Signature Arsenic Detoxification Pathways in Halomonas sp. Strain GFAJ-1.

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.

DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks.

The modification of DNA by phosphorothioate (PT) occurs when the non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur. This DNA backbone modification was recently discovered and is governed by the dndABCDE genes in a diverse group of bacteria and archaea. However, the biological function of DNA PT modifications is poorly understood. In this study, we employed the RNA-seq analysis to characterize the global transcriptional changes in response to PT modifications. Our results show that DNA without PT protection is susceptible to DNA damage caused by the dndFGHI gene products. The DNA double-stranded breaks then trigger the SOS response, cell filamentation and prophage induction. Heterologous expression of dndBCDE conferring DNA PT modifications at GPSA and GPST prevented the damage in Salmonella enterica. Our data provide insights into the physiological role of the DNA PT system.

In vivo mutational characterization of DndE involved in DNA phosphorothioate modification.

DNA phosphorothioate (PT) modification is a recently identified epigenetic modification that occurs in the sugar-phosphate backbone of prokaryotic DNA. Previous studies have demonstrated that DNA PT modification is governed by the five DndABCDE proteins in a sequence-selective and RP stereo-specific manner. Bacteria may have acquired this physiological modification along with dndFGH as a restriction-modification system. However, little is known about the biological function of Dnd proteins, especially the smallest protein, DndE, in the PT modification pathway. DndE was reported to be a DNA-binding protein with a preference for nicked dsDNA in vitro; the binding of DndE to DNA occurs via six positively charged lysine residues on its surface. The substitution of these key lysine residues significantly decreased the DNA binding affinities of DndE proteins to undetectable levels. In this study, we conducted site-directed mutagenesis of dndE on a plasmid and measured DNA PT modifications under physiological conditions by mass spectrometry. We observed distinctive differences from the in vitro binding assays. Several mutants with lysine residues mutated to alanine decreased the total frequency of PT modifications, but none of the mutants completely eliminated PT modification. Our results suggest that the nicked dsDNA-binding capacity of DndE may not be crucial for PT modification and/or that DndE may have other biological functions in addition to binding to dsDNA.