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This minireview highlights the importance of endophytic fungi for sustainable agriculture and horticulture production. Fungal endophytes play a key role in habitat adaptation of plants resulting in improved plant performance and plant protection against biotic and abiotic stresses. They encode a vast variety of novel secondary metabolites including volatile organic compounds. In addition to protecting plants against pathogens and pests, selected fungal endophytes have been used to remove animal toxicities associated with fungal endophytes in temperate grasses, to create corn and rice plants that are tolerant to a range of biotic and abiotic stresses, and for improved management of post-harvest control. We argue that practices used in plant breeding, seed treatments and agriculture, often caused by poor knowledge of the importance of fungal endophytes, are among the reasons for the loss of fungal endophyte diversity in domesticated plants and also accounts for the reduced effectiveness of some endophyte strains to confer plant benefits. We provide recommendations on how to mitigate against these negative impacts in modern agriculture., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Monitoring of pathogenic strains of Fusarium oxysporum (Fox), which cause wilt and rots on agricultural and ornamental plants, is important for predicting disease outbreaks. Since both pathogenic and non-pathogenic strains of Fox are ubiquitous and are able to colonize plant roots, detection of Fox DNA in plant material is not the ultimate proof of an ongoing infection which would cause damage to the plant. We followed the colonization of tomato plants by strains Fox f. sp. radicis-lycopersici ZUM2407 (a tomato foot and root rot pathogen), Fox f. sp. radiciscucumerinum V03-2g (a cucumber root rot pathogen) and Fox Fo47 (a well-known non-pathogenic biocontrol strain). We determined fungal DNA concentrations in tomato plantlets by quantitative PCR (qPCR) with primers complementary to the intergenic spacer region (IGS) of these three Fox strains. Two weeks after inoculation of tomato seedlings with these Fox strains, the DNA concentration of Forl ZUM2407 was five times higher than that of the non-compatible pathogen Forc V03-2g and 10 times higher than that of Fo47. In 3-week-old plantlets the concentration of Forl ZUM2407 DNA was at least 10 times higher than those of the other strains. The fungal DNA concentration, as determined by qPCR, appeared to be in good agreement with data of the score of visible symptoms of tomato foot and root rot obtained 3 weeks after inoculation of tomato with Forl ZUM2407. Our results show that targeting of the multicopy ribosomal operon results in a highly sensitive qPCR reaction for the detection of Fox DNA. Since formae speciales of Fox cannot be distinguished by comparison of ribosomal operons, detection of Fox DNA is not evidence of plant infection by a compatible pathogen. Nevertheless, the observed difference in levels of plant colonization between pathogenic and non-pathogenic strains strongly suggests that a concentration of Fox DNA in plant material above the threshold level of 0.005% is due to proliferation of pathogenic Fox.

Aim: This study was undertaken to isolate Bacillus subtilis strains with biological activity against soil-borne phytopathogenic fungi from the avocado rhizoplane., Methods and Results: A collection of 905 bacterial isolates obtained from the rhizoplane of healthy avocado trees, contains 277 gram-positive isolates. From these gram-positive isolates, four strains, PCL1605, PCL1608, PCL1610 and PCL1612, identified as B. subtilis, were selected on the basis of their antifungal activity against diverse soil-borne phytopathogenic fungi. Analysis of the antifungal compounds involved in their antagonistic activity showed that these strains produced hydrolytic enzymes such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin, and/or iturin A. In biocontrol trials using the pathosystems tomato/Fusarium oxysporum f.sp. radicis-lycopersici and avocado/Rosellinia necatrix, two B. subtilis strains, PCL1608 and PCL1612, both producing iturin A, exhibited the highest biocontrol and colonization capabilities., Conclusions: Diverse antagonistic B. subtilis strains isolated from healthy avocado rhizoplanes have shown promising biocontrol abilities, which are closely linked with the production of antifungal lipopeptides and good colonization aptitudes., Significance and Impact of the Study: This is one of the few reports dealing with isolation and characterization of B. subtilis strains with biocontrol activity against the common soil-borne phytopathogenic fungi F. oxysporum f.sp. radicis-lycopersici and R. necatrix.

Secondary metabolites are important factors for interactions between bacteria and other organisms. Pseudomonas chlororaphis PCL1391 produces the antifungal secondary metabolite phenazine-1-carboxamide (PCN) that inhibits growth of Fusarium oxysporum f. sp. radius lycopersici the causative agent of tomato foot and root rot. Our previous work unraveled a cascade of genes regulating the PCN biosynthesis operon, phzABCDEFGH. Via a genetic screen, we identify in this study a novel TetR/AcrR regulator, named Pip (phenazine inducing protein), which is essential for PCN biosynthesis. A combination of a phenotypical characterization of a pip mutant, in trans complementation assays of various mutant strains, and electrophoretic mobility shift assays identified Pip as the fifth DNA-binding protein so far involved in regulation of PCN biosynthesis. In this regulatory pathway, Pip is positioned downstream of PsrA (Pseudomonas sigma factor regulator) and the stationary-phase sigma factor RpoS, while it is upstream of the quorum-sensing system PhzI/PhzR. These findings provide further evidence that the path leading to the expression of secondary metabolism gene clusters in Pseudomonas species is highly complex.

Pseudomonas fluorescens strain PCL1210, a competitive tomato root tip colonization mutant of the efficient root colonizing wild type strain WCS365, is impaired in the two-component sensor-response regulator system ColR/ColS. Here we show that a putative methyltransferase/wapQ operon is located downstream of colR/colS and that this operon is regulated by ColR/ColS. Since wapQ encodes a putative lipopolysaccharide (LPS) phosphatase, the possibility was studied that the integrity of the outer membrane of PCL1210 was altered. Indeed, it was shown that mutant PCL1210 is more resistant to various chemically unrelated antibiotics which have to pass the outer membrane for their action. In contrast, the mutant is more sensitive to the LPS-binding antibiotic polymyxin B. Mutant PCL1210 loses growth in competition with its wild type when grown in tomato root exudate. Mutants in the methyltransferase/wapQ operon are also altered in their outer membrane permeability and are defective in competitive tomato root tip colonization. A model for the altered outer membrane of PCL1210 is discussed.

Pseudomonas putida strain PCL1445 produces two cyclic lipopeptides, putisolvin I and putisolvin II, which possess surface tension-reducing abilities and are able to inhibit biofilm formation and to break down existing biofilms of several Pseudomonas spp., including P. aeruginosa. Putisolvins are secreted in the culture medium during growth at late exponential phase, indicating that production is possibly regulated by quorum sensing. In the present study, we identified a quorum-sensing system in PCL1445 that is composed of ppuI, rsaL, and ppuR and shows very high similarity with gene clusters of P. putida strains IsoF and WCS358. Strains with mutations in ppuI and ppuR showed a severe reduction of putisolvin production. Expression analysis of the putisolvin biosynthetic gene in a ppuI background showed decreased expression, which could be complemented by the addition of synthetic 3-oxo-C(10)-N-acyl homoserine lactone (3-oxo-C(10)-AHL) or 3-oxo-C(12)-AHL to the medium. An rsaL mutant overproduces AHLs, and production of putisolvins is induced early during growth. Analysis of biofilm formation on polyvinylchloride showed that ppuI and ppuR mutants produce a denser biofilm than PCL1445, which correlates with decreased production of putisolvins, whereas an rsaL mutant shows a delay in biofilm production, which correlates with early production of putisolvins. The results demonstrate that quorum-sensing signals induce the production of cyclic lipopeptides putisolvin I and II and consequently control biofilm formation by Pseudomonas putida.

A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and phenazine-1-carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol ability. The major antifungal activity produced by strain P. fluorescens PCL1606 did not correspond to any of the major classes of antifungal antibiotics produced by Pseudomonas biocontrol strains. This compound was purified and subsequently identified as 2-hexyl 5-propyl resorcinol (HPR). To study the role of HPR in biocontrol activity, two Tn5 mutants of P. fluorescens PCL1606 impaired in antagonistic activity were selected. These mutants were shown to impair HRP production and showed a decrease in biocontrol activity. As far as we know, this is the first report of a Pseudomonas biocontrol strain that produces HPR in which the production of this compound correlates with its biocontrol activity.

Pseudomonas putida PCL1445 produces two cyclic lipopeptides, putisolvins I and II, which possess surfactant activity and play an important role in biofilm formation and degradation. In order to identify genes and traits that are involved in the regulation of putisolvin production of PCL1445, a Tn5luxAB library was generated and mutants were selected for the lack of biosurfactant production using a drop-collapsing assay. Sequence analysis of the Tn5luxAB flanking region of one biosurfactant mutant, strain PCL1627, showed that the transposon had inserted in a dnaK homologue which is located downstream of grpE and upstream of dnaJ. Analysis of putisolvin production and expression studies indicate that dnaK, together with the dnaJ and grpE heat shock genes, takes part in the positive regulation (directly or indirectly) of putisolvin biosynthesis at the transcriptional level. Growth of PCL1445 at low temperature resulted in an increased level of putisolvins, and mutant analyses showed that this requires dnaK and dnaJ but not grpE. In addition, putisolvin biosynthesis of PCL1445 was found to be dependent on the GacA/GacS two-component signaling system. Expression analysis indicated that dnaK is positively regulated by GacA/GacS.

The soilborne fungus Fusarium oxysporum f. sp. radicis-lycopersici causes tomato foot and root rot (TFRR), which can be controlled by the addition of the nonpathogenic fungus F. oxysporum Fo47 to the soil. To improve our understanding of the interactions between the two Fusarium strains on tomato roots during biocontrol, the fungi were labeled using different autofluorescent proteins as markers and subsequently visualized using confocal laser scanning microscopy. The results were as follows. i) An at least 50-fold excess of Fo47over F. oxysporum f. sp. radicis-lycopersici was required to obtain control of TFRR. ii) When seedlings were planted in sand infested with spores of a single fungus, Fo47 hyphae attached to the root earlier than those of F. oxysporum f. sp. radicis-lycopersici. iii) Subsequent root colonization by F. oxysporum f. sp. radicis-lycopersici was faster and to a larger extent than that by Fo47. iv) Under disease-controlling conditions, colonization of tomato roots by the pathogenic fungus was significantly reduced. v) When the inoculum concentration of Fo47 was increased, root colonization by the pathogen was arrested at the stage of initial attachment to the root. vi) The percentage of spores of Fo47 that germinates in tomato root exudate in vitro is higher than that of the pathogen F. oxysporum f. sp. radicis-lycopersici. Based on these results, the mechanisms by which Fo47 controls TFRR are discussed in terms of i) rate of spore germination and competition for nutrients before the two fungi reach the rhizoplane; ii) competition for initial sites of attachment, intercellular junctions, and nutrients on the tomato root surface; and iii) inducing systemic resistance.

The rhizobacterium Pseudomonas chlororaphis PCL1391 produces the antifungal metabolite phenazine-1-carboxamide (PCN), which is a crucial trait in its competition with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici in the rhizosphere. The expression of the PCN biosynthetic gene cluster in PCL1391 is population density-dependent and is regulated by the quorum-sensing genes phzI and phzR via synthesis of the autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL). Here, we describe the identification of an additional regulatory gene of PCN biosynthesis in PCL1391. A mutation in the psrA gene (Pseudomonas sigma regulator), the gene product of which is a member of the TetR/AcrR family of transcriptional regulators, resulted in increased production of autoinducer molecules and PCN. Expression studies showed that inactivation of psrA resulted in increased expression of the phzI and phzR genes and the phz biosynthetic operon and that introduction of functional copies of psrA represses the expression of these genes, resulting in reduced production of autoinducer signal and PCN. Surprisingly, inactivation of psrA in the phzI or phzR quorum-sensing mutants, which do not produce detectable amounts of PCN and autoinducers by themselves, restored PCN biosynthesis. This phenomenon was accompanied by the appearance of compounds with autoinducer activities migrating at the positions of C4-HSL and C6-HSL on C18 reverse phase-thin-layer chromatography. These observations indicate that PsrA also represses at least one silent, yet unidentified, quorum-sensing system or autoinducer biosynthetic pathway in PCL1391. The expression of psrA declines at the onset of the stationary phase at the same moment at which quorum-sensing (-regulated) genes are activated. In addition, expression studies in a psrA- and a multicopy psrA background showed that psrA is autoregulated. Multiple copies of psrA repress its own expression. Mutation of gacS, encoding the sensor kinase member of a two-component global regulatory system significantly reduced production of autoinducers and PCN. We show a novel link between global regulation and quorum sensing via the PsrA regulator.

Pseudomonas sp. strain PCL1171 displays colony phase variation between opaque phase I and translucent phase II colonies, thereby regulating the production of secondary metabolites and exoenzymes. Complementation and sequence analysis of 26 phase II mutants and of 13 wild-type phase II sectors growing out of phase I colonies showed that in all these cases the phase II phenotype is caused by spontaneous mutations in gacA or/and gacS. Mutation of gac reduced both the length of the lag phase and the generation time. Isolation and sequencing of the gacS genes from the phase II bacteria revealed one insertion as well as several random point mutations, deletions, and DNA rearrangements. Most phase II colonies reverted with a high frequency, resulting in wild-type gacA and gacS genes and a phase I phenotype. Some phase II bacteria retained the phase II phenotype but changed genotypically as a result of (re)introduction of mutations in either gacA or gacS. The reversion of gacA or gacS to the wild type was not affected by mutation of recA and recB. We conclude that in Pseudomonas sp. strain PCL1171, mutations in gacA and gacS are the basis for phase variation from phase I to phase II colonies and that, since these mutations are efficiently removed, mutations in gac result in dynamic switches between the "wild-type" population and the subpopulations harboring spontaneous mutations in gacA and or gacS, thereby enabling both populations to be maintained.

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.

Pseudomonas chlororaphis PCL1391 produces the secondary metabolite phenazine-1-carboxamide (PCN), which is an antifungal metabolite required for biocontrol activity of the strain. Identification of conditions involved in PCN production showed that some carbon sources and all amino acids tested promote PCN levels. Decreasing the pH from 7 to 6 or decreasing the growth temperature from 21 to 16 degrees C decreased PCN production dramatically. In contrast, growth at 1% oxygen as well as low magnesium concentrations increased PCN levels. Salt stress, low concentrations of ferric iron, phosphate, sulfate, and ammonium ions reduced PCN levels. Fusaric acid, a secondary metabolite produced by the soilborne Fusarium spp. fungi, also reduced PCN levels. Different nitrogen sources greatly influenced PCN levels. Analysis of autoinducer levels at conditions of high and low PCN production demonstrated that, under all tested conditions, PCN levels correlate with autoinducer levels, indicating that the regulation of PCN production by environmental factors takes place at or before autoinducer production. Moreover, the results show that autoinducer production not only is induced by a high optical density but also can be induced by certain environmental conditions. We discuss our findings in relation to the success of biocontrol in the field.

A recently published procedure to enrich for efficient competitive root tip colonizers (I. Kuiper, G. V. Bloemberg, and B. J. J. Lugtenberg, Mol. Plant-Microbe Interact. 14:1197-1205) after bacterization of seeds was applied to isolate efficient competitive root tip colonizers for both the dicotyledenous plant tomato and the monocotyledenous plant grass from a random Tn5luxAB mutant bank of the good root colonizer Pseudomonas fluorescens WCS365. Unexpectedly, the best-colonizing mutant, strain PCL1286, showed a strongly enhanced competitive root-tip-colonizing phenotype. Sequence analyses of the Tn5luxAB flanking regions showed that the transposon had inserted in a mutY homolog. This gene is involved in the repair of A. G mismatches caused by spontaneous oxidation of guanine. We hypothesized that, since the mutant is defective in repairing its mismatches, its cells harbor an increased number of mutations and therefore can adapt faster to the environment of the root system. To test this hypothesis, we constructed another mutY mutant and analyzed its competitive root tip colonization behavior prior to and after enrichment. As a control, a nonmutated wild type was subjected to the enrichment procedure. The results of these analyses showed (i) that the enrichment procedure did not alter the colonization ability of the wild type, (ii) that the new mutY mutant was strongly impaired in its colonization ability, but (iii) that after three enrichment cycles it colonized significantly better than its wild type. Therefore it is concluded that both the mutY mutation and the selection procedure are required to obtain an enhanced root-tip-colonizing mutant.

Worldwide, contamination of soil and ground water is a severe problem. The negative effects of pollutants on the environment and on human health are diverse and depend on the nature of the pollution. The search for alternative methods for excavation and incineration to clean polluted sites resulted in the application of bioremediation techniques. In this review, we describe some generally accepted bioremediation tools and subsequently focus on the combination of two approaches, phytoremediation and bioaugmentation, resulting in rhizoremediation. During rhizoremediation, exudates derived from the plant can help to stimulate the survival and action of bacteria, which subsequently results in a more efficient degradation of pollutants. The root system of plants can help to spread bacteria through soil and help to penetrate otherwise impermeable soil layers. The inoculation of pollutant-degrading bacteria on plant seed can be an important additive to improve the efficiency of phytoremediation or bioaugmentation.

Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High-performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface-tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface-active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N-terminus of a 12-amino-acid peptide moiety, in which the C-terminal carboxylic acid group forms an ester with the hydroxyl side-chain of Ser9. The difference between the two structures is located in the second amino acid from the C-terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio-synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild-type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.

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.

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.

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 present status of research on the molecular basis of microbe-plant interactions is discussed. Principles and mechanisms which play a role in the interactions of microbial pathogens, biofertilizers, phytostimulators, rhizoremediators and biocontrol agents with the plants are treated. Special emphasis is given to colonization, phase variation, two-component systems, quorum sensing, complex regulation of the syntheses of extracellular enzymes and secondary metabolites, Type 4 pili and Type III and Type IV secretion systems.

Previously, we have described the selection of a plant-bacterium pair that is efficient in rhizoremediating naphthalene pollution in microcosm studies. After repeated selection for efficient root tip colonization upon inoculation of seeds of grass cv. Barmultra and for stable and efficient growth on naphthalene, Pseudomonas putida PCL1444 was selected as the most efficient colonizer of Barmultra roots. Here, we report the analysis of Barmultra root exudate composition and our subsequent tests of the growth rate of the bacterium and of the expression of the naphthalene degradation genes on individual exudate components. High performance liquid chromatography analysis of the organic acid and sugar root-exudate components revealed that glucose and fructose are the most abundant sugars, whereas succinic acid and citric acid are the most abundant organic acids. Tn5luxAB mutants of PCL1444 impaired in naphthalene degradation appeared to be impaired in genes homologous to genes of the upper naphthalene degradation pathway present in various Pseudomonas strains and to genes of the lower pathway genes for naphthalene degradation in P. stutzeri. Highest expression for both pathways involved in naphthalene degradation during growth in minimal medium with the carbon source to be tested was observed at the start of the logarithmic phase. Naphthalene did not induce the upper pathway, but a different pattern of expression was observed in the lower pathway reporter, probably due to the conversion of naphthalene to salicylic acid. Salicylic acid, which is described as an intermediate of the naphthalene degradation pathway in many Pseudomonas strains, did induce both pathways, resulting in an up to sixfold higher expression level at the start of the logarithmic phase. When expression levels during growth on the different carbon sources present in root exudate were compared, highest expression was observed on the two major root exudate components, glucose and succinic acid. These results show an excellent correlation between successful naphthalene rhizoremediation by the Barmultra-P. putida PCL1444 pair and both efficient utilization of the major exudate components for growth and high transcription of the naphthalene catabolic genes on the major exudate components. Therefore, we hypothesize that efficient root colonizing and naphthalene degradation is the result of the applied colonization enrichment procedure.

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 fungus Fusarium oxysporum f. sp. radicis-lycopersici is the causal agent of tomato foot and root rot disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Transformation of F oxysporum f. sp. radicis-lycopersici was very efficient and gfp expression was stable for at least nine subcultures. Microscopic analysis of the transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the chlamydospores and conidia. To our knowledge, this is the first report in which this is shown. The transformation did not affect the pathogenicity. Using confocal laser scanning microscopy, colonization, infection, and disease development on tomato roots were visualized in detail and several new aspects of these processes were observed, such as (i) the complete colonization pattern of the tomato root system; (ii) the very first steps of contact between the fungus and the host, which takes place at the root hair zone by mingling and by the attachment of hyphae to the root hairs; (iii) the preferential colonization sites on the root surface, which are the grooves along the junctions of the epidermal cells; and (iv) the absence of specific infection sites, such as sites of emergence of secondary roots, root tips, or wounded tissue, and the absence of specific infection structures, such as appressoria. The results of this work prove that the use of GFP as a marker for F. oxysporum f. sp. radicis-lycopersici is a convenient, fast, and effective approach for studying plant-fungus interactions.

We developed a novel procedure for the selection of a microbe-plant pair for the stable and efficient degradation of naphthalene. Based on the rationale that root exudate is the best nutrient source available in soil, the grass (Lolium multiflorum) cultivar Barmultra was selected because of its abilities to produce a highly branched root system, root deeply, and carry a high population of Pseudomonas spp. bacteria on its roots. Starting with a mixture of total rhizobacteria from grass-like vegetation collected from a heavily polluted site and selecting for stable naphthalene degradation as well as for efficient root colonization, Pseudomonas putida strain PCL1444 was isolated. The strain's ability to degrade naphthalene was shown to be stable in the rhizosphere. Moreover, it had superior root-colonizing properties because, after the inoculation of grass seedlings, it appeared to colonize the root tip up to 100-fold better than the efficient root colonizer Pseudomonas fluorescens WCS365. Strain PCL1444 uses root exudate as the dominant nutrient source because the presence of grass seedlings in soil results in up to a 10-fold increase of PCL1444 cells. Moreover, the root colonized by strain PCL1444 was able to penetrate through an agar layer, resulting in the degradation of naphthalene underneath this layer. In addition, the inoculation of grass seeds or seedlings with PCL1444 protected them against naphthalene phytotoxicity. Finally, this plant-microbe combination appeared able to degrade naphthalene from soil that was heavily polluted with a complex mixture of polycyclic aromatic hydrocarbons. To our knowledge, this is the first time that a naturally occurring bacterium has been selected for the combination of the abilities to degrade a pollutant and colonize plant roots. We suggest that the principle described here, to select a bacterium which combines efficient root colonization with a beneficial activity, also can be used to improve the selection of other more efficient plant-bacterium pairs for beneficial purposes such as biocontrol, biofertilization, and phytostimulation.

Sequence analysis of the chromosomal Tn5lacZ flanking regions of the Pseudomonas fluorescens WCS365 competitive root colonization mutant PCL1206 showed that the Tn5lacZ is inserted between genes homologous to bioA and potF. The latter gene is the first gene of the potF1F2GHI operon, which codes for a putrescine transport system in Escherichia coli. The position of the Tn5lacZ suggests an effect on the expression of the pot operon. A mutation in the potF1 gene as constructed in PCL1270, however, had no effect on competitive root colonization. The rate of uptake of [1,4-14C]putrescine by cells of mutant PCL1206 appeared to be increased, whereas cells of strain PCL1270 were strongly impaired in the uptake of putrescine. Dansylation of tomato root exudate and subsequent thin-layer chromatography showed the presence of a component with the same Rf value as dansyl-putrescine, which was identified as dansyl-putrescine by mass spectrometric analyses. Other polyamines such as spermine and spermidine were not detected in the root exudate. Growth of mutant strains, either alone or in competition with the wild type, was tested in media containing putrescine, spermine, or spermidine as the sole nitrogen source. The results show that mutant PCL1206 is strongly impaired in growth on putrescine and slightly impaired on spermine and spermidine. The presence of the polyamines had a similar effect on the growth rate of strain PCL1270 in the presence of putrescine but a less severe effect in the presence of spermine and spermidine. We conclude that an increased rate of putrescine uptake has a bacteriostatic effect on Pseudomonas spp. cells. We have shown that putrescine is an important tomato root exudate component and that root-colonizing pseudomonads must carefully regulate their rate of uptake because increased uptake causes a decreased growth rate and, therefore, a decreased competitive colonization ability.

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-L-homoserine lactone (C6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.

Plant-growth-promoting rhizobacteria (PGPRs) are used as inoculants for biofertilization, phytostimulation and biocontrol. The interactions of PGPRs with their biotic environment, for example with plants and microorganisms, are often complex. Substantial advances in elucidating the genetic basis of the beneficial effects of PGPRs on plants have been made, some from whole-genome sequencing projects. This progress will lead to a more efficient use of these strains and possibly to their improvement by genetic modification.

The highly conserved nod box sequence in the promoters of the inducible nodulation genes of rhizobia is required for transcription activation together with NodD, a LysR-type transcriptional regulator, and a flavonoid as a coinducer. DNA fragments containing nod box sequences form two binding complexes when crude preparations of Rhizobium leguminosarum bv. viciae are used: a NodD-dependent and an additional, NodD-independent complex. The role of individual nucleotides in the conserved nod box sequence in complex formation and in nodulation gene expression was investigated by introducing 13 individual base-pair substitutions in the nodF nod box of R. leguminosarum bv. viciae and studying their effect on promoter activity and protein-DNA complex formation. Two mutants showed decreased NodD binding and decreased promoter activity. Five mutants showed a NodD-dependent complex as with the wild-type nodF nod box, whereas their promoter activity was severely reduced after induction. This result is in agreement with earlier observations that NodD DNA binding also occurs in the absence of inducer. Four mutants were impaired in the formation of the NodD-independent retardation complex. Three of them showed no alterations in promoter activity, meaning that no specific role for the protein forming the NodD-independent complex could be established. The two mutants in the highly conserved LysR motif of the nod box were unable to direct coinducer-dependent promoter activity but, unexpectedly, their retardation patterns were not altered. The remaining two mutants showed constitutive promoter activity. The results are discussed in terms of the relevance of conserved nucleotides and motifs identified in the nod box.

Rhizosphere colonization is one of the first steps in the pathogenesis of soilborne microorganisms. It can also be crucial for the action of microbial inoculants used as biofertilizers, biopesticides, phytostimulators, and bioremediators. Pseudomonas, one of the best root colonizers, is therefore used as a model root colonizer. This review focuses on (a) the temporal-spatial description of root-colonizing bacteria as visualized by confocal laser scanning microscopal analysis of autofluorescent microorganisms, and (b) bacterial genes and traits involved in root colonization. The results show a strong parallel between traits used for the colonization of roots and of animal tissues, indicating the general importance of such a study. Finally, we identify several noteworthy areas for future research.

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.

To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.

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.

The role of tomato seed and root exudate sugars as nutrients for Pseudomonas biocontrol bacteria was studied. To this end, the major exudate sugars of tomato seeds, seedlings and roots were identified and quantified using high-performance liquid chromatographic (HPLC) analysis. Glucose, fructose and maltose were present in all studied growth stages of the plant, but the ratios of these sugars were strongly dependent on the developmental stage. In order to study the putative role of exudate sugar utilization in rhizosphere colonization, two approaches were adopted. First, after co-inoculation on germinated tomato seeds, the root-colonizing ability of the efficient root-colonizing P. fluorescens strain WCS365 in a gnotobiotic quartz sand-plant nutrient solution system was compared with that of other Pseudomonas biocontrol strains. No correlation was observed between the colonizing ability of a strain and its ability to use the major exudate sugars as the only carbon and energy source. Secondly, a Tn5lacZ mutant of P. fluorescens strain WCS365, strain PCL1083, was isolated, which is impaired in its ability to grow on simple sugars, including those found in exudate. The mutation appeared to reside in zwf, which encodes glucose-6-phosphate dehydrogenase. The mutant grows as well as the parental strain on other media, including tomato root exudate. After inoculation of germinated sterile tomato seeds, the mutant cells reached the same population levels at the root tip as the wild-type strain, both alone and in competition, indicating that the ability to use exudate sugars does not play a major role in tomato root colonization, despite the fact that sugars have often been reported to represent the major exudate carbon source. This conclusion is supported by the observation that the growth of mutant PCL1083 in vitro is inhibited by glucose, a major exudate sugar, at a concentration of 0.001%, which indicates that the glucose concentration in the tomato rhizosphere is very low.

Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants. Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M. loti nodC gene. The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway. We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides. Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus. The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue. The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase. Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc. Consequently, chain elongation in chitin oligosaccharide synthesis by M. loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions.

We have analyzed the nucleotide sequences of the nodX genes from two strains of Rhizobium leguminosarum bv. viciae able to nodulate Afghan peas (strains A1 and Himalaya) and from two strains of R. leguminosarum bv. trifolii (ANU843 and CSF). The nodX genes of strains A1 and ANU843 were shown to be functional for the induction of nodules on Afghan peas. To analyze the cause of phenotypic differences of strain A1 and strain TOM we have studied the composition of the lipochitin-oligosaccharides (LCOs) produced by strain A1 after induction by the flavonoid naringenin or various pea root exudates. The structural analysis of the LCOs by mass spectrometry revealed that strain A1 synthesizes a family of at least 23 different LCOs. The use of exudates instead of naringenin resulted only in quantitative differences in the ratios of various LCOs produced.

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.

A colonization mutant of the efficient root-colonizing biocontrol strain Pseudomonas fluorescens WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation. The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions. A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to lppL, lysA, dapF, orf235/233, xerC/sss, and the largely incomplete orf238. The transposon insertion in PCL1233 appeared to be present in the orf235/233 homologue, designated orf240. Introduction of a mutation in the xerC/sss homologue revealed that the xerC/sss gene homologue rather than orf240 is crucial for colonization. xerC in Escherichia coli and sss in Pseudomonas aeruginosa encode proteins that belong to the lambda integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. The function of the xerC/sss homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats. Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere. Thus we show the importance of phase variation in microbe-plant interactions.

The nodulation protein NodF of Rhizobium shows 25% identity to acyl carrier protein (ACP) from Escherichia coli (encoded by the gene acpP). However, NodF cannot be functionally replaced by AcpP. We have investigated whether NodF is a substrate for various E. coli enzymes which are involved in the synthesis of fatty acids. NodF is a substrate for the addition of the 4'-phosphopantetheine prosthetic group by holo-ACP synthase. The Km value for NodF is 61 microM, as compared to 2 microM for AcpP. The resulting holo-NodF serves as a substrate for coupling of malonate by malonyl-CoA:ACP transacylase (MCAT) and for coupling of palmitic acid by acyl-ACP synthetase. NodF is not a substrate for beta-keto-acyl ACP synthase III (KASIII), which catalyses the initial condensation reaction in fatty acid biosynthesis. A chimeric gene was constructed comprising part of the E. coli acpP gene and part of the nodF gene. Circular dichroism studies of the chimeric AcpP-NodF (residues 1-33 of AcpP fused to amino acids 43-93 of NodF) protein encoded by this gene indicate a similar folding pattern to that of the parental proteins. Enzymatic analysis shows that AcpP-NodF is a substrate for the enzymes holo-ACP synthase, MCAT and acyl-ACP synthetase. Biological complementation studies show that the chimeric AcpP-NodF gene is able functionally to replace NodF in the root nodulation process in Vicia sativa. We therefore conclude that NodF is a specialized acyl carrier protein whose specific features are encoded in the C-terminal region of the protein. The ability to exchange domains between such distantly related proteins without affecting conformation opens exciting possibilities for further mapping of the functional domains of acyl carrier proteins (i. e., their recognition sites for many enzymes).

We describe the characterization of a novel Tn5lacZ colonization mutant of the efficiently colonizing Pseudomonas fluorescens strain WCS365, mutant strain PCL1210, which is at least 300- to 1,000-fold impaired in colonization of the potato root tip after co-inoculation of potato stem cuttings with a 1:1 mixture of mutant and parental cells. Similarly, the mutant is also impaired in colonization of tomato, wheat, and radish, indicating that the gene involved plays a role in the ability of P. fluorescens WCS365 to colonize a wide range of plant species. A 3.1-kb DNA fragment was found to be able to complement the observed mutation. The nucleotide sequence of the region around the Tn5lacZ insertion showed three open reading frames (ORFs). The transcriptional start site was determined. The operon is preceded by an integration host factor (IHF) binding site consensus sequence whereas no clear -10 and -35 sequences are present. The deduced amino acid sequences of the first two genes of the operon, designated as colR and colS, show strong similarity with known members of two-component regulatory systems. ColR has homology with the response regulators of the OmpR-PhoB subclass whereas ColS, the product of the gene in which the mutation resides, shows similarity to the sensor kinase members of these two-component systems. Hydrophobicity plots show that this hypothetical sensor kinase has two transmembrane domains, as is also known for other sensor kinases. The product of the third ORF, Orf222, shows no homology with known proteins. Only part of the orf222 gene is present in the colonization-complementing, 3.1-kb region, and it therefore does not play a role in complementation. No experimental evidence for a role of the ColR/ColS two-component system in the suspected colonization traits chemotaxis and transport of exudate compounds could be obtained. The function of this novel two-component system therefore remains to be elucidated. We conclude that colonization is an active process in which an environmental stimulus, through this two-component system, activates a so far unknown trait that is crucial for colonization.

In Rhizobium leguminosarum, the nodABC and nodFEL operons are involved in the production of lipo-chitin oligosaccharide signals that mediate host specificity. A nodFE-determined, highly unsaturated C18:4 fatty acid (trans-2, trans-4, trans-6, cis-11-octadecatetraenoic acid) is essential for the ability of the signals to induce nodule meristems and pre-infection thread structures on the host plant Vicia sativa. Of the nod genes, induction of only nodFE is sufficient to modify fatty acid biosynthesis to yield trans-2, trans-4, trans-6, cis-11-octadecatetraenoic acid, with an absorbance maximum of 303 nm. This unusual C18:4 fatty acid is not only found in the lipo-chitin oligosaccharides but is also associated with the phospholipids (O. Geiger, J. E. Thomas-Oates, J. Glushka, H. P. Spaink, and B. J. J. Lugtenberg, 1994, J. Biol. Chem. 269:11090-11097). Here we report that the phospholipids can contain other nodFE-derived fatty acids, a C18:3 trans-4, trans-6, cis-11-octadecatrienoic acid that has a characteristic absorption maximum at 225 nm, and a C18:2 octadecadienoic acid. Neither this C18:3 nor this C18:2 fatty acid has to date been observed attached to lipo-chitin oligosaccharides, suggesting that an as yet unknown acyl transferase (presumably NodA), responsible for the transfer of the fatty acyl chain to the glycan backbone of the lipo-chitin oligosaccharides, does not transfer all fatty acids synthesized by the action of NodFE to the lipo-chitin oligosaccharides. Rather, it must have a preference for alpha-beta unsaturated fatty acids during transfer.

Rhizobia, bacterial symbionts of leguminous plants, produce lipo-chitin oligosaccharide (LCO) signal molecules that can induce nodule organogenesis in the cortex of legume roots in a host-specific way. The multi-unsaturated fatty acyl and the O-acetyl moieties of the LCOs of Rhizobium leguminosarum biovar viciae were shown to be essential for obtaining root nodule induction in Vicia sativa plants. We have used ballistic microtargeting as a novel approach to deliver derivatives of the nodulation signal molecules inside the roots of V. sativa. This method offers the unique ability to introduce soluble compounds into the tissue at a small area. The mitogenic effect of microtargeting of chitin oligosaccharides, including an analysis of the influence of the chain length and modifications, was tested in a qualitative assay. The role of a cell division factor from the root stele, uridine, has also been examined in these experiments. The results show that O-acetylated chitin oligosaccharides can induce root cortical cell divisions when delivered by microtargeting. For this effect it is essential that uridine is co-targeted. The foci of cortical cell division were often similar to root nodule primordia. Anatomical examination also revealed chimeric structures that share characteristics with lateral root and nodule primordia. Our data favour a model in which the oligosaccharide moiety of the rhizobial LCO induces cortical cell division and the fatty acyl moiety plays a role in transport of the LCO into the plant tissue.

The development of sensitive methods for observing individual bacterial cells in a population in experimental models and natural environments, such as in biofilms or on plant roots, is of great importance for studying these systems. We report the construction of plasmids which constitutively express a bright mutant of the green fluorescent protein of the jellyfish Aequorea victoria and are stably maintained in Pseudomonas spp. We demonstrate the utility of these plasmids to detect individual cells in two experimental laboratory systems: (i) the examination of a mixed bacterial population of Pseudomonas aeruginosa and Burkholderia cepacia attached to an abiotic surface and (ii) the association of Pseudomonas fluorescens WCS365 with tomato seedling roots. We also show that two plasmids, pSMC2 and pGB5, are particularly useful, because they are stable in the absence of antibiotic selection, they place an undetectable metabolic burden on cells that carry the plasmids, and cells carrying these constructs continue to fluoresce even after 7 days in culture without the addition of fresh nutrients. The construction of improved Escherichia coli-Pseudomonas shuttle vectors which carry multiple drug resistance markers also is described.

The bioluminescently marked Pseudomonas fluorescens strain 5RL, has been used previously to follow colonisation of soy bean roots (De Weger et al. [1991] Appl. Environ. Microbiol. 57:36-41). In the present paper the method has been further developed and optimized for wheat roots and it is used to get a quick overview of the colonisation patterns of many different root systems at the same time. Colonisation was followed on wheat plants grown in our gnotobiotic sand system (Simons et al., 1996. Mol Plant Microbe Interact 9: 600-607) and the following results were obtained. (i) A spatio-temporal analysis of the colonisation of wheat roots showed that 4 days after planting the highest bacterial activity was observed at the upper part of the root. After 6 days the high bacterial activity at the upper part was further increased, whereas spot-like activities were observed on the lower root parts, possibly due to micro-colonies. (ii) Bacterial mutations causing lack of motility or auxotrophy for amino acids resulted in impaired colonisation of the lower root parts, indicating that motility and prototrophy for the involved amino acid(s) are important factors for wheat root colonisation by strain 5RL. (iii) Coinoculation of strain 5RL with other wild type Pseudomonas strains on the root influenced the colonisation pattern observed for strain 5RL. Colonisation was not visually affected when the competing strain was a poor root coloniser, but was severely reduced when the competing strain was a good root coloniser. The results show that the spatio-temporal colonisation of wheat root by P. fluorescens strain 5RL and derivatives is similar to that of strain WCS365 on tomato. The advantage of the use of lux-marked strains is that the results are obtained much quicker than when conventional methods are used and that the result is supplied as an image of the colonisation pattern of many different roots.

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.

NodA controls transfer of a fatty acid in the biosynthesis of lipochitin oligosaccharides by rhizobia. In an in vitro assay, we used de-N-acetylated chitin oligosaccharides substituted with an O-acetyl moiety as acyl acceptor substrates. We show that acyl-acyl carrier protein is used as a donor in NodA-directed fatty acid transfer.

The nodZ gene, which is present in various soil bacteria such as Bradyrhizobium japonicum, Azorhizobium caulinodans, and Rhizobium loti, is involved in the addition of a fucosyl residue to the reducing N-acetylglucosamine residue of lipochitin oligosaccharide (LCO) signal molecules. Using an Escherichia coli strain that produces large quantities of the NodZ protein of B. japonicum, we have purified the NodZ protein to homogeneity. The purified NodZ protein appears to be active in an in vitro transfucosylation assay in which GDP-beta-fucose and LCOs or chitin oligosaccharides are used as substrates. The products of the in vitro reaction using chitin oligosaccharides as substrate were studied by using mass spectrometry, linkage analysis, and composition analysis. The data show that one fucose residue is added to C6 of the reducing-terminal N-acetylglucosamine residue. The substrate specificity of NodZ protein was analyzed in further detail, using radiolabeled GDP-beta-fucose as the donor. The results show that chitin oligosaccharides are much better substrates than LCOs, suggesting that in Rhizobium NodZ fucosylates chitin oligosaccharides prior to their acylation. The free glycan core pentasaccharides of N-linked glycoproteins are also substrates for NodZ. Therefore, the NodZ enzyme seems to have an activity equivalent to that of the enzyme involved in the addition of the C6-linked fucosyl substituent in the glycan core of N-linked glycoproteins in eukaryotes. Oligosaccharides that contain only one N-acetylglucosamine at the reducing terminus are also substrates for NodZ, although in this case very high concentrations of such oligosaccharides are needed. An example is the leukocyte antigen Lewis-X, which can be converted by NodZ to a novel fucosylated derivative that could be used for binding studies with E-selectin.

Synthesis of chitin oligosaccharides by NodC is the first committed step in the biosynthesis of rhizobial lipochitin oligosaccharides (LCOs). The distribution of oligosaccharide chain lengths in LCOs differs between various Rhizobium species. We expressed the cloned nodC genes of Rhizobium meliloti, R. leguminosarum bv. viciae, and R. loti in Escherichia coli. The in vivo activities of the various NodC proteins differed with respect to the length of the major chitin oligosaccharide produced. The clearest difference was observed between strains with R. meliloti and R. loti NodC, producing chitintetraose and chitinpentaose, respectively. In vitro experiments, using UDP-[14C]GlcNAc as a precursor, show that this difference reflects intrinsic properties of these NodC proteins and that it is not influenced by the UDP-GlcNAc concentration. Analysis of oligosaccharide chain lengths in LCOs produced by a R. leguminosarum bv. viciae nodC mutant, expressing the three cloned nodC genes mentioned above, shows that the difference in oligosaccharide chain length in LCOs of R. meliloti and R. leguminosarum bv. viciae is due only to nodC. The exclusive production of LCOs which contain a chitinpentaose backbone by R. loti strains is not due to NodC but to end product selection by Nod proteins involved in further modification of the chitin oligosaccharide. These results indicate that nodC contributes to the host specificity of R. meliloti, a conclusion consistent with the results of several studies which have shown that the lengths of the oligosaccharide backbones of LCOs can strongly influence their activities on host plants.

The lipo-chitin oligosaccharides (LCO) or nodulation factors synthesized by Rhizobium leguminosarum bv. trifolii were analyzed using positive mode fast atom bombardment and positive and negative mode electrospray ionization mass spectrometry. From their mass spectrometric behavior it is clearly possible to distinguish between the [M + Na]+ pseudomolecular ion of the nodE-independent molecule IV(C18:1,Ac) and the [M + H]+ pseudomolecular ion of the nodE-dependent molecule IV(C20:4,Ac), although they both have the same mass value. The results unequivocally show that the bacterial strain investigated produces nodE-dependent LCOs with highly unsaturated fatty acyl moieties. We further demonstrate that the interpretation of the mass spectrometric data by Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Orgambide, G. G., Bradford, J. J., Smith, D. K., Hollingsworth, R. I., and Dazzo, F. B. (1995) J. Biol. Chem. 270, 20968) is incorrect and that their data do not contradict our hypothesis that the nodE gene determines the host specificity of R. leguminosarum bv. trifolii.