Search

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.

Transcript profiling is crucial to study biological systems, and various platforms have been implemented to survey mRNAs at the genome scale. We have assessed the performance of the CATMA microarray designed for Arabidopsis (Arabidopsis thaliana) transcriptome analysis and compared it with the Agilent and Affymetrix commercial platforms. The CATMA array consists of gene-specific sequence tags of 150 to 500 bp, the Agilent (Arabidopsis 2) array of 60mer oligonucleotides, and the Affymetrix gene chip (ATH1) of 25mer oligonucleotide sets. We have matched each probe repertoire with the Arabidopsis genome annotation (The Institute for Genomic Research release 5.0) and determined the correspondence between them. Array performance was analyzed by hybridization with labeled targets derived from eight RNA samples made of shoot total RNA spiked with a calibrated series of 14 control transcripts. CATMA arrays showed the largest dynamic range extending over three to four logs. Agilent and Affymetrix arrays displayed a narrower range, presumably because signal saturation occurred for transcripts at concentrations beyond 1,000 copies per cell. Sensitivity was comparable for all three platforms. For Affymetrix GeneChip data, the RMA software package outperformed Microarray Suite 5.0 for all investigated criteria, confirming that the information provided by the mismatch oligonucleotides has no added value. In addition, taking advantage of replicates in our dataset, we conducted a robust statistical analysis of the platform propensity to yield false positive and false negative differentially expressed genes, and all gave satisfactory results. The results establish the CATMA array as a mature alternative to the Affymetrix and Agilent platforms.

To visualize simultaneously different populations of pseu-domonads 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. Micro-colonies consisting of mixed populations were frequently observed at the base of the root system, whereas micro-colonies 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.

Four different genes of Rhizobium leguminosarum bv. tri-folii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum bv. viciae VF39 and highly homologous to a family of glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively. The three other genes were found to be closely linked on a different complementing cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an op-eron closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production. Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis. Furthermore, pssD and pssE were homologous to the 5′ and 3′ parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase. Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent.

In the biosynthesis of lipochitin oligosaccharides (LCOs) the Rhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence of nodA in the nodABCIJ operon makes genetic studies difficult to interpret. In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which the nodA, nodB and nodC genes are separately present on different plasmids. Efficient nodulation was only obtained if nodC was present on a low-copy-number vector. Our results confirm the notion that nodA of Rhizobium leguminosarum biovar viciae is essential for nodulation on Vicia. Surprisingly, replacement of R. l. by viciae nodA by that of Bradyrhizobium sp. ANU289 results in a nodulation-minus phenotype on Vicia. Further analysis revealed that the Bradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of the R. l. by, viciae nodFE-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion that nodA is a common nod gene should be revised.

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.

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.

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 bio-control 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 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-β-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-β-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.

Signal molecules which determine the host specificity of rhizobia for particular leguminous plants include flavonoids secreted by the host plant roots and the bacterial lipo-chitin oligosaccharides (LCOs). This presentation will mainly focuss on the role of the LCOs. For instance, a highly unsaturated fattyl moiety of LCOs plays a major role in the specificity of rhizobia which associate with indeterminate-nodulating plants. The genes nodF (acyl carrier protein), nodE (β-ketoacylsynthase) and nodA (acyl transferase) are involved in the production of LCOs containing such a special fatty acids. For determinate nodulating plants a fucosyl residue (located at the reducing N-acetylglucosamine residue of LCOs) plays a major role in determining specificity. The nodZ gene which is present in various rhizobia such as Bradyrhizobium japonicum, Rhizobium loti and R.etli, is a fucosyltransferase involved in the addition of this fucose (Lopez-Lara et al, 1996). The role of other determinants of host specificity, such as nodX and nolL will be discussed.

Thin-layer chromatographic analysis of extracts of D-[1-14C]glucosamine-labelled rhizobia was used to analyze the effects of nodI, nodJ, and nodT on secretion of lipochitin oligosaccharide (LCO) signal molecules. Secretion was analyzed by comparing quantities of radiolabelled LCOs present in the cellular and spent growth medium fractions. A second rapid and sensitive method was introduced to estimate the secreted LCO fractions by using D-[1-14C]glucosamine-labelled cells grown in medium supplemented with chitinase. At various times after induction of LCO synthesis, the quantity of degradation products of LCOs was compared with the amount of nondegraded LCOs. In wild-type strains of Rhizobium leguminosarum biovars viciae and trifolii the nodI and nodJ genes (but not the nodT gene) strongly enhance the secretion of LCOs during the first 5 h after the induction of LCO synthesis. In LCO-overproducing strains the enhancement of secretion was observed only during the first 3 h after induction. At times later than 5 h after induction, a significant influence of the presence of the nodI and nodJ genes on LCO secretion was detectable neither in the wild type nor in LCO-overproducing strains. By using plasmids in which the nodI and nodJ genes are cloned separately under control of a flavonoid-inducible promoter, it was shown that both genes are needed for a wild-type level of LCO secretion. Therefore, these results demonstrate that nodI and nodJ play a role in determining the efficiency of LCO secretion.

The Rhizobium nodulation genes nodABC are involved in the synthesis of lipo-chitin oligosaccharides. We have analysed the metabolites which are produced in vivo and in vitro by Rhizobium strains which express the single nodA, nodB and nodC genes or combinations of the three. In vivo radioactive labelling experiments, in which D-[1-14C]-glucosamine was used as a precursor, followed by mass spectrometric analysis of the purified radiolabelled metabolic products, showed that Rhizobium strains that only express the combination of the nodB and nodC genes do not produce lipo-chitin oligosaccharides but instead produce chitin oligomers (mainly pentamers) which are devoid of the N-acetyl group on the non-reducing terminal sugar residue (designated NodBC metabolites). Using the same procedure we have shown that when the nodL gene is expressed in addition to the nodBC genes the majority of metabolites contain an additional O-acetyl substituent on the non-reducing terminal sugar residue (designated NodBCL metabolites). The NodBC and NodBCL metabolites purified after in vivo labelling were compared with the radiolabelled metabolites produced in vitro by Rhizobium bacterial cell lysates to which UDP-N-acetyl-D-[U-14C]-glucosamine was added using thin-layer chromatography. The results show that the lysates of strains which expressed the nodBC or nodBCL genes can also produce NodBC and NodBCL metabolites. The same results were obtained when the NodB and NodC proteins were produced separately in two different strains. On the basis of these and other recent results, we propose that NodB is a chitin oligosaccharide deacetylase, NodC an N-acetylglucosaminyltransferase and, by default, NodA is involved in lipid attachment.

Bradyrhizobium japonicum produces lipo-oligosaccharide signal molecules that induce deformation of root hairs and meristematic activity on soybeans. B. japonicum USDA135 (a Type I strain) produces modified chitin pentasaccharide molecules with either a terminal N-C16:0- or N-C18:1-glucosamine with and without an O-acetyl group at C-6 and with 2-O-methylfucose linked to C-6 of the reducing N-acetylglucosamine. An additional molecule has N-C16:1-glucosamine and no O-acetyl group. All of these molecules cause root hair deformation on Vicia sativa and Glycine soja. The C18:1-containing molecules were tested and found to induce meristem formation on G. soja. USDA61 (a Type II strain) produces eight additional molecules. Five have a carbamoyl group on the terminal N-acylglucosamine. Six have chitin tetrasaccharide backbones. Three have a terminal N-acyl-N-methylglucosaminosyl residue. In four molecules, the reducing-end N-acetylglucosamine is glycosidically linked to glycerol and has a branching fucosyl, rather than a 2-O-methylfucosyl, residue. One molecule has a terminal N-acylglucosamine that has both acetyl and carbamoyl groups (one each).

All tested strains of the genera Rhizobium,Bradyrhizobium. and Azorhizobium are able to produce lipo-oligosaccharide signals upon activation of the flavonoid-inducible nod genes. R.leguminosarum was chosen as a model system to study the biochemical function of the Nod proteins in the synthesis of the lipooligosaccharides. In R.leguminosarum bv. viciae, the operons nodABC and nodFEL encode for the Nod proteins which are minimally required for synthesis of the wild type lipo-oligosaccharides and have therefore been studied in most detail. The NodE protein determines host-specificity by its involvement in the synthesis of highly unsaturated fatty acid moieties. The role of the lipo-oligosaccharides in the host plant will be emphasized. Results are presented which tempt us to believe that lipo-oligosaccharides, similar to those produced by rhizobia, are naturally occurring in the uninfected plant

In a Sym plasmid-cured strain of R.leguminosarum the presence of the NodA,B,C,F,E,L proteins is sufficient for production and secretion of the wild type lipo-oligosaccharides. Using PCR technology we have constructed plasmids of various incompatibility groups (IncW, IncP, IncQ) which contain the separated single genes, under control of a common promoter (nodA- or T7-derived) and a synthetic ribosomal binding site. Rhizobium strains harbouring various combinations of the single nod gene-containing plasmids were tested for the production of lipooligosaccharides and the results show that: (i) The absence of either of the NodA, NodB or NodC proteins prevents the production of lipo-oligosaccharide molecules, (ii) absence of NodF or NodE proteins prevents the production of lipooligosaccharides which contain a highly unsaturated (C18:4) fatty acyl moiety, (iii) absence of the NodL protein prevents the addition of the O-acetyl group on the non-reducing terminal sugar. The biochemical function of these Nod proteins is further studied by the analysis of biosynthetic intermediates of the lipooligosaccharides in strains producing the various combinations of Nod proteins. Furthermore, we have set up assays for in vitro biochemical activity of the Nod proteins at present focusing on NodA, NodB, NodC and NodL. The obtained results suggest that NodC functions as an N-acetylglucosaminyltransferase involved in the synthesis of the sugar backbone whereas NodA and NodB are involved in the de-N-acetylation and/or acylation of the non-reducing terminal Nacetylglucosamine. The NodL protein is an O-acetyl transferase which can use non-O-acetylated lipo-oligosaccharides or chitin oligomers as substrates.

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.

The region of the Rhizobium leguminosarum biovar viciae Sym plasmid pRL1JI, responsible for the production and secretion of a previously described 50-kilodalton protein (R. A. de Maagd, C. A. Wijffelman, E. Pees, and B. J. J. Lugtenberg, J. Bacteriol. 170:4424-4427, 1988), was cloned and its nucleotide sequence was determined. A new nod gene, nodO, preceded by a poorly conserved nod box, was identified and its transcriptional start site was determined. Comparison of its predicted protein product with the N-terminal amino acid sequence of the isolated secreted protein showed that nodO is the structural gene of this protein, although the nucleotide sequence predicted a protein only 30,002 daltons in size. This comparison also showed that the secreted protein is not the product of N-terminal processing of a larger precursor. A conventional N-terminal signal sequence was not detected in the NodO protein. The NodO protein has significant homology with a part (residues 720 to 920) of the hemolysin protein (HlyA) of Escherichia coli. Analysis of the transcriptional regulation of the nodO gene revealed that, in contrast with other nod promoters in this species, activity of the nodO promoter is greatly enhanced in the presence of multiple copies of the nodD gene.

Rhizobia, bacterial symbionts of leguminous plants, produce lipo-chitin oligosaccharide (LCO) signal molecules after induction of their nodulation(nod)genes by flavonoids secreted by the plant host. These LCOs have various effects on the roots of the host plants. One of these is the dedifferentiation of groups of cells located in the cortex of the root which leads to the formation of a nodule primordium. Host specificity of nodulation is mediated by the LCOs. In this paper we present an overview of the results of our recent studies on LCOs, mainly focusing on the molecular basis of theRhizobium leguminosarum -plant interaction.