Since the original report that Halomonas sp. strain GFAJ-1 was capable of using arsenic instead of phosphorus to sustain growth, additional studies have been conducted, and GFAJ-1 is now considered a highly arsenic-resistant but phosphorus-dependent bacterium. However, the mechanisms supporting the extreme arsenic resistance of the GFAJ-1 strain remain unknown. In this study, we show that GFAJ-1 has multiple distinct arsenic resistance mechanisms. It lacks the genes to reduce arsenate, which is the essential step in the well-characterized resistance mechanism of arsenate reduction coupled to arsenite extrusion. Instead, GFAJ-1 has two arsenic resistance operons, arsH1-acr3-2-arsH2 and mfs1-mfs2-gapdh, enabling tolerance to high levels of arsenate. mfs2 and gapdh encode proteins homologous to Pseudomonas aeruginosa ArsJ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively, which constitute the equivalent of an As(V) efflux system to catalyze the transformation of inorganic arsenate to pentavalent organoarsenical 1-arseno-3-phosphoglycerate and its subsequent extrusion. Surprisingly, the arsH1-acr3-2-arsH2 operon seems to consist of typical arsenite resistance genes, but this operon is sufficient to confer both arsenite and arsenate resistance on Escherichia coli AW3110 even in the absence of arsenate reductase, suggesting a novel pathway of arsenic detoxification. The simultaneous occurrence of these two unusual detoxification mechanisms enables the adaptation of strain GFAJ-1 to the particularly arsenic-rich environment of Mono Lake.IMPORTANCEHalomonas sp. strain GFAJ-1 was previously reported to use arsenic as a substitute for phosphorus to sustain life under phosphate-limited conditions. Although this claim was later undermined by several groups, how GFAJ-1 can thrive in environments with high arsenic concentrations remains unclear. Here, we determined that this ability can be attributed to the possession of two arsenic detoxification operons, arsH1-acr3-2-arsH2 and mfs1-mfs2-gapdhmfs2 and gapdh encode proteins homologous to ArsJ and GAPDH in Pseudomonas aeruginosa; these proteins create an arsenate efflux pathway to reduce cellular arsenate accumulation. Interestingly, the combination of acr3-2 with either arsH gene was sufficient to confer resistance to both arsenite and arsenate in E. coli AW3110, even in the absence of arsenate reductase, suggesting a new strategy for bacterial arsenic detoxification. This study concludes that the survival of GFAJ-1 in high arsenic concentrations is attributable to the cooccurrence of these two unusual arsenic detoxification mechanisms.

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