Departamento de Biología, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain
Correspondence
Rafael Rivilla
rafael.rivilla{at}uam.es
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ABSTRACT |
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INTRODUCTION |
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The pseudomonads possess orthologues of genes encoding these site-specific recombinases. A gene from Pseudomonas aeruginosa affecting pyoverdin production, and named sss, was shown to be homologous to the E. coli xerC gene (Hofte et al., 1994). The sss gene has also been cloned from different strains of Pseudomonas fluorescens (Dekkers et al., 1998
; Sánchez-Contreras et al., 2002
). An sss mutant of P. fluorescens WCS365 was affected in competitive rhizosphere colonization, and was displaced by the wild-type from the root tip of a variety of plants (Dekkers et al., 1998
). It has also been shown that introduction of extra copies of the sss gene can improve rhizosphere colonization (Dekkers et al., 2000
) and biocontrol abilities of different pseudomonads (Chin-a-Woeng et al., 2000
). A second recombinase that might function in conjunction with Sss is also present in pseudomonads, since the sequences of the P. aeruginosa PAO1 and P. fluorescens Pf0-1 genomes have shown the presence of genes homologous to the E. coli xerD genes. To date, these xerD homologues have not been cloned or analysed.
Phenotypic (phase) variation in pseudomonads occurs during rhizosphere colonization, and phenotypic variants showing a translucent and diffuse colony morphology have been isolated in laboratory cultures (Van Den Broek et al., 2003), and after rhizosphere passage (Sánchez-Contreras et al., 2002
; Achouak et al., 2004
). In P. fluorescens F113, the phenotypic variants isolated from the alfalfa rhizosphere showed enhanced motility, and their numbers were significantly reduced by a mutation in the sss gene (Sánchez-Contreras et al., 2002
). Most of the few variants observed among the sss mutants also carried a secondary mutation affecting the Gac system (Sánchez-Contreras et al., 2002
). The Gac system, forming a two-component regulatory system encoded by the gacA and the gacS (lemA) genes, regulates the production of secondary metabolites such as exoprotease and cyanide (Blumer et al., 1999
). This system has also been implicated in phase variation in Pseudomonas sp. PCL1171 (Van Den Broek et al., 2003
), since mutants in the gacS gene were locked in phase II, which is morphologically equivalent to the phenotypic variants produced by P. fluorescens F113 after rhizosphere colonization.
Due to the importance of phenotypic variation for rhizosphere colonization, and to improve biotechnological applications of these bacteria in biocontrol and rhizoremediation, the aim of this work was to characterize the xerD gene of P. fluorescens F113, and to determine its possible role in phenotypic variation and rhizosphere colonization.
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METHODS |
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DNA manipulations and sequence analysis.
Standard methods were used for DNA extraction and gene cloning (Sambrook et al., 1989). Southern blotting and colony hybridization were performed with a non-radioactive detection kit, and a chemiluminescence method was used to detect hybridization signals according to the instructions of the manufacturer (Boehringer Mannheim). PCR reactions were performed using the Tth enzyme (Biotools) under standard conditions. Primer sequences are available on request. DNA was custom sequenced by Centro Nacional de Investigaciones Oncológicas (CNIO; Madrid, Spain) and Sistemas Genómicos (Valencia, Spain). Sequence analysis was performed with software from the Genetics Computer Group (Madison, WI, USA) and the BLAST programs.
Protein sequences were aligned by using the multiple-sequence-alignment tool CLUSTAL W (Thompson et al., 1994). The aligned sequences were studied by using genetic distance and bootstrap NJ tree for phylogenetic inference (1000 iterations). Ambiguous characteristics (where a deletion, insertion or unidentified state was recorded for any strain) were removed from the alignment data.
Rhizosphere colonization experiments.
Alfalfa seeds were sterilized in 70 % ethanol for 2 min, diluted bleach (1 : 5) for 15 min, and then rinsed thoroughly with sterile distilled water. Seed vernalization was performed at 4 °C overnight, and germination was for 1 day at 28 °C. Germinated alfalfa seeds were sown in Leonard jar gnotobiotic systems (Villacieros et al., 2003) using perlite as the solid substrate, and 8 mM KNO3-supplemented Fahraeus (FP) medium (Fahraeus, 1957
) as the mineral solution. After 2 days, alfalfa seeds were inoculated with
108 cells of the appropriate strains. In competition experiments, strains were inoculated at a 1 : 1 ratio. Plants were maintained in controlled conditions (16 h light at 25 °C, and 8 h dark at 18 °C) for 3 weeks. Bacteria were recovered from the rhizosphere by vortexing the root tips (last centimetre of the main root) for 2 min in a tube containing 5 ml 0·9 % NaCl, and plating the appropriate dilutions on SA agar containing antibiotics. Each experiment was performed in triplicate, and each replicate contained at least 20 plants. Statistical analysis was done with Sigma Plot 4 software (Windows).
Gene expression analysis.
Transcriptional lacZ fusions of the sss and xerD genes were constructed by directionally cloning an internal fragment of the gene into the pVIK112 suicide vector (Kalogeraki & Winans, 1997). The construct was integrated into the F113 genome by triparental mating, and single homologous recombinants were checked by PCR and Southern blotting.
Cells harbouring these constructs were grown in LB medium, and samples were taken at intervals. -Galactosidase activity was determined according to Miller (1972)
.
Aqueous and methanolic root exudates were prepared by extracting roots from 100 5-day-old seedlings, growing on agar plates, in 10 ml solvent (distilled water or methanol) by shaking for 30 min. Exudates were used at a concentration of 10 µl ml1.
The reporter strains were also lawn plated on SA agar supplemented with X-Gal (40 µg ml1). Alfalfa seedlings, and wooden toothpicks impregnated with exudates, were laid on top of the bacterial lawn, and after 2 days incubation at 28 °C, plates were inspected microscopically.
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RESULTS |
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The plasmids overexpressing the genes encoding the site-specific recombinases were also mobilized into the sss and xerD mutants to generate strains mutated in one of the recombinases, but overexpressing the other. As shown in Table 2, overexpression of the sss gene in a XerD background resulted in a high proportion of phenotypic variants after prolonged cultivation. Therefore, overexpression of sss suppressed the phenotype of a xerD mutant, indicating that the XerD recombinase is necessary for the wild-type level of phenotypic variation when Sss is produced at a physiological (wild-type) level, but that it plays a marginal role when sss is expressed at higher levels. Conversely, the overexpressed xerD gene could not suppress the phenotype of the sss mutant, as the number of phenotypic variants after prolonged cultivation and rhizosphere passage remained very low. These results show that the Sss recombinase plays a more important role in phenotypic variation than the XerD recombinase, although the latter is necessary for wild-type level of phenotypic variation when sss is expressed at a physiological level.
The Gac system is affected by site-specific recombinase activity
The colony morphology of phenotypic variants resembles the morphology of mutants in the GacA/GacS two-component system (Sánchez-Contreras et al., 2002). To test the Gac phenotype of the variants produced by overexpression of both recombinases, 17 variants from the sss overexpression experiment, 15 variants from the xerD overexpression experiment, and 10 colonies with wild-type morphology obtained from the overexpression experiments were randomly selected and tested for exoprotease and pyoverdin production. gac mutants in P. fluorescens do not produce exoprotease, and they produce higher levels of the siderophore pyoverdin than the wild-type (Sánchez-Contreras et al., 2002
; Duffy & Defago, 2000
; Blumer et al., 1999
; Sacherer et al., 1994
). None of the variants in this study produced a halo in plates containing skim milk, indicating the lack of exoprotease activity (Fig. 3
B, D), while all the wild-type colonies produced a halo. Similarly, all the variants produced greater fluorescence when plates were observed under UV light, indicating that they produce more pyoverdin (Fig. 3A, C
). All the wild-type colonies showed a normal level of pyoverdin production. These results indicate that the Gac system is affected by site-specific recombinase activity, and that the variant phenotype is, at least partially, due to mutations in the genes encoding this system. A complementation analysis (Fig. 3
) using the cloned gacS gene from Pseudomonas syringae pv. syringae B728a (Hrabak & Willis, 1992
), and the cloned gacA gene from P. fluorescens CHA0 (Laville et al., 1992
), showed that 13 of the variants from the sss overexpression experiment, and 11 of the variants from the xerD overexpression experiment, were complemented for exoprotease and pyoverdin production by the cloned gacS gene (Fig. 3A, B
). Three variants from each experiment were complemented by the gacA gene (Fig. 3C, D
), and one variant from each experiment was not complemented by either gene (gacA or gacS). These results suggest that both gacA and gacS are affected by site-specific recombinase activity.
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As shown in Fig. 5(A), the expression level of the sss gene was low, and was dependent on the growth phase (Fig. 5C
), being lower during exponential growth and increasing during the stationary phase. When bacteria harbouring this fusion were grown in the presence of aqueous or methanolic root extracts, no differences in expression were observed, indicating that expression of the sss gene does not depend on diffusible compounds exuded by the plant root. Fig. 5(B, C)
shows that the expression level and pattern of the xerD gene were similar to those of sss, also being independent of compounds present in the root extracts.
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DISCUSSION |
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A mutant affected in the xerD gene showed the same phenotype as an sss mutant with regard to phenotypic variation, producing a very low number of phenotypic variants after rhizosphere colonization. This indicates that not only Sss, but also that XerD is necessary to produce the genetic rearrangements leading to the appearance of variants. Considering that in E. coli the XerC/XerD heterotetramer is necessary for recombination (Ferreira et al., 2003), it seems reasonable to assume that in pseudomonads, Sss and XerD form a heterotetramer responsible for an increase in phenotypic variation. However, we have found differences in the phenotype of strains overproducing either recombinase. Overexpression of either of the genes encoding the two recombinases led to an increase in the proportion of phenotypic variants, although the impact of overproduction of Sss was much higher than overproduction of XerD. Furthermore, overproduction of Sss could overcome the phenotype of a xerD mutant, indicating that at high cellular concentration, Sss alone (forming either homodimers or homotetramers) can catalyse recombination events that give rise to variants. Conversely, overexpression of the xerD gene was not enough to suppress the sss mutation, suggesting that there is a strict requirement of the Sss recombinase for the increase in phenotypic variation. However this interpretation is unlikely because of the increase in the number of phenotypic variants obtained after overproduction of XerD at constant and possibly low levels (see below) of Sss. Furthermore, we have been unable to construct a stable double mutant affecting both recombinases, suggesting that one or other of them is necessary for chromosome segregation after replication. The finding that, in contrast to E. coli (Hendricks et al., 2000
), single mutants grew at the same rate as the wild-type (data not shown), and with a normal cell morphology, indicate that either of the recombinases can function independently.
Analysis of the genotype of phenotypic variants has shown that all of them carry mutations in the Gac system. Mutations in different variants affected either the gacS or the gacA genes, although in two variants, the affected gene could not be determined. It is likely that these two variants are affected in both genes, although it is also possible that they are affected in genes acting downstream in the gac regulatory cascade. These results could suggest that the gacA and gacS genes are a target for the site-specific recombinases. However, considering that these genes are located in different parts of the genome, it is unlikely that the activity of the recombinases would result in the accumulation of diverse mutations in both genes. Therefore, we propose an indirect effect of the site-specific recombinases on the Gac system. The relationship between the Gac system and phenotypic variation has been shown by Van Den Broek et al. (2003). These authors demonstrated that Pseudomonas sp. PCL1171 transposon-generated mutants in the gacS gene were locked in the variant colony morphology. It has also been shown that a phenotypic variant of P. fluorescens F113, isolated from the alfalfa rhizosphere, carried a point mutation in the gacA gene (Sánchez-Contreras et al., 2002
). In the same report, the few phenotypic variants appearing in the rhizosphere after colonization by an sss mutant were defective in the Gac system.
The Sss recombinase has been shown to be essential for competitive root colonization of the potato, radish, wheat and tomato rhizospheres by P. fluorescens WCS365 (Dekkers et al., 1998). Here, we show the need of this recombinase for competitive colonization of the alfalfa rhizosphere by another biocontrol strain, P. fluorescens F113. This strain is an efficient colonizer of all plants tested so far, including alfalfa (Villacieros et al., 2003
), sugarbeet (Delany et al., 2001
; Shanahan et al., 1992
), pea (Naseby & Lynch, 1999
) and tomato (Simons et al., 1996
). It is likely that the activity of site-specific recombinases is a requirement for competitive rhizosphere colonization of a large variety of plants by P. fluorescens. We have also shown that an additional recombinase, XerD, is required for competitive rhizosphere colonization. The finding that the proportion of phenotypic variants depends directly on the cellular amount of recombinases, and the higher proportion of variants after rhizosphere passage, indicates that phenotypic variants are selected in the rhizosphere, and it makes a clear link between recombinase activity, phenotypic variation and competence for rhizosphere colonization. It is interesting to note that rhizosphere colonization has been shown to be necessary for biocontrol (Chin-a-Woeng et al., 2000
), and that biocontrol traits have been shown to be regulated by phase variation in pseudomonads (Van Den Broek et al., 2003
).
Expression of the genes encoding the two recombinases under laboratory growth conditions was low, as expected for genes whose products might alter the genome structure. The expression pattern showed that the lower level of expression occurred during the exponential phase, probably reflecting the low amounts of recombinases required for chromosome segregation, even in an actively growing population. The expression level was higher during the stationary phase, when cell division is restricted. It is possible that larger amounts of recombinases during this period are related to the stress-induced adaptive mutagenesis that appears during stationary phase in bacteria (Wright, 2004). It is important to note that it has been shown that after prolonged growth, gacA and gacS mutants accumulate in P. fluorescens CHA0 batch cultures (Duffy & Defago, 2000
). It is likely that this accumulation of mutants, with a colony morphology and genotype resembling recombinase-induced variants, is partially due to the overproduction of the recombinases during long-term stationary-phase conditions.
Genes encoding the two recombinases are induced in the alfalfa rhizosphere, although no diffusible inducer could be detected in root exudates. Since attachment to toothpicks impregnated with exudates did not induce these genes, it is likely that a non-diffusible inducer present in the plant root is responsible for the increased gene expression. Following a strategy to identify plant-induced genes of P. syringae, Marco et al. (2003) found that one of the five plant-induced genes detected corresponded to a xerD homologue, suggesting that site-specific recombinases might also play a role in pathogenic plantmicrobe interactions. Rhizosphere induction also indicates that besides phenotypic selection of variants during rhizosphere colonization, there is also an increase in the production of variants, since we have shown that the proportion of variants is dependent on the expression level of the recombinases. Dekkers et al. (2000)
have shown that overexpression of the sss gene can improve root colonization by different P. fluorescens strains. Our results show that the xerD gene offers new possibilities to improve rhizosphere colonization and, possibly, biotechnological applications of pseudomonads.
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ACKNOWLEDGEMENTS |
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Received 24 August 2004;
revised 3 November 2004;
accepted 8 November 2004.
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