Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization

Silvia Capdevila, Francisco M. Martínez-Granero, María Sánchez-Contreras, Rafael Rivilla and Marta Martín

Departamento de Biología, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain

Correspondence
Marta Martín
m.martin{at}uam.es


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The ability of plant-associated micro-organisms to colonize and compete in the rhizosphere is specially relevant for the biotechnological application of micro-organisms as inoculants. Pseudomonads are one of the best root colonizers and they are widely used in plant-pathogen biocontrol and in soil bioremediation. This study analyses the motility mechanism of the well-known biocontrol strain Pseudomonas fluorescens F113. A 6·5 kb region involved in the flagellar filament synthesis, containing the fliC, flaG, fliD, fliS, fliT and fleQ genes and part of the fleS gene, was sequenced and mutants in this region were made. Several non-motile mutants affected in the fliC, fliS and fleQ genes, and a fliT mutant with reduced motility properties, were obtained. These mutants were completely displaced from the root tip when competing with the wild-type F113 strain, indicating that the wild-type motility properties are necessary for competitive root colonization. A mutant affected in the flaG gene had longer flagella, but the same motility and colonization properties as the wild-type. However, in rich medium or in the absence of iron limitation, it showed a higher motility, suggesting the possibility of improving competitive root colonization by manipulating the motility processes.


The GenBank/EMBL/DDBJ accession number for the 6·5 kb region sequence reported in this paper is AF399739.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The study of rhizosphere colonization by micro-organisms is crucial for the efficient application of bacteria as inoculants, both in agricultural and in environmental biotechnology processes. Pseudomonas spp. can colonize the roots of a wide range of plants (Simons et al., 1996; Naseby & Lynch, 1998; Villacieros et al., 2003), being one of the best root colonizers, and are used as a model in root-colonization studies (Bloemberg et al., 2000; Chin-a-Woeng et al., 2000). The rhizosphere is a complex environment that supports a large and metabolically active microbial population, several orders of magnitude higher than the non-rhizospheric soil. Many bacterial genes and traits have been shown to be involved in plant-root colonization (Lugtenberg & Dekkers, 1999; Rainey, 1999; Lugtenberg et al., 2001). However, not only colonization but also the pseudomonads' ability to compete with the indigenous microbial population are essential to improve their biotechnological applications in the rhizosphere environment.

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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
The strains and plasmids used in this study are described in Table 1. P. fluorescens F113 was originally isolated from the sugarbeet rhizosphere (Shanahan et al., 1992). The F113 gene bank was constructed with partially EcoRI-digested genomic DNA cloned into plasmid pLAFR3 in Escherichia coli LE392. Pseudomonas strains were grown on SA medium (Scher & Baker, 1982) at 28 °C; solid growth media contained 1·5 % (w/v) purified agar. When appropriate, kanamycin, gentamicin and rifampicin were supplemented for antibiotic selection to a final concentration of 50, 10 and 100 µg ml–1, respectively. E. coli strains were grown at 37 °C in Luria–Bertani (LB) medium (Bertani, 1951), and antibiotics were added at the following concentrations when required: kanamycin, 25 µg ml–1; gentamicin, 10 µg ml–1.


View this table:
[in this window]
[in a new window]
 
Table 1. Strains and plasmids used in this study

 
DNA techniques.
Standard techniques for subcloning procedures, plasmid preparations and agarose gel electrophoresis were used as described by Sambrook et al. (1989). Southern blot hybridizations were performed with a non-radioactive detection kit, and a chemiluminiscence method was used to detect hybridization bands according to the manufacturer's instructions (Roche Diagnostics). DNA sequencing was done by the chain-termination method using DyeDeoxy terminator cycle sequencing kit protocol as described by the manufacturer (Applied Biosystems). Homology search and sequence analysis were done using the software from the Genetics Computer Group.

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.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Characterization of a genetic region containing genes involved in flagellar filament synthesis
Fig. 1 shows the genetic organization of a 6·5 kb DNA region from the P. fluorescens F113 genome that was isolated from a cosmid that contained the fliC gene, from an F113 gene bank (Sanchez-Contreras et al., 2002). This region also contains another five ORFs and a partial ORF. Two of these complete ORFs and the partial ORF show high homology and synteny with the previously described genes fliD, encoding the filament cap protein, fleQ (adnA), encoding a master transcriptional regulator, and fleS, encoding a two-component sensor protein, respectively. These genes have been found in every pseudomonad analysed (Arora et al., 2000; Dasgupta et al., 2002, 2003; Robleto et al., 2003) and have been thoroughly characterized. Downstream of the fliC gene, there is a small ORF homologous to ORFs with the same gene context in other pseudomonads. This ORF shows homology with the flaG gene of Vibrio anguillarum (McGee et al., 1996), which affects filament length through an unknown mechanism. Downstream of the fliD gene and after an AT-rich intergenic region there are two other small ORFs that show limited homology with the PA1095 and PA1096 genes in the Pseudomonas aeruginosa PAO1 genome (Stover et al., 2000). The first ORF sequence shows high homology (77–79 % identity) with the FliS protein in P. fluorescens Pf0-1 and Pseudomonas syringae, and lower homology with other pseudomonads including P. aeruginosa and Pseudomonas putida (58–62 %). It also shows limited but significant homology (37 %) with the enterobacterial FliS proteins. Similarly to the enterobacterial FliS proteins, the F113 FliS has a putative amphipathic alpha-helix in the carboxy-terminus. The second ORF (fliT) is similar in size to ORFs with the same location in other genomes, although homology between them is very low (Table 2). This ORF has been assigned to the orf96 gene in P. aeruginosa PAK (Arora et al., 1998), the fleP gene in P. aeruginosa PAO1 strain (Dasgupta et al., 2003), and fliT in Salmonella (Bennett et al., 2001). Overall, the genetic organization of this region is identical to that of P. fluorescens Pf0-1, P. aeruginosa PAO1, P. putida KT2440 and several pathovars of P. syringae (Nelson et al., 2002). However, it differs from the gene order in P. aeruginosa strains containing type a flagellin such as PAK (GenBank accession no. L81176) and DG1 (GenBank accession no. L43064), which contain an extra copy of a gene similar to fliS, called fliS', located downstream of fliS.



View larger version (5K):
[in this window]
[in a new window]
 
Fig. 1. Physical map of the 6·5 kb DNA region containing the genes implicated in P. fluorescens F113 flagellar filament synthesis.

 

View this table:
[in this window]
[in a new window]
 
Table 2. FliT protein sequence comparison between P. fluorescens F113 and other bacteria

 
Mutants affected in each of these genes were generated by insertional mutagenesis by cloning an internal fragment of the gene in plasmid pVIK112 (Kalogeraki & Winans, 1997) and homologous recombination in the wild-type strain; this resulted in non-polar mutations, as determined by genetic complementation (see below). Despite several efforts, we have been unable to obtain mutants in the fliD gene, since besides the insertion, a wild-type copy of the gene was generated.

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.



View larger version (116K):
[in this window]
[in a new window]
 
Fig. 2. Electron microscopy images of F113 wild-type and flagellar filament mutants: (A) F113 wild-type; (B) F113-fliC; (C) F113-flaG; (D) F113-fliS. The arrow points to a thin, short flagellar filament produced by the fliS mutant. Flagella of the fliT mutant are identical to wild-type. The fleQ mutant does not produce flagella.

 
Analysis of the motility phenotype
In order to study the motility characteristics of the mutants, we analysed their ability to swim (Fig. 3). After 18 h the wild-type strain produces a 6–7 mm diameter swimming halo. Mutations located within the fliC, fleQ and fliS genes resulted in non-motile mutants as they did not produce swimming haloes. Complementation of these mutants with the wild-type genes restored motility to 100 %, 85 % and 85 % of the wild-type, respectively. The mutant affected in the flaG gene, which had longer flagellar filaments than the F113 wild-type, produced haloes similar to those of the wild-type strain. However, when swimming experiments were performed in richer medium such as LB or iron-supplemented SA, this mutant produced swimming haloes with a diameter 50–100 % wider than those from the wild-type strain. The fliT mutant, whose filament morphology was similar to the wild-type strain, produced swimming haloes 50 % smaller than the wild-type. Complementation of the fliT mutant restored motility to 83 % of the wild-type. It is important to note that the fliT mutant haloes, although smaller, presented clear concentric circles inside them, typical of the chemotactic swimming movement.



View larger version (50K):
[in this window]
[in a new window]
 
Fig. 3. Swimming haloes produced on 0·3 % agar plates by: F113 on SA (A); F113-fliC on SA (B); F113-flaG on SA (C); F113-flaG on LB (D); F113-fliS on SA (E); F113-fliT on SA (F); and F113-fleQ on SA (G). The bar represents the diameter of the wild-type strain halo in the same swimming conditions.

 
Flagellin synthesis and export
Total bacterial proteins and exported proteins were analysed by Western blotting with an antiflagellin antiserum (De Weger et al., 1987) (Fig. 4). The F113 wild-type strain gave a band corresponding to the FliC flagellin, both in the total protein and in the exported protein preparations. The same results were obtained with the fliT mutant. As expected, the fliC mutant did not produce flagellin and no band was detected in either of the protein preparations. The mutation affecting the fleQ gene produced very low levels of flagellin in the experiment done with the total proteins; no band appeared in the case of exported proteins. In the flaG mutant, the Western blot revealed a normal level of flagellin in the total protein extract and a higher level of exported flagellin, in accordance with its longer flagellar filament morphology. In the case of the fliS mutant, we did not observe a band in the total protein Western blot analysis, although a very faint band appeared in the exported proteins extract. In order to understand the results obtained with the fliS mutant Western blot experiment, a dot-blot analysis with a whole-cell lysate (soluble and non-soluble proteins) was performed. The antiflagellin antiserum gave a very strong reaction with this bacterial lysate (data not shown). These results indicate that in the fliS mutant, most of the FliC protein is probably being accumulated in inclusion bodies formed inside the cell cytoplasm.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4. Western blot analysis of total proteins (A) and external proteins (B) from flagellar filament mutants and wild-type F113, reacted with an antiflagellin antiserum. The observed band is approximately 35 kDa and corresponds to FliC.

 
Colonization analysis of flagellar mutants
Colonization experiments with each of the mutants inoculated individually showed that all the mutants constructed in this work were able to colonize the alfalfa rhizosphere (data not shown). In order to study the importance of bacterial motility in the rhizosphere colonization process, we analysed the competitiveness between the flagellar filament mutants and the wild-type F113 strain. A wild-type F113 strain tagged in a neutral part of the genome with the same integration plasmid (pVIK112) that had been used to generate the mutants was used as the competitor strain. As shown in Fig. 5, all the non-motile mutants, fliC, fliS and fleQ, were very poor competitors and were displaced by the wild-type strain from the last centimetre of the root. In a similar way, the fliT mutant that showed reduced motility was displaced by the wild-type F113 strain. No significant differences were observed between the competitive colonization ability of non-motile and reduced-motility mutants. The control strain and the flaG mutant competed at the same level as the wild-type strain F113 under our laboratory gnotobiotic competitive assay conditions.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5. Competitive root tip colonization analysis of flagellar filament mutants and wild-type F113 competitor strain. Black bars represent percentage of competitor colonies and grey bars represent percentage of colonies from each tested strain recovered from the last centimetre of the main root after competitive colonization assays. Means and standard deviations from three independent assays, performed in triplicate, are shown.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The study of bacterial motility and its influence on root colonization and competition in the rhizosphere may eventually result in improved efficacy of biotechnological applications. It is already known that bacterial motility is important in the colonization of the rhizosphere, since different non-motile mutants from P. fluorescens strain WCS374 were severely impaired in colonization (Dekkers et al., 1998a). In fact, the non-motile mutants belong to the most defective competitive class of colonization mutants (Dekkers et al., 1998b; Chin-a-Woeng et al., 2000).

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.


   ACKNOWLEDGEMENTS
 
We are grateful to Ine Mulders and Ben Lugtenberg for the antiflagellin antiserum.

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.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Arora, S. K., Ritchings, B. W., Almira, E. C., Lory, S. & Ramphal, R. (1998). The Pseudomonas aeruginosa flagellar cap protein, FliD, is responsible for mucin adhesion. Infect Immun 66, 1000–1007.[Abstract/Free Full Text]

Arora, S. K., Dasgupta, N., Lory, S. & Ramphal, R. (2000). Identification of two distinct types of flagellar cap proteins, FliD, in Pseudomonas aeruginosa. Infect Immun 68, 1474–1479.[Abstract/Free Full Text]

Auvray, F., Thomas, J., Fraser, G. M. & Hughes, C. (2001). Flagellin polymerization control by a cytosolic export chaperone. J Mol Biol 308, 221–229.[CrossRef][Medline]

Bennett, J. C. Q., Thomas, J., Fraser, G. M. & Hughes, C. (2001). Substrate complexes and domain organization of the Salmonella flagellar export chaperones FlgN and FliT. Mol Microbiol 39, 781–791.[CrossRef][Medline]

Bertani, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62, 293–300.

Bloemberg, G. V., Wijfjes, A. H. M., Lamers, G. E. M., Stuurman, N. & Lugtenberg, B. J. J. (2000). Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere: new perspectives for studying microbial communities. Mol Plant–Microbe Interact 13, 1170–1176.[Medline]

Brazil, G. M., Kenefick, L., Callanan, M., Haro, A., de Lorenzo, V., Dowling, D. N. & O'Gara, F. (1995). Construction of a rhizosphere pseudomonad with potential to degrade polychlorinated-biphenyls and detection of bph gene-expression in the rhizosphere. Appl Environ Microbiol 61, 1946–1952.[Abstract]

Casaz, P., Happel, A., Keithan, J., Read, D. L., Strain, S. R. & Levy, S. B. (2001). The Pseudomonas fluorescens transcription activator AdnA is required for adhesion and motility. Microbiology 147, 355–361.[Medline]

Chin-a-Woeng, T. F. C., Bloemberg, G. V., Mulders, I. H. M., Dekkers, L. C. & Lugtenberg, B. J. J. (2000). Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot. Mol Plant–Microbe Interact 13, 1340–1345.[Medline]

Dasgupta, N., Ferrell, E. P., Kanack, K. J., West, S. E. H. & Ramphal, R. (2002). fleQ, the gene encoding the major flagellar regulator of Pseudomonas aeruginosa, is sigma(70) dependent and is downregulated by Vfr, a homolog of Escherichia coli cyclic AMP receptor protein. J Bacteriol 184, 5240–5250.[Abstract/Free Full Text]

Dasgupta, N., Wolfgang, M. C., Goodman, A. L., Arora, S. K., Jyot, J., Lory, S. & Ramphal, R. (2003). A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa. Mol Microbiol 50, 809–824.[CrossRef][Medline]

Dekkers, L. C., Phoelich, C. C., Van der Fits, L. & Lugtenberg, B. J. J. (1998a). A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365. Proc Natl Acad Sci U S A 95, 7051–7056.[Abstract/Free Full Text]

Dekkers, L. C., Van der Bij, A. J., Mulders, I. H. M., Phoelich, C. C., Wentwoord, R. A. R., Glandorf, D. C. M., Wijffelman, C. A. & Lugtenberg, B. J. J. (1998b). Role of the O-antigen of lipopolysaccharide, and possible roles of growth rate and of NADH : ubiquinone oxidoreductase (Nuo) in competitive tomato root-tip colonization by Pseudomonas fluorescens WCS365. Mol Plant–Microbe Interact 11, 763–771.[Medline]

de Weert, S., Vermeiren, H., Mulders, I. H. M., Kuiper, I., Hendrickx, N., Bloemberg, G. V., Vanderleyden, J., De Mot, R. & Lugtenberg, B. J. J. (2002). Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens. Mol Plant–Microbe Interact 15, 1173–1180.[Medline]

De Weger, L. A., Van der Vlugt, C. I., Wijfjes, A. H., Bakker, P. A., Schippers, B. & Lugtenberg, B. J. J. (1987). Flagella of a plant-growth-stimulating Pseudomonas fluorescens strain are required for colonization of potato roots. J Bacteriol 169, 2769–2773.[Medline]

Dowling, D. N. & O'Gara, F. (1994). Metabolites of Pseudomonas involved in the biocontrol of plant-disease. Trends Biotechnol 12, 133–141.

Fahraeus, G. (1957). The infection of clover root hairs by nodule bacteria studied by simple glass technique. J Genet Microbiol 16, 374–381.

Figurski, D. H. & Helinski, D. R. (1979). Replication of an origin containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 1648–1652.[Abstract]

Fraser, G. M., Bennett, J. C. Q. & Hughes, C. (1999). Substrate-specific binding of hook-associated proteins by FlgN and FliT, putative chaperones for flagellum assembly. Mol Microbiol 32, 569–580.[CrossRef][Medline]

Höflich, G., Wiehe, W. & Hecht-Buchholz, C. (1995). Rhizosphere colonization of different crops with growth promoting Pseudomonas and Rhizobium bacteria. Microbiol Res 150, 139–147.

Jyot, J., Dasgupta, N. & Ramphal, R. (2002). fleQ, the major flagellar gene regulator in Pseudomonas aeruginosa, binds to enhancer sites located either upstream or atypically downstream of the RpoN binding site. J Bacteriol 184, 5251–5260.[Abstract/Free Full Text]

Kalogeraki, V. S. & Winans, S. C. (1997). Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene 188, 69–75.[CrossRef][Medline]

Karlson, U., Dowling, D., O'Gara, F., Rivilla, R., Bittens, M., Francesconi, S., Pritchard, H. & Pedersen, H. C. (1998). Development of self-contained plant/GMM systems for soil bioremediation, In Past, Present and Future Risk Assessment when using GMOs, pp. 23–31. Edited by G. E. de Vries. Overschild, NL: ProBio Partners.

Labes, M., Pühler, A. & Simon, R. (1990). A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for Gram-negative bacteria. Gene 89, 37–46.[CrossRef][Medline]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680–685.

Lugtenberg, B. J. J. & Dekkers, L. C. (1999). What makes Pseudomonas bacteria rhizosphere competent? Environ Microbiol 1, 9–13.[CrossRef][Medline]

Lugtenberg, B. J. J., Dekkers, L. & Bloemberg, G. V. (2001). Molecular determinants of rhizosphere colonization by Pseudomonas. Annu Rev Phytopathol 39, 461–490.[CrossRef][Medline]

Marshall, B., Robleto, E. A., Wetzler, R., Kulle, P., Casaz, P. & Levy, S. B. (2001). The adnA transcriptional factor affects persistence and spread of Pseudomonas fluorescens under natural field conditions. Appl Environ Microbiol 67, 852–857.[Abstract/Free Full Text]

McGee, K., Hörstedt, P. & Milton, D. L. (1996). Identification and characterization of additional flagellin genes from Vibrio anguillarum. J Bacteriol 178, 5188–5198.[Abstract]

Naseby, D. C. & Lynch, J. M. (1998). Impact of wild-type and genetically modified Pseudomonas fluorescens on soil enzyme activities and microbial population structure in the rhizosphere of pea. Mol Ecol 7, 617–625.[CrossRef]

Nelson, K. E., Weinel, C., Paulsen, I. T. & 40 other authors (2002). Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4, 799–808.[CrossRef][Medline]

Ozin, A. J., Claret, L., Auvray, F. & Hughes, C. (2003). The FliS chaperone selectively binds the disordered flagellin C-terminal domain central to polymerisation. FEMS Microbiol Lett 219, 219–224.[CrossRef][Medline]

Rainey, P. B. (1999). Adaptation of Pseudomonas fluorescens to the plant rhizosphere. Environ Microbiol 1, 243–257.[CrossRef][Medline]

Ramos, J. L., Duque, E. & Ramos-González, M. I. (1991). Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant Tol plasmid. Appl Environ Microbiol 57, 260–266.[Medline]

Robleto, E. A., López-Hernández, I., Silby, M. W. & Levy, S. B. (2003). Genetic analysis of the AdnA regulon in Pseudomonas fluorescens: nonessential role of flagella in adhesion to sand and biofilm formation. J Bacteriol 185, 453–460.[Abstract/Free Full Text]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory.

Sanchez-Contreras, M., Martin, M. Villacieros M., O'Gara, F., Bonilla, I. & Rivilla, R. (2002). Phenotypic selection and phase variation occur during alfalfa root colonization by Pseudomonas fluorescens F113. J Bacteriol 184, 1587–1596.[Abstract/Free Full Text]

Scher, F. M. & Baker, R. (1982). Effects of Pseudomonas putida and a synthetic iron chelator on induction of soil supressiveness to Fusarium wilt pathogens. Phytopathology 72, 1567–1573.

Shanahan, P., Borro, A., O'Gara, F. & Glennon, J. D. (1992). Isolation, trace enrichment and liquid-chromatographic analysis of diacetylphloroglucinol in culture and soil samples using UV and amperometric detection. J Chromatogr 606, 171–177.[CrossRef]

Simons, M., Vanderbij, A. J., Brand, I., Deweger, L. A., Wijffelman, C. A. & Lugtenberg, B. J. J. (1996). Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria. Mol Plant–Microbe Interact 9, 600–607.[Medline]

Stover, K. C., Pham, X. Q., Erwin, A. L. & 28 other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1: an opportunistic pathogen. Nature 406, 959–964.[CrossRef][Medline]

Villacieros, M., Power, B., Sanchez-Contreras, M. & 8 other authors (2003). Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere. Plant Soil 251, 47–54.[CrossRef]

Walsh, U. F. J. P., Morrissey, J. P. & O'Gara, F. (2001). Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation. Curr Opin Biotechnol 12, 289–295.[CrossRef][Medline]

Wattiau, P., Woestyn, S. & Cornelis, G. R. (1996). Customized secretion chaperones in pathogenic bacteria. Mol Microbiol 20, 255–262.[Medline]

Yee, D. C., Maynard, J. A. & Wood, T. K. (1998). Rhizoremediation of trichloroethylene by a recombinant, root-colonizing Pseudomonas fluorescens strain expressing toluene ortho-monooxygenase constitutively. Appl Environ Microbiol 64, 112–118.[Abstract/Free Full Text]

Received 27 May 2004; revised 30 June 2004; accepted 2 July 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Capdevila, S.
Articles by Martín, M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Capdevila, S.
Articles by Martín, M.
Agricola
Articles by Capdevila, S.
Articles by Martín, M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.