Extracellular Protease of Pseudomonas fluorescens CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode Meloidogyne incognita

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlledaprA gene (encoding the major extracellular protease) or thegacA regulatory gene resulted in reduced biocontrol activityagainst the root-knot nematode Meloidogyne incognita duringtomato and soybean infection. Culture supernatants of strainCHA0 inhibited egg hatching and induced mortality of M. incognitajuveniles more strongly than did supernatants of aprA and gacAmutants, suggesting that AprA protease contributes to biocontrol.

INTRODUCTION

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Introduction

Plant diseases caused by soilborne root pathogens account formajor crop losses worldwide. Yet in a small number of environments,i.e., in suppressive soils, little or no disease is observed,despite the presence of pathogens. Disease suppression depends,in part, on microorganisms that are able to antagonize pathogens(5, 10, 14, 28). The root-colonizing bacterium Pseudomonas fluorescensCHA0, which was isolated from a suppressive soil, has been studiedin detail as a model strain for the biological control of severalfungal plant diseases, such as black root rot of tobacco andtake-all disease of wheat (5, 27). In this strain, as well asin other biocontrol pseudomonads, antifungal secondary metabolites,e.g., 2,4-diacetylphloroglucinol, hydrogen cyanide, and pyoluteorin,are important for biocontrol activity. These biocontrol factorsare synthesized in response to environmental conditions andto population densities of the producer strain, whereby theGacS/GacA two-component system exerts a crucial role as a positivecontrol element (6, 8, 9, 11, 26). Some rhizosphere microorganisms,including P. fluorescens CHA0, can also act as antagonists ofplant-pathogenic nematodes (23). For antagonistic fungi, thisbiological control has been shown to involve extracellular proteases(2, 21). In strain CHA0, the production of the major extracellularEDTA-sensitive protease, AprA, is controlled by the GacS/GacAsignal transduction pathway (8, 17, 26, 29). The present studywas undertaken to find out whether this enzyme contributes tothe biocontrol properties of strain CHA0 in plant-nematode interactions.

Characterization of the aprA-aprI-aprD gene region involved in production of the major exoprotease of strain CHA0.

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Characterization of the apra...

Strain CHA803, a Tn5 insertion mutant derivative of wild-typeCHA0 (20), lacked proteolytic and lipolytic activities on indicatoragar plates (17, 18) but showed wild-type production of antifungalmetabolites, indicating that the Tn5 insertion was not in gacSor gacA (9). The Tn5 insertion was mapped to the 3' end of theaprD gene (Fig. 1), whose deduced amino acid sequence has 56%identity with the ATP-driven translocator AprD, a componentof the type I secretion machinery required for the secretionof alkaline protease AprA in P. aeruginosa (1, 3). By a chromosomewalking approach (7), the genes located upstream of aprD, thatis, an open reading frame coding for an amino acid transporter,dmpA (for a putative aminopeptidase), aprA (for extracellularprotease), and aprI (for the cognate protease inhibitor), werecloned and sequenced in strain CHA0 (Fig. 1). The genomic sequenceof P. fluorescens Pf-5, which is phenotypically and genotypicallyvery similar to P. fluorescens CHA0 (4, 15), predicts that theaprAID genes are the proximal part of an aprAIDEF operon, whichincludes the lipA gene (for extracellular lipase) at the 3'end (Fig. 1).

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FIG. 1. Genetic organization of the region surrounding aprA in P. fluorescens CHA0 and Pf-5. The 6.7-kb SacI-BamHI fragment of strain CHA0, which was sequenced in this study (GenBank accession no. AY644718), is aligned with the homologous region of strain Pf-5 (http://www.tigr.org) shown above. The sites where a translational 'lacZ fusion and a Tn5 element are inserted in the chromosome of strains CHA805 and CHA803, respectively, are shown above the aprA and aprD genes. Papr, promoter of aprA; aph, kanamycin resistance gene.

The deduced aprA gene product shows 62% identity with the AprAalkaline protease of P. aeruginosa (3) and contains Zn²⁺- andCa²⁺-binding motifs. The calculated molecular mass of 49.9 kDafor the secreted form of AprA is in reasonable agreement withthe value (47.1 kDa) previously determined for the EDTA-sensitive,major exoprotease of strain CHA0 (17). Between the aprA andaprD genes lies the aprI gene (Fig. 1) coding for a predicted13.8-kDa protein which shows 40% amino acid sequence identitywith the P. aeruginosa AprI protein, an AprA-specific inhibitor(3).

A nonpolar aprA mutation was constructed by the insertion ofa 'lacZ cassette into the unique XhoI site of the chromosomalaprA gene (Fig. 1) in the wild type and in a gacS background,using the suicide plasmid pME6063 (Table 1). This resulted instrains CHA805 and CHA806 (Table 1), respectively. Strain CHA805was exoprotease negative, as expected, but lipase positive,in keeping with the nonpolar nature of the 'lacZ insertion.ß-Galactosidase activities of the aprA'-'lacZ translationalfusion in strain CHA805 showed a marked cell density-dependentexpression profile. In contrast, in the gacS mutant CHA806,almost no ß-galactosidase activity was measured (Fig.2).

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TABLE 1. P. fluorescens strains and plasmids

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FIG. 2. GacS control of an aprA::'lacZ fusion in P. fluorescens grown in liquid King's B medium. ß-Galactosidase activities were determined by the Miller method (13) for aprA::'lacZ in the wild-type derivative CHA805 ( ${circ}$ ) and in the gacS mutant CHA806 (•). The growth rates of both strains were similar (data not shown). Each value is the average ± standard deviation from three different cultures.

Impact of the aprA gene product on nematode populations.

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Impact of the apra...

Meloidogyne spp., the root-knot nematodes, are sedentary endoparasitesof a wide range of plants, including many of agronomical importance.Meloidogyne incognita belongs to a group of nematodes that causeimportant crop losses in developing countries (12, 19). Culturesupernatants of wild-type strain CHA0 grown in 1/20-strengthKing's B medium (0.1% [wt/vol] Oxoid proteose peptone, 0.05%[wt/vol] glycerol, 0.3 mM MgSO₄, 0.3 mM K₂HPO₄) inhibited egghatching and caused mortality of the juveniles of M. incognitain vitro, in comparison with the uninoculated controls (P $≤$ 0.05)(Table 2). The protease-negative mutants CHA805 (aprA) and CHA89(gacA) failed to inhibit egg hatching and to kill M. incognitajuveniles (Table 2). The addition of the protease inhibitorEDTA (4 mM) to a culture supernatant of strain CHA0 grown inKing's B medium markedly reduced (P $≤$ 0.05) the juvenile killingactivity of strain CHA0 but had little effect on the supernatantsof the mutants CHA805 and CHA89 (Table 3). These data supportthe involvement of AprA protease in the inhibition of egg hatchingand in killing of juveniles. However, AprA protease may notbe the only antinematode factor of strain CHA0, in that antibioticcompounds produced under GacA control may also have a role innematode control (23; I. A. Siddiqui and S. S. Shaukat, unpublisheddata).

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TABLE 2. In vitro effects of culture filtrates of P. fluorescens strains on M. incognita egg hatching and juvenile mortality

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TABLE 3. In vitro effects of the addition of 4 mM EDTA to P. fluorescens culture filtrates on the mortality of M. incognita juveniles

In comparison to nonbacterized controls, P. fluorescens CHA0applied to unsterilized sandy loam soil suppressed (P $≤$ 0.05)root-knot development and nematode final population densitieson both tomato and soybean under greenhouse conditions (Table4). Carbofuran (Furadan) treatment, however, was more effectivein reducing nematode population densities in soil and rootsand subsequent root-knot development in both crops (Table 4).Strains CHA805 and CHA89 had no significant impact on nematodepopulation densities in soil and root-knot disease in eithercrop (Table 4). Application of strain CHA805 resulted in a reduction(P $≤$ 0.05), but not a complete loss, of nematode final populationdensities in soybean roots (Table 4). In these experiments,bacterial colonization of the tomato and soybean rhizosphereswas not significantly different between the three strains tested(data not shown).

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TABLE 4. Effects of carbofuran and P. fluorescens strains on gall formation caused by M. incognita and on soil and root populations in tomato and soybean grown under glasshouse conditions^a

In conclusion, these findings are consistent with the notionthat AprA protease of strain CHA0 contributes, directly or indirectly,to biocontrol of M. incognita. This study also extends previousobservations that P. fluorescens CHA0 has biological controlactivity against root-knot nematodes (23-25).

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The 6.7-kb SacI-BamHI fragment of strain CHA0 was sequencedin this study and was deposited in GenBank under accession no.AY644718.

ACKNOWLEDGMENTS

We thank Karin Heurlier for determining the lipase phenotypeof P. fluorescens strains.

Support from the Swiss National Foundation for Scientific Research(project 3100A0-100180) is gratefully acknowledged.

FOOTNOTES

* Corresponding author. Present address: Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom. Phone: 44 (0115) 951 50 89. Fax: 44 (0115) 846 79 51. E-mail: Stephan.Heeb{at}nottingham.ac.uk.

REFERENCES

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