Department of Biology, Temple University, Philadelphia, PA 19122, USA1
Author for correspondence: F. N. Chang. Tel: +1 215 204 8843. Fax: +1 215 204 6646. e-mail: fchang{at}nimbus.temple.edu
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ABSTRACT |
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Keywords: protease, protease inhibitor, insect nematode, inhibitorprotease interaction
Abbreviations: CBZ-Lys-pNP, N-CBZ-L-lysine p-nitrophenyl ester
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INTRODUCTION |
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Ph. luminescens bacteria occur in two phases, primary and secondary, which can be distinguished by biochemical tests, bioluminescence and colony morphology (Akhurst, 1980 ; Boemare & Akhurst, 1988
). Previous investigators have shown that the primary-phase bacteria are present predominantly in the gut of third-instar infective juvenile nematodes (Akhurst, 1980
). It was also reported that only the primary-phase and not the secondary-phase bacteria possess the protease and this characteristic may be used to differentiate these two phases (Schmidt et al., 1988
; Nealson et al., 1990
; Forst & Nealson, 1996
). Little is known about the regulation of protease activity in these bacteria. In this paper, we show that, contrary to previous reports, both primary- and secondary-phase bacteria synthesize protease. However, in the secondary-phase bacteria the protease activity is suppressed by this newly discovered protease inhibitor. We have purified and partially characterized this protease inhibitor with the aim of providing an explanation of its possible functions.
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METHODS |
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Purification of protease from primary-phase Ph. luminescens.
The primary-phase bacteria were distinguished from the secondary-phase bacteria by colony morphology, presence of luminescence and the ability of the primary form to absorb neutral red from MacConkey agar (Boemare & Akhurst, 1988 ). Secondary-phase bacteria were isolated from in vitro cultures initiated from the primary-phase bacteria.
A single Ph. luminescens primary-phase colony was selected from a MacConkey agar plate and grown overnight in 5 ml LuriaBertani (LB) medium (Sambrook et al., 1989 ). One millilitre of the inoculum was transferred into 1 l LB medium and incubated for 7 d at 25 °C in a shaker at 150 r.p.m. Unless otherwise stated, all subsequent operations were carried out in the cold (4 °C). Cells were removed by centrifugation at 7500 g for 60 min and the supernatant fraction was carefully collected into a large beaker. Ammonium sulfate was gradually added with stirring to the culture supernatant to 90% saturation. The precipitate was collected by centrifugation at 7000 g and then dissolved in 50 mM sodium phosphate buffer, pH 7·0. The ammonium sulfate precipitate was dialysed against distilled water overnight. The dialysate was then applied to a Bio-Rad Mini-Prep cell under native conditions and run at 100 V overnight with a flow rate of 0·75 ml min-1. The fractions containing protease activity (as assayed in the next section) were collected and analysed for purity by PAGE in the presence of SDS. Protease from the X. nematophila primary-phase bacteria was also prepared according to the same procedure.
Assay of protease activity.
Protease activity was determined initially using Azocoll as the substrate as described by Schmidt et al. (1988) . Eppendorf tubes containing a reaction mixture (total volume 1·2 ml) consisting of 2 mg Azocoll ml-1 in 50 mM sodium phosphate buffer (pH 7·0) were prepared. Enzyme solutions (10200 µl) were added to initiate the reaction and the mixture was then incubated at 37 °C. To terminate the reaction, the contents of each tube were centrifuged in an Eppendorf microcentrifuge for 2 min to remove any undigested Azocoll. The A520 of the liberated azo dye from the reaction supernatant was read using a Beckman DU-20 spectrophotometer. One unit of activity was defined as the amount of enzyme that caused an increase in the A520 of 0·01 min-1 (Schmidt et al., 1988
).
Purification of protease inhibitor from secondary-phase Ph. luminescens.
A tube containing 5 ml LB medium was inoculated with a single colony of secondary-phase Ph. luminescens isolated from a MacConkey agar plate and grown for 8 h at 28 °C with shaking at 150 r.p.m. One millilitre of this inoculum was transferred to a flask containing 500 ml LB medium and the culture was grown for 14 d at 28 °C with shaking at 150 r.p.m. The cells were removed from the culture medium by centrifugation at 7000 g for 1 h at 4 °C. Unless otherwise stated, all subsequent operations were carried out at 4 °C. The supernatant was collected and concentrated by adding ammonium sulfate with stirring to 90% saturation. The ammonium sulfate precipitate was collected by centrifugation at 9000 g for 1 h. The pellet was resuspended in 30 ml 50 mM sodium phosphate buffer (pH 7·0) and dialysed against 4 l distilled water overnight with two changes of water. The dialysate was then loaded into a Bio-Rad Rotofor unit, which separates proteins according to their isoelectric points in a liquid medium, using a pH gradient of pH 310. The collected fractions were analysed for inhibitor activity by mixing 50 µl of each fraction with 50 µl purified protease from the primary-phase Ph. luminescens. The fractions having the highest amount of inhibitor activity were incubated with trypsin-acrylic beads. The bound protease inhibitor was then eluted using the Electro-eluter. (In the presence of SDS, the Electro-eluter elutes the negatively charged proteins toward the anode, where they are trapped by a 3500 Mr cut-off dialysis membrane.) SDS was removed from the preparation by exchanging the lower buffer halfway through the run according to the manufacturers protocol. The purity of the inhibitor was analysed using discontinuous SDS-PAGE. Protein concentration was determined by the Bradford microassay method (Bradford, 1976 ).
Stoichiometry of proteaseprotease inhibitor interaction.
Ten microlitres of a 100 nM purified protease from primary-phase Ph. luminescens was incubated with various amounts of purified protease inhibitor from secondary-phase Ph. luminescens and allowed to react for 20 min at room temperature. Since the azocoll method used previously was rather insensitive, a different substrate (CBZ-Lys-pNP) was used for the stoichiometry study. Twenty microlitres of 50 µM CBZ-Lys-pNP substrate was then added to the reaction mixture and further incubated for 10 min at 37 °C. The reaction was terminated by adding 700 µl ice-cold 0·2 M sodium acetate buffer (pH 4·0) and the A326 was read immediately in a Beckman DU-20 spectrophotometer (Twining, 1994 ). Protease inhibitor activity was determined as the percentage of protease activity remaining when compared to the control tube containing buffer alone.
Activity spectrum of purified protease inhibitor.
To study the activity spectrum of the purified protease inhibitor, it was necessary to use a general protease substrate, and caseinFITC (Twining, 1994 ) was found to be an ideal substrate for this purpose. The following proteases were used: primary-phase proteases purified from both Ph. luminescens and X. nematophila, proteinase A, cathepsin B, cathepsin D, cathepsin G, chymotrypsin, elastase, thermolysin, subtilisin and trypsin. Each protease was diluted in 50 mM sodium phosphate buffer (pH 6·5) and adjusted to its pH optimum according to the protocol suggested by Sigma Chemicals, except for cathepsin D, which was diluted in 50 mM sodium acetate buffer at pH 3·5. The assay mixture contained 5 µl 20 nM protease and 5 µl 100 nM protease inhibitor. After incubating at room temperature for 20 min, 20 µl caseinFITC (1 mg ml-1) and 100 µl 50 mM buffer at the appropriate pH were added. The mixture was then incubated at 37 °C for 30 min and the reaction was stopped by adding 100 µl 5% TCA. The undigested caseinFITC was removed by centrifugation at 14000 r.p.m. in an Eppendorf microfuge for 3 min. Two hundred microlitres of supernatant was transferred to a tube containing 500 µl 0·2 M sodium phosphate buffer (pH 7·8) to neutralize the TCA. Inhibitor activity was determined as percentage of activity remaining when compared to a control tube containing only buffer, using an Amicon fluorimeter (excitation at 492 nm and emission at 515 nm).
pH stability of the protease inhibitor.
Five microlitres of protease inhibitor was diluted in 50 µl of buffers of overlapping pH ranges from pH 3·5 to 11·0 and incubated at room temperature for 1 h. Microcon 3 was then used to exchange buffers to 50 mM sodium phosphate buffer, pH 6·8. Residual inhibitor activity was assayed against purified protease from primary-phase Ph. luminescens. The assay contained 10 µl 10 nM enzyme and 50 µl 10 nM pH-adjusted protease inhibitor and the reaction mixture was incubated at room temperature for 20 min. CaseinFITC (20 µl) (1 mg ml-1) and 40 µl 50 mM sodium phosphate buffer (pH 6·8) were then added and the reaction mixture was further incubated at 37 °C for 30 min. The reaction was then stopped with 100 µl 5% TCA and the undigested caseinFITC was removed by centrifugation at 14000 r.p.m. in an Eppendorf microfuge for 3 min. Two hundred microlitres of supernatant was transferred to a tube containing 500 µl 0·2 M sodium phosphate buffer (pH 7·8) to neutralize the TCA. Protease inhibitor activity was determined fluorimetrically relative to the control as described previously.
Thermal stability of protease inhibitor.
Eppendorf tubes containing 5 µl protease inhibitor were incubated over a range of temperatures between 25 and 100 °C for 1 h and subsequently adjusted to 37 °C. The adjusted protease inhibitor was mixed with 10 µl primary-phase protease and the activity was assayed as described previously.
N-terminal peptide sequencing of purified inhibitor from Ph. luminescens.
Protein sequence determination was performed by Dr Joe Lykam of Michigan State University Department of Macromolecular Biochemistry (East Lansing, MI, USA).
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RESULTS |
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Determination of the N-terminal amino acid sequence of the protease inhibitor
After localization of inhibitory activity, the protease inhibitor band was excised from the polyacrylamide gel and subjected to amino acid sequence determination. The N-terminal 16 amino acids of the purified protease inhibitor were determined as STGIVTFKND(X)GEDIV (residue 11 appears to be modified). A sequence search was performed and it was found that it bears high sequence homology to the N-terminal region of an endogenous inhibitor (IA-1) from the fruiting bodies of an edible mushroom, Pleurotus ostreatus (Dohmae et al., 1995 ). As shown below, 11 of the first 15 amino acids (73%) were identical between these two protease inhibitors.
Interestingly, both protease inhibitors start with the serine residue, although in the mushroom it is acetylated, suggesting its cytosolic location (Odani et al., 1999 ). The Ph. luminescens protease inhibitor, on the other hand, is a secretory protein.
Proteaseprotease inhibitor interaction
In order to study the nature of the interaction between the newly isolated protease inhibitor and its substrates, it was necessary to purify proteases from different nematode-symbiotic bacteria. The culture medium from the primary-phase Ph. luminescens was subjected to ammonium sulfate precipitation, dialysis and preparative PAGE under native conditions as described in Methods. A single band containing protease activity was observed with an apparent Mr of 56000 (figure not shown). The ability of protease inhibitor from Ph. luminescens to inhibit its homologous protease was then examined. Fig. 3 shows that at a molar ratio of one to one, 90% of the protease activity was inhibited, indicating an almost stoichiometric interaction between the protease and its inhibitor.
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DISCUSSION |
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Since Ph. luminescens is a symbiont of insect-parasitic nematodes, several properties of the protease inhibitor suggest that it may participate in the parasitization process. First, the protease inhibitor is rather unusual in that it retains full activity in the pH range tested (3·511·0). This broad pH range not only covers the pH of insect haemolymph, where the bacteria are initially released by insect nematodes, but also the gut pHs of various insects. It is known that both the lepidopteran and dipteran insects have rather alkaline guts whereas the coleopteran insects have acidic guts (McFarlane, 1985 ). The fact that Ph. luminescens protease inhibitor is active at these gut pHs indicates that it may interfere with the insects digestive system and speed up the parasitization process. The exceedingly high homology between Ph. luminescens protease inhibitor and that produced by the fruiting bodies of an edible mushroom, Pl. ostreatus, used to defend against ingestion by insects, lends further support to the role of protease inhibitor in controlling the digestive processes of the insect host. Second, the protease inhibitor was found to suppress insect proteases that are required to activate the prophenoloxidase cascade essential for the insect defensive system (Kanost, 1999
; Park et al., 2000
). Although we have not tested the activity of Ph. luminescens protease inhibitor against various insect proteases, the broad spectrum of the protease inhibitor (Table 3
) suggests that it will inactivate the insect proteases.
The protease inhibitor may also be involved in regulating the bacterial phase shift during the development of insect nematodes. It is known that the primary-phase bacteria are present predominantly in the gut of insect-parasitic nematodes (Akhurst, 1980 ) and it is clearly advantageous to have high protease activity during the initial stage of the infection process inside a target insect host rich in proteinaceous nutrients. At later stages of infection when the nutrients are depleted, it will be necessary to control or curtail this protease activity. One way to regulate the protease activity is to convert some primary-phase bacteria, which apparently do not produce protease inhibitor or may have produced so small an amount that it escaped detection, into the secondary phase. It has been documented that when cultured in vitro, primary-phase Ph. luminescens can easily be converted to the secondary phase and this process appears to be irreversible (Bleakley & Nealson, 1988
; Forst & Nealson, 1996
). An alternative method for controlling the protease activity is to suppress the growth of the primary-phase bacteria at later stages of parasitization, thereby allowing the secondary-phase bacteria to populate. In this regard, Smigielski et al. (1994)
showed that the secondary-phase bacteria can grow twice as fast in vitro when compared to the primary-phase bacteria. It has also been reported that in Streptomyces albogriseolus, overproduction of protease (due to a deletion mutation in the protease inhibitor gene) leads to a marked decrease in growth rate (Taguchi et al., 1998
). It is likely that the secondary-phase Ph. luminescens bacteria without the burden of high protease activity can outgrow the primary phase and be dominant in a mixed culture when nutrients become limiting. Our results indicate that rather than being perceived primarily as an in vitro artefact, secondary-phase Ph. luminescens may actually be quite critical to the development of insect nematodes.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 20 April 2000;
revised 14 August 2000;
accepted 21 August 2000.