Acid and Iron Experimental Evolution of Halobacterium sp. NRC-1

Halobacterium sp. NRC-1 (NRC-1) is an extremely halophilic archaeon that is well adapted to multiple stressors such as UV, ionizing radiation, and arsenic exposure. We conducted experimental evolution of NRC-1 under acid and iron stress to expand the stressors. NRC-1 was serially cultured in CM+ medium modified by four stress conditions, with four evolving populations per condition. At 500 generations the conditions were: optimal pH (pH 7.5), acid stress (pH 6.3), iron stress (600 μM ferrous sulfate, pH 7.5), and acid plus iron stress (600 μM ferrous sulfate, pH 6.3). 16 clones from the 500th generation were isolated and characterized for phenotypic changes, and the genomes of the evolved clones were sequenced. Genotypic analysis of all 16 clones revealed 378 mutations, with patterns of high variability arising from movement of insertion sequences (ISH elements) and large deletions. One minichromosome (megaplasmid) pNRC100 had increased copy number. The 500-generation clones had frequent loss of gas vesicles and arsenic resistance. An acid-evolved clone had increased fitness compared to the ancestral stock, when cultured at low pH. Seven of eight acid-evolved clones had a mutation in or upstream of nhaC3 , encoding a sodium-proton antiporter that exports sodium and takes in protons; no non-acid adapted strains had nhaC3 mutations. Two acid-adapted strains shared a common mutation in bop , encoding the bacteriorhodopsin light-driven proton pump. Mutations also affected the arcR regulator of arginine catabolism, which can mediate proton transport. Thus, in the haloarchaeon NRC-1, as in bacteria, pH adaptation was associated with genes affecting proton transport.