Departamento de Biología, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain
Correspondence
Marta Martín
m.martin{at}uam.es
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ABSTRACT |
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INTRODUCTION |
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The soil-borne fluorescent pseudomonads are used as biocontrol inoculants because of their ability to produce some antifungal metabolites (Dowling & O'Gara, 1994; Walsh et al., 2001
). Other applications of pseudomonads include soil biofertilization and rhizoremediation (Ramos et al., 1991
; Brazil et al., 1995
; Höflich et al., 1995
; Yee et al., 1998
).
The strain Pseudomonas fluorescens F113 was isolated from the sugarbeet rhizosphere and it is used as a biocontrol agent against the fungal pathogen Pythium ultimum, which causes damping-off disease in sugarbeet seedlings. The biocontrol abilities of this strain are due mainly to the production of the antifungal metabolite DAPG (2,4-diacetylphloroglucinol) (Shanahan et al., 1992). P. fluorescens F113 has also been genetically modified, by introducing the bph genes that encode the biphenyl degradative pathway, to be used in rhizoremediation of polychlorinated biphenyls (Brazil et al., 1995
; Karlson et al., 1998
). The efficacy of P. fluorescens F113 as inoculant clearly depends on its capacity to compete and efficiently colonize the rhizosphere.
Motility seems to be very important in colonization since non-motile mutants of different P. fluorescens strains are severely affected in the root colonization. The defect was larger at sites more distant from the inoculation site, in the root systems formed after the bacterial inoculation (De Weger et al., 1987; Dekkers et al., 1998b
; Chin-a-Woeng et al., 2000
). Furthermore motility-impaired mutants of Pseudomonas chlororaphis PCL1391 do not reduce the disease produced by Fusarium oxysporum on tomato plants (Chin-a-Woeng et al., 2000
). Therefore, motility is required to colonize growing roots successfully and to maintain the biocontrol capacities.
The objective of this work was to study the phenotype of mutations affecting the flagellar filament synthesis in P. fluorescens F113 and their influence on motility and root competitive colonization. Reports to date refer to mutants that are either aflagellate (De Weger et al., 1987) or deficient in chemotaxis (de Weert et al., 2002
). The mutants obtained in this work are not affected in chemotaxis but they are affected in motility to different degrees. We show that wild-type motility properties are necessary for competitive rhizosphere colonization.
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METHODS |
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Construction of mutants.
Insertional mutagenesis has been used to generate mutants by single homologous recombination. Amplified internal fragments from the different flagellar filament synthesis genes were cloned into the kanamycin-resistant plasmid pVIK112 (Kalogeraki & Winans, 1997) and introduced into wild-type F113 by triparental mating using pRK2013 as the helper plasmid (Figurski & Helinski, 1979
). Mutants resulting from single homologous recombination were checked by Southern blotting using probes from the interrupted genes, and by PCR using primers designed from the genes and the pVIK112 plasmid sequences (the primer sequences are available on request). Mutant complementation analysis was done by cloning each intact gene under the control of the nptII strong promoter into plasmid pML122 (Labes et al., 1990
) and introducing the recombinant plasmid into the corresponding mutant strain by triparental mating. Then, in order to correlate the strain phenotype with the interrupted gene, disappearance of the mutant phenotype was analysed.
Transmission electron microscopy.
Formvar-coated grids were placed on the top of a drop of bacterial cells for 30 s to allow bacterial adhesion. Grids were stained for 1 min with a 1 % solution of potassium phosphotungstate and washed for 1 min with a drop of water. Flagellum length was measured with the Q-Win software (Leica).
Swimming assays.
SA (Scher & Baker, 1982), LB (Bertani, 1951
) and iron-supplemented SA medium plates containing 0·3 % purified agar were used to test the swimming abilities of wild-type F113 and the different mutants. The cells were inoculated in the middle of the plate, in triplicate, using a toothpick, from exponentially growing cultures. Swimming haloes were measured after 18, 24 and 42 h inoculation. Every assay was done at least three times.
Colonization experiments.
Alfalfa seeds were sterilized in 70 % ethanol for 2 min and in diluted bleach (1 : 5, v/v) for 15 min and rinsed thoroughly with sterile distilled water. Seeds were germinated at 4 °C for 16 h followed by incubation in darkness, at 28 °C for 1 day. Germinated alfalfa seeds were sown in Leonard jar gnotobiotic systems using Perlite as the solid substrate and 8 mM KNO3-supplemented FP (Fahraeus, 1957) as the mineral solution. After 2 days, alfalfa seedlings were inoculated with
108 cells of the appropriate strain. For the competitive colonization experiments, the tested strain and the competitor were inoculated at a ratio of 1 : 1. Plants were maintained for 3 weeks in a plant growth cabinet in the following controlled conditions: 16 h of light at 25 °C and 8 h of dark at 18 °C. Bacteria were recovered from the last centimetre of the main root by vortexing for 2 min in 5 ml of 0·9 % NaCl and appropriate dilutions were plated in SA supplemented with the selective antibiotics. The mean of recovered bacteria per g of root tip was 2·48x107, the range being from 1·35x106 to 2·96x108. Colonization experiments were done three times in triplicate with at least 20 plants per replica.
Protein extraction and Western blots.
Proteins were extracted from 200 ml cultures grown for 2 days. In order to detach the flagellar filaments, the cultures were agitated by vortexing for 2 min and then centrifuged for 20 min at 12 000 r.p.m. Total proteins were extracted from the pellet with Laemmli buffer (Laemmli, 1970) and extracellular proteins were extracted from the supernatant, by precipitation for 16 h at 4 °C with 10 % (w/v) TCA, followed by two washes with acetone, and were finally resuspended in Laemmli buffer. Proteins were electrophoresed in 12 % acrylamide gels and stained with Coomassie blue. The same electrophoretic conditions were used for Western blotting. Gels were transferred to nitrocellulose membranes and incubated with 1 : 10 000 dilution of an antiflagellin antiserum (Dekkers et al., 1998a
) and with a peroxidase-tagged secondary antibody (anti-rabbit immunoglobulin). In the dot-blot experiments, the culture was agitated by vortexing and was centrifuged to separate the flagellar filaments. A drop from the pellets obtained was transferred to the nitrocellulose membrane and incubated with the antiflagellin antiserum in the same conditions as described above.
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RESULTS |
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All the mutants obtained were grown in SA and LB liquid media and showed no differences in growth parameters from the wild-type strain.
The 6·5 kb region sequence has been deposited in GenBank with the accession number AF399739.
Morphological analysis of mutants
Flagellar and cell morphology of P. fluorescens F113 and the mutants in the flagellar synthesis region was studied by transmission electron microscopy (Fig. 2). The F113 wild-type strain possesses one or two polar flagella of mean length 2·4 µm (Sanchez-Contreras et al., 2002
). The mutations located within the fliC and the fleQ genes resulted in completely aflagellate bacterial cells. The presence of flagella was restored by the introduction of plasmids containing the wild-type genes under the control of the nptII promoter. The mutant affected in the fliS gene had a single thin and very short flagellum of about 0·8 µm in length. Introduction of the wild-type gene into this mutant resulted in normal flagellate cells. Conversely, the mutation in the flaG gene resulted in bacterial cells with one or two very long (more than 5 µm) flagellar filaments. Finally, the mutation affecting the fliT gene had no visible effect, and bacterial cells had flagella with identical morphology to those of the wild-type strain.
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DISCUSSION |
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In this work we have characterized a genetic region of P. fluorescens F113 implicated in the synthesis of the flagellar filament and we have shown that the genetic organization is similar to other pseudomonads but differs from the P. aeruginosa PAK and DG1 strains because they have two copies of the fliS gene. The first gene in the region, fliC, encodes a type b flagellin, the main structural protein of the flagellar filament. As expected, the mutation of this gene yields completely non-motile and aflagellate bacteria. In the mutation affecting the fleQ gene we have found the same morphological and non-motile phenotype. These results are in agreement with the already described function for the FleQ protein, which is the major flagellar regulator in P. aeruginosa (Dasgupta et al., 2002; Jyot et al., 2002
). In P. fluorescens Pf0-1, a fleQ homologue gene called adnA encodes a transcriptional factor that affects persistence and spread, also being required for bacterial adhesion and motility (Casaz et al., 2001
; Marshall et al., 2001
). From the F113 fleQ mutant phenotype (Figs 2 and 3
) and the results from the Western blot analysis (Fig. 4
), it can be concluded that in P. fluorescens F113 the fleQ gene is necessary for the production and secretion of the flagellin FliC.
Another mutation causing non-motile cells affects the fliS gene. In pseudomonads the role of the FliS protein remains unknown. Its distant homologue in enterobacteria has been described as a substrate-specific cytosolic chaperone that facilitates FliC secretion and contributes to the stabilization of the flagellin subunits during polymerization (Auvray et al., 2001; Ozin et al., 2003
). The F113 fliS mutant has a very short and thin flagellum (Fig. 2
), probably because FliC is not well stabilized and is undergoing wrong polymerization and limited secretion, thus impairing the formation of a normal flagellar filament. To our knowledge, this is the first description of such a phenotype. The results from the fliS mutant Western blot analysis confirm the FliS putative function as a FliC chaperone. These results indicate that, instead of being secreted, the FliC flagellin is accumulated in the cytoplasm and as these proteins cannot be extracted together with the total soluble bacterial proteins, the flagellin might be accumulated inside the cytoplasm inclusion bodies. Moreover, the structural analysis of the P. fluorescens F113 FliS protein shows that it is homologous to other FliS proteins, being a small peptide, with an acidic isoelectric point (5·18) and having an amphipathic alpha-helix in the C-terminal domain, typical characteristics for most cytoplasmic chaperones (Wattiau et al., 1996
; Fraser et al., 1999
).
Downstream of fliS, the pseudomonads contain a small ORF showing very low homology with the fliT genes of enterobacteria (Table 2). In these bacteria, the FliT protein has been described as the FliD substrate-specific chaperone (Fraser et al., 1999
), although motility studies done with the Salmonella typhimurium fliT mutant concluded that there were no differences in the swimming ability compared with the wild-type strain (Bennett et al., 2001
). In P. fluorescens F113, the morphological phenotype of the fliT mutant was identical to the wild-type strain and the Western blot analysis revealed that the FliC protein is exported to form the flagellar filament. Therefore the putative protein FliT cannot be acting as a FliD cytosolic chaperone. Furthermore, the structural characteristics of the FliT protein are different to those described for cytoplasmic chaperones. In P. aeruginosa PAK, an ORF similar in size and gene location to fliT has been designed as fleP, encoding a hypothetical protein FleP. The swimming haloes produced by a mutation affecting the fleP gene are much smaller than those produced by the P. aeruginosa wild-type strain (Dasgupta et al., 2003
). This fleP mutant motility phenotype is similar to the P. fluorescens F113 fliT mutant phenotype, which produced swimming haloes 50 % smaller than the wild-type strain. However, electron microscopy studies of the mutant affected in the fleP gene in P. aeruginosa PAK revealed that its flagella were mostly detached from the cells and the length of polar type IV pili was significantly longer than those from the wild-type strain (Dasgupta et al., 2003
). Based on these results, the authors concluded that fleP represents a novel flagellar gene specific for Pseudomonas, responsible for maintaining the length of type IV pili and stable flagellar attachment to the bacterial pole. These results do not correlate with the normal flagellar morphology observed for our P. fluorescens F113 fliT mutant (Fig. 2
). Such differences, together with the lack of homology between them (Table 2
), indicate that the fleP gene in P. aeruginosa and the fliT gene in P. fluorescens are different and possess different functions.
A mutant affected in the flaG gene had longer flagella than the wild-type F113 (Fig. 2) and flagellin was clearly exported in a higher quantity than in the wild-type strain as observed by Western blot analysis (Fig. 4
). These characteristics did not result in higher motility properties in an iron-limited minimal medium, and the flaG mutant produced swimming haloes with a diameter similar to that of the wild-type strain (Fig. 3
). However, in rich medium or in the absence of iron limitation, this mutant showed a substantially higher motility (Fig. 3
). In V. anguillarum, an ORF 3 showing a 57 % identity to FlaG from Vibrio parahaemolyticus and 34 % identity to FlaG from P. fluorescens F113 has been described, and a mutant affected in this gene had elongated flagella, the same morphological phenotype as in the P. fluorescens F113 flaG mutant, although it showed an 11 % decrease compared with the wild-type motility (McGee et al., 1996
). These results indicate that in addition to filament length, FlaG could influence swimming speed, at least under certain conditions.
Root colonization is a complex and crucial process for the use of micro-organisms for agricultural and environmental biotechnology applications, since an improvement in colonization could result in an improvement in the efficacy of these applications, as has been shown for biocontrol (Chin-a-Woeng et al., 2000). Most of the already described non-motile mutants are severely affected in root colonization, especially at sites most distant from the inoculation site (De Weger et al., 1987
). We have also observed the predominance of flagellar variants with enhanced surface motility, in the distal parts of the rhizosphere that are not easily reached by the wild-type strain (Sanchez-Contreras et al., 2002
).
All the non-motile P. fluorescens F113 mutants studied in this work fliC, fleQ, fliS although able to colonize when inoculated independently, were very poor competitors, compared with the wild-type F113. These results are in agreement with previously published work that shows that aflagellate (De Weger et al., 1987) or non-chemotactic mutants (de Weert et al., 2002
) are very poor competitors. We have isolated and tested a mutant affected in the fliT gene that, although still motile and chemotactic, showed a reduced motility phenotype. This mutant was as poor a competitor as aflagellate mutants, showing that not only flagella and chemotactic motility (de Weert et al., 2002
), but also a wild-type level of motility are necessary for competitive rhizosphere colonization. Furthermore, although no differences in motility or colonization were observed for a flaG mutant under the standard conditions used, the fact that this mutant showed higher motility under certain conditions and the preferred location of hypermotile variants in distal parts of the root (Sanchez-Contreras et al., 2002
) suggest the possibility of improving competitive root colonization by manipulating the motility processes.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the European Union (GM-RHIZOREMEDIATION QLK3-CT-2001-00101), Comunidad Autónoma de Madrid (07 M-0062-2000) and Spanish Ministry of Science and Technology BIO 2003-03412. M. M. is the recipient of a Ramón y Cajal contract from the Spanish Ministry of Science and Technology.
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Received 27 May 2004;
revised 30 June 2004;
accepted 2 July 2004.
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