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A Hyperthermoactive-Cas9 Editing Tool Reveals the Role of a Unique Arsenite Methyltransferase in the Arsenic Resistance System of Thermus thermophilus HB27.Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to humans. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pulldown assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-l-methionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM-dependent arsenite methylation with formation of monomethylarsenites (MMAs) and dimethylarsenites (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions. IMPORTANCE We here describe the discovery of an unknown protein by using a proteomics approach with a transcriptional regulator as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcriptional regulator controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite-producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65°C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole-cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.