Isolation and Characterization of an acid sensitive Tn5 mutant of the alfalfa nodulating Or191-like rhizobia

DEL PAPA, M. E., DRAGHI, W.O., PISTORIO, M. Y LAGARES, A.

5 al 8 de diciembre de 2004, Puerto Iguazú, Misiones, Argentina

Congreso; XL Reunión Nacional de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2004

Soil acidity is one of the environmental factors that more strongly hampers the establishment of an effective symbiotic interaction between rhizobia and Medicago sativa (alfalfa). In the recent past years a new group of alfalfa-nodulating rhizobia (Or191-like rhizobia) has been identified and genetically characterized, mainly due to their remarkable tolerance to acidity, inefficiency to fix nitrogen, and strong ability to outcompete S. meliloti during root nodulation at low pH. To advance on the genetics of acid tolerance in the Or191-like rhizobia, we generated a collection of Tn5B20 mutants and we screen the collection for acid sensitive phenotypes. One mutant out of 1,500 clones failed to grow on minimal medium at pH 5.0 but not at neutral pH. The acid-sensitive mutant, namely LPU833, also showed to be sensitive to oxidative stress, and to the presence of 100μM Cu++ and 40μM Zn++. Genetic analysis showed that mutant LPU833 carries a single transposon insertion within an ORF with sequence similarity to a family of bacterial oxidoreductases, located immediately upstream of an ubiHhomolog involved in ubiquinone metabolism. The mutant LPU833 kept the ability to nodulate alfalfa. However, the symbiotic relevance of the affected locus was evident in coinoculation experiements where mutant LPU833 resulted fully outcompeted by the wild type strain at both acidic and neutral pH.