INTRODUCTION

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Yersinia ruckeri is known to be the etiological agent responsible for the enteric red mouth (ERM) disease of fish. ERM disease is spread throughout the world and affects mainly intensive aquaculture of trout and salmon. Disease outbreaks have a relationship with stress conditions, but little information is available about the virulence mechanisms involved in the disease progression. Several factors contribute to the pathogenic potential of this bacterium. Y. ruckeri produces protease, lipase, and hemolysin as extracellular factors which when injected into fish lead to the appearance of symptoms associated with pathogenicity (38). Furones et al. (15, 16) have shown that virulence of Y. ruckeri is related to the activity of a heat-sensitive factor present in cell extracts. Iron availability influences the virulence of many pathogens, and in Y. ruckeri several outer membrane proteins regulated by iron have also been identified (11, 37).

Bacterial proteases are mainly involved in providing peptide nutrients for the microorganism. However, the production of bacterial proteases could contribute to the pathogenesis of infections, and therefore they could be considered virulence factors. In fact, some authors regard proteases as the main virulence factors present among the extracellular factors. Although direct evidence revealing the molecular mechanisms by which bacterial proteases participate in the development of the pathology is still lacking, it has been suggested that proteolytic enzymes of fish pathogens, such as Aeromonas hydrophila (26), Vibrio anguillarum (33), Vibrio vulnificus (24), Aeromonas salmonicida (19, 39), Flexibacter columnaris (18), and Flexibacter psychrophilus (2), play an important role in causing massive tissue damage in the host, which may aid the establishment of infection. Most of the characterized proteases from fish pathogens are metalloproteases requiring zinc for enzymatic activity (19, 24, 32). For the pathogen Pseudomonas aeruginosa extensive evidence suggests that elastase, a metalloprotease, is required for maximal virulence (23, 35).

The effect of environmental conditions on the production of extracellular proteolytic enzymes could play an important role in the induction or repression of the enzyme by specific compounds. Production of extracellular proteases has been shown to be sensitive to repression by different carbohydrate and nitrogen sources (21, 27). Catabolic enzymes responded to both carbon control and nitrogen control in enteric bacteria (14, 17). In the bacteria A. hydrophila (34), A. salmonicida (9), and P. aeruginosa (23) protease production is influenced by carbon and nitrogen sources. Additionally, the temperature can influence the protease production, as occurs in A. hydrophila (31, 34).

In this paper, we report the finding of a protease with caseinolytic activity produced by Y. ruckeri strains from different origins. The protease was detected at the end of the exponential growth phase under different culture conditions, and it was repressed by the presence of NH₄Cl as well as glucose and other sugars in the medium. It was purified and biochemically characterized. We also obtained mutants that showed either no caseinolytic activity or overproduction which will be useful for further studies of this protease as a virulence factor.

	MATERIALS AND METHODS

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Bacterial strains and culture conditions. The origins of the Y. ruckeri strains are as follows: strains 146, 147, 148, 149, and 150 had been isolated during naturally occurring outbreaks of ERM disease at a Danish fish farm and were kindly provided by J. L. Larsen; strains A189, N190, R191, and C192 had been isolated during different outbreaks of the disease at a Spanish fish farm and were taxonomically characterized by I. Marquez; strains 35/85 and 13/86 were kindly provided by C. J. Rodgers; and strains 11.4, 11.29, and ATCC 29473 were from the CECT (Spanish Type Culture Collection).

Assay of proteolytic activity and protein content. Proteolytic activity was assayed by using azocasein (Sigma) as a substrate. Briefly, 120 µl of a suitable dilution of enzyme solution was added to 480 µl of azocasein (1%, wt/vol) in reaction buffer (Tris buffer containing MgCl₂ [final concentration, 5 mM]), and the mixture was incubated at 30°C for 30 min. The reaction was terminated by adding 600 µl of 10% (vol/wt) trichloroacetic acid and left for 30 min on ice, followed by centrifugation at 15,000 × g, at 4°C for 10 min. Eight hundred microliters of the supernatant was neutralized by adding 200 µl of 1.8 N NaOH, and the absorbance at 420 nm (A₄₂₀) was measured using a spectrophotometer (lambda 3A; Perkin-Elmer). One unit of enzyme activity was defined as the amount which yielded an increase in A₄₂₀ of 0.01 in 30 min at 30°C.

Protease purification. Among the different strains tested, Y. ruckeri 150 was chosen for further studies because it showed the highest hydrolysis activity on casein plates (NA supplemented with 1.25% skim milk powder; Oxoid).

Characterization of the enzyme activity. The caseinolytic activity was assayed at different pH values ranging from 4 to 11 by using azocasein as a substrate. The buffers used were the following: for pH 4, 25 mM piperazine; for pHs 6.1 and 6.7, 25 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)]; for pHs 7.0, 7.6, 8.1, 8.5, and 9.5, 25 mM Tris-HCl; and for pHs 10 and 11, 25 mM CAPS [3-(cyclohexylamino)-1-propanesulfonic acid]. For thermostability testing, the purified protein was incubated at 4, 12, 18, 25, 30, 37, 42, 47, and 55°C for 30 min in Tris buffer containing 5 mM MgCl₂ and 1% azocasein. The activation energy (E_a) was determined from the slope (E_a/R) of Arrhenius plots of ln k (k = 100 × enzyme units [EU]) against the reciprocal of the temperature. The effects of different ions (Ca²⁺, Mg²⁺, and K⁺) on protease thermostability were studied by incubating purified protein at 42°C in Tris buffer containing 10 µM to 5 mM ion concentrations for 0, 10, 20, and 30 min. Afterwards, the residual activities were assayed with 1% azocasein as the substrate at 30°C for 30 min.

Electrophoresis and zymograms. The method used for SDS-PAGE was essentially the one described by Laemmli (25). For zymogram analysis, sodium caseinate (1%) copolymerized with the gels was used. Samples were loaded into the gel without heating, and electrophoresis was performed at 4°C at a constant current of 25 mA. Following electrophoresis, gels were washed successively, twice with deionized water and then twice with Tris buffer, each for 30 min at 4°C. Then, gels were incubated overnight in Tris buffer containing 5 mM MgCl₂ at room temperature. Finally, gels were stained with 0.1% Coomassie brilliant blue R 250 in 4:1:5 methanol/acetic acid/water (vol/vol/vol) and destained in the same solution without the dye, to reveal zones of substrate hydrolysis.

Preparation of antiserum and immunodetection. A 2-ml mixture of equal parts of Freund complete adjuvant and purified protease (80 µg) was injected subcutaneously in 50-µl aliquots into a New Zealand White rabbit (2.5 kg). Twenty days after the injections, the rabbit was exsanguinated and serum was separated and stored in fractions. For immunodetection experiments, proteins were separated electrophoretically as described above and transferred onto nitrocellulose membranes (Hybond-P; Amersham) for 1 h at 50 volumes in 10 mM CAPS (pH 11) containing 20% methanol by using a Trans-blots cell (Bio-Rad). The nitrocellulose filters were then washed by incubation at room temperature in 20 mM Tris-HCl (pH 7.6)-137 mM NaCl containing 0.1% Tween 20 (TBS-T) for 10 min, and immunodetection was carried out by following the procedure for the ECL Western blotting system from Amersham (ECL kit) (RPN2108) by using rabbit antiserum raised against the protease (1:500) and anti-rabbit immunoglobulin G-peroxidase conjugate (Amersham) (1:2,500). Excess of ligand was washed away in TBS-T for 15 min, and detection of the proteins was performed according to the manufacturer's instructions. Membranes were exposed for different times to Hyperfilm ECL (Amersham) until a suitable signal was obtained.

Mutagenesis. Y. ruckeri 150 was cultured in 100 ml of NB in a 250-ml flask at 20°C, with constant agitation (250 rpm). Cells were harvested by centrifugation from 20-ml cultures in early stationary phase (16.5 × 10⁸ cells/ml) and washed twice with Tris buffer. The pellets were then suspended in 5 ml of Tris buffer containing 1 mM EDTA and incubated for 30 min at room temperature. Cells were washed twice with Tris buffer, resuspended in 1 ml of the same buffer containing 100 µg of N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and incubated for 90 min at room temperature. Under those conditions 99% cell mortality was achieved. Then, cells were washed twice with Tris buffer, resuspended in 1 ml of the same buffer, and smeared on NA plates supplemented with 1.25% (wt/vol) skim milk powder. After incubation at 20°C for 3 days, about 2,000 colonies were screened for mutants displaying both extremes of protease activity, i.e., deficient and hyperproducing mutants. Therefore, colonies with a major hydrolysis zone as well as those without a clear proteolytic one were selected and subcultured several times in order to obtain stable mutants.

	RESULTS

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Effects of culture conditions on protease production. Caseinolytic activity in NB culture supernatants of Y. ruckeri 150 was detected in the late logarithmic growth phase (Fig. 1A and B). Approximately 12 h after the initiation of protease production activity reached a maximum, and this was followed by a slow but progressive decrease (Fig. 1B). Two mutant strains (CS1 and E1) of Y. ruckeri 150 were isolated after treatment with NTG. The mutant CS1 produced 1.5 to 2 times more protease activity than the parent strain, and the mutant strain E1 did not produce any detectable caseinolytic activity (Fig. 1B).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Production of caseinolytic protease during Y. ruckeri growth. Cells were grown in NB medium, and at different incubation times 120 µl of the cell-free supernatants was used to measure the caseinolytic activity, with azocasein used as a substrate. (A) Absorbance at 600 nm as a measure of cell density. (B) Caseinolytic activity. Symbols: , Y. ruckeri wild-type strain; , CS1 mutant; and , E1 mutant.

Purification and biochemical properties of exocellular protease from Y. ruckeri. Y. ruckeri protease was purified from NB culture supernatant as indicated in Materials and Methods. The purification results are shown in Table 2. Ammonium sulfate precipitation followed by dialysis resulted in a 1.6-fold increase of specific activity. The protein was then adsorbed onto DEAE-Sephacel, and the caseinolytic activity was recovered as a sharp peak, with the greater proportion of proteolytic activity eluting at 0.27 M NaCl. The SDS-PAGE analysis of the molecular mass of the purified enzyme revealed a single band, of 47 kDa (Fig. 2). The process yielded about 80-fold purification.

View larger version (38K): [in this window] [in a new window]   FIG. 2.   SDS-12% PAGE of purified caseinolytic protease produced by Y. ruckeri. Protein (~7.2 µg) pooled at the DEAE-Sephacel purification step was gel loaded and, after electrophoresis, stained with Coomassie brilliant blue R-250. Protein molecular mass markers (expressed in kilodaltons) are indicated on the left. Lane 1, Purified 47-kDa protein.

View larger version (10K): [in this window] [in a new window]   FIG. 3.   Effect of temperature on the activity of the 47-kDa protease. (A) The enzyme (~2.1 µg) was incubated in 600 µl of Tris buffer containing 5 mM MgCl2 and 1% azocasein for 30 min at various temperatures, and caseinolytic activity was measured as described. The values obtained at 37°C were taken as 100%. Relative activities are the averages for two independent experiments. (B) Arrhenius plot showing thermal deactivation of the 47-kDa protease. The ln of specific activity (k) (100 × EU) was plotted against the reciprocal of absolute temperature (T).

Caseinolytic activities of different Y. ruckeri strains and mutants. In order to discern if the caseinolytic activity is a constant characteristic in this bacterium, the presence of the enzyme as indicated by SDS-PAGE analysis, sodium caseinate zymograms, and quantitative azocasein assays was determined for 14 Y. ruckeri strains grown in NB. The culture supernatants of stationary-phase cultures of these strains had different caseinolytic activities as measured by the azocasein method. Thus, the strains 150, 148, 4319, 13/86, 149, A189, N190, E191, and C192 showed strong activity (Azo⁺), whereas negligible levels were found in the strains 146, 147, 955, 956 and 35/85 (Azo) (data not shown). In order to establish a correlation between azocasein hydrolysis and the presence of a particular protein in the culture supernatant, SDS-PAGE or the 14 different strains was performed. Only those strains defined as Azo⁺ produced and excreted the 47-kDa protein, whereas Azo strains lacked this protein (Fig. 4A). Furthermore, two different protein patterns, indicated as Azo⁺ and Azo (lane 1 and lane 2, respectively, in Fig. 4A), were observed. The 47-kDa protein was the predominant protein in the strains with the Azo⁺ pattern, whereas Azo strains presented two different major proteins of approximately 42 and 52 kDa. At the same time, zymograms obtained with sodium caseinate as the substrate showed a unique degradation area for Azo⁺ strains while no degradation was observed for cell-free supernatant obtained from Azo strains (Fig. 4B). Immunoblot analysis using antibodies raised against the 47-kDa protein showed that it was present in the Azo⁺ strains and that there was no immunological reaction with any protein from the Azo group, confirming that the 47-kDa protein was absent in this group of strains (Fig. 4C).

View larger version (47K): [in this window] [in a new window]   FIG. 4.   Analysis of the presence of the 47-kDa enzyme in cell-free supernatants of representative Azo+ (Y. ruckeri 150) and Azo (Y. ruckeri 146) strains. Culture supernatants (20 ml) from cells grown on NB for 24 h were dialyzed, freeze-dried, and resuspended in 0.5 ml of Tris buffer (protein concentration, 50 µg/ml). Aliquots (10 to 40 µl) were used to analyze the presence of the 47-kDa protein by SDS-12% PAGE, (A) caseinolytic activity by zymograms by using 1% sodium caseinate (B) and the immunoblot probed with antibodies (1:500) raised against the 47-kDa protein (C). The details are described in the Materials and Methods section. Molecular mass markers (expressed in kilodaltons) are indicated on the left. Lanes 1, Azo+ strain (Y. ruckeri 150); lanes 2: Azo strain (Y. ruckeri 146).

View larger version (49K): [in this window] [in a new window]   FIG. 5.   Comparison of the 47-kDa protease production by the parental strain and production by the mutant (E1 and CS1) strains. The different strains were grown in NB; after 24 h of incubation 20 ml of each supernatant was dialyzed, freeze-dried, and resuspended in 0.5 ml of Tris buffer (protein concentration, 50 µg/ml), and 10- to 40-µl aliquots were used to analyze the presence of the 47-kDa protein by SDS-12% PAGE (A), caseinolytic activity by zymograms by using 1% sodium caseinate (B), and the immunoblot probed with antibodies (1:500) raised against the 47-kDa protein (C). Molecular mass markers (expressed in kilodaltons) are indicated on the left. Lanes 1, parent strain (Y. ruckeri 150); lanes 2, mutant strain E1; and lanes 3, mutant strain CS1.

	DISCUSSION

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Proteolytic activity was first detected in Y. ruckeri culture supernatants towards the end of the exponential growth phase. This phenomenon of protease synthesis occurring at the end of the exponential growth phase is a common feature of many bacteria, including Vibrio alginolyticus (29) and Clostridium sporogenes (1). By contrast, A. hydrophila (34), Vibrio cholerae (45), Vibrio strain SA1 (43), and Erwinia chrysanthemi (41) display proteolytic activity during the exponential growth phase.

The composition of the culture medium had no effect on the onset of protease production, including that for the hyperproteolytic strain CS1. However, protease synthesis seemed to be regulated in some way, as it occurred only after active growth was about to cease. Protease activity was absent in the mutant E1, suggesting that protease is not essential for growth.

The production of the 47-kDa protease was influenced by the composition of the culture medium: growth and protease production were optimum in peptone medium whereas activity was not detected when the microorganism was grown in the presence of Casamino Acids, suggesting that intact peptides are necessary in the induction process. A similar behavior has been observed for A. salmonicida (9), Aeromonas liquefaciens (9), Serratia marcescens (4), Vibrio species (12), and E. chrysanthemi (41). By contrast, in the case of A. hydrophila, peptone was the best medium for protease production, although growth was not too prolific (34). After 24 h of incubation, Y. ruckeri did not grow in the presence of sodium caseinate or gelatin. This fact could suggest that the basal level of protease was insufficient, during early exponential growth, to release enough peptides and that therefore there was a lack of induction of enzyme production and hence a lack of growth. Alternatively, low levels of particular amino acids in those proteins could explain the inability of bacteria to grow on these substrates.

Growth was increased when carbohydrates were present in the culture media, but they had negative effects on protease production. A similar catabolic repression mechanism for extracellular enzyme production has been described for V. alginolyticus (29), Pseudomonas maltophilia (3), and Staphylococcus aureus (44), suggesting that in the absence of the sugar (i.e., glucose) the protease plays a role in supplying peptides or amino acids as the carbon or energy source in addition to being the nitrogen source. Thus, protease synthesis may be repressed when the energy status of the cells is high. This kind of regulatory mechanism has been postulated for proteases of other pathogens like P. aeruginosa (42) and Vibrio strain SA1 (43). However, it should be considered that a study of the effect of carbohydrates on enzyme production may not reveal the in vivo situation, where amino acids, peptides, lipids, and host proteins are more likely to be the carbon and energy sources.

The presence of ammonium significantly decreased protease production. Thus, inhibition was found when 50 mM NH₄Cl was present in the medium. This NH₄⁺ effect was similar to that described for A. hydrophila (34), A. salmonicida (28), and Vibrio strain SA1 (43), giving evidence to support the idea that ammonium-specific repression is likely to be the explanation for the results found.

The inhibition of protease production observed at 28°C seems to be a specific effect not related to the growth rate. This kind of enzyme regulation dependent on temperature and independent of growth could indicate that protease production (as a putative virulence factor) in Y. ruckeri is a process adapted to the aquatic environmental conditions needed for infecting and survival in fish. Other enteropathogenic Yersinia species (Y. enterocolitica and Y. pseudotuberculosis) also express temperature-dependent proteins at 37°C but not at 26°C (7, 8). Among these proteins are the regulators or effectors of virulence which allow the expression at host temperature but not at room temperature.

The fact that azocasein protease activities were found in 8 of 14 strains of Y. ruckeri tested shows that this characteristic is not general in this species. The absence of the 47-kDa protein in the Azo strains indicated that the protease does not occur in any of these strains. These results were confirmed by the zymogram and immunoblot analyses. There was also a correlation between the presence or absence of caseinolytic activity and the protein profile observed by SDS-PAGE. Thus, two different exocellular protein patterns with no apparent common components were found, each one corresponding with the Azo⁺ or Azo characteristic, and all the strains analyzed in this work could be classified into one of them. Since all the strains with the exception of strain 956 (which was serotype 2) were serotype 1, the most virulent and common serotype, these two patterns had no relationship with serotype. Nevertheless, it is possible that the presence of this protease could be a factor that enhances virulence. On the other hand, the growth curve of E1, which lacked the protease, probably because it was affected at a regulatory level, showed a shallower slope than that for the wild-type strain. This could suggest that the presence of the protease means a better adaptation to competitive growth.

At this point, the role of this protease is unclear. Although it was not necessary for growth, it could digest proteins as a nutrient source and support in vivo proliferation from digested tissues. In addition to providing nutrients for the bacteria, the protease could have potential toxicity for fish as a virulence factor which helps the bacteria to produce ERM disease, as it seems to occur in other fish pathogens such as V. anguillarum (33) and A. salmonicida (19).

Ammonium sulfate precipitation followed by ion exchange was an effective procedure in increasing the specific activity of this protease by more than 7,633-fold and the recovery of protease activity was 2.07%. It also yielded a pure homogeneous protein as evidenced by detection of a single band by SDS-PAGE.

The profile of enzyme temperature dependence, having the maximum at 37°C, shows that this is a cold-adapted enzyme, like other enzymes from different bacteria, including lipases from Pseudomonas sp. (6), an amylase from a psychrophilic bacterium isolated from Japan Sea sediments (20), and subtilisin from Bacillus strain TA41 (10). Cold-adapted enzymes exhibit lower activation temperatures than their mesophilic counterparts but also exhibit an increased heat lability. The instability at temperatures of 42°C and above, together with the energy of activation (E_a = 15.53 ± 0.23 kcal/mol), defines this 47-kDa protease as a thermolabile enzyme similar to enzymes from other psychrotrophs, such as Moraxella (13) and Acinetobacter (5). The protease can be classified as an alkaline metalloprotease (optimum pH near 8) dependent on Mg²⁺ for its activity, since EDTA and 1,10-phenanthroline did inhibit the enzyme's action. EGTA inhibited the enzyme activity by approximately 50%, and the presence of Ca²⁺ was shown to be more effective than that of Mg²⁺ in conferring thermostability. Thus, the protease seems to be dependent on Mg²⁺ for its activity and Ca²⁺ for its thermostability. In this way, for further enzyme purification MgCl₂ (5 mM) might be included in the culture medium as well as in the purification buffers in order to increase the total activity and yield. CaCl₂ should also be included for the final purification step in order to improve the enzyme stability. On the other hand, phenylmethylsulfonyl fluoride, which irreversibly and specifically reacts with serine residues in the active site, had no effect on protease activity. Furthermore, inhibition by dithiothreitol suggests that disulfide bonds could be important in maintaining the molecular conformation required for activity.

The pure protease is a valuable tool for testing the effect of the enzyme on fish tissues and also for studying the immunogenic ability of heat-denatured protease in the context of subsequent infections. Fifty percent lethal dose experiments, using Azo⁺ and Azo strains, as well as the mutants, will be especially useful to directly test the role of the 47-kDa protease from Y. ruckeri in ERM disease.