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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.
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The ArsQ permease and transport of the antibiotic arsinothricin
Abstract The pentavalent organoarsenical arsinothricin (AST) is a natural product synthesized by the rhizosphere bacteriumBurkholderia gladioliGSRB05.AST is a broad‐spectrum antibiotic effective against human pathogens such as carbapenem‐resistantEnterobacter cloacae.It is a non‐proteogenic amino acid and glutamate mimetic that inhibits bacterial glutamine synthetase. The AST biosynthetic pathway is composed of a three‐gene cluster,arsQML.ArsL catalyzes synthesis of reduced trivalent hydroxyarsinothricin (R‐AST‐OH), which is methylated by ArsM to the reduced trivalent form of AST (R‐AST). In the culture medium ofB. gladioli, both trivalent species appear as the corresponding pentavalent arsenicals, likely due to oxidation in air. ArsQ is an efflux permease that is proposed to transport AST or related species out of the cells, but the chemical nature of the actual transport substrate is unclear. In this study,B. gladioli arsQwas expressed inEscherichia coliand shown to confer resistance to AST and its derivatives. Cells ofE. coliaccumulate R‐AST, and exponentially growing cells expressingarsQtake up less R‐AST. The cells exhibit little transport of their pentavalent forms. Transport was independent of cellular energy and appears to be equilibrative. A homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R‐AST‐OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy‐independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.
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- Award ID(s):
- 1817962
- PAR ID:
- 10406524
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Microbiology
- Volume:
- 119
- Issue:
- 4
- ISSN:
- 0950-382X
- Page Range / eLocation ID:
- p. 505-514
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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