Signature Arsenic Detoxification Pathways in <i>Halomonas</i> sp. Strain GFAJ-1

Abstract

Since the original report that <i>Halomonas</i> 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, <i>arsH1</i>-<i>acr3</i>-<i>2</i>-<i>arsH2</i> and <i>mfs1</i>-<i>mfs2</i>-<i>gapdh</i>, enabling tolerance to high levels of arsenate. <i>mfs2</i> and <i>gapdh</i> encode proteins homologous to <i>Pseudomonas aeruginosa</i> 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 <i>arsH1</i>-<i>acr3</i>-<i>2</i>-<i>arsH2</i> operon seems to consist of typical arsenite resistance genes, but this operon is sufficient to confer both arsenite and arsenate resistance on <i>Escherichia coli</i> 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.<b>IMPORTANCE</b><i>Halomonas</i> 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, <i>arsH1</i>-<i>acr3</i>-<i>2</i>-<i>arsH2</i> and <i>mfs1</i>-<i>mfs2</i>-<i>gapdh</i><i>mfs2</i> and <i>gapdh</i> encode proteins homologous to ArsJ and GAPDH in <i>Pseudomonas aeruginosa</i>; these proteins create an arsenate efflux pathway to reduce cellular arsenate accumulation. Interestingly, the combination of <i>acr3</i>-<i>2</i> with either <i>arsH</i> gene was sufficient to confer resistance to both arsenite and arsenate in <i>E. coli</i> 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.