Ine H. M. Mulders

Flagella-Driven Chemotaxis Towards Exudate Components Is an Important Trait for Tomato Root Colonization by Pseudomonas fluorescens

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.

Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot

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.

The sss Colonization Gene of the Tomato-Fusarium oxysporum f. sp. radicis-lycopersici Biocontrol Strain Pseudomonas fluorescens WCS365 Can Improve Root Colonization of Other Wild-type Pseudomonas spp. Bacteria

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.

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

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.

Biocontrol Traits of Pseudomonas spp. Are Regulated by Phase Variation

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.

A Rhizobium leguminosarum Biovar trifolii locus not localized on the sym plasmid hinders effective nodulation on plants of the pea cross-inoculation group

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.

Characterization of NADH dehydrogenases of Pseudomonas fluorescens WCS365 and their role in competitive root colonization.

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.

Induction of Rhizobium Nod Genes by Flavonoids: Differential Adaptation of Promoter, nodD Gene and Inducers for Various Cross-Inoculation Groups

The nodulation of leguminous plants by the soil bacterium Rhizobium is a multi-step process in which both plant genes and bacterial genes are involved (Vincent 1980). Many bacterial genes involved in the nodulation process have been shown to reside on plasmids, which are generally called Sym plasmids (Hooykaas et al 1981). These nod genes can be divided in common and host-specific genes, depending on whether they can be complemented by the corresponding genes of other fast-growing Rhizobia (Djordjevic et al 1985a; Fisher et al 1985; Wijffelman et al 1985). The genetic organization and the function of the common nod genes A, B, C, D, I, J of R. leguminosarum, R. trifolii and R. meliloti is very homologous, whereas the organization of the host-specific nodulation functions is more different (Djordjevic et al 1985b; Egelhoff et al 1985; Rossen et al 1984; Schofield et al 1986; Spaink et al, this volume; Torok et al 1984). The nodD gene of all three species is the only nod gene which is transcribed constitutively, whereas the other nod operons are not transcribed when cells are grown in the standard culture media (Innes et al 1985; Mulligan et al 1985; Rossen et al 1985; Spaink et al, this volume). Recent data in several laboratories using the nod promoters fused to the E.coli lacZ gene show that the induction of these nod operons requires both a functional nodD gene product and a substance present in root exudate (Innes et al 1985; Mulligan et al 1985; Rossen et al 1985; Shearman et al 1986; Spaink et al, this volume). These inducible common or host-specific nod operons are preceeded by a very strongly conserved sequence of DNA, the nod box, which is located about 200 bp upstream the start codon of the first gene (Rostas et al 1986; Schofield et al 1986; Scott et al 1986; Shearman et al 1985; Spaink et al, this volume). The complete inducibility of a 114 bp clone of the nodA promoter of R. leguminosarum, which contains an intact nod box strongly suggest that these nod boxes are important elements of nod promoters (Spaink et al, this volume).

Isolation of ropB, a gene encoding a 22-kDa Rhizobium leguminosarum outer membrane protein.

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.

Visualisation of Rhizosphere Interactions of Pseudomonas and Bacillus Biocontrol Strains

This chapter provides hands-on protocols as well as theoretical background information for the selection of Pseudomonas and Bacillus strains from the rhizosphere antagonistic to phytopathogens. These strains can be evaluated in a bioassay for their beneficial properties. The strains can be marked with a reporter gene after selection and used to study cellular and molecular interactions between one or more beneficial strains and a soil-borne phytopathogen in the rhizosphere of a host plant.Autofluorescent proteins can be used for the non-invasive study of rhizosphere interactions using epifluorescence and confocal laser scanning microscopy (CLSM). Autofluorescent proteins have become an outstanding and convenient tool for studying rhizosphere and other in situ environmental interactions and have allowed microbiologists to visualise the spatial distribution of various microorganisms. Intricate molecular mechanisms and relationships in the rhizosphere can now be studied. Methods to mark rhizosphere bacteria as well as fungi are provided.