Selective Methylation by an ArsM S-Adenosylmethionine Methyltransferase from Burkholderia gladioli GSRB05 Enhances Antibiotic Production

Abstract

Arsenic methylation contributes to the formation and diversity of environmental organoarsenicals, an important process in the arsenic biogeochemical cycle. The arsM gene encoding an arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferase is widely distributed in members of every kingdom. A number of ArsM enzymes have been shown to have different patterns of methylation. When incubated with inorganic As(III), Burkholderia gladioli GSRB05 has been shown to synthesize the organoarsenical antibiotic arsinothricin (AST) but does not produce either methylarsenate (MAs(V)) or dimethylarsenate (DMAs(V)). Here, we show that cells of B. gladioli GSRB05 synthesize DMAs(V) when cultured with either MAs(III) or MAs(V). Heterologous expression of the BgarsM gene in Escherichia coli conferred resistance to MAs(III) but not As(III). The cells methylate MAs(III) and the AST precursor, reduced trivalent hydroxyarsinothricin (R-AST-OH) but do not methylate inorganic As(III). Similar results were obtained with purified BgArsM. Compared with ArsM orthologs, BgArsM has an additional 37 amino acid residues in a linker region between domains. Deletion of the additional 37 residues restored As(III) methylation activity. Cells of E. coli co-expressing the BgarsL gene encoding the noncanonical radical SAM enzyme that catalyzes the synthesis of R-AST-OH together with the BgarsM gene produce much more of the antibiotic AST compared with E. coli cells co-expressing BgarsL together with the CrarsM gene from Chlamydomonas reinhardtii, which lacks the sequence for additional 37 residues. We propose that the presence of the insertion reduces the fitness of B. gladioli because it cannot detoxify inorganic arsenic but concomitantly confers an evolutionary advantage by increasing the ability to produce AST.

Arsenite Methyltransferase Diversity and Optimization of Methylation Efficiency

“…Other ArsM orthologs have only A and B domains, and some of those methylate both As­(III) and MAs­(III), while others methylate only MAs­(III). For example, Figure shows a model of a typical three-domain ArsM, Burkholderia gladioli BgArsM . The radical SAM enzyme BgArsL forms reduced AST-OH.…”

“…BgArsM has A, B, and C domains as well as a linker­(D) between the A and B domains that contains 37 more residues than most other ArsMs (Figure and Figure S2). We previously reported that deletion of the additional residues restores As­(III) methylation, leading to the hypothesis that the extended linker modulates As­(III) methylation . This increases the rate of methylation of reduced AST-OH, which benefits host organisms for arsenical antibiotic biosynthesis.…”

“…For example, Figure 1 shows a model of a typical three-domain ArsM, Burkholderia gladioli BgArsM. 29 The radical SAM enzyme BgArsL forms reduced AST-OH. BgArsM transfers a methyl group from SAM to reduced AST-OH, forming the trivalent form of the antibiotic AST.…”

“…We previously reported that deletion of the additional residues restores As(III) methylation, leading to the hypothesis that the extended linker modulates As(III) methylation. 29 This increases the rate of methylation of reduced AST-OH, which benefits host organisms for arsenical antibiotic biosynthesis.…”

Reduction of Dimethylarsenate to Highly Toxic Dimethylarsenite in Paddy Soil and Rice Plants

“…Arsenic (As) biomethylation is an important component of As biogeochemistry . Many organisms including some bacteria, archaea, fungi, algae, and animals are able to methylate inorganic arsenite [As­(III)] into different methylated As species such as monomethylarsenate [MMAs­(V)], dimethylarsenate [DMAs­(V)], and trimethylarsine [TMAs­(III)]. − Enzymes catalyzing As biomethylation in microbes (arsenite S -adenosylmethionine methyltransferase, ArsM) and humans (AS3MT, As­(III) methyltransferase) have been identified and characterized. , Interestingly, higher plants such as rice, tomato, and red clover do not appear to be able to methylate As­(III) . Rice growing in flooded paddy soil often accumulates substantial amounts of methylated As species, especially DMAs­(V), in addition to inorganic As. − These methylated As species are derived from microbial As biomethylation in soil, which is enhanced by soil flooding. − DMAs­(V) is the most common methylated As species found in paddy soils. , Synthetic DMAs­(V) (also called dimethylarsinic acid or cacodylic acid) has also been widely used as a herbicide or defoliant in the past …”