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Genetic Analysis of a pH-Regulated Operon from Rhizobium tropici CIAT899 Involved in Acid Tolerance and Nodulation Competitiveness

¹FB Biologie der Philipps-Universität, FG für Zellbiologie und Angewandte Botanik, Karl von Frisch Str., D-35032 Marburg, Germany, ²Leiden University, Institute for Molecular Plant Sciences, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands; ³Centro de Investigación sobre Fijación de Nitrógeno-UNAM, Programa de Ecología Molecular y Microbiana, Aptdo. 565A, Cuernavaca, Morelos, Mexico

Rhizobium tropici CIAT899 is highly acid tolerant and a good competitor for Phaseolus vulgaris nodule occupancy at low pH values. Using Tn5 mutagenesis, we identified an operon required for acid tolerance and nodulation competitiveness. The insertion was mapped to the 5′ end of atvA, encoding a product with high sequence identity to the agrobacterial AcvB virulence protein. Complementation analyses indicated that atvA is an ortholog of acvB, both genes being required for acid tolerance. A Ser/Ala substitution in the LIPASE_SER motif of AtvA resulted in an acid sensitive Fix⁺ but very poorly competing strain, demonstrating that Ser-313 is essential for AtvA function. atvA is the second gene in an operon that is transcriptionally upregulated by acid shock. The acid-responsive promoter was mapped to a 469-bp intergenic region located upstream of lpiA, the first gene in the operon. lpiA-like genes are found in several α, β, and γ Proteobacteria that interact with eukaryotic host cells, and they are predicted to encode membrane proteins related to the FmtC/MprF family from low G+C Firmicutes. The latter proteins are involved in resistance to cationic antimicrobial peptides. A nonpolar deletion in lpiA caused a sevenfold decrease in relative nodulation competitiveness.

Additional keywords: gusA, lipolytic enzymes, symbiosis.