Characterisation of Phaseolus symbionts isolated from Mediterranean soils and analysis of genetic factors related to pH tolerance

The ultimate objective of PhIMED, in which two European (Germany, Italy) an d two Mediterranean (Morocco, Egypt) countries collaborate, is to improve t he cultivation of French bean (Phaseolus vulgaris) under and and semi-arid conditions by analysing and enhancing stress tolerance of the nitrogen fixi ng rhizobial microsymbionts. Rhizobial strains nodulating P. vulgaris (RP s trains) isolated from areas in Morocco frequently subjected to drought were analysed for their salt and pH tolerance and their phylogenetic. relations hip. Strain RP163, exhibiting high nodulation efficiency and a broad pH tol erance was mutagenised by Tn5 and mutants unable to grow on extreme pH medi a were isolated. Some of the mutants affected in low pH tolerance were foun d to be mutated in genes related to cobalmin biosynthesis. and in succinate dehydrogenase (sdhA). In a parallel approach, promoters and genes inducibl e under extreme pH values were identified in Rhizobium leguminosarum bv. vi ciae VF39, among them gabT, which encodes the GABA transaminase and which i s induced under acidic conditions. The same gene is present and similarly r egulated in RP163. The actSR gene region was cloned from VF39, sequenced an d mutants generated in this region were found to be impaired in growth at l ow pH, but also under neutral conditions. The Agrobacterium rhizogenes 'pro mintron' promoter, reported to be activated in stationary phase, was found to be also strongly induced under acidic conditions in rhizobia and it is c urrently being characterised to construct a system allowing the expression of stress tolerance genes in bacteroids and free-living bacteria. (C) 2001 Elsevier Science B.V. All rights reserved.