Ellen Luyten

Indole-3-acetic acid-regulated genes in Rhizobium etli CNPAF512

In the rhizosphere and their interaction with plants rhizobia encounter many different plant compounds, including phytohormones like auxins. Moreover, some rhizobial strains are capable of producing the auxin, indole-3-acetic acid (IAA). However, the role of IAA for the bacterial partner in the legume-Rhizobium symbiosis is not known. To identify the effect of IAA on rhizobial gene expression, a transposon (mTn5gusA-oriV) mutant library of Rhizobium etli, enriched for mutants that show differential gene expression under microaerobiosis and/or addition of nodule extracts as compared with control conditions, was screened for altered gene expression upon IAA addition. Four genes were found to be regulated by IAA. These genes appear to be involved in plant signal processing, motility or attachment to plant roots, clearly demonstrating a distinct role for IAA in legume-Rhizobium interactions.

Mutations in lipopolysaccharide biosynthetic genes impair maize rhizosphere and root colonization of Rhizobium tropici CIAT899

Three transposon mutants of Rhizobium tropici CIAT899 affected in lipopolysaccharide (LPS) biosynthesis were characterized and their maize rhizosphere and endophytic root colonization abilities were evaluated. The disrupted genes coded for the following putative products: the ATPase component of an O antigen ABC-2 type transporter (wzt), a nucleotide-sugar dehydratase (lpsbeta2) and a bifunctional enzyme producing GDP-mannose (noeJ). Electrophoretic analysis of affinity purified LPS showed that all mutants lacked the smooth LPS bands indicating an O antigen minus phenotype. In the noeJ mutant, the rough LPS band migrated faster than the parental band, suggesting a truncated LPS core. When inoculated individually, the wzt and noeJ mutants colonize the rhizosphere and root to a lower extent than the parental strain while no differences were observed between the lpsbeta2 mutant and the parental strain. All mutants were impaired in competitive rhizosphere and root colonization. Pleiotropic effects of the mutations on known colonization traits such as motility and growth rate were observed, but they were not sufficient to explain the colonization behaviours. It was found that the LPS mutants were sensitive to the maize antimicrobial 6-methoxy-2-benzoxazolinone (MBOA). Only the combined effects of altered growth rate and susceptibility to maize antimicrobials could account for all the observed colonization phenotypes. The results suggest an involvement of the LPS in protecting R. tropici against maize defence response during rhizosphere and root colonization.

Survey of genes identified in Sinorhizobium meliloti spp., necessary for the development of an efficient symbiosis.

The symbiosis between the soil bacteria Rhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium or Bradyrhizobium and leguminous plants is characterised by a specific multistep signal exchange. Only when a compatible rhizobial strain encounters its leguminous host, nodules will be formed on the roots of the host. During infection of this nodule, the microsymbiont evolves into a bacteroid form which, when provided with plant-derived carbon sources, is able to convert atmospheric nitrogen to ammonia that subsequently is supplied to the plant. The developmental programme underlying nodule organogenesis and functioning has been studied intensively for several decades. In this review, several observed plant phenotypes resulting from an ineffective symbiosis between plants and mutant rhizobial strains are represented. Besides the influence of the bacterial nodulation, nitrogen fixation and surface polysaccharide genes on symbiosis, the role of other genes important for the formation of effective nitrogen fixing nodules will be explained.

Rhizobium sp. BR816 produces a complex mixture of known and novel lipochitooligosaccharide molecules.

Rhizobial lipochitooligosaccharide (LCO) signal molecules induce various plant responses, leading to nodule development. We report here the LCO structures of the broadhost range strain Rhizobium sp. BR816. The LCOs produced are all pentamers, carrying common C18:1 or C18:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing/terminal residue. A second acetyl group can be present on the penultimate N-acetylglucosamine from the nonreducing terminus. Two novel characteristics were observed: the reducing/terminal residue can be a glucosaminitol (open structure) and the degree of acetylation of this glucosaminitol or of the reducing residue can vary.

Analysis of a Symbiosis-Specific Cytochrome P450 Homolog in Rhizobium sp. BR816

Sequence analysis of the DNA region upstream of nodO in Rhizobium sp. BR816 revealed an open reading frame in which the deduced amino acid sequence shows homology with cytochrome P450. Because the BR816 P450 homolog shows 73% amino acid similarity with CYP127A1(Y4vG), which is identified on the symbiotic plasmid of Rhizobium sp. NGR234, it is named CYP127A2. Transcriptional analysis of CYP127A2 revealed high expression in bacteroids, whereas no or hardly any expression was observed under free-living conditions. Low-level, free-living expression, however was noticed when cells were grown microoxically at acid pH levels. A number of possible substrates that may induce P450 gene expression were analyzed, but only the addition of short-chain alcohols to cultures slightly increased CYP127A2 expression. High levels of CYP127A2 expression observed in bacteroids of a nifH mutant strain, which formed non-fixing nodules on bean, indicated that the genuine substrate for CYP127A2 is not a metabolite resulting from N2-fixation. Nevertheless, expression analysis pointed to a NifA- and sigma54-dependent transcription.

Characterization of a GroESL Homologue in Rhizobium sp. BR816: A Possible Role in the Host Range Extension of Various Rhizobium Strains to Leucaena leucocephala

In our search for different determinants responsible for host specificity, we are analysing the broad host range strain Rhizobium sp. BR816 isolated from the nodules of a tropical tree, Leucaena lencocephala. pBRF2, a 24 kb clone from the megaplasmids cosmid bank of R. sp. BR816 confers host range extension of the narrow hostrange bean symbionts Rhizobium etli CE3 and CNPAF512 to Leucaena leucocephala. Deletion and sequence analysis of this clone revealed open reading frames of which the putative products show homology with the NodO protein of R. leguminosamm bv. viciae and the GroESL in Rhizobium meliloti. Similar to the role of certain chaperonins in R. meliloti and Bradyrhizobium japonicum (Fischer et al 1993, Ogawa, Long 1996) the GroESL homologue in R. sp. BR816 appears to have a function in the symbiotic nitrogen fixation. While nodO alone is sufficient for the Nod+ phenotype of R. etli on Leucaena, the full length pBRF2 clone containing groESL is required for the Fix+ phenotype on Leucaena (van Rhijn et al 1996). A similar hostrange extension has been demonstrated for Azorhizobium caulinodans ORS571. This strain normally nodulates L. leucocephala with a very low efficiency and the resulting nodules do not fix nitrogen. A nodS mutant of ORS571 does not show any nodulation on Leucaena (Waelkens et al 1995). Interestingly, introduction of the full length pBRF2 clone into this mutant strain resulted in the formation of nitrogen fixing nodules on L. leucocephala while the mutant containing nodO alone formed merely non-fixing nodules.To further investigate the significance of chaperonins in R. sp. BR816, we screened the overall genome of R. sp. BR816 for the presence of groEL homologous sequences.