Revealing the roles of y4wF and tidC genes in Rhizobium tropici CIAT 899: biosynthesis of indolic compounds and impact on symbiotic properties.

Rhizobium tropici CIAT 899 is a strain known by its ability to nodulate a broad range of legume species, to synthesize a variety of Nod factors, its tolerance of abiotic stresses, and its high capacity to fix atmospheric N2, especially in symbiosis with common bean (Phaseolus vulgaris L.). Genes putatively related to the synthesis of indole acetic acid (IAA) have been found in the symbiotic plasmid of CIAT 899, in the vicinity of the regulatory nodulation gene nodD5, and, in this study, we obtained mutants for two of these genes, y4wF and tidC (R. tropiciindole-3-pyruvic acid decarboxylase), and investigated their expression in the absence and presence of tryptophan (TRP) and apigenin (API). In general, mutations of both genes increased exopolysaccharide (EPS) synthesis and did not affect swimming or surface motility; mutations also delayed nodule formation, but increased competitiveness. We found that the indole-3-acetamide (IAM) pathway was active in CIAT 899 and not affected by the mutations, and-noteworthy-that API was required to activate the tryptamine (TAM) and the indol-3-pyruvic acid (IPyA) pathways in all strains, particularly in the mutants. High up-regulation of y4wF and tidC genes was observed in both the wild-type and the mutant strains in the presence of API. The results obtained revealed an intriguing relationship between IAA metabolism and nod-gene-inducing activity in R. tropici CIAT 899. We discuss the IAA pathways, and, based on our results, we attribute functions to the y4wF and tidC genes of R. tropici.

Hydroxylated ornithine lipids increase stress tolerance in Rhizobium tropici CIAT899.

Ornithine lipids (OLs) are widespread among Gram-negative bacteria. Their basic structure consists of a 3-hydroxy fatty acyl group attached in amide linkage to the α-amino group of ornithine and a second fatty acyl group ester-linked to the 3-hydroxy position of the first fatty acid. OLs can be hydroxylated within the secondary fatty acyl moiety and this modification has been related to increased stress tolerance. Rhizobium tropici, a nodule-forming α-proteobacterium known for its stress tolerance, forms four different OLs. Studies of the function of these OLs have been hampered due to lack of knowledge about their biosynthesis. Here we describe that OL biosynthesis increases under acid stress and that OLs are enriched in the outer membrane. Using a functional expression screen, the OL hydroxylase OlsE was identified, which in combination with the OL hydroxylase OlsC is responsible for the synthesis of modified OLs in R. tropici. Unlike described OL hydroxylations, the OlsE-catalysed hydroxylation occurs within the ornithine moiety. Mutants deficient in OlsE or OlsC and double mutants deficient in OlsC/OlsE were characterized. R. tropici mutants deficient in OlsC-mediated OL hydroxylation are more susceptible to acid and temperature stress. All three mutants lacking OL hydroxylases are affected during symbiosis.

Degradation of trehalose by rhizobia and characteristics of a trehalose-degrading enzyme isolated from Rhizobium species NGR234.

AIMS: This study was designed to examine the breakdown of trehalose by rhizobia and to characterize the trehalose-degrading enzyme isolated from Rhizobium sp. NGR234. METHODS AND RESULTS: Rhizobium sp. NGR234, Rhizobium fredii USDA257, R. phaseoli RCR3622, R. tropici CIAT899 and R. etli CE3 showed good growth in the presence of carbohydrate. Validamycin A did not prevent the growth of NGR234 on trehalose. The expression of a trehalose-degrading enzyme by NGR234 was intracellular and inducible by trehalose. The isolated enzyme digested other disaccharides, p-nitrophenyl-alpha-d-glucopyranoside and the substrate. The enzyme showed optimum activities at pH 7.0 and 30 degrees C. Its pI was 4.75 and the V(max) of the enzyme occurred at 35.7 micromol s(-1) mg(-1) protein with the K(m) of 23 mmol when trehalose was hydrolysed. CONCLUSIONS: An enzyme capable of breaking down trehalose was produced. Some of the properties of the trehalose-degrading enzyme are similar to those isolated from other organisms but, this enzyme was validamycin resistant. These rhizobia like other trehalose-degrading microbes use trehalose by enzymatic catabolic action. SIGNIFICANCE AND IMPACT OF THE STUDY: Trehalose which accumulates during legume-rhizobia symbiosis is toxic to plants. Detoxification by trehalose-degrading enzymes is important for the progress of symbiosis.