES, MOPS and Tricine were used at pH 6.5, 7.5 and 8.5, respectively. All strains were inoculated and incubated in VNSS media with 5, 10 and 15 WM CCCP at pH 6.5, 7.5 and 8.5, respectively. These values of CCCP corresponded to the minimum concentrations that inhibited the growth of V. anguillarum M93 at each pH. Cultures were incubated at 20‡C with shaking (rpm 180). As for a control, bacterial growth was estimated by measuring optical density at 450 nm (OD450 ) when each strain reached early stationary phase without CCCP. All strains grew well in VNSS media without CCCP. Growth values with CCCP were expressed as a percentage to OD450 at early stationary phase in VNSS media without CCCP at each pH. Thus, %growth = (OD450 with CCCP)U100/ (OD450 without CCCP). 2.3. Preparation of membrane fraction and NADH oxidase assay Marine minimal medium (MMM), a chemically de¢ned medium, was used [26]. The composition of MMM was as
follows : 0.2% glucose, 9.52 mM NH4 Cl, 1.32 mM K2 HPO4 , 40 mM MOPS in NSS (pH 8.0; [24]). Log phase cells were harvested and membrane fractions were prepared by an osmotic lysis method [27]. The membrane fractions were suspended in 10 mM NaCl, 20 mM Tris^ HCl (pH 8.0) and 10% glycerol, and kept at 20‡C until measurements of NADH oxidase activity. NADH oxidase activity was measured from the decrease in absorbance at 340 nm at 30‡C. The standard assay mixture contained 20 mM Tris^HCl (pH 8.0), 0.2 mM NADH, 0.2 M salt (NaCl, KCl or LiCl), and 10^30 Wg of membrane protein in a ¢nal volume of 1 ml. HQNO was used to observe the inhibitory e¡ects on Naþ extruding segments [7,28,29]. Protein concentration was determined as described by Lowry et al. [30] by using a Modi¢ed Lowry Protein Assay 23240 Kit (Pierce).
3. Results 3.1. E¡ect of CCCP on bacterial growth The growth of various Vibrio strains with CCCP under Table 1 List of bacterial strains used Bacteria
Serotype
Source
Strain no.
V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. mimicus V. pelagius V. splendidus V. parahaemolyticus V. natriegens V. mediterranei V. ¢scheri V. harveyi V. carchariae V. campbellii V. damselae V. £uvialis V. proteolyticus V. vulni¢cus ‘V. angustum’ S14 V. alginolyticus
O-1 O-2 O-3 O-4 O-5 O-6 O-7 O-8 O-9 O-10 J-O-1(O-2) O-2
ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC
43305 43306 43307 43308 43309 43310 43311 43312 43313 43314 M93 13266T 33653T 25916T 33125T 12711T 2575T 43341T 7744T 14126T 35084T 25920T 33539T 11327T 1326T 27562T 15956 138-2T
IFO ATCC ATCC ATCC IFO CCM ATCC ATCC ATCC ATCC ATCC ATCC NCTC NCMB ATCC CCUG
ATCC, American type culture collection ; CCM, Czechoslovak collection of microorganisms; CCUG, Culture collection of University of Go«teborg; IFO, Institute for fermentation, Osaka; NCMB, National collection of marine bacteria; NCTC, National collection of type cultures.
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Fig. 1. E¡ect of CCCP on growth of various Vibrio species at pH 6.5, 7.5, and 8.5. Bacterial growth was measured by optical density at 450 nm (OD450 ). Growth values were expressed as the percentage to OD450 in the early stationary phase in VNSS without CCCP at each pH. CCCP concentrations were 5 WM at pH 6.5 (light gray box), 10 WM at pH 7.5 (dark gray box), and 15 WM at pH 8.5 (black box). Experiments were repeated at least three times, and standard deviation bars are shown.
various pH conditions is summarized in Fig. 1. With the addition of CCCP, there is a tendency that Vibrio strains grow at pH 8.5 but not at pH 6.5. Compared with other Vibrio species, V. anguillarum strains with the exception of serotype O-4 were more sensitive to CCCP. Even at pH 8.5, no V. anguillarum strains except serotype O-4 could grow well. Other Vibrio species, except V. mimicus, V. splendidus and V. ¢scheri could grow well at pH 8.5 even in the presence of CCCP. V. angustum S14 and V. alginolyticus, which were used for further NADH oxidase assay, also showed a typical pattern of marine bacteria. Even with CCCP, both strains grew well at pH 8.5. 3.2. E¡ects of monovalent cations on NADH oxidase of bacterial membrane fractions NADH oxidase in membrane fractions prepared from V. angustum S14 required Naþ as speci¢cally as did those of V. alginolyticus (Fig. 2C and D). NADH oxidase in membrane fractions from V. anguillarum M93 and serotype O-4 also required Naþ for maximum activity (Fig. 2A and B). However, with the V. anguillarum strains, Kþ also
stimulated the NADH oxidase activities. The NADH activities of these strains with Kþ reached 60^70% of the activities with Naþ (Fig. 2A and B). The NADH oxidase activity of V. anguillarum M93 reached the maximum level at 0.1 M NaCl, as in saline (Fig. 2A). The NADH oxidase activity of V. anguillarum serotype O-4 reached the maximum level at 0.1^0.2 M NaCl (Fig. 2B). On the other hand, NADH oxidase activities of V. angustum S14 and V. alginolyticus reached their maxima at 0.4 M NaCl, as in seawater (Fig. 2C and D). 3.3. E¡ects of inhibitor HQNO on NADH oxidase of bacterial membrane fractions The respiratory inhibitor, HQNO, strongly inhibits the Naþ -translocating activity of Naþ -NQR. When more than 0.5 WM of HQNO was added to the membrane fractions, the NADH oxidase activities of V. anguillarum M93, V. anguillarum serotype O-4, V. angustum S14 and V. alginolyticus were totally inhibited (Fig. 3). This means that all tested Vibrio strains require active Naþ extrusion sites for their active NADH oxidation in membrane fractions.
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Fig. 2. E¡ects of monovalent cations on NADH oxidase activity of V. anguillarum M93 (A), V. anguillarum O-4 (B), V. angustum S14 (C), and V. alginolyticus (D). The assay mixture (1 ml) contained 20 mM Tris^HCl (pH 8.0), 0.2 mM NADH, 10^30 Wg of membrane protein and various concentrations of KCl (F), NaCl (b) or LiCl (R). Experiments were repeated at least three times, and standard deviation bars are shown.
4. Discussion The presented enzymatic experiments showed that V. anguillarum M93, V. anguillarum serotype O-4, and V. angustum S14 possess Naþ -dependent NADH oxidase (Fig. 2). Naþ -NQR (primary Naþ pump) of V. alginolyticus consists of three major di¡erent subunits, K, L and Q, and catalyzes the initiatory reaction of the respiratory chain. The L subunit functions as NADH oxidase and its activity was measured in this study. The K and Q subunits are regarded as components to extrude Naþ across the cell membrane [23]. These three subunits are linked together and an inhibition of any of the sites stops all other functions [31]. The NADH oxidase activities in V. anguillarum M93 and O-4 and V. angustum S14 were all stimulated by Naþ (Fig. 2A, B and C) and inhibited by HQNO (Fig. 3). This indicates that the three strains possess Naþ -dependent NQR like other Vibrios. The existence of genes for the Naþ -NQR in V. anguillarum M93 and V. angustum S14 was also con¢rmed by polymerase chain reaction with primers of Naþ -NQR from V. alginolyticus (Kita-Tsukamoto, unpublished data). While the Naþ -dependent NADH oxidase did exist in V. anguillarum M93 and O-4, the speci¢city for monova-
lent cations (Naþ , Kþ and Liþ ) was di¡erent from that in V. angustum S14 and V. alginolyticus. V. angustum S14 and V. alginolyticus showed more than two-fold increase in the activity when Naþ was added, compared with Kþ and Liþ (Fig. 2). According to Oh et al. [10], NADH oxidase is considered to be speci¢c for a certain cation when that cation activates the NADH oxidase more than two-fold compared with other cations. Judging from this criterion, it is clear that V. angustum S14 possesses NADH oxidase that is speci¢cally dependent on Naþ , just as other Vibrios do. The NADH oxidase of V. anguillarum M93 and serotype O-4 was also highly activated by Naþ , however Kþ also enhanced the activity up to 60^70% of that with Naþ . This tendency exhibited by V. anguillarum has never been recognized in any other Vibrios. This indicates a qualitative di¡erence in the Naþ dependent NADH oxidase of V. anguillarum and those of other Vibrios such as V. angustum S14 and V. alginolyticus. The response of the Naþ -dependent NADH oxidase to the Naþ concentration was also di¡erent among V. anguillarum strains and other Vibrios. The NADH oxidase activities of V. angustum S14 and V. alginolyticus reached their maxima at a concentration of 0.4 M NaCl, the natural seawater level (Fig. 2C and D). In contrast, the NADH oxidase of V. anguillarum M93 showed maximum activity at a lower NaCl concentration (0.1 M), and its activity slightly decreased in accordance with the increase of NaCl concentration (V0.4 M) (Fig. 2A). V. anguillarum O-4 also reached the maximum Naþ -NADH oxidase activity at lower concentrations of NaCl (0.1^0.2 M). Different from strain M93, strain O-4 maintained the same level of the activity at higher NaCl concentrations (Fig. 2B). As strain M93 was isolated from a freshwater ¢sh and strain O-4 was isolated from natural seawater, the di¡er-
Fig. 3. E¡ect of HQNO on NADH oxidases of V. anguillarum M93, O-4, V. angustum S14, and V. alginolyticus. The activity of the NADH oxidases in the presence of 0.2 M NaCl was determined as described in Fig. 2 as a function of HQNO concentration. Membranes examined were V. anguillarum M93 (b), V. anguillarum O-4 (8), V. angustum S14 (F), V. alginolyticus (R). Experiments were repeated at least three times, and standard deviation bars are shown.
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ence in the NADH oxidase activity between these two strains probably re£ects the di¡erence in their original habitats of freshwater and seawater. Even without added NaCl (0 M), NADH oxidase of strains M93 and O-4 showed some activity (around 0.5 Wmol min31 mg protein31 ) (Fig. 2A and B). This is probably caused by the small amount of NaCl (0.1 mM) in the bu¡er solution of bacterial membrane fractions. Marine Vibrios did not show such a tendency (Fig. 2C and D). It was reported that V. cholerae also possessed Naþ dependent NQR [6]. Naþ speci¢cally activated NADH oxidase of V. cholerae, but neither Kþ nor Liþ did. This tendency is similar to that of marine-type Vibrios such as V. angustum S14 and V. alginolyticus. However, the activity of the Naþ -dependent NADH oxidase in V. cholerae reached the maximum at relatively lower Naþ concentration, around 0.1 M [6]. This tendency is similar to that of V. anguillarum. From this point, Naþ -NQR of V. cholerae seems to be similar to that of V. anguillarum. When CCCP, a proton conductor, collapses the electrical potential (vi) produced by the Hþ gradient across a cell membrane, bacterial cells that depend only on proton motive force are unable to acquire energy through respiration and die [12]. At pH 8.5, CCCP could not destroy the vi generated by V. alginolyticus. This is because V. alginolyticus uses a Naþ motive force instead of a Hþ motive force at pH 8.5 [32]. With the addition of CCCP, even at pH 8.5, V. anguillarum strains other than serotype O-4 could not grow well (Fig. 1). This means that, even at higher pH, V. anguillarum is probably more dependent on vi produced by Hþ than by Naþ . Luminescent marine Vibrios, such as V. ¢scheri and V. harveyi, also possessed a primary Naþ pump (Naþ -NQR) and their luminescence seemed to link to the sodium motive force bioenergetically [33]. With the addition of CCCP at pH 8.5, V. harveyi could grow well and luminesce, but V. ¢scheri could not (Fig. 1; [33]). Wada and Kogure [33] suggested two possible reasons for this. One was a qualitative di¡erence in their primary Naþ pumps, and the other was a quantitative di¡erence in the number of functional Naþ pumps. The present study has shown that at least there are qualitative di¡erences among the primary Naþ pumps in Vibrios. In this study, it was revealed that (1) V. anguillarum M93 from a freshwater area possesses a Naþ -dependent NADH oxidase that is characteristic of marine bacteria; however, (2) V. anguillarum M93 is less dependent on the Naþ motive force than are marine bacteria; and furthermore, (3) the Naþ -dependent NADH oxidase of V. anguillarum M93 is adapted to lower salinity environments. V. anguillarum often infects salmon group ¢sh, and this type of ¢sh migrates between freshwater and seawater. Thus, V. anguillarum that attaches to the ¢sh experiences various salt concentrations, di¡erent from other marine bacteria. The characteristics of the Naþ -dependent respiratory chain of V. anguillarum probably re£ect this eco-
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logical background with relating lower Naþ concentrations. The present results highlight the possibility that a respiratory chain, one of the essential metabolic pathways, evolutionary adapts to the microbial life styles and habitats. We are now sequencing the full gene (V7 kb) of Naþ NQR in V. anguillarum to produce Naþ -NQR deleted mutants. The Naþ -NQR mutants of V. anguillarum will be helpful and useful to clarify the eco-physiological functions of Naþ -NQR, especially under natural starvation conditions.
Acknowledgements We are grateful to T. Nishino, K. Yao and T. Yamane for their useful suggestions on this study. We thank the reviewers for their particularly constructive critiques. This work was partly supported by funds granted by the Japanese Society for the Promotion of Science (grant no. 10660191) and Grant-in-Aid for Creative Basic Research, #12NP0201 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
References [1] Dimroth, P. (1987) Sodium ion transport decarboxylases and other aspects of sodium ion cycling in bacteria. Microbiol. Rev. 51, 320^ 340. [2] Ha«se, C.C., Fedorova, N.D., Galperin, M.Y. and Dibrov, P.A. (2001) Sodium ion cycle in bacterial pathogens: Evidence from cross-genome comparisons. Microbiol. Mol. Biol. Rev. 65, 353^370. [3] Laddaga, R.A. and MacLeod, R.A. (1982) E¡ects of wash treatments on the ultrastructure and lysozyme penetrability of the outer membrane of various marine and two terrestrial gram-negative bacteria. Can. J. Microbiol. 28, 318^324. [4] McCarter, L.L. (2001) Polar £agellar motility of the Vibrionaceae. Microbiol. Mol. Biol. Rev. 65, 445^462. [5] Unemoto, T., Tsuruoka, T. and Hayashi, M. (1973) Role of Naþ and Kþ in preventing lysis of a slightly halophilic Vibrio alginolyticus. Can. J. Microbiol. 19, 563^571. [6] Barquera, B., Hellwig, P., Zhou, W., Morgan, J.E., Ha«se, C.C., Gosink, K.K., Nilges, M., Brueseho¡, P.J., Roth, A., Lancaster, C.R.D. and Gennis, R.B. (2002) Puri¢cation and characterization of the recombinant Naþ -translocating NADH:quinone oxidoreductase from Vibrio cholerae. Biochemistry 41, 3781^3789. [7] Cho, K.H. and Kim, Y.J. (2000) Enzymatic and energetic properties of the aerobic respiratory chain-linked NADH oxidase system in the marine bacterium Pseudomonas nautica. Mol. Cell 10, 432^436. [8] Dimroth, P. (1997) Primary sodium ion translocating enzymes. Biochim. Biophys. Acta 1318, 11^51. [9] Kato, S. and Yumoto, I. (2000) Detection of the Naþ -translocating NADH-quinone reductase in marine bacteria using a PCR technique. Can. J. Microbiol. 46, 325^332. [10] Oh, S., Kogure, K., Ohwada, K. and Simidu, U. (1991) Correlation between possession of a respiration-dependent Naþ pump and Naþ requirement for growth of marine bacteria. Appl. Environ. Microbiol. 57, 1844^1846. [11] Tokuda, H. and Kogure, K. (1989) Generalized distribution and common properties of Naþ -dependent NADH :quinone oxidoreductases in gram-negative marine bacteria. J. Gen. Microbiol. 135, 703^709.
FEMSEC 1498 8-4-03
230
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[12] Kogure, K. (1998) Bioenergetics of marine bacteria. Environ. Biotech. 9, 278^282. [13] Larsen, J.L. (1984) Vibrio anguillarum : in£uence of temperature, pH, NaCl concentration and incubation time on growth. J. Appl. Bacteriol. 57, 237^246. [14] Muroga, K. and Egusa, S. (1967) Vibrio anguillarum from an endemic disease of ayu in lake Hamana. Bull. Jpn. Soc. Sci. Fish. 33, 636^640. [15] Miyamoto, N. and Eguchi, M. (1997) Direct detection of a ¢sh pathogen, Vibrio anguillarum serotype J-O-1, in freshwater by £uorescent antibody technique. Fish. Sci. 63, 253^257. [16] Muroga, K., Iida, M. and Matsumoto, H. (1986) Detection of Vibrio anguillarum from waters. Bull. Jpn. Soc. Sci. Fish. 52, 641^647. [17] Miyamoto, N. and Eguchi, M. (1996) Development of monoclonal antibodies that speci¢cally react with a ¢sh pathogen, Vibrio anguillarum serotype J-O-1. Fish. Sci. 62, 710^714. [18] Eguchi, M., Fujiwara, E. and Miyamoto, N. (2000) Survival of Vibrio anguillarum in freshwater environments: adaptation or debilitation ? J. Infect. Chemother. 6, 126^129. [19] Ho¡, K.A. (1989) Survival of Vibrio anguillarum and Vibrio salmonicida at di¡erent salinities. Appl. Environ. Microbiol. 55, 1775^1786. [20] Miyamoto, N. and Eguchi, M. (1997) Response to low osmotic stress in a ¢sh pathogen, Vibrio anguillarum. FEMS Microbiol. Ecol. 22, 225^231. [21] Garcia, T., Otto, K., Kjelleberg, S. and Nelson, D.R. (1997) Growth of Vibrio anguillarum in salmon intestinal mucus. Appl. Environ. Microbiol. 63, 1034^1039. [22] SZrensen, U.B.S. and Larsen, J.L. (1986) Serotyping of Vibrio anguillarum. Appl. Environ. Microbiol. 51, 593^597. [23] Hayashi, M., Nakayama, Y. and Unemoto, T. (2001) Recent progress in the Naþ -translocating NADH-quinone reductase from the marine Vibrio alginolyticus. Biochim. Biophys. Acta 1505, 37^44. [24] Eguchi, M., Nishikawa, T., MacDonald, K., Cavicchioli, R., Gottschal, J.C. and Kjelleberg, S. (1996) Responses to stress and nutrient
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
availability by the marine ultramicrobacterium Sphingomonas sp. strain RB2256. Appl. Environ. Microbiol. 62, 1287^1294. Nystro«m, T., Marden, P. and Kjelleberg, S. (1986) Relative changes in incorporation rates of leucine and methionine during starvation of two bacteria isolated from marine waters. FEMS Microbiol. Ecol. 38, 285^292. º stling, J., Goodman, A. and Kjelleberg, S. (1991) Behaviour of O IncP-1 plasmids and a miniMu transposon in a marine Vibrio sp.: isolation of starvation inducible lac operon fusions. FEMS Microbiol. Ecol. 86, 83^94. Unemoto, T., Hayashi, M. and Hayashi, M. (1977) Naþ -dependent activation of NADH oxidase in membrane fractions from halophilic Vibrio alginolyticus and V. costicolus. J. Biochem. 82, 1389^1395. Ha«se, C.C. and Mekalanos, J.J. (1999) E¡ects of changes in membrane sodium £ux on virulence gene expression in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 96, 3183^3187. Unemoto, T. and Hayashi, M. (1993) Naþ -translocating NADH-quinone reductase of marine and halophilic bacteria. J. Bioenerg. Biomembr. 25, 385^391. Lowry, O.H., Rosebrough, N.J., Farr, L. and Radall, R.J. (1951) Protein measurement with the Folin Phenol Reagent. J. Biol. Chem. 193, 267^275. Hayashi, M. and Unemoto, T. (1987) Subunit component and their roles in the sodium-transport NADH:quinone reductase of a marine bacterium, Vibrio alginolyticus. Biochim. Biophys. Acta 890, 47^54. Tokuda, H. and Unemoto, T. (1983) Growth of a marine Vibrio alginolyticus and moderately halophilic V. costicola becomes uncoupler resistant when the respiration-dependent Naþ pump functions. J. Bacteriol. 156, 636^643. Wada, M. and Kogure, K. (2000) Membrane bioenergetics in reference to marine bacterial culturability. In: Nonculturable Microorganisms in the Environment, pp. 47^55. American Society for Microbiology, Washington, DC.
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Characteristics of Naþ-dependent respiratory chain in Vibrio anguillarum, a ¢sh pathogen, in comparison with other marine Vibrios Erina Fujiwara-Nagata a , Kazuhiro Kogure b , Kumiko Kita-Tsukamoto b , Minoru Wada b , Mitsuru Eguchi a; b
a Department of Fisheries, Kinki University, Nara 631-8505, Japan Ocean Research Institute, The University of Tokyo, Tokyo 164-8639, Japan
Received 27 September 2002; received in revised form 13 December 2002; accepted 29 December 2002 First published online 12 February 2003
Abstract The activity of membrane-bound NADH oxidase of Vibrio anguillarum M93 (serotype J-O-1), which causes vibriosis in freshwater area was activated by Naþ in the same manner as other marine Vibrios. However, in addition to Naþ , Kþ was also found to positively enhance the NADH oxidase activity of strain M93. This tendency has not been recognized in other marine Vibrios. Furthermore, the Naþ dependent NADH oxidase of strain M93 required less Naþ (0.1 M) for its maximum activity than those of other Vibrios such as Vibrio alginolyticus and ‘Vibrio angustum’ S14, which were in the range of 0.4 M NaCl, similar as seawater. Destruction of Hþ motive force by a proton conductor carbonylcyanide m-chlorophenylhydrazone (CCCP) revealed high dependency of V. anguillarum on the primary Hþ pump. Even at pH 8.5, V. anguillarum strains other than serotype O-4 could not grow well with the addition of CCCP. In contrast, marine-type Vibrios such as V. alginolyticus and V. angustum S14 can grow well at pH 8.5 even with the addition of CCCP. The lower requirement for Naþ in V. anguillarum probably reflects the salinity of their original habitats. > 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Naþ -dependent NADH oxidase; Marine bacteria ; Primary Naþ pump; Fish pathogen; Vibrio anguillarum
1. Introduction Why do marine bacteria require Naþ for growth? This has long been a major question for marine microbiologists, and studies have uncovered a few Naþ functions in marine bacteria, such as Naþ stabilization of the cell membrane, Naþ -dependent active transport, and £agellar motility [1^5]. Besides these physiological Naþ functions, it was reported that marine bacteria possessed a Naþ -dependent respiratory chain (Naþ -NQR, Naþ -NADH :quinone oxidoreductase; primary Naþ pump) in addition to the Hþ -dependent respiratory chain [2,6^12,23]. From a bioenergetic point of view, the Naþ -rich and alkaline environment of the sea must make it advantageous for marine
* Corresponding author. Tel. : +81 (742) 431511; Fax : +81 (742) 431316. E-mail address : [email protected] (M. Eguchi).
bacteria to possess a primary Naþ pump in addition to the primary Hþ pump [12]. Vibrio anguillarum is a well-known ¢sh pathogen that requires around 2% NaCl for optimal growth [13,14]. This pathogen has caused serious damage to aquaculture systems worldwide, especially salmon cultures in brackish and coastal water regions. An unusual case is a vibriosis by V. anguillarum that occurs to landlocked salmonoid in freshwater areas in Japan [15,16]. Although this pathogen was detected from bottom sediments of freshwater areas by an indirect immuno£uorescence method with a speci¢c monoclonal antibody [15,17], it has never been detected by conventional culturing methods. The infectious process and ecology of the pathogen in freshwater regions remain unclear. When V. anguillarum cells were exposed to sterilized freshwater directly, their ability to form colonies on agar plates and their pathogenicity to ¢sh disappeared rapidly [18^20]. The main reason for this rapid loss of culturability seems to be the lack of Naþ in freshwater [18].
0168-6496 / 03 / $22.00 > 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-6496(03)00031-X
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In this report, we demonstrate the presence and describe the characteristics of a Naþ -dependent respiratory chain in V. anguillarum isolated from freshwater ¢sh killed by vibriosis. The relationship between the characteristics of the primary Naþ pump and the salinity of the original habitat is also discussed.
2. Materials and methods 2.1. Bacterial strains In the bacterial growth experiments under the presence of a proton conductor, carbonylcyanide m-chlorophenylhydrazone (CCCP, Sigma), 28 strains of Vibrio species were used as shown in Table 1. V. anguillarum M93 was originally isolated from diseased freshwater ¢sh, Plecoglossus altivelis (Salmoniforms) in Lake Biwa, Japan. It causes vibriosis in freshwater and seawater ¢sh [20,21]. Its Japanese serotype is J-O-1, which probably corresponds to the Danish serotype O-2 [17]. Naþ -NADH oxidase activities and inhibitory e¡ects on the activities by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO, Sigma) were measured with V. anguillarum M93, and V. anguillarum serotype O-4. V. anguillarum serotype O-4 is more common in the marine environment [22]. ‘Vibrio angustum’ S14 (Vibrio sp. CCUG15956) and Vibrio alginolyticus were used as representatives of marine Vibrio. V. alginolyticus was known to possess a marinetype Naþ -NQR [23]. 2.2. Growth with CCCP Strains were pre-incubated in a VNSS medium, a complex medium for marine bacteria [24,25]. For pH control, 40 mM of MES, MOPS and Tricine were used at pH 6.5, 7.5 and 8.5, respectively. All strains were inoculated and incubated in VNSS media with 5, 10 and 15 WM CCCP at pH 6.5, 7.5 and 8.5, respectively. These values of CCCP corresponded to the minimum concentrations that inhibited the growth of V. anguillarum M93 at each pH. Cultures were incubated at 20‡C with shaking (rpm 180). As for a control, bacterial growth was estimated by measuring optical density at 450 nm (OD450 ) when each strain reached early stationary phase without CCCP. All strains grew well in VNSS media without CCCP. Growth values with CCCP were expressed as a percentage to OD450 at early stationary phase in VNSS media without CCCP at each pH. Thus, %growth = (OD450 with CCCP)U100/ (OD450 without CCCP). 2.3. Preparation of membrane fraction and NADH oxidase assay Marine minimal medium (MMM), a chemically de¢ned medium, was used [26]. The composition of MMM was as
follows : 0.2% glucose, 9.52 mM NH4 Cl, 1.32 mM K2 HPO4 , 40 mM MOPS in NSS (pH 8.0; [24]). Log phase cells were harvested and membrane fractions were prepared by an osmotic lysis method [27]. The membrane fractions were suspended in 10 mM NaCl, 20 mM Tris^ HCl (pH 8.0) and 10% glycerol, and kept at 20‡C until measurements of NADH oxidase activity. NADH oxidase activity was measured from the decrease in absorbance at 340 nm at 30‡C. The standard assay mixture contained 20 mM Tris^HCl (pH 8.0), 0.2 mM NADH, 0.2 M salt (NaCl, KCl or LiCl), and 10^30 Wg of membrane protein in a ¢nal volume of 1 ml. HQNO was used to observe the inhibitory e¡ects on Naþ extruding segments [7,28,29]. Protein concentration was determined as described by Lowry et al. [30] by using a Modi¢ed Lowry Protein Assay 23240 Kit (Pierce).
3. Results 3.1. E¡ect of CCCP on bacterial growth The growth of various Vibrio strains with CCCP under Table 1 List of bacterial strains used Bacteria
Serotype
Source
Strain no.
V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. anguillarum V. mimicus V. pelagius V. splendidus V. parahaemolyticus V. natriegens V. mediterranei V. ¢scheri V. harveyi V. carchariae V. campbellii V. damselae V. £uvialis V. proteolyticus V. vulni¢cus ‘V. angustum’ S14 V. alginolyticus
O-1 O-2 O-3 O-4 O-5 O-6 O-7 O-8 O-9 O-10 J-O-1(O-2) O-2
ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC
43305 43306 43307 43308 43309 43310 43311 43312 43313 43314 M93 13266T 33653T 25916T 33125T 12711T 2575T 43341T 7744T 14126T 35084T 25920T 33539T 11327T 1326T 27562T 15956 138-2T
IFO ATCC ATCC ATCC IFO CCM ATCC ATCC ATCC ATCC ATCC ATCC NCTC NCMB ATCC CCUG
ATCC, American type culture collection ; CCM, Czechoslovak collection of microorganisms; CCUG, Culture collection of University of Go«teborg; IFO, Institute for fermentation, Osaka; NCMB, National collection of marine bacteria; NCTC, National collection of type cultures.
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Fig. 1. E¡ect of CCCP on growth of various Vibrio species at pH 6.5, 7.5, and 8.5. Bacterial growth was measured by optical density at 450 nm (OD450 ). Growth values were expressed as the percentage to OD450 in the early stationary phase in VNSS without CCCP at each pH. CCCP concentrations were 5 WM at pH 6.5 (light gray box), 10 WM at pH 7.5 (dark gray box), and 15 WM at pH 8.5 (black box). Experiments were repeated at least three times, and standard deviation bars are shown.
various pH conditions is summarized in Fig. 1. With the addition of CCCP, there is a tendency that Vibrio strains grow at pH 8.5 but not at pH 6.5. Compared with other Vibrio species, V. anguillarum strains with the exception of serotype O-4 were more sensitive to CCCP. Even at pH 8.5, no V. anguillarum strains except serotype O-4 could grow well. Other Vibrio species, except V. mimicus, V. splendidus and V. ¢scheri could grow well at pH 8.5 even in the presence of CCCP. V. angustum S14 and V. alginolyticus, which were used for further NADH oxidase assay, also showed a typical pattern of marine bacteria. Even with CCCP, both strains grew well at pH 8.5. 3.2. E¡ects of monovalent cations on NADH oxidase of bacterial membrane fractions NADH oxidase in membrane fractions prepared from V. angustum S14 required Naþ as speci¢cally as did those of V. alginolyticus (Fig. 2C and D). NADH oxidase in membrane fractions from V. anguillarum M93 and serotype O-4 also required Naþ for maximum activity (Fig. 2A and B). However, with the V. anguillarum strains, Kþ also
stimulated the NADH oxidase activities. The NADH activities of these strains with Kþ reached 60^70% of the activities with Naþ (Fig. 2A and B). The NADH oxidase activity of V. anguillarum M93 reached the maximum level at 0.1 M NaCl, as in saline (Fig. 2A). The NADH oxidase activity of V. anguillarum serotype O-4 reached the maximum level at 0.1^0.2 M NaCl (Fig. 2B). On the other hand, NADH oxidase activities of V. angustum S14 and V. alginolyticus reached their maxima at 0.4 M NaCl, as in seawater (Fig. 2C and D). 3.3. E¡ects of inhibitor HQNO on NADH oxidase of bacterial membrane fractions The respiratory inhibitor, HQNO, strongly inhibits the Naþ -translocating activity of Naþ -NQR. When more than 0.5 WM of HQNO was added to the membrane fractions, the NADH oxidase activities of V. anguillarum M93, V. anguillarum serotype O-4, V. angustum S14 and V. alginolyticus were totally inhibited (Fig. 3). This means that all tested Vibrio strains require active Naþ extrusion sites for their active NADH oxidation in membrane fractions.
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Fig. 2. E¡ects of monovalent cations on NADH oxidase activity of V. anguillarum M93 (A), V. anguillarum O-4 (B), V. angustum S14 (C), and V. alginolyticus (D). The assay mixture (1 ml) contained 20 mM Tris^HCl (pH 8.0), 0.2 mM NADH, 10^30 Wg of membrane protein and various concentrations of KCl (F), NaCl (b) or LiCl (R). Experiments were repeated at least three times, and standard deviation bars are shown.
4. Discussion The presented enzymatic experiments showed that V. anguillarum M93, V. anguillarum serotype O-4, and V. angustum S14 possess Naþ -dependent NADH oxidase (Fig. 2). Naþ -NQR (primary Naþ pump) of V. alginolyticus consists of three major di¡erent subunits, K, L and Q, and catalyzes the initiatory reaction of the respiratory chain. The L subunit functions as NADH oxidase and its activity was measured in this study. The K and Q subunits are regarded as components to extrude Naþ across the cell membrane [23]. These three subunits are linked together and an inhibition of any of the sites stops all other functions [31]. The NADH oxidase activities in V. anguillarum M93 and O-4 and V. angustum S14 were all stimulated by Naþ (Fig. 2A, B and C) and inhibited by HQNO (Fig. 3). This indicates that the three strains possess Naþ -dependent NQR like other Vibrios. The existence of genes for the Naþ -NQR in V. anguillarum M93 and V. angustum S14 was also con¢rmed by polymerase chain reaction with primers of Naþ -NQR from V. alginolyticus (Kita-Tsukamoto, unpublished data). While the Naþ -dependent NADH oxidase did exist in V. anguillarum M93 and O-4, the speci¢city for monova-
lent cations (Naþ , Kþ and Liþ ) was di¡erent from that in V. angustum S14 and V. alginolyticus. V. angustum S14 and V. alginolyticus showed more than two-fold increase in the activity when Naþ was added, compared with Kþ and Liþ (Fig. 2). According to Oh et al. [10], NADH oxidase is considered to be speci¢c for a certain cation when that cation activates the NADH oxidase more than two-fold compared with other cations. Judging from this criterion, it is clear that V. angustum S14 possesses NADH oxidase that is speci¢cally dependent on Naþ , just as other Vibrios do. The NADH oxidase of V. anguillarum M93 and serotype O-4 was also highly activated by Naþ , however Kþ also enhanced the activity up to 60^70% of that with Naþ . This tendency exhibited by V. anguillarum has never been recognized in any other Vibrios. This indicates a qualitative di¡erence in the Naþ dependent NADH oxidase of V. anguillarum and those of other Vibrios such as V. angustum S14 and V. alginolyticus. The response of the Naþ -dependent NADH oxidase to the Naþ concentration was also di¡erent among V. anguillarum strains and other Vibrios. The NADH oxidase activities of V. angustum S14 and V. alginolyticus reached their maxima at a concentration of 0.4 M NaCl, the natural seawater level (Fig. 2C and D). In contrast, the NADH oxidase of V. anguillarum M93 showed maximum activity at a lower NaCl concentration (0.1 M), and its activity slightly decreased in accordance with the increase of NaCl concentration (V0.4 M) (Fig. 2A). V. anguillarum O-4 also reached the maximum Naþ -NADH oxidase activity at lower concentrations of NaCl (0.1^0.2 M). Different from strain M93, strain O-4 maintained the same level of the activity at higher NaCl concentrations (Fig. 2B). As strain M93 was isolated from a freshwater ¢sh and strain O-4 was isolated from natural seawater, the di¡er-
Fig. 3. E¡ect of HQNO on NADH oxidases of V. anguillarum M93, O-4, V. angustum S14, and V. alginolyticus. The activity of the NADH oxidases in the presence of 0.2 M NaCl was determined as described in Fig. 2 as a function of HQNO concentration. Membranes examined were V. anguillarum M93 (b), V. anguillarum O-4 (8), V. angustum S14 (F), V. alginolyticus (R). Experiments were repeated at least three times, and standard deviation bars are shown.
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ence in the NADH oxidase activity between these two strains probably re£ects the di¡erence in their original habitats of freshwater and seawater. Even without added NaCl (0 M), NADH oxidase of strains M93 and O-4 showed some activity (around 0.5 Wmol min31 mg protein31 ) (Fig. 2A and B). This is probably caused by the small amount of NaCl (0.1 mM) in the bu¡er solution of bacterial membrane fractions. Marine Vibrios did not show such a tendency (Fig. 2C and D). It was reported that V. cholerae also possessed Naþ dependent NQR [6]. Naþ speci¢cally activated NADH oxidase of V. cholerae, but neither Kþ nor Liþ did. This tendency is similar to that of marine-type Vibrios such as V. angustum S14 and V. alginolyticus. However, the activity of the Naþ -dependent NADH oxidase in V. cholerae reached the maximum at relatively lower Naþ concentration, around 0.1 M [6]. This tendency is similar to that of V. anguillarum. From this point, Naþ -NQR of V. cholerae seems to be similar to that of V. anguillarum. When CCCP, a proton conductor, collapses the electrical potential (vi) produced by the Hþ gradient across a cell membrane, bacterial cells that depend only on proton motive force are unable to acquire energy through respiration and die [12]. At pH 8.5, CCCP could not destroy the vi generated by V. alginolyticus. This is because V. alginolyticus uses a Naþ motive force instead of a Hþ motive force at pH 8.5 [32]. With the addition of CCCP, even at pH 8.5, V. anguillarum strains other than serotype O-4 could not grow well (Fig. 1). This means that, even at higher pH, V. anguillarum is probably more dependent on vi produced by Hþ than by Naþ . Luminescent marine Vibrios, such as V. ¢scheri and V. harveyi, also possessed a primary Naþ pump (Naþ -NQR) and their luminescence seemed to link to the sodium motive force bioenergetically [33]. With the addition of CCCP at pH 8.5, V. harveyi could grow well and luminesce, but V. ¢scheri could not (Fig. 1; [33]). Wada and Kogure [33] suggested two possible reasons for this. One was a qualitative di¡erence in their primary Naþ pumps, and the other was a quantitative di¡erence in the number of functional Naþ pumps. The present study has shown that at least there are qualitative di¡erences among the primary Naþ pumps in Vibrios. In this study, it was revealed that (1) V. anguillarum M93 from a freshwater area possesses a Naþ -dependent NADH oxidase that is characteristic of marine bacteria; however, (2) V. anguillarum M93 is less dependent on the Naþ motive force than are marine bacteria; and furthermore, (3) the Naþ -dependent NADH oxidase of V. anguillarum M93 is adapted to lower salinity environments. V. anguillarum often infects salmon group ¢sh, and this type of ¢sh migrates between freshwater and seawater. Thus, V. anguillarum that attaches to the ¢sh experiences various salt concentrations, di¡erent from other marine bacteria. The characteristics of the Naþ -dependent respiratory chain of V. anguillarum probably re£ect this eco-
229
logical background with relating lower Naþ concentrations. The present results highlight the possibility that a respiratory chain, one of the essential metabolic pathways, evolutionary adapts to the microbial life styles and habitats. We are now sequencing the full gene (V7 kb) of Naþ NQR in V. anguillarum to produce Naþ -NQR deleted mutants. The Naþ -NQR mutants of V. anguillarum will be helpful and useful to clarify the eco-physiological functions of Naþ -NQR, especially under natural starvation conditions.
Acknowledgements We are grateful to T. Nishino, K. Yao and T. Yamane for their useful suggestions on this study. We thank the reviewers for their particularly constructive critiques. This work was partly supported by funds granted by the Japanese Society for the Promotion of Science (grant no. 10660191) and Grant-in-Aid for Creative Basic Research, #12NP0201 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
References [1] Dimroth, P. (1987) Sodium ion transport decarboxylases and other aspects of sodium ion cycling in bacteria. Microbiol. Rev. 51, 320^ 340. [2] Ha«se, C.C., Fedorova, N.D., Galperin, M.Y. and Dibrov, P.A. (2001) Sodium ion cycle in bacterial pathogens: Evidence from cross-genome comparisons. Microbiol. Mol. Biol. Rev. 65, 353^370. [3] Laddaga, R.A. and MacLeod, R.A. (1982) E¡ects of wash treatments on the ultrastructure and lysozyme penetrability of the outer membrane of various marine and two terrestrial gram-negative bacteria. Can. J. Microbiol. 28, 318^324. [4] McCarter, L.L. (2001) Polar £agellar motility of the Vibrionaceae. Microbiol. Mol. Biol. Rev. 65, 445^462. [5] Unemoto, T., Tsuruoka, T. and Hayashi, M. (1973) Role of Naþ and Kþ in preventing lysis of a slightly halophilic Vibrio alginolyticus. Can. J. Microbiol. 19, 563^571. [6] Barquera, B., Hellwig, P., Zhou, W., Morgan, J.E., Ha«se, C.C., Gosink, K.K., Nilges, M., Brueseho¡, P.J., Roth, A., Lancaster, C.R.D. and Gennis, R.B. (2002) Puri¢cation and characterization of the recombinant Naþ -translocating NADH:quinone oxidoreductase from Vibrio cholerae. Biochemistry 41, 3781^3789. [7] Cho, K.H. and Kim, Y.J. (2000) Enzymatic and energetic properties of the aerobic respiratory chain-linked NADH oxidase system in the marine bacterium Pseudomonas nautica. Mol. Cell 10, 432^436. [8] Dimroth, P. (1997) Primary sodium ion translocating enzymes. Biochim. Biophys. Acta 1318, 11^51. [9] Kato, S. and Yumoto, I. (2000) Detection of the Naþ -translocating NADH-quinone reductase in marine bacteria using a PCR technique. Can. J. Microbiol. 46, 325^332. [10] Oh, S., Kogure, K., Ohwada, K. and Simidu, U. (1991) Correlation between possession of a respiration-dependent Naþ pump and Naþ requirement for growth of marine bacteria. Appl. Environ. Microbiol. 57, 1844^1846. [11] Tokuda, H. and Kogure, K. (1989) Generalized distribution and common properties of Naþ -dependent NADH :quinone oxidoreductases in gram-negative marine bacteria. J. Gen. Microbiol. 135, 703^709.
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[12] Kogure, K. (1998) Bioenergetics of marine bacteria. Environ. Biotech. 9, 278^282. [13] Larsen, J.L. (1984) Vibrio anguillarum : in£uence of temperature, pH, NaCl concentration and incubation time on growth. J. Appl. Bacteriol. 57, 237^246. [14] Muroga, K. and Egusa, S. (1967) Vibrio anguillarum from an endemic disease of ayu in lake Hamana. Bull. Jpn. Soc. Sci. Fish. 33, 636^640. [15] Miyamoto, N. and Eguchi, M. (1997) Direct detection of a ¢sh pathogen, Vibrio anguillarum serotype J-O-1, in freshwater by £uorescent antibody technique. Fish. Sci. 63, 253^257. [16] Muroga, K., Iida, M. and Matsumoto, H. (1986) Detection of Vibrio anguillarum from waters. Bull. Jpn. Soc. Sci. Fish. 52, 641^647. [17] Miyamoto, N. and Eguchi, M. (1996) Development of monoclonal antibodies that speci¢cally react with a ¢sh pathogen, Vibrio anguillarum serotype J-O-1. Fish. Sci. 62, 710^714. [18] Eguchi, M., Fujiwara, E. and Miyamoto, N. (2000) Survival of Vibrio anguillarum in freshwater environments: adaptation or debilitation ? J. Infect. Chemother. 6, 126^129. [19] Ho¡, K.A. (1989) Survival of Vibrio anguillarum and Vibrio salmonicida at di¡erent salinities. Appl. Environ. Microbiol. 55, 1775^1786. [20] Miyamoto, N. and Eguchi, M. (1997) Response to low osmotic stress in a ¢sh pathogen, Vibrio anguillarum. FEMS Microbiol. Ecol. 22, 225^231. [21] Garcia, T., Otto, K., Kjelleberg, S. and Nelson, D.R. (1997) Growth of Vibrio anguillarum in salmon intestinal mucus. Appl. Environ. Microbiol. 63, 1034^1039. [22] SZrensen, U.B.S. and Larsen, J.L. (1986) Serotyping of Vibrio anguillarum. Appl. Environ. Microbiol. 51, 593^597. [23] Hayashi, M., Nakayama, Y. and Unemoto, T. (2001) Recent progress in the Naþ -translocating NADH-quinone reductase from the marine Vibrio alginolyticus. Biochim. Biophys. Acta 1505, 37^44. [24] Eguchi, M., Nishikawa, T., MacDonald, K., Cavicchioli, R., Gottschal, J.C. and Kjelleberg, S. (1996) Responses to stress and nutrient
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
availability by the marine ultramicrobacterium Sphingomonas sp. strain RB2256. Appl. Environ. Microbiol. 62, 1287^1294. Nystro«m, T., Marden, P. and Kjelleberg, S. (1986) Relative changes in incorporation rates of leucine and methionine during starvation of two bacteria isolated from marine waters. FEMS Microbiol. Ecol. 38, 285^292. º stling, J., Goodman, A. and Kjelleberg, S. (1991) Behaviour of O IncP-1 plasmids and a miniMu transposon in a marine Vibrio sp.: isolation of starvation inducible lac operon fusions. FEMS Microbiol. Ecol. 86, 83^94. Unemoto, T., Hayashi, M. and Hayashi, M. (1977) Naþ -dependent activation of NADH oxidase in membrane fractions from halophilic Vibrio alginolyticus and V. costicolus. J. Biochem. 82, 1389^1395. Ha«se, C.C. and Mekalanos, J.J. (1999) E¡ects of changes in membrane sodium £ux on virulence gene expression in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 96, 3183^3187. Unemoto, T. and Hayashi, M. (1993) Naþ -translocating NADH-quinone reductase of marine and halophilic bacteria. J. Bioenerg. Biomembr. 25, 385^391. Lowry, O.H., Rosebrough, N.J., Farr, L. and Radall, R.J. (1951) Protein measurement with the Folin Phenol Reagent. J. Biol. Chem. 193, 267^275. Hayashi, M. and Unemoto, T. (1987) Subunit component and their roles in the sodium-transport NADH:quinone reductase of a marine bacterium, Vibrio alginolyticus. Biochim. Biophys. Acta 890, 47^54. Tokuda, H. and Unemoto, T. (1983) Growth of a marine Vibrio alginolyticus and moderately halophilic V. costicola becomes uncoupler resistant when the respiration-dependent Naþ pump functions. J. Bacteriol. 156, 636^643. Wada, M. and Kogure, K. (2000) Membrane bioenergetics in reference to marine bacterial culturability. In: Nonculturable Microorganisms in the Environment, pp. 47^55. American Society for Microbiology, Washington, DC.
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