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Of 214 Pseudomonas strains isolated from maize rhizosphere, 46 turned out to be antagonistic, of which 43 displayed clear colony phase variation. The latter strains formed both opaque and translucent colonies, designated as phase I and phase II, respectively. It appeared that important biocontrol traits, such as motility and the production of antifungal metabolites, proteases, lipases, chitinases, and biosurfactants, are correlated with phase I morphology and are absent in bacteria with phase II morphology. From a Tn5luxAB transposon library of Pseudomonas sp. strain PCL1171 phase I cells, two mutants exhibiting stable expression of phase II had insertions in gacS. A third mutant, which showed an increased colony phase variation frequency was mutated in mutS. Inoculation of wheat seeds with PCL1171 bacteria of phase I morphology resulted in efficient suppression of take-all disease, whereas disease suppression was absent with phase II bacteria. Neither the gacS nor the mutS mutant was able to suppress take-all, but biocontrol activity was restored after genetic complementation of these mutants. Furthermore, in a number of cases, complementation by gacS of wild-type phase II sectors to phase I phenotype could be shown. A PCL1171 phase I mutant defective in antagonistic activity appeared to have a mutation in a gene encoding a lipopeptide synthetase homologue and had lost its biocontrol activity, suggesting that biocontrol by strain PCL1171 is dependent on the production of a lipopeptide. Our results show that colony phase variation plays a regulatory role in biocontrol by Pseudomonas bacteria by influencing the expression of major biocontrol traits and that the gacS and mutS genes play a role in the colony phase variation process. Therefore phase variation not only plays a role in escaping animal defense but it also appears to play a much broader and vital role in the ecology of bacteria producing exoenzymes, antibiotics, and other secondary metabolites.

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.

The excellent-root-colonizing Pseudomonas fluorescens WCS365 was selected previously as the parental strain for the isolation of mutants impaired in root colonization. Transposon mutagenesis of WCS365 and testing for root colonization resulted in the isolation of mutant strain PCL1201, which is approximately 100-fold impaired in competitive tomato root colonization. In this manuscript, we provide evidence that shows that the lack of NADH dehydrogenase I, an enzyme of the aerobic respiratory chain encoded by the nuo operon, is responsible for the impaired root-colonization ability of PCL1201. The complete sequence of the nuo operon (ranging from nuoA to nuoN) of P. fluorescens WCS365 was identified, including the promoter region and a transcriptional terminator consensus sequence downstream of nuoN. It was shown biochemically that PCL1201 is lacking NADH dehydrogenase I activity. In addition, the presence and activity of a second NADH dehydrogenase, encoded by the ndh gene, was identified to our knowledge for the first time in the genus Pseudomonas. Since it was assumed that low-oxygen conditions were present in the rhizosphere, we analyzed the activity of the nuo and the ndh promoters at different oxygen tensions. The results showed that both promoters are up-regulated by low concentrations of oxygen and that their levels of expression vary during growth. By using lacZ as a marker, it was shown that both the nuo operon and the ndh gene are expressed in the tomato rhizosphere. In contrast to the nuo mutant PCL1201, an ndh mutant of WCS365 appeared not to be impaired in competitive root tip colonization.

The phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of phenazine-1-carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce phenazine-1-carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a biocontrol strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.

Colonization-defective, transposon-induced mutants of the efficient root colonizer Pseudomonas fluorescens WCS365 were identified with a gnotobiotic system. Most mutants were impaired in known colonization traits, i.e., prototrophy for amino acids, motility, and synthesis of the O-antigen of LPS (lipopolysaccharide). Mutants lacking the O-antigen of LPS were impaired in both colonization and competitive growth whereas one mutant (PCL1205) with a shorter O-antigen chain was defective only in colonization ability, suggesting a role for the intact O-antigen of LPS in colonization. Eight competitive colonization mutants that were not defective in the above-mentioned traits colonized the tomato root tip well when inoculated alone, but were defective in competitive root colonization of tomato, radish, and wheat, indicating they contained mutations affecting host range. One of these eight mutants (PCL1201) was further characterized and contains a mutation in a gene that shows homology to the Escherichia coli nuo4 gene, which encodes a subunit of one of two known NADH:ubiquinone oxidoreductases. Competition experiments in an oxygen-poor medium between mutant PCL1201 and its parental strain showed a decreased growth rate of mutant PCL1201. The requirement of the nuo4 gene homolog for optimal growth under conditions of oxygen limitation suggests that the root-tip environment is micro-aerobic. A mutant characterized by a slow growth rate (PCL1216) was analyzed further and contained a mutation in a gene with similarity to the E. coli HtrB protein, a lauroyl transferase that functions in lipid A biosynthesis.

Introduction of the Sym plasmid pRL1JI into the cured Rhizobium leguminosarum bv. trifolii strain RCR5 resulted in a strain, designated RBL5523, that was expected to nodulate plants of the pea cross-inoculation group. However, effective nodulation occurred only on Vicia sativa plants, not on V. hirsuta or Pisum sativum. After random Tn5 mutagenesis, a derivative of RBL5523 was isolated that effectively nodulated and fixed nitrogen on P. sativum and V. hirsuta. Characterization of the mutant, RBL5787, indicated the cell surface components, extracellular polysaccharides, lipopolysaccharides, and outer membrane proteins, as well as the pattern of Nod metabolites, were indistinguishable from those of the parental strain. To obtain an indication of the function of the mutated locus, the flanking regions were sequenced and used to perform searches in protein and nonredundant nucleotide data-bases. No significant similarity or homology with any known sequence was detected.

As judged from immunochemical detection, the levels of outer membrane antigen groups II and III of Rhizobium leguminosarum bv. viciae strain 248 decrease during bacteroid differentiation (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J. J. Lugtenberg, J. Bacteriol. 171:1136-1142, 1989). Using a newly developed colony blot assay, a cosmid clone expressing the Mab8 epitope of the outer membrane antigen group II of R. l. bv. viciae strain 248 was selected in Rhizobium meliloti LPR2120. From this cosmid the gene encoding this epitope was cloned and characterized. An open reading frame of 636 nucleotides was found and predicted to encode a protein with a calculated molecular mass of 22.5 kDa. After subtraction of the predicted 23 amino acid signal peptide, a M(r) of 20.3 kDa was calculated for the mature protein. This gene, designated ropB, was not active in Escherichia coli under the control of its own promoter. The C-terminal amino acid of the protein is a phenylalanine residue which is required for efficient translocation of outer membrane proteins. Membrane spanning amphipathic beta-sheets are predicted to represent a major part of the secondary structure of the protein. A model of the topology of the protein is presented. We determined the start of transcription in order to analyze the promoter region. No homology was found with other known promoter sequences. The ropB gene appeared to be well-conserved in R. leguminosarum and Agrobacterium tumefaciens strains. An attempt was made to mimic the immunochemical decrease of RopB ex planta. Neither the various growth conditions tested nor the addition of nodule or plant extracts resulted in a reduction of the Mab8 epitope to bacteroid levels.

We describe the cloning of a gene from Rhizobium leguminosarum biovar viciae strain 248 encoding protein IIIa, the 36-kDa outer membrane protein forming a part of the outer membrane protein antigen group III. The expression of this antigen group is repressed in the bacteroid form during symbiosis (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J. J. Lugtenberg, J. Bacteriol. 171:1136-1142, 1989). A cosmid clone expressing the strain 248-specific MAb38 epitope of this antigen group in a nonrelated strain was selected by a colony blot assay. Sequencing revealed one large open reading frame encoding a 39-kDa protein. N-terminal amino acid sequencing of the purified 36-kDa outer membrane protein IIIa revealed that the isolated gene, now designated ropA, is the structural gene for this protein and that the mature protein was formed by processing of the 22-residue N-terminal signal sequence. The gene is preceded by a promoter that was active in R. leguminosarum but not in Escherichia coli. This promoter, which showed no homology to known promoter sequences, was located approximately by determination of the transcription start site. The region upstream of the putative promoter was shown to contain two potential binding sites for integration host factor protein. Expression of protein IIIa under control of the inducible lac promoter in E. coli shows that, of its earlier described properties, the peptidoglycan linkage of protein IIIa is specific for R. leguminosarum but that outer membrane localization and calcium-stabilized oligomer formation can to a large extent also occur in E. coli.

Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies. With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS). Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens. The observation that individual monoclonal antibodies react with sets of related proteins is discussed. Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen. This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells. Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids.

Effects of alterations in lipopolysaccharide (LPS) structure of Rhizobium leguminosarum bv. viciae on effective symbiosis and on a number of cell surface characteristics were studied. Tn5 mutants with altered LPSs were screened for their inability to bind monoclonal antibody 3, one of three monoclonal antibodies to the tentative O-antigenic part of the wild-type LPS of strain 248. Ten class I LPS mutants completely lacked the O-antigen-containing LPS species. The class II LPS mutant had a severely diminished amount of an antigenically altered O-antigen-containing LPS. The class III LPS mutant had normal amounts of an altered, O-antigen-containing LPS. Class I and II mutants, but not the class III mutant, showed abnormal nodule development (i.e., blocked in the stage of bacterial release from the infection thread) resulting in nodules in which very few, at the most, plant cells contained bacteroids and which were unable to fix nitrogen. Class I and II mutants were nonmotile and were more sensitive to hydrophobic compounds than the parent strain. The most striking difference between the symbiotically defective class I and II LPS mutants on one hand and the wild-type strain and the class III mutant on the other hand was that the class I and II mutants have a more hydrophobic cell surface and a higher electrophoretic mobility. A role for an O-antigen-containing LPS in bacterial release from the infection thread, through its effects on general physicochemical cell surface characteristics, is proposed.

Rhizobium promoters involved in the formation of root nodules on leguminous plants are activated by flavonoids in plant root exudate. A series of Rhizobium strains which all contain the inducible Rhizobium leguminosarum nodA promoter fused to the Escherichia coli lacZ gene, and which differ only in the source of the regulatory nodD gene, were recently used to show that the regulatory nodD gene determines which flavonoids are able to activate the nodA promoter (HP Spaink, CA Wijffelman, E Pees, RJH Okker, BJJ Lugtenberg 1987 Nature 328: 337-340). Since these strains therefore are able to discriminate between various flavonoids, they were used to determine whether or not plants that are nodulated by R. leguminosarum produce different inducers. After chromatographic separation of root exudate constituents from Vicia sativa L. subsp. nigra (L.), V. hirsuta (L.) S.F. Gray, Pisum sativum L. cv Rondo, and Trifolium subterraneum L., the fractions were tested with a set of strains containing a nodD gene of R. leguminosarum, R. trifolii, or Rhizobium meliloti, respectively. It appeared that the source of nodD determined whether, and to what extent, the R. leguminosarum nodA promoter was induced. Lack of induction could not be attributed to the presence of inhibitors. Most of the inducers were able to activate the nodA promoter in the presence of one particular nodD gene only. The inducers that were active in the presence of the R. leguminosarum nodD gene were different in each root exudate.

A previously described (R. A. de Maagd, C. A. Wijffelman, E. Pees, and B. J. J. Lugtenberg, J. Bacteriol. 170:4424-4427, 1988) Sym plasmid-dependent, naringenin-inducible 50-kilodalton protein of Rhizobium leguminosarum biovar viciae is further characterized in this paper. The protein was overproduced by constructing a strain containing multiple copies of the R. meliloti nodD gene, which facilitated its purification. An antiserum was used to screen Tn5 insertion mutants located in the pRL1JI region found to be responsible for the production of the 50-kilodalton protein. These inserts define a new nod locus left of the nod genes identified previously. Mutations in this region affect the nodulation ability in a way which is dependent on the bacterial background as well as on the host plant. The mutants nodulate normally in a strain RBL1532 (R. leguminosarum biovar viciae strain 248, cured of its Sym plasmid) background on all three tested host plant species. In contrast, in a strain RBL5045 (R. leguminosarum biovar trifolii strain RCR5, cured of its Sym plasmid) background, nodulation on Vicia sativa is severely impaired, whereas nodulation on Vicia hirsuta and Trifolium subterraneum is apparently unaltered.