1 Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Stigboejlen 4, DK-1870 Frederiksberg C, Denmark
2 Marine Biological Laboratory, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark
Correspondence
John E. Olsen
jeo{at}kvl.dk
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ABSTRACT |
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INTRODUCTION |
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Flagellation and motility are of relative high cost to the bacterial metabolism and the stable maintenance of this system indicates that it is important for survival of the bacteria. As an inhabitant of the natural marine environment, V. anguillarum must survive changes in environmental factors such as salinity, temperature and availability of nutrients. Bordas et al. (1998) showed that V. anguillarum has a reduced chemotactic response towards fish mucus when exposed to 3·5 % NaCl compared to 1·7 % NaCl, although this was dependent on the source of the mucus. It is, however, not fully understood how environmental factors influence motility and/or chemotaxis of V. anguillarum, or whether environmentally induced changes in motility influence the ability of the bacterium to cause disease.
The aim of this study was to determine the influence of temperature, salinity and starvation on the motility and chemotaxis of V. anguillarum. Serine was chosen as the attractant as it has previously been found in relatively high concentrations in mucus from rainbow trout and is a good attractant for V. anguillarum compared to other carbohydrates and amino acids present in mucus (O'Toole et al., 1999). The role of pre-adaptation to different conditions on the chemotactic response was also investigated. The flagellar motor of other marine vibrios (e.g. Vibrio cholerae, Vibrio parahaemolyticus and Vibrio alginolyticus) is powered by an electrochemical gradient of Na+ across the cytoplasmic membrane (Atsumi et al., 1992
; Chernyak et al., 1983
; Kawagishi et al., 1995
; Kojima et al., 1999a
). If this were also the case in V. anguillarum, it would provide a direct mechanism whereby a change in Na+ concentration would influence chemotactic motility. We therefore investigated the dependence of V. anguillarum flagellum rotation on Na+ during growth and starvation.
The experiments show that V. anguillarum is motile and chemotactic even after starvation. Since motility and chemotaxis are important virulence factors, it was interesting to test whether the ability of the organism to cause disease was also unaffected by this treatment. We therefore tested whether starvation, which is a common situation for marine bacteria (Östling et al., 1993), affects virulence of V. anguillarum.
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METHODS |
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Growth and stress conditions.
To study the role of temperature on chemotaxis, the bacteria were cultured and the chemotaxis assay (see below) was performed at 5, 15 and 25 °C. All equipment and liquids were adjusted to the actual temperature before use. The role of salinity on motility and chemotaxis was investigated on cells grown in TSB-1·5 and TSB-3·0 at 25 °C. Bacteria used for measurements of motility and chemotaxis were harvested from exponentially growing cultures by centrifugation at 4000 r.p.m. for 5 min, washed twice in PBS [130 mM NaCl (0·8 %), 10 mM NaPO4, pH 7·2] and resuspended in PBS to 1x108 c.f.u. ml1. Cells grown in TSB-3·0 were washed and resuspended in PBS with 2·0 % NaCl. Thereafter the bacteria were diluted 1 : 9 in chemotaxis buffer (PBS, 0·01 mM EDTA) containing 0·8, 2·0, 3·0 and 4·0 % (w/v) NaCl. For the starvation experiments, the bacteria were diluted in PBS (multiple nutrient limitations) and thereafter left at 25 °C for 2 and 8 days. The number of c.f.u. was determined in all bacterial suspensions by spreading serial dilutions on TSA-1·5 prior to and after 1 h incubation in chemotaxis buffer with different salinity and temperature. Total cell numbers were counted by using a Zeiss Axioplane epifluorescence microscope after polycarbonate membrane filtration and acridine orange staining (Binnerup et al., 1993
).
Chemotaxis assay.
The technique used was the capillary assay (Adler, 1973) with a few modifications as described previously (Larsen et al., 2001
). Briefly, suspensions of washed cells were dispensed in 0·4 ml quantities to 1·5 ml Eppendorf tubes. Capillaries (disposable 5 µl pre-calibrated pipettes) (Vitrex) filled with 10 mM serine diluted in chemotaxis buffer were immersed into the bacterial suspension. Capillary tubes filled with the same buffer as used for dilution of the bacteria were included as controls. Data shown represent the mean of at least three separate experiments, each done in duplicate. The same number of c.f.u. ml1 was used in all assays, except for cells starved for 8 days and exposed to 2·7 % NaCl. To normalize differences in motility and experimental variations, the chemotactic response is expressed as the ratio of the number of bacteria in attractant capillaries to that in control capillaries. A ratio of 2 or greater is considered significant (Hazen et al., 1984
). The data were analysed statistically by analysis of variance (one-way ANOVA). Bacterial counts were log transformed before the data were analysed in order to achieve a uniform variance.
Motility assay.
Motility was measured by digitized image analysis. A standard video camera (JAI 2040) was mounted on a Zeiss Axioplan microscope. To study the role of salinity and starvation, V. anguillarum was sucked up in a microslides capillary tube, viewing path length 0·4 mm (Camlab) and examined by dark-field microscopy (10x/0·30 plan neofluar objective). Ten seconds of each video sequence was subsequently digitized (25 images s1, PAL format). Mean run, and mean and maximum velocity were determined using the tracking system LABTRACK (Dimedia) (Thar et al., 2000). Swimming tracks were recorded two-dimensionally but all tracks were detected. Thus, to ensure that the tracks used for calculations are almost parallel to the optical plane of the microscope (to avoid interference of bacteria swimming through the focal plane), only tracks above a certain length and velocity were used. The threshold levels for minimum length per track and minimum velocity were set to 0·36 s and 8 µm s1, respectively. Number of motile cells analysed per video sequence ranged from 148 to 282 cells with exponential phase cells and from 32 to 192 with starved cells. To study the role of temperature on swimming speed, V. anguillarum cells growing exponentially in TSB-1·5 at 5, 15 and 25 °C were examined by phase-contrast microscopy (100x/1·30 plan neofluar objective). A video was recorded and 15 s subsequently digitized (12·5 images s1, PAL format). The observation temperature was the same as the incubation temperature. The mean swimming speed was obtained by measuring at least 20 runs of cells by use of the AXIOVISION 3.0 image analysis program (Carl Zeiss).
Na+-dependence of the flagellar motor.
Phenamil was used to study the dependence of rotation of the flagellum on Na+ as described by Atsumi et al. (1990) and Kojima et al. (1999a
, b)
. Different concentrations (550 µM) of phenamil (Sigma) were added to growing bacteria after wash and resuspension in PBS and to bacteria starved in PBS at 25 °C for 2 and 8 days. Phenamil was added immediately before motility analysis. The swimming speed was measured as described above to study the role of temperature.
Fish infections.
Rainbow trout (Oncorhynchus mykiss L.) with a body weight of 1·33·2 g (mean 2·4 g) were kept in 100 l capacity tanks containing aerated, re-circulating tap water supplemented with 0·6 % NaCl at 12 °C. The fish were infected by immersion with either exponentially growing cells washed twice in PBS or bacteria starved for 2 days in PBS. The bacteria were added in 10-fold dilutions to 1 litre of diluted M9 minimal medium [g (l tap water)1 after dilution: Na2HPO4, 3·94; KH2PO4, 0·923; NaCl, 0·31; NH4Cl, 0·31]. Seven fish were exposed to each bacterial dilution. The fish were removed after 30 min, dipped briefly in tap water before they were transferred to a tank containing 30 l of aerated, re-circulating water. Fish were monitored daily. Dead fish were removed and subjected to bacteriological examination. Bacteria recovered from the kidney on Marine agar (Difco) supplemented with 5 % bovine blood were subjected to a slide agglutination test to confirm identity. The antiserum used was a polyclonal antiserum raised in rabbits against V. anguillarum serotype O1 (Larsen et al., 1994). The 50 % lethal dose (LD50) was calculated according to the statistical method of Reed & Muench (1938)
. The experiment was repeated once. The experiment was done in accordance with the legal notices of the Danish Animal Experiments Inspectorate.
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RESULTS |
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Chemotactic responses during starvation
The capillary assay demands that approximately the same number of culturable cells is used in each assay. To ensure that this was achievable in studies using starved cells, the total number of cells (TC) and the number of c.f.u. was initially followed during starvation. There was no difference between TC and the number of c.f.u. at the onset of starvation and after 2 days starvation. During this period, numbers increased approximately 17 % to 2·2x108 most likely because V. anguillarum is known to perform reductive cell divisions in the transition from growth to starvation, i.e. the number of bacteria increases without a corresponding increase in biomass (Kjelleberg et al., 1987; Nelson et al., 1997
). From day 2 to day 8, TC was reduced by 27 % to 1·5x108 and the number of c.f.u. was reduced by 91 % to 1·7x107. Thus, the fraction of culturable cells (TC/c.f.u.) was 11 % after 8 days starvation. In general, the number of c.f.u. after 8 days varied between 3 and 11 % of the maximum number of culturable cells (day 2).
The starved bacteria showed a high chemotactic response, but the response was significantly lower than the response of non-starved cells (one-way ANOVA, P=0·04) (Table 3). Although the chemotactic response (measured as ratio) was decreased, the number of c.f.u. in the capillary (serine and control) was higher after 2 days starvation. The reason for this is unknown but changed surface properties of the bacteria may be involved. Bacteria starved for 2 days in PBS were not able to make a significant chemotactic response at 2·7 % NaCl (ratio <2) and after 8 days starvation the chemotactic response at 2·7 % NaCl was below the level of detection (Table 3
). The culturability of V. anguillarum was not influenced by incubation for 1 h in chemotaxis buffer with 2·7 % NaCl (data not shown).
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Effects of NaCl on motility
The maximum velocity of the motile cells was higher when measured in the growth medium than after washes and resuspension in PBS (Table 4). However, only a few bacteria in each sample reached the maximum velocity (data not shown). The velocity seemed unaffected by the concentration of NaCl in the medium. The relatively high standard deviation between cells in one sample showed that the velocity of cells within the same population is very heterogeneous (Table 4
). The mean time of runs was 0·51 s in all cases.
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DISCUSSION |
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The swimming speed of V. anguillarum was reduced at lower temperatures. A similar temperature dependency has been described for Salmonella enterica serovar Typhimurium (Maeda et al., 1976; Miller & Koshland, 1977
). The relationship between viscosity and temperature may account for this. The viscosity of pure water at 5 and 25 °C is 1·519 and 0·890 centipoises, respectively, a change by a factor of 1·6 (Weast & Astle, 1983
). This corresponds to the difference in swimming velocity found at the two temperatures in the present study, and most likely flagellar power-generation is the same at the different temperatures.
The decrease in chemotactic response at decreasing temperatures was not only due to reduced swimming speed as shown by the change in chemotactic ratio between attractant and control capillaries. This allowed us to conclude that temperature has a more pronounced effect on chemotaxis than on motility. This is in accordance with reports on Escherichia coli, where a raise in temperature from 20 to 30 °C results in a 20-fold increase in chemotactic response, but only a two- to threefold increase in motility (Adler, 1973). The reason for the temperature dependency of chemotaxis is not known. However, the rate of phosphorylation and/or (de-)methylation of signal transduction proteins and receptors may increase with higher temperature, like the kinetics of other enzymic reactions. Furthermore, the fluidity of membranes depends on temperature. This may also include the sheath of the flagellum of V. anguillarum, as the flagellar sheath is a distinct outer-membrane domain (McCarter, 2001
).
The chemotactic responses to serine were also influenced by salinity, and primarily by the salinity of the chemotaxis buffer compared to the salinity of the growth medium. Thus, the chemotactic responses were significantly reduced when the salinity of the chemotaxis buffer increased. Likewise, data from another study indicate that the highest chemotactic response of V. anguillarum to fish mucus was never obtained at a salinity of 3·5 % compared to 1·0 and 1·7 % (Bordas et al., 1998). V. anguillarum is a halophilic bacterium causing infections in the marine environment and survives starvation in sea water microcosm with 1·03·5 % NaCl for up to 4 weeks (Hoff, 1989
). Padan & Krulwich (2000)
speculated that Na+ interferes with optimal protein folding, and in some bacteria Na+-dependent gene expression has been shown, for example in V. cholerae (Hase & Mekalanos, 1999
). A plausible explanation for the dependency on salinity of chemotactic responses of V. anguillarum is that proteins of the signal transduction pathway are affected in a Na+-dependent manner. However, more detailed studies are needed to elucidate the role of Na+ in chemotaxis.
Pre-adaptation to low temperature or high osmolarity did not alter the influence of these parameters on chemotaxis. This was investigated by pre-incubation for 2·5 h. This incubation time was chosen based on information from other bacteria, as the synthesis of specific stress proteins of V. anguillarum has not been investigated. Most of the stress and starvation proteins in Salmonella, Pseudomonas putida and Vibrio sp. strain S14 are synthesized during the first 2 h following exposure to stress conditions (Nyström et al., 1990; Spector, 1998
; Givskov et al., 1994
), and many of the structural and physiological changes which are triggered by osmotic shifts in E. coli, such as cell dehydration, decreased cytoplasmic water activity and reduced cell respiration, are restored within the first hour after a shift (Wood, 1999
). However, the starvation-stress response of V. anguillarum has been shown to differ from these bacteria (Nelson et al., 1997
) and it may be that the time chosen does not correspond to the time that V. anguillarum requires to induce the stress response.
By video analysis it was found that individual cells of V. anguillarum had similar motility characteristics with respect to velocity, acceleration and duration of runs in PBS containing 0·83·6 % NaCl. This is in accordance with a study of V. alginolyticus showing that the swimming speed is independent of the NaCl concentration when it is above 100 mM (0·6 %) (Yoshida et al., 1990). The results indicate that the rotation rate of the flagellum is independent of a concentration of NaCl between 0·8 and 3·6 %, the concentrations used in this study.
The swimming speed of bacteria that use Na+ to drive their flagellar motors is reduced by the addition of phenamil, which binds specifically to the Na+ channel of the flagellar motor and inhibits its rotation (Atsumi et al., 1990; Kojima et al., 1999a
). The swimming speed of non-starved V. anguillarum cells was decreased in the presence of phenamil, indicating that Na+ drives the flagellum of V. anguillarum as in other marine vibrios, for example V. cholerae, V. parahaemolyticus (Atsumi et al., 1992
) and V. alginolyticus (Chernyak et al., 1983
; Kawagishi et al., 1995
; Kojima et al., 1999a
). Here we also observed that the swimming speed of starved V. anguillarum cells was decreased by phenamil, indicating that V. anguillarum is able to maintain a Na+ gradient sufficient to rotate the flagellum at maximum speed during 8 days starvation. Maintenance of a sizeable membrane potential, at least during short-term starvation (2 days), has previously been shown in Vibrio fluvialis (Smigielski et al., 1989
).
During starvation, the culturable and motile fractions of V. anguillarum were reduced, and after 8 days the motile fraction roughly corresponded to the culturable fraction. Furthermore, the starved cells displayed a strong chemotactic response to serine. However, in marine Vibrio sp. strain S14 no chemotactic response could be detected after 24 h starvation, and after 3 days the motile fraction was only 5 % although the culturable fraction was approximately 100 % (Malmcrona-Friberg et al., 1990). This loss of motility during starvation corresponded to a loss of flagella (Malmcrona-Friberg et al., 1990
). Thus, it seems that these two marine vibrios have different starvation survival strategies.
We showed that transferring cells of V. anguillarum prior to the chemotaxis assay from 0·8 to 2·7 % NaCl in chemotaxis buffer gave a larger reduction in chemotactic ability after 2 days starvation than at the onset of starvation. In other words, the chemotactic response of starved cells of V. anguillarum was more sensitive to high concentrations of NaCl than growing cells. This suggests that V. anguillarum does not develop cross-resistance to osmotic stress during starvation or that the resistance is transient. This is in accordance with a previous report that has shown that V. anguillarum does not develop cross-resistance against heat shock during starvation and that the cross-resistance to oxidative stress disappears after 5 days starvation (Nelson et al., 1997). Also, motility of cells starved for 8 days was affected by 2·7 % NaCl. There seems to be a complex relationship between Na+ and other stresses such as temperature and pH (Padan & Krulwich, 2000
).
Cells of V. anguillarum that maintain motility during starvation had the same motility characteristic with respect to velocity, acceleration and duration of runs as growing cells. This indicates that at least a fraction of starved cells of V. anguillarum maintain a motive force that is sufficient to drive the rotation of the flagellum at levels corresponding to the rate of rotation during growth. This is different from E. coli, where a decrease in swimming speed was found when the cells entered the stationary phase, most likely due to a decreased proton motive force (Amsler et al., 1993).
A hallmark of bacteria is their ability to survive and respond to changes in their surroundings. Motility and chemotaxis are also influenced by such changes. This study showed that V. anguillarum is motile and chemotactic at many different conditions of relevance to its natural environment. Temperature and salinity, however, affect the phenotype of V. anguillarum, and the growth conditions prior to the assay were of less importance than the incubation conditions at the time of the measurements of the chemotactic ability. V. anguillarum was able to elicit a high chemotactic response during starvation. In nutrient-deprived environments, it may be important for the survival of bacteria that they are able to localize themselves in microenvironments with the best conditions for growth (Blackburn et al., 1998). Furthermore, we showed that V. anguillarum cells starved for 2 days were as virulent as exponentially growing cells after bath challenge. This is similar to what has been shown in Aeromonas hydrophila to a degree, where cells starved for 1 day were more virulent than cells in exponential growth phase after intraperitoneal injections (Rahman et al., 1997
). Also, starved cells of V. vulnificus and Pasteurella piscicida have been shown to retain a degree of virulence similar to that of non-starved cells after intraperitoneal inoculations into eels and turbot, respectively (Biosca et al., 1996
; Magarinos et al., 1994
). The role of starvation for regulation of virulence genes in V. anguillarum has not been studied yet. However, the stationary-phase sigma factor RpoS is known to control a large group of genes including genes important for virulence in other species, for example V. cholerae and S. enterica serotype Typhimurium (Spector, 1998
; Yildiz & Schoolnik, 1998
). Thus, it seems that under certain conditions non-growth prior to contact with the host does not interfere with the ability of the bacterium V. anguillarum to cause infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 March 2003;
revised 23 December 2003;
accepted 7 January 2004.
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