Search for: All records

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.

 

Arsenic (As) and mercury (Hg) were examined in the Yellowstone Lake food chain, focusing on two lake locations separated by approximately 20 km and differing in lake floor hydrothermal vent activity. Sampling spanned from femtoplankton to the main fish species, Yellowstone cutthroat trout and the apex predator lake trout. Mercury bioaccumulated in muscle and liver of both trout species, biomagnifying with age, whereas As decreased in older fish, which indicates differential exposure routes for these metal(loid)s. Mercury and As concentrations were higher in all food chain filter fractions (0.1‐, 0.8‐, and 3.0‐μm filters) at the vent‐associated Inflated Plain site, illustrating the impact of localized hydrothermal inputs. Femtoplankton and picoplankton size biomass (0.1‐ and 0.8‐μm filters) accounted for 30%–70% of total Hg or As at both locations. By contrast, only approximately 4% of As and <1% of Hg were found in the 0.1‐μm filtrate, indicating that comparatively little As or Hg actually exists as an ionic form or intercalated with humic compounds, a frequent assumption in freshwaters and marine waters. Ribosomal RNA (18S) gene sequencing of DNA derived from the 0.1‐, 0.8‐, and 3.0‐μm filters showed significant eukaryote biomass in these fractions, providing a novel view of the femtoplankton and picoplankton size biomass, which assists in explaining why these fractions may contain such significant Hg and As. These results infer that femtoplankton and picoplankton metal(loid) loads represent aquatic food chain entry points that need to be accounted for and that are important for better understanding Hg and As biochemistry in aquatic systems.Environ Toxicol Chem2023;42:225–241. © 2022 SETAC

 

The emergence and spread of antimicrobial resistance highlights the urgent need for new antibiotics. Organoarsenicals have been used as antimicrobials since Paul Ehrlich’s salvarsan. Recently a soil bacterium was shown to produce the organoarsenical arsinothricin. We demonstrate that arsinothricin, a non-proteinogenic analog of glutamate that inhibits glutamine synthetase, is an effective broad-spectrum antibiotic against both Gram-positive and Gram-negative bacteria, suggesting that bacteria have evolved the ability to utilize the pervasive environmental toxic metalloid arsenic to produce a potent antimicrobial. With every new antibiotic, resistance inevitably arises. ThearsN1gene, widely distributed in bacterial arsenic resistance (ars) operons, selectively confers resistance to arsinothricin by acetylation of the α-amino group. Crystal structures of ArsN1N-acetyltransferase, with or without arsinothricin, shed light on the mechanism of its substrate selectivity. These findings have the potential for development of a new class of organoarsenical antimicrobials and ArsN1 inhibitors.

 

Arsenic is the most ubiquitous environmental toxin. Here, we demonstrate that bacteria have evolved the ability to use arsenic to gain a competitive advantage over other bacteria at least twice. Microbes generate toxic methylarsenite (MAs(III)) by methylation of arsenite (As(III)) or reduction of methylarsenate (MAs(V)). MAs(III) is oxidized aerobically to MAs(V), making methylation a detoxification process. MAs(V) is continually re‐reduced to MAs(III) by other community members, giving them a competitive advantage over sensitive bacteria. Because generation of a sustained pool of MAs(III) requires microbial communities, these complex interactions are an emergent property. We show that reduction of MAs(V) byBurkholderiasp. MR1 produces toxic MAs(III) that inhibits growth ofEscherichia coliin mixed culture. There are three microbial mechanisms for resistance to MAs(III). ArsH oxidizes MAs(III) to MAs(V). ArsI degrades MAs(III) to As(III). ArsP confers resistance by efflux. Cells ofE. coliexpressingarsI,arsHorarsPgrow in mixed culture withBurkholderiasp. MR1 in the presence of MAs(V). Thus MAs(III) has antibiotic properties: a toxic organic compound produced by one microbe to kill off competitors. Our results demonstrate that life has adapted to use environmental arsenic as a weapon in the continuing battle for dominance.

 

Mushrooms have unique properties in arsenic metabolism. In many commercial and wild-grown mushrooms, arsenobetaine (AsB), a non-toxic arsenical, was found as the dominant arsenic species. The AsB biosynthesis remains unknown, so we designed experiments to study conditions for AsB formation in the white button mushroom, Agaricus bisporus. The mushrooms were treated with various arsenic species including arsenite (As(III)), arsenate (As(V)), methylarsenate (MAs(V)), dimethylarsenate (DMAs(V)) and trimethylarsine oxide (TMAsO), and their accumulation and metabolism were determined using inductively coupled mass spectrometer (ICP-MS) and high-pressure liquid chromatography coupled with ICP-MS (HPLC-ICP-MS), respectively. Our results showed that mycelia have a higher accumulation for inorganic arsenicals while fruiting bodies showed higher accumulation for methylated arsenic species. Two major arsenic metabolites were produced in fruiting bodies: DMAs(V) and AsB. Among tested arsenicals, only MAs(V) was methylated to DMAs(V). Surprisingly, AsB was only detected as the major arsenic product when TMAsO was supplied. Additionally, AsB was only detected in the fruiting body, but not mycelium, suggesting that methylated products were transported to the fruiting body for arsenobetaine formation. Overall, our results support that methylation and AsB formation are two connected pathways where trimethylated arsenic is the optimal precursor for AsB formation.

 

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. 

Arsenicals are one of the oldest treatments for a variety of human disorders. Although infamous for its toxicity, arsenic is paradoxically a therapeutic agent that has been used since ancient times for the treatment of multiple diseases. The use of most arsenic-based drugs was abandoned with the discovery of antibiotics in the 1940s, but a few remained in use such as those for the treatment of trypanosomiasis. In the 1970s, arsenic trioxide, the active ingredient in a traditional Chinese medicine, was shown to produce dramatic remission of acute promyelocytic leukemia similar to the effect of all-trans retinoic acid. Since then, there has been a renewed interest in the clinical use of arsenicals. Here the ancient and modern medicinal uses of inorganic and organic arsenicals are reviewed. Included are antimicrobial, antiviral, antiparasitic and anticancer applications. In the face of increasing antibiotic resistance and the emergence of deadly pathogens such as the severe acute respiratory syndrome coronavirus 2, we propose revisiting arsenicals with proven efficacy to combat emerging pathogens. Current advances in science and technology can be employed to design newer arsenical drugs with high therapeutic index. These novel arsenicals can be used in combination with existing drugs or serve as valuable alternatives in the fight against cancer and emerging pathogens. The discovery of the pentavalent arsenic-containing antibiotic arsinothricin, which is effective against multidrug-resistant pathogens, illustrates the future potential of this new class of organoarsenical antibiotics. 

We report two routes of chemical synthesis of arsinothricin (AST), the novel organoarsenical antibiotic. One is by condensation of the 2-chloroethyl(methyl)arsinic acid with acetamidomalonate, and the second involves reduction of the N -acetyl protected derivative of hydroxyarsinothricin (AST-OH) and subsequent methylation of a trivalent arsenic intermediate with methyl iodide. The enzyme AST N -acetyltransferase (ArsN1) was utilized to purify l -AST from racemic AST. This chemical synthesis provides a source of this novel antibiotic for future drug development.