mrpA, a gene with roles in resistance to Na+ and adaptation to alkaline pH in the cyanobacterium Anabaena sp. PCC7120

1 Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid 28049, Spain 2 Departamento de Química Física Aplicada, Universidad Autónoma de Madrid, Madrid 28049, Spain Correspondence F. Fernández-Piñas francisca.pina{at}uam.es Transposon mutagenesis of Anabaena sp. PCC7120 led to the isolation of a mutant strain, PHB11, which grew poorly at pH values above 10. The mutant strain exhibited pronounced Na + sensitivity; this sensitivity was higher under basic conditions. Mutant PHB11 also showed an inhibition of photosynthesis that was much more pronounced at alkaline pH. Reconstruction of the transposon mutation of PHB11 in the wild-type strain reproduced the phenotype of the original mutant. The wild-type version of the mutated gene was cloned and the mutation complemented. In mutant strain PHB11, the transposon had inserted within an ORF that is part of a seven-ORF operon with significant sequence similarity to a family of bacterial operons that are believed to code for a novel multiprotein cation/proton antiporter primarily involved in resistance to salt stress and adaptation to alkaline pH. The Anabaena operon was denoted mrp (multiple resistance and pH adaptation) following the nomenclature of the Bacillus subtilis operon; the ORF mutated in PHB11 corresponded to mrpA . Computer analysis suggested that all seven predicted Anabaena Mrp proteins were highly hydrophobic with several transmembrane domains; in fact, the predicted protein sequences encoded by mrpA , mrpB and mrpC showed significant similarity to hydrophobic subunits of the proton pumping NADH : ubiquinone oxidoreductase. In vivo expression studies indicated that mrpA is induced with increasing external Na + concentrations and alkaline pH; mrpA is also upregulated under inorganic carbon (Ci) limitation. The biological significance of a putative cyanobacterial Mrp complex is discussed. Abbreviations: Chl, chlorophyll; Ci, inorganic carbon; Em, erythromycin; Km, kanamycin sulphate; Nm, neomycin sulfate; pH ex , external pH; pH in , internal pH; Sp, spectinomycin dihydrochloride The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF239979 . Characteristics of the putative gene products of the Anabaena mrp locus and their predicted similarity to known protein sequences are available in Supplementary Table S1, an alignment of MrpA from Anabaena sp. strain PCC7120 and MrpA from other bacterial species in Supplementary Fig. S1, a schematic dia