Coping with Environmental Eukaryotes; Identification of Pseudomonas syringae Genes during the Interaction with Alternative Hosts or Predators.

Understanding the molecular mechanisms underpinning the ecological success of plant pathogens is critical to develop strategies for controlling diseases and protecting crops. Recent observations have shown that plant pathogenic bacteria, particularly Pseudomonas, exist in a range of natural environments away from their natural plant host e.g., water courses, soil, non-host plants. This exposes them to a variety of eukaryotic predators such as nematodes, insects and amoebae present in the environment. Nematodes and amoeba in particular are bacterial predators while insect herbivores may act as indirect predators, ingesting bacteria on plant tissue. We therefore postulated that bacteria are probably under selective pressure to avoid or survive predation and have therefore developed appropriate coping mechanisms. We tested the hypothesis that plant pathogenic Pseudomonas syringae are able to cope with predation pressure and found that three pathovars show weak, but significant resistance or toxicity. To identify the gene systems that contribute to resistance or toxicity we applied a heterologous screening technique, called Rapid Virulence Annotation (RVA), for anti-predation and toxicity mechanisms. Three cosmid libraries for P. syringae pv. aesculi, pv. tomato and pv. phaseolicola, of approximately 2000 cosmids each, were screened in the susceptible/non-toxic bacterium Escherichia coli against nematode, amoebae and an insect. A number of potential conserved and unique genes were identified which included genes encoding haemolysins, biofilm formation, motility and adhesion. These data provide the first multi-pathovar comparative insight to how plant pathogens cope with different predation pressures and infection of an insect gut and provide a foundation for further study into the function of selected genes and their role in ecological success.

The Essential Genome of Burkholderia cenocepacia H111.

The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera.IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of "core essential genes" that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.

Microbial trench-based optofluidic system for reagentless determination of phenolic compounds.

Phenolic compounds are one of the main contaminants of soil and water due to their toxicity and persistence in the natural environment. Their presence is commonly determined with bulky and expensive instrumentation (e.g. chromatography systems), requiring sample collection and transport to the laboratory. Sample transport delays data acquisition, postponing potential actions to prevent environmental catastrophes. This article presents a portable, miniaturized, robust and low-cost microbial trench-based optofluidic system for reagentless determination of phenols in water. The optofluidic system is composed of a poly(methyl methacrylate) structure, incorporating polymeric optical elements and miniaturized discrete auxiliary components for optical transduction. An electronic circuit, adapted from a lock-in amplifier, is used for system control and interfering ambient light subtraction. In the trench, genetically modified bacteria are stably entrapped in an alginate hydrogel for quantitative determination of model phenol catechol. Alginate is also acting as a diffusion barrier for compounds present in the sample. Additionally, the superior refractive index of the gel (compared to water) confines the light in the lower level of the chip. Hence, the optical readout of the device is only altered by changes in the trench. Catechol molecules (colorless) in the sample diffuse through the alginate matrix and reach bacteria, which degrade them to a colored compound. The absorbance increase at 450 nm reports the presence of catechol simply, quickly (~10 min) and quantitatively without addition of chemical reagents. This miniaturized, portable and robust optofluidic system opens the possibility for quick and reliable determination of environmental contamination in situ, thus mitigating the effects of accidental spills.

Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins.

In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots.

Genome sequence reveals that Pseudomonas fluorescens F113 possesses a large and diverse array of systems for rhizosphere function and host interaction.

BACKGROUND: Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) isolated from the sugar-beet rhizosphere. This bacterium has been extensively studied as a model strain for genetic regulation of secondary metabolite production in P. fluorescens, as a candidate biocontrol agent against phytopathogens, and as a heterologous host for expression of genes with biotechnological application. The F113 genome sequence and annotation has been recently reported. RESULTS: Comparative analysis of 50 genome sequences of strains belonging to the P. fluorescens group has revealed the existence of five distinct subgroups. F113 belongs to subgroup I, which is mostly composed of strains classified as P. brassicacearum. The core genome of these five strains is highly conserved and represents approximately 76% of the protein-coding genes in any given genome. Despite this strong conservation, F113 also contains a large number of unique protein-coding genes that encode traits potentially involved in the rhizocompetence of this strain. These features include protein coding genes required for denitrification, diterpenoids catabolism, motility and chemotaxis, protein secretion and production of antimicrobial compounds and insect toxins. CONCLUSIONS: The genome of P. fluorescens F113 is composed of numerous protein-coding genes, not usually found together in previously sequenced genomes, which are potentially decisive during the colonisation of the rhizosphere and/or interaction with other soil organisms. This includes genes encoding proteins involved in the production of a second flagellar apparatus, the use of abietic acid as a growth substrate, the complete denitrification pathway, the possible production of a macrolide antibiotic and the assembly of multiple protein secretion systems.

Effects of harmful cyanobacteria on the freshwater pathogenic free-living amoeba Acanthamoeba castellanii.

Grazing is a major regulating factor in cyanobacterial population dynamics and, subsequently, considerable effort has been spent on investigating the effects of cyanotoxins on major metazoan grazers. However, protozoan grazers such as free-living amoebae can also feed efficiently on cyanobacteria, while simultaneously posing a major threat for public health as parasites of humans and potential reservoirs of opportunistic pathogens. In this study, we conducted several experiments in which the freshwater amoeba Acanthamoeba castellanii was exposed to pure microcystin-LR (MC-LR) and six cyanobacterial strains, three MC-producing strains (MC-LR, MC-RR, MC-YR, MC-WR, [Dha7] MC-RR) and three strains containing other oligopeptides such as anabaenopeptins and cyanopeptolins. Although the exposure to high concentrations of pure MC-LR yielded no effects on amoeba, all MC-producing strains inflicted high mortality rates on amoeba populations, suggesting that toxic effects must be mediated through the ingestion of toxic cells. Interestingly, an anabaenopeptin-producing strain caused the greatest inhibition of amoeba growth, indicating that toxic bioactive compounds other than MCs are of great importance for amoebae grazers. Confocal scanning microscopy revealed different alterations in amoeba cytoskeleton integrity and as such, the observed declines in amoeba densities could have indeed been caused via a cascade of cellular events primarily triggered by oligopeptides with protein-phosphatase inhibition capabilities such as MCs or anabaenopeptins. Moreover, inducible-defense mechanisms such as the egestion of toxic, MC-producing cyanobacterial cells and the increase of resting stages (encystation) in amoebae co-cultivated with all cyanobacterial strains were observed in our experiments. Consequently, cyanobacterial strains showed different susceptibilities to amoeba grazing which were possibly influenced by the potentiality of their toxic secondary metabolites. Hence, this study shows the importance of cyanobacterial toxicity against amoeba grazing and, that cyanobacteria may contain a wide range of chemical compounds capable of negatively affect free-living, herbivorous amoebae. Moreover, this is of high importance for understanding the interactions and population dynamics of such organisms in aquatic ecosystems.

Genome sequence of the biocontrol strain Pseudomonas fluorescens F113.

Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere colonization. Here, we present its complete genome sequence, which shows that besides a core genome very similar to those of other strains sequenced within this species, F113 possesses a wide array of genes encoding specialized functions for thriving in the rhizosphere and interacting with eukaryotic organisms.

Influence of the Photorhabdus luminescens phosphomannose isomerase gene, manA, on mannose utilization, exopolysaccharide structure, and biofilm formation.

Extracellular polysaccharide (EPS) is produced by diverse bacterial pathogens and fulfills assorted roles, including providing a structural matrix for biofilm formation and more specific functions in virulence, such as protection against immune defenses. We report here the first investigation of some of the genes important for biofilm formation in Photorhabdus luminescens and demonstrate the key role of the phosphomannose isomerase gene, manA, in the structure of functional EPS. Phenotypic analyses of a manA-deficient mutant showed the importance of EPS in motility, insect virulence, and biofilm formation on abiotic surfaces as well as the requirement of this gene for the use of mannose as the sole carbon source. Conversely, this defect had no apparent impact on symbiosis with the heterorhabditid nematode vector. A more detailed analysis of biofilm formation revealed that the manA mutant was able to attach to surfaces with the same efficiency as that of the wild-type strain but could not develop the more extended biofilm matrix structures. A compositional analysis of P. luminescens EPS reveals how the manA mutation has a major effect on the formation of a complete, branched EPS.

Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.

BACKGROUND: Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. RESULTS: A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28 degrees C) and human (37 degrees C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. CONCLUSIONS: We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of EPS properties. Despite its abundance and conservation in the genus, we find no evidence for a role of Pam in either virulence or symbiosis.

Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with the insect pathogen Photorhabdus luminescens.

BACKGROUND: The Gram-negative bacterium Photorhabdus asymbiotica (Pa) has been recovered from human infections in both North America and Australia. Recently, Pa has been shown to have a nematode vector that can also infect insects, like its sister species the insect pathogen P. luminescens (Pl). To understand the relationship between pathogenicity to insects and humans in Photorhabdus we have sequenced the complete genome of Pa strain ATCC43949 from North America. This strain (formerly referred to as Xenorhabdus luminescens strain 2) was isolated in 1977 from the blood of an 80 year old female patient with endocarditis, in Maryland, USA. Here we compare the complete genome of Pa ATCC43949 with that of the previously sequenced insect pathogen P. luminescens strain TT01 which was isolated from its entomopathogenic nematode vector collected from soil in Trinidad and Tobago. RESULTS: We found that the human pathogen Pa had a smaller genome (5,064,808 bp) than that of the insect pathogen Pl (5,688,987 bp) but that each pathogen carries approximately one megabase of DNA that is unique to each strain. The reduced size of the Pa genome is associated with a smaller diversity in insecticidal genes such as those encoding the Toxin complexes (Tc's), Makes caterpillars floppy (Mcf) toxins and the Photorhabdus Virulence Cassettes (PVCs). The Pa genome, however, also shows the addition of a plasmid related to pMT1 from Yersinia pestis and several novel pathogenicity islands including a novel Type Three Secretion System (TTSS) encoding island. Together these data suggest that Pa may show virulence against man via the acquisition of the pMT1-like plasmid and specific effectors, such as SopB, that promote its persistence inside human macrophages. Interestingly the loss of insecticidal genes in Pa is not reflected by a loss of pathogenicity towards insects. CONCLUSION: Our results suggest that North American isolates of Pa have acquired virulence against man via the acquisition of a plasmid and specific virulence factors with similarity to those shown to play roles in pathogenicity against humans in other bacteria.

Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with the insect pathogen Photorhabdus luminescens.

BACKGROUND: The gram-negative bacterium Photorhabdus asymbiotica (Pa) has been recovered from human infections in both North America and Australia. Recently, Pa has been shown to have a nematode vector that can also infect insects, like its sister species the insect pathogen P. luminescens (Pl). To understand the relationship between pathogenicity to insects and humans in Photorhabdus we have sequenced the complete genome of Pa strain ATCC43949 from North America. This strain (formerly referred to as Xenorhabdus luminescens strain 2) was isolated in 1977 from the blood of an 80 year old female patient with endocarditis, in Maryland, USA. Here we compare the complete genome of Pa ATCC43949 with that of the previously sequenced insect pathogen P. luminescens strain TT01 which was isolated from its entomopathogenic nematode vector collected from soil in Trinidad and Tobago. RESULTS: We found that the human pathogen Pa had a smaller genome (5,064,808 bp) than that of the insect pathogen Pl (5,688,987 bp) but that each pathogen carries approximately one megabase of DNA that is unique to each strain. The reduced size of the Pa genome is associated with a smaller diversity in insecticidal genes such as those encoding the Toxin complexes (Tc's), Makes caterpillars floppy (Mcf) toxins and the Photorhabdus Virulence Cassettes (PVCs). The Pa genome, however, also shows the addition of a plasmid related to pMT1 from Yersinia pestis and several novel pathogenicity islands including a novel Type Three Secretion System (TTSS) encoding island. Together these data suggest that Pa may show virulence against man via the acquisition of the pMT1-like plasmid and specific effectors, such as SopB, that promote its persistence inside human macrophages. Interestingly the loss of insecticidal genes in Pa is not reflected by a loss of pathogenicity towards insects...

Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts.

Current sequence databases now contain numerous whole genome sequences of pathogenic bacteria. However, many of the predicted genes lack any functional annotation. We describe an assumption-free approach, Rapid Virulence Annotation (RVA), for the high-throughput parallel screening of genomic libraries against four different taxa: insects, nematodes, amoeba, and mammalian macrophages. These hosts represent different aspects of both the vertebrate and invertebrate immune system. Here, we apply RVA to the emerging human pathogen Photorhabdus asymbiotica using "gain of toxicity" assays of recombinant Escherichia coli clones. We describe a wealth of potential virulence loci and attribute biological function to several putative genomic islands, which may then be further characterized using conventional molecular techniques. The application of RVA to other pathogen genomes promises to ascribe biological function to otherwise uncharacterized virulence genes.

Quorum-sensing-regulated transcriptional initiation of plasmid transfer and replication genes in Rhizobium leguminosarum biovar viciae.

Transfer of the Rhizobium leguminosarum biovar viciae symbiosis plasmid pRL1JI is regulated by a cascade of gene induction involving three LuxR-type quorum-sensing regulators, TraR, BisR and CinR. TraR induces the plasmid transfer traI-trb operon in a population-density-dependent manner in response to N-acylhomoserine lactones (AHLs) made by TraI. Expression of the traR gene is primarily induced by BisR in response to AHLs made by CinI, and expression of cinI is induced by CinR and repressed by BisR. Analysis of transcription initiation of cinI, traR and traI identified potential regulatory domains recognized by the CinR, BisR and TraR regulators. Deletion and mutation of the cinI promoter identified potential recognition motifs for activation by CinR and repression by BisR. Analysis of the DNA sequence upstream of traI and expression of transcriptional gene fusions revealed a predicted TraR-binding (tra-box) domain. Two transcript initiation sites were identified upstream of the plasmid replication gene repA, which is divergently transcribed from traI; one of these repA transcripts requires the quorum-sensing cascade mediated via BisR and TraR, showing that the pRL1JI plasmid replication genes are co-regulated with the plasmid transfer genes.

Quorum-sensing regulation in rhizobia and its role in symbiotic interactions with legumes.

Legume-nodulating bacteria (rhizobia) usually produce N-acyl homoserine lactones, which regulate the induction of gene expression in a quorum-sensing (or population-density)-dependent manner. There is significant diversity in the types of quorum-sensing regulatory systems that are present in different rhizobia and no two independent isolates worked on in detail have the same complement of quorum-sensing genes. The genes regulated by quorum sensing appear to be rather diverse and many are associated with adaptive aspects of physiology that are probably important in the rhizosphere. It is evident that some aspects of rhizobial physiology related to the interaction between rhizobia and legumes are influenced by quorum sensing. However, it also appears that the legumes play an active role, both in terms of interfering with the rhizobial quorum-sensing systems and responding to the signalling molecules made by the bacteria. In this article, we review the diversity of quorum-sensing regulation in rhizobia and the potential role of legumes in influencing and responding to this signalling system.

Polychlorinated biphenyl rhizoremediation by Pseudomonas fluorescens F113 derivatives, using a Sinorhizobium meliloti nod system to drive bph gene expression.

Rhizoremediation of organic chemicals requires high-level expression of biodegradation genes in bacterial strains that are excellent rhizosphere colonizers. Pseudomonas fluorescens F113 is a biocontrol strain that was shown to be an excellent colonizer of numerous plant rhizospheres, including alfalfa. Although a derivative of F113 expressing polychlorinated biphenyl (PCB) biodegradation genes (F113pcb) has been reported previously, this strain shows a low level of bph gene expression, limiting its rhizoremediation potential. Here, a high-level expression system was designed from rhizobial nod gene regulatory relays. Nod promoters were tested in strain F113 by using beta-galactosidase transcriptional fusions. This analysis showed that nodbox 4 from Sinorhizobium meliloti has a high level of expression in F113 that is dependent on an intact nodD1 gene. A transcriptional fusion of a nodbox cassette containing the nodD1 gene and nodbox 4 fused to a gfp gene was expressed in the alfalfa rhizosphere. The bph operon from Burkholderia sp. strain LB400 was cloned under the control of the nodbox cassette and was inserted as a single copy into the genome of F113, generating strain F113L::1180. This new genetically modified strain has a high level of BphC activity and grows on biphenyl as a sole carbon and energy source at a growth rate that is more than three times higher than that of F113pcb. Degradation of PCBs 3, 4, 5, 17, and 25 was also much faster in F113L::1180 than in F113pcb. Finally, the modified strain cometabolized PCB congeners present in Delor103 better than strain LB400, the donor of the bph genes used.

Two site-specific recombinases are implicated in phenotypic variation and competitive rhizosphere colonization in Pseudomonas fluorescens.

The biocontrol agent Pseudomonas fluorescens F113 undergoes phenotypic variation during rhizosphere colonization, and this variation has been related to the activity of a site-specific recombinase encoded by the sss gene. Here, it is shown that a second recombinase encoded by the xerD gene is also implicated in phenotypic variation. A putative xerD gene from this strain was cloned, and sequence analysis confirmed that it encoded a site-specific recombinase of the lambda integrase family. Mutants affected in the sss or xerD genes produced a very low quantity of phenotypic variants compared to the wild-type strain, both under prolonged cultivation in the laboratory and after rhizosphere colonization, and they were severely impaired in competitive root colonization. Overexpression of the genes encoding either recombinase resulted in a substantial increment in the production of phenotypic variants under both culture and rhizosphere colonization conditions, implying that both site-specific recombinases are involved in phenotypic variation. Overexpression of the sss gene suppressed the phenotype of a xerD mutant, but overexpression of the xerD gene had no effect on the phenotype of an sss mutant. Genetic analysis of the phenotypic variants obtained after overexpression of the genes encoding both the recombinases showed that they carried mutations in the gacA/S genes, which are necessary to produce a variety of secondary metabolites. These results indicate that the Gac system is affected by the activity of the site-specific recombinases. Transcriptional fusions of the sss and xerD genes with a promoterless lacZ gene showed that both genes have a similar expression pattern, with maximal expression during stationary phase. Although the expression of both genes was independent of diffusible compounds present in root exudates, it was induced by the plant, since bacteria attached to the root showed enhanced expression.

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

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.

Phenotypic selection and phase variation occur during alfalfa root colonization by Pseudomonas fluorescens F113.

During colonization of the alfalfa rhizosphere, Pseudomonas fluorescens F113 undergoes phenotypic variation, resulting in the appearance of colonies with different morphology. Among phenotypic variants, three isolates, C, F, and S were selected, with the C variant showing colony morphology identical to that of the inoculated wild-type strain and F and S having a translucent and diffuse morphology. Phenotypic variants F and S were shown to preferentially colonize distal parts of the roots and showed alterations in motility, swimming faster than the C variant and swarming under conditions that did not allow swarming of the C variant. The motility behavior correlated with overproduction of the fliC-encoded protein flagellin but not with hyperflagellation. Flagella of the F and S variants were several times longer than those of the C variant, and overproduction of flagellin was regulated at the transcriptional level. Variant F showed alterations in traits that have been shown to be important for rhizosphere colonization, such as siderophore, cyanide, and exoprotease production, and these phenotypes were complemented by a cloned gacA. Sequence analysis of the gacA alelle in variant F suggested selection of the phenotype in the rhizosphere. Variant F was also affected in other phenotypes, such as lipopolysaccharide structure and flocculation in unshaken liquid medium, which were not complemented by the gacA or gacS gene. Mutation of the F113 sss gene, encoding a site-specific recombinase, showed that most of the phenotypic variation was due to the activity of this recombinase, indicating that phase variation occurs during rhizosphere colonization.