arsenic and the gastrointestinal tract microbiome

Minireview

Arsenic and the gastrointestinal tract microbiome

Timothy R. McDermott, 1* John F. Stolz2** andRonald S. Oremland3

1Department of Land Resources and EnvironmentalSciences, Montana State University, Bozeman, MT,59717, USA.2Department of Biological Sciences and Center forEnvironmental Research and Education, DuquesneUniversity, Pittsburgh, PA, USA.3United States Geological Survey, Menlo Park, CA,94025, USA.

Summary

Arsenic is a toxin, ranking first on the Agency for ToxicSubstances and Disease Registry and the Environmen-tal Protection Agency Priority List of Hazardous Sub-stances. Chronic exposure increases the risk of a broadrange of human illnesses, most notably cancer; how-ever, there is significant variability in arsenic-induceddisease among exposed individuals. Human genetics isa known component, but it alone cannot account for thelarge inter-individual variability in the presentation ofarsenicosis symptoms. Each part of the gastrointestinaltract (GIT) may be considered as a unique environmentwith characteristic pH, oxygen concentration, andmicrobiome. Given the well-established arsenic redoxtransformation activities of microorganisms, it is rea-sonable to imagine how the GIT microbiome composi-tion variability among individuals could play asignificant role in determining the fate, mobility and tox-icity of arsenic, whether inhaled or ingested. This is arelatively new field of research that would benefit fromearly dialogue aimed at summarizing what is known andidentifying reasonable research targets and concepts.Herein, we strive to initiate this dialogue by reviewingknown aspects of microbe–arsenic interactions andplacing it in the context of potential for influencing hostexposure and health risks. We finish by consideringfuture experimental approaches that might be of value.

Introduction

The influence of microbiomes on human health and diseaseis well established (e.g., the NIH Human Microbiome Project;Chatelier et al., 2013), with new studies continuing to demon-strate the potential for important advancements in our under-standing of the basis for various human diseases andconditions. One such disease condition concerns arsenic(As), a toxin and carcinogen, ranking first on the UnitedStates Environmental Protection Agency Priority List of Haz-ardous Substances (Agency for Toxic Substances and Dis-ease Registry, n.d.). Based on hair analyses of Chinchorrosculture mummies, excess As exposure dates to prehistoricpopulations of present-day Chile (Atacama Desert) (Byrneet al., 2010). In today’s world more than 200 million peopleare exposed to toxic levels of As in drinking water(Bhattacharjee et al., 2013; Naujokas et al., 2013), with arse-nic contamination of drinking water in Bangladesh beingreferred to as the largest mass poisoning event in human his-tory (Smith et al., 2000). Chronic As exposure has beenlinked to a range of diseases (arsenicosis), including lung,skin, bladder and liver cancers (Liu et al., 2008; Faita et al.,2013). Interestingly, symptoms vary among individuals shar-ing similar exposure (Naujokas et al., 2013; Cubadda et al.,2015). Some variability is attributed to host genetics and otherfactors (e.g., diet), but other causes remain to be determined.

Primary As exposure is via ingestion of either food or,more commonly, As-contaminated water. In every environ-ment thus far studied, microbial activities significantly influ-ence As redox speciation (trivalent vs. pentavalent), whichdictates toxicity and mobility. Therefore, it is reasonable topredict that the initial fate of As in the gastrointestinal tract(GIT) will likewise be influenced by microbiome As metabo-lism. Since microbiome composition and diversity variesbetween individuals, there is potential for functional linkagesbetween GIT microbiome As metabolism, host exposure,relative toxicity and disease likelihood, and thus a factor toexplain symptom variability among individuals.

Much of the basics regarding microbe–As interactionsoriginally derived from work using Escherichia coli orStaphylococcus aureus as models. This was followed bynear countless studies using environmental isolates (soil,marine, rhizosphere, etc.). Together, these studies havebeen invaluable for understanding what is possible

Received 27 September, 2019; accepted 25 November, 2019. Forcorrespondence. *E-mail [email protected]; **E-mail [email protected]

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd

Environmental Microbiology Reports (2020) 12(2), 136–159 doi:10.1111/1758-2229.12814

https://orcid.org/0000-0001-8293-5106

mailto:[email protected]



elsewhere, including the GIT, and establish a frameworkfor future work in the GIT microbiome arena. It is worthnoting that the first As-GIT microbiome study was con-ducted by an environmental microbiology group thatdeveloped SHIME® (Simulator of the Human IntestinalMicrobial Ecosystem), which is a modular bioreactor gutmodel that mimics the entire GIT, incorporating the stom-ach, small intestine and different colon regions as ahuman intestinal microbial ecosystem (Van de Wieleet al., 2004; Van de Wiele et al., 2015). It was first usedto examine arsenic metabolism in the context of the GIT(Van de Wiele et al., 2010), exposing a colon microbialcommunity to either inorganic As (iAs) or As-contaminated soil, recording significant levels of As meth-ylation and thiolation.

Arsenic research in the GIT microbiome is in a relativestate of infancy and can benefit from an early exchangeof ideas, concepts and views. Chi et al. (2018) reviewhow human genetics, lifestyle and diet will influence indi-vidual susceptibility to As-related diseases. We offer tocontinue this dialogue but explicitly from the perspectiveof the GIT microbiome. There are now growing GITmicrobiome databases for healthy or diseased individ-uals, including the oral cavity (e.g., nasal, buccal, gingivaand tongue), stomach, small intestine and large intestine(Fig. 1). We consider each as a unique environment withrespect to pH and oxygen concentration, as well as theresidence time (i.e., how quickly ingested arsenic movesthrough the GIT and hence influencing potential hostexposure).

We begin by summarizing how and why microbes reactto As to establish a context with respect to the range ofmicrobial biochemistries that may be occurring in theGIT. We discuss how these activities may reduce orenhance toxicity and host exposure, and highlight thecomplexities of microbial arsenic transformations inregards to sorting out cause and effect, as well as poten-tial influence(s) on the host. This is followed by theresults of our GIT metagenomes surveys and a review ofthe GIT microbiome As metabolism. We finish bysuggesting lines of investigation that may provide greaterinsight into how the GIT microbiome contributes topathology as well as potential preventative healthstrategies.

Microbe–arsenic interactions

Crucial factors affecting As cycling in any environment andhuman exposure (i.e., toxicity, bioavailability and bio-accumulation) are directly related to its chemical speciation(Stolz and Oremland, 1999; Inskeep et al., 2001; Oremlandand Stolz, 2005). The oxyanions arsenite [As(OH)3, hereaf-ter referred to as As(III)] and its oxidized counterpart arse-nate [HnAsO4

-(3-n), As(V)] are the prevalent forms of arsenic

in nature. As(V) normally dominates in well-aerated environ-ments and most likely to be consumed. As(III) is both moretoxic and mobile than As(V), making As(III) more problem-atic, especially in the context of drinking water from sub-oxicaquifers. In all environments where arsenic and microbescoexist, microbes are the principal drivers of this speciationand thus are an integral part of understanding arseniccycling (Stolz and Oremland, 1999; Inskeep et al., 2001;Oremland and Stolz, 2005; Huang et al., 2014). In simplestterms, microbial catalysed arsenic transformations can besummarized as being either oxidation, reduction, and/or(de)methylation. From the perspective of the microbe, allthese reactions are ‘motivated’ by self-interest, such asdetoxification of a poison, or in some cases the generationof cellular energy. Table 1 and Figs 2 and 3 are provided asreference material for the different transformation functionsdiscussed below.

Arsenate resistance and transformations

ars gene-based resistance

Microbes reduce As(V) as part of a resistance mecha-nism and/or for energy generation (Stolz and Oremland,1999; Oremland and Stolz, 2005). While genetically dis-tinct (ars genes encode resistance; arr genes encoderespiratory activity), these processes are not necessarilymutually exclusive and can occur in the same organism(reviewed by Andres and Bertin, 2016). They are mostoften found in the genome, but the literature documentsnumerous instances where they are also found on plas-mids (e.g., San Francisco et al., 1990; Bruhn et al., 1996;Uhrynowski et al., 2019).

Arsenic resistance is encoded by the ars genes(Table 1) expressed in response to As exposure. The arsoperon is comprised of at least three genes arsRBC(Rosen et al., 1992), typically expressed as one transcrip-tional unit. ArsR is a repressor that exerts on/off controlof the ars operon. ArsC is a reductase that convertsAs(V) to As(III) (Fig. 2A) and ArsB facilitates As(III) extru-sion (Figs 2A and 3). Acr3 is another extrusion mecha-nism and its encoding gene is actually found morefrequently than arsB in ars operons (Yang et al., 2015).Just recently, Shi et al. (2018) characterized ArsK, whichalso facilitates extrusion of As(III) as well as other triva-lent metalloids (Fig. 3). Importantly, ars gene/operonexpression is independent of redox conditions.

When As(V) enters the GIT, a bacterium will take it upvia a phosphate transporter, with two recognized fates. Itcan be reduced by background levels of ArsC to formAs(III), which is the inducer that triggers up-regulation ofthe ars genes resulting in considerably enhancedAs(V) reductase activity and the As(III) efflux pumpingmechanism (ArsB, ArsK or Acr3). Thus, somewhat

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 12, 136–159

Arsenic and the gastrointestinal tract microbiome 137

paradoxically, the primary As(V) resistance strategy is totransform As(V) into a more toxic form, but which is thesubstrate for an active and efficient mechanism thatremoves it from the cell. We also note that Chauhanet al. (2009) first described ArsN as a putativeacetyltransferase and inferred from bioassays that it hasArsC-like activity. Just recently, the acetyltransferaseactivity has been confirmed, but now ArsN is shown toselectively confer resistance to organoarsenical antibiotic,arsinothricin by acetylation of the α-amino group (Nadaret al., 2019).As(V) can also be extruded back out from the cell. Many

ars operons contain genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and arsJ, with bothup-regulated in response to As(III). In Pseudomonasaeruginosa (Chen et al., 2016) and Halomonassp. (Wu et al., 2018), GAPDH incorporates As(V) intoglyceraldehyde-3-phosphate, generating 1-arseno-3-pho-sphoglycerate that is a transport substrate for the exporter ArsJ(Figs 2B and 3). Presumably, 1-arseno-3-phosphoglycerate is

structurally similar enough to glyceraldehyde-3-phosphateto be recognized byArsJ, facilitating extrusion. This amountsto an As(V) import–export cycle. As(III) can enter the cell viaan aquaglyceroporin (Sanders et al., 1997) and will subse-quently trigger the same set of regulatory and extrusionresponses.

Relevance to the GIT: Ars-based resistance results inextrusion from the microbial cell (Figs 2 and 3). The host-relevant outcome is to extrude As(V) (via GAPDH/ArsJ),which is neutral with respect to influencing toxicity to hostor neighbouring microbiome members. Alternatively,As(V) is reduced to As(III) and extruded (ArsB, ArsK orAcr3), which could increase toxin exposure for the hostand neighbouring microbiome members.

Arsenic methylation

Arsenicmethyltransferase is encoded by arsM and has beenstudied to some degree in Rhodopseudomonas (Qin et al.,2006), Clostridium (Wang et al. 2015a), the archaeon

Fig. 1. The human gastrointestinal tract (GIT) beginning in the oral cavity and ending at the rectum. Major phyla and genera recognized as impor-tant members of the GIT microbiome are displayed. GIT microbiome databases can be accessed at https://img.jgi.doe.gov/cgibin/m/main.cgi?section=TaxonList&domain=Metagenome&seq_center=all&page=metaCatList&phylum=Host-associated&ir_class=Human


138 T. R. McDermott, J. F. Stolz and R. S. Oremland

https://img.jgi.doe.gov/cgibin/m/main.cgi?section=TaxonListanddomain=Metagenomeandseq_center=allandpage=metaCatListandphylum=Host-associatedandir_class=Human

https://img.jgi.doe.gov/cgibin/m/main.cgi?section=TaxonListanddomain=Metagenomeandseq_center=allandpage=metaCatListandphylum=Host-associatedandir_class=Human

Methosarcina (Wang et al., 2014) and fungi (Chen et al.,2017; Li et al., 2018). However, by far the best-studied andunderstood enzyme was cloned from the acidothermophilicalga Cyanidioschyzon (Qin et al., 2009) and has been usedextensively to model this reaction. ArsM generates trivalentmethylated species (Ajees et al., 2012; Ajees and Rosen,2015) (Fig. 2C), not pentavalent species as originally hypoth-esized (Challenger, 1945). Methylarsenite [MAs(III)] anddimethylarsenite [DMAs(III)] are the most toxic arsenicalsknown, but in aerobic culture conditions where these methyl-ation reactions are most often studied, the pentavalent spe-cies MAs(V) and DMAs(V) are the major arsenicalsobserved, likely due to autooxidation of the trivalent species(Qin et al., 2006; Qin et al., 2009). In addition to ArsM, somemethanogens will methylate metalloids (As, Se, Sb, Te andBi) using the methyltransferase MtaA to catalyse methyl

transfer from methylcobalamin [CH3Cob(III)] to 2-mercap-toethanesulfonic acid (CoM) (Thomas et al., 2011).

In an aerobic environment where oxygen is available,the function of ArsM inmicroorganisms appears to be detox-ification, where methylated As(III) becomes oxidized toeither a less toxic pentavalent methylated species or the vol-atile trimethylarsine (TMAs), which would spontaneouslyrelease from the cell (Qin et al., 2006; Qin et al., 2009). Forthe microbe catalysing this reaction, MAs(III) synthesis isproblematic because of its extreme toxicity. This can besolved byArsP, a specificMAs(III) efflux permease or detox-ified via ArsH, which is an organoarsenical oxidase capableof oxidizing trivalent methylated and aromatic arsenicals(Chen et al. 2015a) or ArsI, which cleaves the carbon-arsenicbonds in methylated As(III) derivatives (Figs 2D and 3)(Yoshinaga andRosen, 2014).

Table 1. List of microbial genes that have been characterized to varying degrees with respect to their encoded functions, and documented to beinvolved in some aspect of arsenic biogeochemistry and or mobility.

Function Gene Encoded function References

Resistance arsR Transcriptional repressor involved inregulating genes involved in Asresistance, As(III) oxidation and As(V)respiration

Wu and Rosen (1991); Slyemi andBonnefoy (2012); Kang et al. (2012);Zargar et al. (2010)

arsB/acr3 Transmembrane carrier pumps (extrusion) Tisa and Rosen (1990); Rosen (2002)arsC Arsenate reductase Mukhopadhyay and Rosen (2002)arsA Anion stimulated ATPase Tisa and Rosen (1990); Rosen et al. (1999)arsD Transcriptional repressor and As(III)

chaperoneWu and Rosen (1993); Lin et al. (2006)

arsH Oxidizes MMAs(III) to MMAs(V) Chen et al. (2015a)arsJ Extrudes 1-arseno-3-phosphoglycerate Chen and Rosen (2016)arsK Facilitates As(III) extrusion Shi et al. (2018)arsN Confers resistance to the organoarsenical

arsinothricinNadar et al. (2019)

Putative acetyltransferase having ArsCactivity

Chauhan et al. (2009)

arsP methylarsenite efflux Chen et al. (2015b)arsM Arsenite methyltransferase Qin et al. (2006); Qin et al. (2009)arsI C-As&$$$; lyase Yoshinaga and Rosen (2014)

As(III) oxidation aioB Arsenite oxidase small subunit Lett et al. (2012)aioA Arsenite oxidase large subunit Lett et al. (2012)aioC c-type cytochrome Santini et al. (2007); Branco et al. (2009)aioD Molybdenum cofactor Branco et al. (2009)aioX Periplasmic As(III)-binding protein Liu et al. (2012)aioS Two-component signal transduction

histidine kinaseKashyap et al. (2006)

aioR Response regulator Kashyap et al. (2006)arxA Arsenite oxidase large subunit

(Photoarsenotrophy)Zargar et al. (2010)

arxB Arsenite oxidase large subunit(Photoarsenotrophy)

Zargar et al. (2010)

phoR Phosphate stress response two-componentsignal transduction histidine kinase

Slyemi and Bonnefoy (2012); Kang et al.(2012)

phoB Phosphate Stress response Responseregulator

Slyemi and Bonnefoy (2012); Kang et al.(2012); Wang et al. (2018)

Anaerobic As(V) Respiration arrA Respiratory arsenate reductase largesubunit

Saltikov and Newman (2003)

arrB Respiratory arsenate reductase smallsubunit

Saltikov and Newman (2003)

arrC Membrane subunit involved in anchoringand electron transfer

Duval et al. (2008); van Lis et al. (2013)

arrD Chaperone van Lis et al. (2013)



Relevance to the GIT: Arsenic methylation to the trivalentspecies has negative consequences, creating an evenmoretoxic situation that potentially interrupts normal host func-tions as well as microbiome functions that are beneficial to

the host. Microbiome members exposed to extrudedMAs(III) but that lack ArsP, ArsH or ArsI, will be selectedagainst, contributing to shifts in microbiome composition.Indeed, occurrence, abundance and expression of all of the

A

B

C

D

Fig. 2. Potential primary GIT-relevant microbiome arsenic transformations.A. Oxidation or reduction of the principal oxyanions As(III) and As(V). B. Arsenate incorporation into glyceraldehyde 3-phosphate to synthesizethe extrusion substrate for ArsJ.C. Arsenic methylation catalysed by ArsM, resulting in three possible products: MAs(III), DMAs(III) and TMAs(III). Also shown are the spontane-ous oxidation products for each: MAs(V), DMAs(V) and TMAO, respectively.D. Detoxification of methylarsenite by the organoarsenical oxidase ArsH, and ArsI, which cleaves carbon-arsenic bonds.



above ars genes anywhere along the GIT represent animportant example of how these microbiomes can serve todecrease or substantially enhance the toxicity of ingestedarsenic.

As(V) reduction for energy generation

Many microorganisms will reduce As(V) as part of anaer-obic respiration (Stolz and Oremland, 1999; Oremlandand Stolz, 2003). Regulatory control of dissimilatoryAs(V) reductase genes (arrAB) is more complex than arsC(Glasser et al., 2018), requiring both arsenic exposure andanaerobic conditions (Murphy and Saltikov, 2009). Recently,Switzer Blum et al., 2018 described a Citrobacter sp. isolatedfrom a termite hindgut that displays As(V) dependent growth,but without ArrAB. Initial evidence suggests ArsC may besomehow involved and thus represents somewhat of a hybridin a physiologic sense.

Relevance to the GIT: The lumen is viewed to be alargely anaerobic environment (Fig. 4) (except seebelow), therefore, the appropriate redox conditions pre-vail for arrAB expression. Thus, As(V) reduction viaArrAB and ArsC could be additive with regards to the netAs transformation capacity of an organism or

microbiome. Unlike the cytoplasmic ArsC, ArrAB islocated in the periplasm and hence the toxic As(III)formed is outside the bacterial cytoplasm and does not

Fig. 3. Potential microbiome arsenic transformations of relevance to the host. Abbreviations are linked to definitions in the text.

Fig. 4. Cartoon depiction of the lumen epithelia, illustrating the transi-tion from the anaerobic lumen environment to the inner and outermucus environments that are oxygenated via the villi. Adapted fromhttps://en.wikipedia.org/wiki/Intestinal_epithelium



https://en.wikipedia.org/wiki/Intestinal_epithelium

require extrusion. Therefore, microbiome As(V) respirationshould not be considered a positive outcome in the contextof host toxicity and exposure.

As(III) oxidation

As(III) oxidation can serve as a detoxification mechanismor can be used to generate energy, using As(III) as anelectron donor. The structural genes encoding the As(III)oxidase heterodimer are aioB (small sub-unit, Reiske type)and now referred to as aioA (large sub-unit). ACentibacterium arsenoxidans aox (aoi) mutant is moresensitive to As(III) than the wild-type parental strain (Mulleret al., 2003) and thus an example where As(III) oxidationwas shown to serve a detoxification purpose. The firstreproducible report of As(III) chemolithotrophy was in anAgrobacterium/Rhizobium-like organism (Santini et al.,2000). As(III) oxidation is typically found linked to oxygenas an electron acceptor, but anaerobic As(III) oxidationhas also been documented in support of anoxygenic pho-tosynthesis (Kulp et al., 2008; Hernandez-Maldonadoet al., 2016) or where nitrate is the alternative electronacceptor (e.g., Oremland et al., 2002; Rhine et al., 2006).Consequently, As(III) oxidation can proceed under eitheraerobic or anaerobic conditions and so, like ArsC-basedAs(V) reduction, is not tied to prevailing redox conditions.The aioBA genes have been described for a range ofmicrobes (Cai et al., 2009) and are phylogenetically dis-tinct from the more recently discovered ArxAB that cou-ples As(III) oxidation with photosynthesis (Zargar et al.,2010; Zargar et al., 2012) or nitrate reduction (Hoeftet al., 2007).In the best-studied bacteria, aioBA expression is regu-

lated by a signal transduction system that includes a peri-plasmic As(III) binding protein, AioX (Liu et al., 2012), thesensor kinase AioS and response regulatory protein AioR(Kashyap et al., 2006; Koechler et al., 2010) (Table 1).Expression of aioXSR requires As(III) exposure, but in addi-tion, aioSR are ultimately controlled by another signal trans-duction system, PhoR-PhoB, which controls the phosphatestarvation response in bacteria (Kang et al., 2012; Wanget al., 2018). AioXSR is not a universal requirement, assome As(III) oxidizing bacteria lack aioXSR (Koechler et al.,2015; Li et al., 2013). Wang et al. (2018) suggested thatPhoRB is essential for regulating As(III) oxidation in most, ifnot all, microorganisms that do so.Relevance to the GIT: Oxidation of As(III) to As(V) will

have a detoxifying effect on both the host as well as themicrobiome.

Thiolated arsenicals

In terms of potential microbial biogenesis, thiolatedarsenicals are the least understood naturally occurring

arsenic compounds. Offering a chemical perspective, Fanet al. (2018) offered a new hypothesis that suggests thio-lation is integrated with methylation, involving protein-boundintermediates. Here, we adopt the thioarsenical definitionused in a recent review of methylated thioarsenical com-pounds (Sun et al., 2016), where glutathione or protein thiolconjugates are not included in this category. Mono-, di-, tri-and tetrathioarsenicals are primarily of interest here and areshown in Fig. 5. The degree of sulphur substitution is criticalto toxicity and an example where a pentavalent arsenicspecies can be highly toxic. DMAs(V) is of low relative tox-icity, but thiolation to dimethylthioarsenate (DMAs(V)S)greatly increases its toxicity, approaching that of As(III) andDMAs(III) (Naranmandura et al., 2007). However, furthersulfidication to form dimethyldithioarsenate (DMAs(V)S2)reduces toxicity significantly (Ochi et al., 2008; Kim et al.,2016). Consequently, one cannot generalize regarding thetoxicity of thiolated arsenicals.

Factors important in determining the extent of forma-tion, solubility and stability of the thioarsenicals includepH, redox conditions, and prevailing concentrations andratios of As to SO4

−2 and or H2S (Wilkin et al., 2003;Bostick et al., 2005; Stauder et al., 2005). Abiotic reac-tions appear to control arsenic thiolation (Planer-Friedrichet al., 2007; Stauder et al., 2005; Bostick et al., 2005. Asthe HS−:arsenic ratio increases, so does the degree ofthiolation (Wilkin et al., 2003; Bostick et al., 2005;Stauder et al., 2005), although there is some disagree-ment whether the thioarsenical formed is trivalent (Wilkinet al., 2003) or pentavalent (Stauder et al., 2005). AbioticH2S reduction of As(V) is highly pH-dependent, reactingpoorly around pH 7.0, even with H2S:As ratios as high as100:1, but reaction rates increase markedly at low pH(Rochette et al., 2000). As(III) and H2S interactions canresult in poorly soluble arsenic solid phases such asamorphous orpiment (As(III)2S3), realgar (As4S4) (Fig. 5)or even elemental As—all of which would be expected tobe poorly bioavailable in the GIT (Liu et al., 2008). This toois highly pH-dependent and oxygen-sensitive. Maximum pHwhere arsenical sulphide precipitation might occur is ~8.0,which is still within the range found in the GIT and can bequantitative at pH 6.1 and increasing with more acidic pH(Rodriguez-Freire et al., 2014). Under neutral to alkalineconditions, abiotic As(III)2S3 dissolution is rapid (Hartig andPlaner-Friedrich, 2012; Suess and Planer-Friedrich, 2012),although transformation kinetics slow with progression fromtrithioarsenate As(V)S3 to thioarsenate (As(V)S). Withintemporal scales of hours (i.e., GIT transit time), As(V)S ischemically stable, even in the absence of H2S (Hartig et al.,2014), but can be rapidly decomposed by capable microor-ganisms leading to As(V) with intermediate accumulation ofAs(III) (Hartig and Planer-Friedrich, 2012). Organisms suchas Thermocrinis ruber will use As(V)S as a chemo-lithotrophic electron donor with O2 as an electron acceptor



(yielding As(V) and SO42−) (Hartig and Planer-Friedrich,

2012), but this activity has not been examined with GIT-relevant samples.

Evidence of direct microbial enzymatically catalysedthioarsenical synthesis is lacking. However, by defaultSO4

2− reducing bacteria (SRB) are involved, which arecommon in the gut (Gibson et al., 1993; Carbonero andGaskins, 2013). A Shewanella putrefaciens isolate maybe an exception; it converts MAs(V) to methythioarsenate(MAs(V)S), and DMAs(V) to dimethythioarsenate (DMAs(V)S) (Chen and Rosen, 2016). SRB activity providesopportunity for the strictly anaerobic chemoautotrophicDeltaproteobacterium (strain MLMS-1) that uses H2S asits e-donor and As(V) as its e-acceptor (Planar-Friederichet al., 2015), and can also result in significant As(III)2S3

and/or As4S4 precipitation or as co-precipitates with ferroussulphide or pyrite (Altun et al., 2014; Rodriguez-Freire et al.,2016). These mineral phase arsenicals are stable at acidicpH, but less so with increasing pH, being soluble at highpH (9.8) (Oremland et al., 2000). SRBs such asDesulfosporosinus (aka Desulfotomaculum) auripigmentum(Newman et al., 1997) and Desulfovibrio sp. str. Ben-RA(Macy et al., 2000) will utilize As(V) and SO4

2− as electronacceptors, resulting in solid-phase As4S4 or As(III)2S3

occurring both intracellularly and extracellularly (Newmanet al., 1997), occurring as inclusion bodies (Newman et al.,1997) or as extracellular/intracellular As-S nanotubes inShewanella sp. (Lee et al., 2007; Jiang et al., 2009).

Relevance to the GIT: The above are all examples ofwhat is possible in terms of GIT microbiome activities

Fig. 5. Major sulphur-arsenic compounds discussed in review. Orpiment and realgar are poorly soluble and of low bioavailability in the GIT (Liuet al., 2008), whereas the other thiolated arsenicals shown have been documented for mammalian urine (Raml et al., 2007; Suzuki et al., 2010,blood (Naranmandura et al., 2007b; Naranmandura and Suzuki, 2008) and saliva (Wang et al. 2015b), or in mouse cecum contents incubatedanaerobically with As(V) (Pinyayev et al., 2011).



relevant to arsenic thiolation. When considering the GITin its entirety, the lowest pH conditions may allow for abi-otic H2S reduction of As(V) if H2S:As ratios are highenough to drive the reaction. Diet (e.g., sulphate levels),pH and SRB activity in the GIT will influence whether As:S precipitation reactions will occur, and consequentlyinfluence the relative toxicity of the arsenic passingthrough the GIT. Arsenic precipitation will reduce solubleAs loads circulating in the GIT, and thus would befavourable to the host although it is uncertain if or howthe microbiome would be influenced. Information regard-ing host uptake of thiolated arsenicals is quite limited,documented only with in vitro studies using cultivated celllines (Naranmandura et al., 2007; Naranmandura et al.,2009; Hinrichsen et al., 2015). Relevance to the in situ isdifficult to judge at this juncture.

Arsenic bioaccumulation

Recent studies have illustrated As(V) uptake and incorpo-ration into microbial biomass (Takeuchi et al., 2007; Pan-dey and Bhatt, 2015; Wang et al., 2018), implying thatAs(V) is not inert in a biochemical context and that micro-bial bioaccumulation can reduce host exposure. Wanget al. (2018) demonstrated As(V) enhanced growth of Pistressed Agrobacterium tumefaciens. The specific bio-chemical pathways and enzymes involved are unknown,although cellular localization work suggests lipid incorpora-tion (Wang et al. 2015; Wang et al., 2017; Wang et al.,2018). Arsenolipids are well documented and naturallyoccurring in marine animals (Dembitsky and Levitsky,2004), macroalgae (Francesconi, 2010; Garcia-Salgadoet al., 2012), as well as cyanobacteria (Xue et al., 2017;Xue et al., 2019), providing sufficient precedent forAs(V) incorporation into specific cellular structures of vari-ous organisms, including GIT microbiome biomass. This willlikely involve stable C As bonds as in arsenohydrocarbonsor phosphonolipids, or the recently discovered 2-O-methylriboside (Glabonjat et al., 2017). Under Pi stress con-ditions, bacteria may replace up to 40% of cellular Pi by:(i) recycling phospholipids (Geiger et al., 1999); (ii) down-regulating genes encoding synthesis of Pi-rich teichoicacids (Lang et al., 1982; Salzberg et al., 2015); and(iii) synthesizing and utilizing sulfolipids (Souza et al., 2008;Zavaleta-Pastor et al., 2010). It is possible that Pi sparingby As(V) may be a fate for As(V) in the GIT microbiomes,but not yet examined at any level. As currently understood,this would require low phosphorus conditions (e.g., <5 μM)and As(V):P ratios of at least 1:1 in the GIT environment.Another mechanism to explain As bioaccumulation in

microorganisms involves arsenic binding to proteins (Shenet al., 2013) due to the strong attraction of trivalent arseni-cals for sulfhydryl groups (Shen et al., 2013) and is the pri-mary basis for As(III) being more toxic than As(V). Some

marine microbes have evolved this into an arsenic defenceresponse; e.g., Phaeodacylum tricornutum will synthesize aphytochelatin in reaction to arsenic exposure. These pep-tides have the general structure of (γ-Glu–Cys)n-Gly, wheren = 2–6 (Grill et al., 1985; Morelli et al., 2005) and in plantsare derived from glutathione (Grill et al., 1989). Thesephytochelatins sequester the trivalent arsenical, avoidingthe inactivation of critical cellular processes. A well-characterized arsenic–protein interaction involves ArsR,which outcompetes As(III) extrusion (via ArsB or Acr3)(Paez-Espino et al., 2009) so as to activate the ars-baseddetoxification system (discussed above). ArsR proteins varyin As(III) affinity (Ke et al., 2018). When expressed asrecombinant proteins in Escherichia coli, ArsR can removeup to 82% of the As(III) in a 10 ppb solution.

Relevance to the GIT: Most ars-based activities (dis-cussed above) result in As being extruded (resistancemechanisms) or other activities are employed in energy-generating transformations that take place exterior to thecytoplasm (As(III) oxidation and or Arr-basedAs(V) reduction). These activities maintain similar levels ofhost exposure, with the reduction or methylation activitiesincreasing toxicity. Conversely, microbial bioaccumulationof arsenic in the GIT may be an important outcome foringested arsenic and could conceivably be viewed as a pro-phylactic strategy of therapeutic value to reduce host expo-sure. Indeed, microbial bioaccumulation has beensuggested to represent an environmental bioremediationstrategy (Paez-Espino et al., 2009), and by extension couldbe of clinical importance, particularly for developing nationswhere filtration systems are unavailable or too expensive.In a human trial involving pregnant women in Tanzania,yogurt treatment (containing Lactobacillus rhamnosus)reduced arsenic and mercury bioaccumulation in the testsubjects (Bisanz et al., 2014), an example of how probioticscould function to reduce host exposure.

Metabolic disruption

Arsenic will seriously disrupt bacterial cell metabolism.RNASeq-based transcriptomic studies showed theeffects are global, altering the expression (+/−) of nearly500 genes encoding a broad range of cellular functionsin A. tumefaciens (Rawle et al., 2019). Prominent exam-ples include elements of the phosphorus stressresponse, carbohydrate metabolism, oxidoreductases/electron transport, as well as copper tolerance. Interest-ingly, As(III) exposure was associated with an organizeddown-regulation of >40 genes linked with iron acquisition,indicating some bacteria may close down iron uptake aspart of an As(III) response, and an example of collateraleffects of arsenic ingestion.

Such broad sweeping transcriptional changes would beexpected to result in metabolism changes, which is exactly



what has been observed. Based on mass spectrometry andnuclear magnetic resonance analyses of the same A.tumefaciens strain mentioned directly above for RNASeqwork, As(III) perturbation of metabolism is likewise global innature (Tokmina-Lukaszewska et al., 2017), involving �changes in >300 polar metabolites and ~600 non-polarmetabolites. Primary influences were documented as inter-rupted glycolysis, and disruption of two key reactions asso-ciated with carbon flow into and within the tricarboxylic acidcycle; i.e., pyruvate dehydrogenase and α-ketoglutaratedehydrogenase, as well as branched-chain α-ketoaciddehydrogenases. These enzymes contain a dihydro-lipoamide dehydrogenase subunit that is sensitive to As(III)inactivation (Bergquist et al., 2009; Afzal et al., 2012),resulting in carbon flow being diverted to amino acids(e.g., glutamate, glutamine, aspartate and alanine)(Tokmina-Lukaszewska et al., 2017).

Relevance to the GIT: A recent Pubmed search identi-fied 347 reviews discussing the importance of micro-biome metabolism to human health. Consequently, it iseasy to propose that arsenic disruption of microbiomemetabolism will have direct, significant effects on hostmetabolism, with a significant likelihood of leading to dis-ease. Below, we now review these effects in more detail.

Arsenic and the GIT microbiome

The above sections summarize known microbial interac-tions and responses to arsenic in pure cultures or in variousenvironments. However, it is reasonable to predict that ifthese genes are present in the GIT, their regulation andencoded transformations would be similar. Owing to the com-plexity of the GIT microbiome (NIH HMPWorking Group et al.,2009), the genetic and functional potential for all of the abovetransformations may be present and occurring, perhaps simul-taneously. This could result in altered community compositionand potentially lead to microbiome dysfunction, depending onthe extent of the change(s). Furthermore, the relative abun-dance and expression of specific functional genes will dictatenet community arsenical output and thus ultimately whether themicrobiome activities will be of positive or negative influence forthe host. The discussion below relates to transformationsshown in Fig. 2 and potentialmicrobiomeactivities summarizedin Fig. 3. Documented functions and genes in GIT-specific set-tings, the controlling factors, and the pros and cons of eachwithrespect to host exposure andwell-being are assessed.

The GIT microbiome will encounter ingested arsenic inthe oral cavity, stomach, small intestine and large intestine(i.e., colon) (Fig. 1). The oral cavity offers both oxic andanoxic environments and a complex community dominatedby species belonging to six major phyla, Firmicutes, Actino-bacteria, Proteobacteria, Fusobacteria, Bacteroidetes andSpirochetes (Verma et al., 2018). The Human Oral Micro-biome Database currently lists over 770 bacterial species,

including the dental plaque forming Staphylococcus and avariety of Campylobacter species. The nasal cavity wouldbe the route for inhaling volatile arsenicals or arsenic-laceddust (e.g., chicken litter and feed). The oral and posteriornasal flora differ, with the latter dominated byActinobacteriaand Firmicutes (Bassis et al., 2014; Koskinen et al., 2018).The stomach microbiome has five major phyla: Firmicutes,Bacteroidetes, Actinobacteria, Fusobacteria and Prote-obacteria, with species of Prevotella, Streptococcus,Veillonella, Rothia and Haemophilus, and the occasionalHelicobacter pylori (Nardone and Compare, 2015) Thelarge intestine has the greatest species richness (Qin et al.,2010). Major phyla are Firmicutes, Bacteroidetes,Actinobacteria, Fusobacteria andProteobacteria, however,the number of genera represented is far greater andincludes a wide range of physiological and biochemicaldiversity (Agioutantisa and Koumandou, 2018). Initial highthroughput 16S rRNA gene sequence analysis provided aglimpse of the overall species diversity of the human micro-biome, but more recentmetagenomic studies have revealedthe full genetic diversity and rich gene pool. This is particu-larly true for arsenic resistance genes.

As described above, both As(III) oxidation andAs(V) reduction can and will occur independent of redoxconditions and thus can occur anywhere along the GIT.Respiratory As(V) reduction requires anaerobic condi-tions and thus should be viewed as more relevant to thelumen interior. Oxygen is secreted from the gut epithe-lium, especially the small intestine (Fig. 4), generatingsteep oxygen gradients extending out roughly 1 mm(Espey, 2013), providing a niche for O2-linked As(III) oxi-dation and O2-linked metabolisms (Donaldson et al.,2016). Thus it is reasonable to imagine how O2 availabil-ity at this precise location could be of significant value tothe host, allowing conversion of more toxic trivalentarsenicals [e.g., As(III) or MAs(III)] to the less toxic penta-valent species [e.g., As(V) or MAs(V)] (Figs 2C and 4).As(III) oxidation is clearly possible in the GIT, not only byO2 but also via alternative electron acceptors such asnitrate, which is easily possible and illuminates the role ofdiet when attempting to fully understand causes andgene penetrance of arsenicosis. Whether diet can ulti-mately alter the redox balance towards As(III) or As(V) isa topic worthy of examination.

Arsenic perturbation of the microbiome

Functional and phylogenetic change. Several studies haveexamined how and to what extent dietary arsenic impactsthe gut microbiome, documenting change in at least two per-spectives. In the functional context, a pioneering rumen-based study found fermentation rates were inhibited ~30%by the addition of ~30 μM As(III) to rumen microflora enrich-ments; levels of As(III) required to inhibit fermentation wereless than that toxic to the animal itself (Forsberg, 1978). In a



phylogenetic context, studies examining insects, fish,rodents and ruminants (Schramm et al., 2003; Pass et al.,2015; Dahan et al., 2018; Gaulke et al., 2018; Liu et al.,2018; Xue et al., 2019b) are congruent in illustrating thatarsenic alters the microbiome community composition.Changes corresponded to increases in Bacteroidetes anddecreases of Firmicutes. Although, when assessed at thesub-phylum taxonomic level the classes Bacilli and Clos-tridia actually increase (Lu et al., 2014), illustrating howreporting community responses at only the phylum level ismisleading and fails to recognize potentially importantchanges in key populations as defined at the genus level oreven as OTUs.

Arsenic effects on human gut microbiomes show similartrends, and in some cases begin to shed light on how suchchanges may relate to the manifestation of humanarsenicosis symptoms. The New England region of theUnited States has elevated arsenic due principally to naturalgeologic inputs. Analysis of stool microbiome composition of204 New Hampshire infants found several genera within theFirmicutes to be suppressed; 15 genera were negatively cor-related with urine arsenic levels, including Bacteroides andBifidobacterium (Hoen et al., 2018). A comparison of stoolmicrobiomes of Bangladesh children exposed to 10 ppb ver-sus >50 ppb arsenic in drinking water (Dong et al., 2017)revealed microbiome compositional shifts, with Prote-obacteria and arsB and arsC copy number increasing at thehigher arsenic exposure (Dong et al., 2017). To our knowl-edge, this is the only example where relevant microbial func-tional genes were examined or recorded as shifting inresponse to host exposure to arsenic and provides the firsthint of the microbiome physiologic responses to be expectedor predicted. In another Bangladesh study (250 human vol-unteers), arsenic-associated microbiome perturbations rev-ealed that the relative abundances of the familyAeromonadaceae and genus Citrobacter were significantlyassociated with intima-media thickness, a surrogate markerfor the cardio-vascular disease atherosclerosis (Wu et al.,2019). Obviously, correlative data are only suggestive butnevertheless may provide important clues for future workthat links the microbiome with host health.

Basis for change. In the context of arsenic exposure,changes in microbiome community composition can derivefrom several factors. Variable As tolerance and detoxificationcapacity among genera and species are well documentedand could result in selective sweeps of the microbiome, per-haps becoming permanent if selection pressure is constant.The ability to utilize As(III) or As(V) for energy generationwould provide a distinct advantage. Furthermore, productionof a toxin such as MAs(III) by even a low abundance organ-ism could have devastating effects on microbiome neigh-bours (Li et al., 2016) that lack the ability to eliminate it fromthe cell (ArsP) or detoxify it (ArsI or ArsH) (Chen et al.,2018). Production of arsenic-based antibiotics such asarsinothricin (Kuramata et al., 2016) could have similareffects, though moderated by ArsN that inactivates it (Nadaret al., 2019) and arsenic resistance acquired from spontane-ous mutations in vivo will also account for some changes.These properties can vary at the species level or evenbetween strains of the same species (Macur et al., 2004),

and so caution must be exercised when attempting to relatecommunity changes to function following arsenic exposure.Associating cause and effect can be elusive and potentiallynear meaningless, particularly if the analyses are at highertaxonomic levels.

Arsenic-associated microbiome compositional changesmay also relate to modified host metabolism that thenimposes selection effects on the microbiome, but that do notinvolve direct arsenic toxicity effects on the microbial cell. Itis reasonable to predict that microbiome community changesderive from host selection can, in turn, result in altered arse-nic transformations and net microbiome arsenic species out-put. Arsenic-induced mouse microbiome perturbations havebeen related to significant changes in host serum profiles offatty acids, phospholipids, sphingolipids, cholesterols andtryptophan (Xue et al., 2019b). Other mouse gutmicrobiome-related changes included downregulation of hostgenes encoding one-carbon and glutathione metabolisms,significantly decreased liver S-adenosylmethionine levels,and numerous genes encoding various functions associatedwith hepatocellular carcinoma were significantly altered (Chiet al., 2019). Arsenic exposed adult mice display higherlevels of CC chemokines, and pro-inflammatory and anti-inflammatory cytokine secretion in the intestine accompaniedby significant changes in the gut microbiome (Gokulan et al.,2018). It is also reasonable to assume microbiome change/differences relate to host genotype as shown in significantmicrobiome compositional differences between wild-type andinterleukin-10 knockout mice that in turn was tied to alteredarsenic metabolism (Lu et al., 2014b), supporting the linkbetween microbiome composition and pre-systemic arseni-cal metabolism. When the murine pathogen Helicobactertrogontum was used to ‘perturb’ a mouse microbiome, sub-sequent arsenic exposure resulted in generally higher levelsof As(V) and lower levels of MAs(III) and DMAs(V) relative to‘uninfected’ mice (Lu et al., 2013). This is an excellent exam-ple where microbiome composition influences arsenic bio-transformation potential, particularly methylation, and in thiscase resulted in reduced host exposure to the more toxicarsenicals.

Genetic potential for microbiome As transformations inthe GIT

Microbiome characterization studies use high throughputsequencing approaches that generate high-level taxondescriptors. Taxonomic resolution can often be limited touncharacterized OTUs within a phylum. This provides lit-tle information about the organism identified and con-strains any understanding of whether the representedorganism has any As metabolic/transformation potential.By contrast, functional gene analysis will provide informa-tion regarding transformation potentials, although inter-pretations or predictions become difficult for microbiomescontaining multiple functionalities. For example, co-existence of As(III) oxidizers and As(V) reducers in thesame environment can favour either As(III) oxidation orAs(V) reduction (Macur et al., 2001; Macur et al., 2004;



Hoeft et al., 2010). However, unless utilizing radiolabeledAs (Hoeft et al., 2010), quantifying each activity is difficultbecause one or the other will dominate the HPLC-ICP-MS output that measures the net/dominant redox activity,but does not account for As(III) $ As(V) cycling. Further-more, being able to define specific genera is not neces-sarily reliable for predicting which type of activity ispresent. These properties can vary at the species level oreven between strains of the same species (Macur et al.,2004). Further complicating matters, the net arsenictransformation phenotype of an organism may be As(III)oxidation if it also carries the aioBA genes (Kashyapet al., 2006). In fact, all organisms isolated and character-ized as As(III) oxidizers also carry the ars genetic deter-minates for resistance-based As(V) reduction.

The above caveats and complexities notwithstanding,a functional biomarker approach to assess the presenceof key genes (Dietert and Silbergeld, 2015) shouldbecome the standard for examining GIT microbiome Asmetabolic potential. Gene selection ideally would encom-pass as many functions as possible (see Figs 2 and 3).Very few human microbiome relevant microorganismscarry a full suite of ars genes (arsA, arsB, arsC, arsD,arsR, aqp and usp) (Isokpehi et al., 2014), but in realityan organism would only need an arsC and arsB/acr3 andperhaps a means of regulation (typically arsR) in order tocontribute to pre-systemic host As metabolism.

We conducted BLASTp searches of stool microbiomemetagenomes reported for 25 randomly selected healthyindividuals (Table 2), focusing on functions depicted inFigs2and 3. For ArsC, we considered the two major ArsC func-tional classifications; grx type and trx type based on theiractivity linked to glutaredoxin or thioredoxin, and that repre-sent Gram-negatives and positives that are well docu-mented to occur and/or particularly abundant in the GIT(E. coli grx type and Bacteroides trx type) and also a goodrepresentative Gram-positive (Faecalibacterium trx type).Similarly for ArsB/Acr3, the search queries represented wellknown and abundant GIT community genera: e.g.,Escherichia, Bacteroides and Faecalibacterium. Usingboth query sequences from Gram+ and Gram– organismsallowed us to capture some degree of phylogeneticdiversity. However, we note that there is a fair amount ofphylogenetic diversity within each well-known arsenic trans-formation activity (e.g., Dunivin et al., 2019) and thus a com-prehensive search would require many different querysequences. For other functions for which less is known,queries used amino acid sequences of known and charac-terized proteins. Hits were screened by the expected proba-bility of a random hit (E-value >10−20) and identity matchscores. The encoding genes were then examined to deter-mine if they are physically associated with other ars genes,which would indicate they are part of an organized arsenicresponse. When available for specific proteins, amino acid

sequences of hits from across the search results spectrum(i.e., best to worst) were examined for conserved aminoacids or amino acid motifs. The searched metagenomesvaried with respect to the total number of identified genes(i.e., ~36 000–300 000; Table S1), which contributed to thevariation of average hits observed per metagenome.

Arsenate reduction. ArsC was most abundant and found atthe greatest frequency (Table 2). Escherichia. coli is not adominant GIT organism, which was reflected by the lowerfrequency of its ArsC in contrast to the more abundantBacteroides and Faecalibacterium type ArsC. The respira-tory As(V) reductase, ArrAB, was far less prevalent, but nev-ertheless represented in two separate individuals andverified by amino acid alignments, phylogenetic analysis andmotif verification. Finding both ArsC and ArrA is an exampleof how this transformation activity can be redundant andpotentially additive. However, an important caveat concernsrelative expression levels of each. For example, transcrip-tional analysis of arsC and arrA genes in Shewanella ANA-3showed that the expression of arrA was induced by 100 nMAs(III), while arsC was not induced until the As(III) levelswere increased by three orders of magnitude (Saltikov et al.,2005). The level of As(III) required in the GIT for arsC to beactivated is currently unknown. Another factor contributing tothis regulatory quagmire is that arrA expression is repressedby other electron acceptors such as nitrate and fumarate.

Arsenite oxidation. As(III) oxidase belongs to the largedimethylsulfoxide (DMSO) reductase family of molyb-doenzymes (McEwan et al., 2002). There were no hits forAioA, but searches did identify other DMSO family members(e.g., formate dehydrogenase), illustrating the sensitivity ofthe search process but also illustrates that one must beaware of how similar protein sequences that turn up in suchsearches actually represent very different functions. Thegene encoding the small subunit of the arsenite oxidaseenzyme (aioB) has been reported in stool microbiomes ofalcoholic cirrhosis patients (Chen et al., 2014). It is notimmediately clear why aioB would be abundant enough todetect in the microbiomes of such individuals, although winehas drawn considerable attention in this context because ofthe arsenic content (Wilson, 2015; Vacchina et al., 2018).Higher abundances of As-relevant genes might be expectedif wines were a beverage of choice for these particular testindividuals. No ArxA proteins were found.

Other functions. ArsM was found in the stool microbiome ofseven individuals and annotated as either Bacteroides orParabacteroides proteins. In one instance the encodingarsM was found adjacent to an arsC, suggesting that thisparticular ArsM is part of an organized arsenic resistanceresponse for a (Para)Bacteroides organism. ArsM functionalvariability would contribute to inter-individual microbiomeuniqueness (Eckburg et al., 2005; O’Toole, 2012; Ukhanovaet al., 2012; Pérez-Cobas et al., 2013), which is supportedby a SHIME-based study that concluded arsenic methylationvaries between individuals and particularly when comparingadults to children (Yin et al., 2017). Again, however, thereare caveats. This study and others (Juhasz et al., 2011;Smith et al., 2014) inoculated the system with soil as a



Tab

le2.

Frequ

ency

ofva

rious

ars,

arran

daioge

nesin

themetag

enom

esof

25he

althyindividu

als

Categ

ory

Protein

andor

source

(que

ry)

Gen

ome

occu

rren

cefreq

uenc

y(of2

5)Ave

rage

hits

per

metag

enom

eE-value

rang

eID

Sco

rerang

e(%

)Adjac

enttoars

oraioge

nes?

Com

men

ts

Red

ox/ene

rgy

ArrA

20.08

�0.06

−77

to0

47–53

Not

expe

cted

Sev

eral

hits

base

don

e-va

lue

andiden

tity,

butd

idno

thav

etherequ

ired

GSPNNISHSSVCAEAHKMG

Mo-bind

ingdo

main

ArxA

00

NA

NA

NA

AioA

00

NA

NA

NA

Que

riesfoun

don

lyform

ate

dehy

drog

enas

eArs

ArsH

10.04

6×10

−39

63%

Yes

ArsI

00

NA

NA

NA

ArsJ

00

NA

NA

NA

ArsJfrom

Ano

xybac

illus

and

Pse

udom

onas

used

asqu

eries

ArsN

00

NA

NA

ArsP

255�

2.9

−20

to−51

24–42

Yes

Verified

‘TPFCSCSXXP’as

define

dby

Che

net

al.

(201

5b)

ArsB/Acr3(As(III)ex

trus

ion)

E.c

oliA

rsB

20.08

�0.27

−63

to−65

97–98

Yes

Bac

teroides

Acr3-2

254.4�

2.4

−20

to0

60–10

0Yes

Allhits

≥60

%ID

toqu

ery

Fae

calib

acteriu

mAcr3

255.0�

2.6

−20

tp0

60–10

0Yes

Allhits

≥60

%ID

toqu

ery

ArsC

(As(V)redu

ctas

e)E.c

oli

10.04

−22

to−24

40Yes

Bac

teroides

2513

.8�

7.8

−20

to−81

51–99

No

Adjac

enttomismatch

repa

irDNAglycos

ylas

eFae

calib

acteriu

m24

8.8�

6.1

−20

to0

51–10

0No

Adjac

enttoice-structuring

protein

ArsM

(As(III)

methy

ltran

sferas

e)Bac

illus

sp.C

X-1

00

00

NA

Clostrid

ium

Rho

dop

seud

omon

asHalob

acteriu

mMetha

noce

lla

71.1�

0.35

−39

to−42

37–46

One

hita

djac

ent

toan

arsC

Allqu

eriesge

neratedthesa

me

hits

andallB

lastpiden

tified

asBac

teroides

orParab

acteroides

arse

nite

methy

ltran

sferas

e

Hum

anmicrobial

commun

ities

from

theNationa

lIns

tituteof

Hea

lth,U

SA.



source of arsenic to simulate soil-borne exposure routes.Consequently, a fair degree of caution must be used in inter-preting such results; i.e. did the ArsM activity originate fromthe faecal inoculants or the soils?

ArsP was found in all microbiomes. Very few ArsP pro-teins have been characterized and so besides E-values andidentity scores, our searches relied heavily on finding the‘TPFCSCSXXP’ motif (Chen et al. 2015b) in amino acidalignments. At present, it is not clear if this motif alone wouldsuffice to enable MAs(III) extrusion, but Chen et al. (2015b)showed that it is an essential property of a functional ArsP.Genes encoding Acr3 were abundant for the dominant gen-era Bacteroides and Faecalibacterium (found in all25 metagenomes), but much less so for E. coli and againconsistent with the lower prevalence of the latter (Table 2).Of the other Ars functions, ArsH was observed in a singleindividual, but no hits were found for ArsI, ArsJ and ArsN,even when using less selective E-values (Table 2).

We also surveyed nares, buccal and gingiva. There was alimited degree of overlap as only a few individuals had all com-ponents of their GIT sampled. Thus, searches of the upperGIT had to be expanded to include additional individuals. Ofthese, 21 buccal, eight nares and one gingiva metagenomesyielded positive hits for arsenic genes. BLASTp searches werenegative for ArrA, ArxA, AioA and ArsM, however as with thelower GIT, apparent ArsP homologues were present. Again,ArsC and ArsB were identified in nares, buccal and gingivametagenomes. Three individuals had arsenic resistancegenes (i.e. arsRBC) in at least one component of the upperGIT and lower GIT, and one individual had resistance genesthroughout (e.g., buccal, nares and lower GIT). Although themetagenomes used were believed to be from healthy individ-uals (based on the annotation), it is noteworthy that in additionto E. coli and Actinobacillus sp., the ArsC homologues identi-fied included those from Neisseria, Moraxella, Klebsiella andHaemophilus species.

Evidence of microbiome As interactions

In theory, almost any known microbial As transformationshould be at least possible in the GIT microbiome envi-ronment (an exception presumably being phototrophy-linked As(III) oxidation as mentioned above). Residencetime in the oral cavity is relatively short, but evidencesuggests that it is still relevant for assessing the overallbudget of arsenic ingestion, absorption and excretion.Methylarsines are garlicky in odour and garlic odours inbreath are often associated with acute arsenic exposure,and thus suggests arsenic metabolism in the oral cavity.Simulated digestions of certified reference seafoods withartificial saliva resulted in substantial release of arsenic(Leufroy et al., 2012). In a modified SHIME experimentthat included a simulated oral chamber containing sali-vary bacteria, partial digestion of arsenic-containing rice,mussels and seaweed resulted in significant arsenicrelease (primarily arsenosugars) from the mussels andseaweed (Calatayud et al., 2018).

Considering the lower regions of the GIT, microbiomemethanogens (Miller et al., 1982, 1986) should be capableof reductive methylation of arsenate (McBride and Wolfe,1971), attributable to a minor methyl transferase side reac-tion occurring between a methyl-cobalamin donor withAs(III) [and other metal(oid)s like Sb, Se, Bi, Te] (Thomaset al., 2001). Bovine rumen studies documented quantitativereduction of millimolar levels of As(V) to As(III), and that wasfaster and more complete with the provision of H2 as anexogenous electron donor as opposed to incubation underonly N2 (Herbel et al., 2002). Similarly, As(V) reduction wasobserved in hamster faecal pellets from As(V)-watered andcontrol animals; however, samples from As(V)-watered ani-mals were obviously primedwith respect to gene expressionor selection for As(V) tolerance/utilization, resulting in signifi-cantly faster As(V) reduction than controls. Use of 73/74As(V) radiotracers in similar experiments but at low concentra-tion As(V) incubations (20 μM) achieved greater temporalsensitivity, demonstrating immediate As(V) reduction toAs(III). In this instance, arsenic was mostly recovered in thesolid phase, likely as an 73/74As(III)2S3-precipitate facilitatedby concurrent sulphate-reduction as the source of sulphide(Herbel et al., 2002). These experiments are important inillustrating how SRB activity can ameliorate toxicity;i.e. precipitating the arsenic as a poorly soluble solid phase(likely orpiment), which is excreted and thus reducing hostexposure.

In a comparison of wild type and As3mt− knockout (lackshost arsenic methyltransferase) mouse genotypes fedAs(V) (Naranmandura et al., 2013), As(III) was clearly thedominant arsenical found in the mutant mouse liver andurine. These mice were not germ-free and so the relativecontribution of the GIT microbiomes to As(V) reduction isunknown but is a reasonable expectation that GIT microor-ganisms could contribute in this manner to pre-systemicmetabolism. This is supported by a SHIME-based study,wherein the colon component registered near-completeAs(V) reduction (Yin et al., 2015), implying the capacity to doso in theGIT.

Aerobic incubation of colon material collected from aSHIME system resulted in extensive As(III) oxidation (Alavaet al., 2011), illustrating that the genetic potential (presum-ably aio genes) was present and could be activated. How-ever, under anaerobic conditions no As(III) oxidation wasapparent relative to initial levels of As(V) and thus the capac-ity to oxidize As(III) anaerobically was not detectable. Knownalternate electron acceptors that would support anaerobicAs(III) oxidation (i.e. NO3

−) would not be expected to be pre-sent in a typical SHIME system (Molly et al., 1993) unlessspecifically tested by adding them into themix.

At present, it is uncertain whether microbiome As(III)methylation activity is of positive or negative value to thehost, but this is an example of a GITmicrobiomeAs transfor-mation activity that requires significant scrutiny in order to



understand the consequences for host arsenic exposure.SHIME-based human microbiome studies have demon-strated As methylation [DMAs(V) and MMAs(V)] in simu-lated transverse and descending colon sections (Yin et al.,2015; Yu et al., 2016), showing the genetic and physiologiccapacity to contribute this type of activity. The preponder-ance of pentavalent methylated species in various studiesmay be an artefact of abiotic auto-oxidation of trivalent spe-cies under aerobic cultivation conditions, in aerobic purifiedenzyme reaction conditions, and or handling of metaboliteextracts prior to analysis. Discussion and interpretation ofthese and future experiments involving microbiome ArsMactivity is not trivial nor simply an interesting biochemicalpuzzle. Depending on whether microbiome methylationactivity generates trivalent or pentavalent arsenicals in largepart dictates the relative toxicity of this reaction series andoutcomes, and potentially account for variability in arsenic-related disease (Valenzuela et al., 2009; Engström et al.,2011, 2013). In the GIT environment, with the exception oflumen epithelial (Fig. 4), the predominance of anaerobicconditions would be predicted to not favour the spontaneousoxidation of trivalent methylated arsenicals, and thus thequestion: Is microbiome As(III) methylation of any value tothe host or is it a source of significant toxicity? By contrast,would the availability of oxygen near the lumen epitheliumoxidize these trivalent species prior to uptake (Fig. 4)?Detection of arsH (one individual) and arsP (allmetagenomes) in our survey (Table 2) further complicatesthis question. While we did not detect ArsI in the survey(Table 2), an in vitro dynamic gut simulator study (Rubinet al., 2014) involving human faecal inoculant documenteddemethylation of MMAV to AsIII, indicating ArsI type activity.Extrusion ofMAs(III) from the bacterial cell (via ArsP), oxida-tion of MAs(III) to MAs(V) (via ArsH), or conversion of triva-lent organo-arsenicals back to As(III) (via ArsI) (YoshinagaandRosen, 2014) could be of critical importance. The occur-rence of such enzymes in the microbial world argues thatmicrobial encounters with trivalent methylated species arenot a rare event. ArsP, ArsH and ArsI detoxification activitiesoffer clear advantages to the bacterial cell. But what aboutthe host? As is always the case, the occurrence, relativeabundances and expression of thesemicrobiome genes arethe key considerations.A recent study by Coryell et al. (2018) used a combination

of antibiotic-disturbed mouse microbiomes and germ-freemice to directly address the question of whether a gut micro-biome can be of value to the host. In this study and similarlyin a just recently reported study by Chi et al. (2019), themouse gut microbiomes performed a bioaccumulation func-tion, resulting in increased arsenic concentrations in mousestool while simultaneously reducing arsenic uptake andaccumulation in the mouse liver, spleen, heart and lung(Coryell et al., 2018). Further, humanizing germ-free As3mt−

knockout mice (hypersensitive to arsenic) with stool

homogenates from different human donors clearly demon-strated how the GIT microorganisms provide a protectivemeasure by significantly reducing arsenic-induced mortality,although varying between human stool donors (Coryellet al., 2018). Illumina 16S metagenome analysis of thesearsenic-exposed humanized mouse microbiomes suggestedFaecalibacterium plays an important role, and indeed miceco-colonized with F. prausnitzii and E. coli (E. coli helps sta-bilize F. prausnitzii) mice were afforded significantlyincreased protection against arsenic relative to mice mono-associated with E. coli (Coryell et al., 2018).

Evidence of potential microbiome arsenic thiolation activ-ity stems from studies of rat caecal contents. Anaerobicin vitro incubations of samples fed As(V) resulted in the gen-eration of As(V)S, dithioarsenate (As(V)S2), and As(V)S3,MAs(V), methyldithioarsenate (MAs(V)S2), DMAs(V)S2,and methyltrithioarsenate (MAs(V)S3) (Pinyayev et al.,2011). Similar experiments documented transformation ofDMAs(V) to DMAs(V)S, DMAs(V)S2, and trimethylarsinesulphide (TMAs(V)S) (Kubachka et al., 2009). Presumably,SRB were present and responsible for generating the H2S(discussed above) and indeed evidence of SRB involve-ment is strong. SHIME-based studies with human faecaland colon microbiome inoculants fed MAs(V) resulted inextensive MAs(V)S synthesis (Rubin et al., 2014). Produc-tion of H2S and MAs(V)S were greatly enhanced by theaddition of sulphate to the incubations and correlated with asubstantial increase in Desulfovibrio desulfuricans (piger).By contrast, H2S production and MAs(V)S synthesis wereabolished by the addition of sodium molybdate, a well-known SRB inhibitor (Kiene et al., 1986). Other interestingobservations derived from this study include (i) H2S genera-tion in these experiments varied significantly between indi-vidual humans (healthy individuals range from 120 to450 μM) (Pitcher et al., 2000); and (ii) MAs(V)S wasundetectable when H2S productionwas <~75 μM.Based onthese data and the arsenic thiolation literature reviewedabove, one might conclude that the H2S:As ratio will likelyneed to be high (~100 H2S:1 As) to facilitate the formation ofthese thiolated arsenicals. Relative differences in micro-biome SRB abundance and activity, As exposure, and die-tary sulphate would be expected to greatly influence arsenicthiolation.

Mineralization of soluble arsenic as As(III)2S3 and As4S4

serves to constrain As mobility, solubility (Cavalca et al.,2013; Altun et al., 2014; Le Pape et al., 2017) and toxicity,reducing overall host exposure and toxicity. Once formed,metal(loid) precipitates or solid forms can be quite stable.For instance, in clinical trials where human volunteers wereadministered colloidal bismuth, it was estimated that ~99%was excreted in faeces and only traces (pg/ml) were foundas CH3BiH2 in blood (Boertz et al., 2009). Long-term(2 months) administration of realgar (As4S4) to rats resultedin the accumulation of DMAs(V) in livers, although there was



no overt liver pathology (Zhou et al., 2019). In a case involv-ing a reported attempted suicide presented in an emergencyroom (Buchanan et al., 2012), the individual ingested 84 g ofAs2S3 (‘chicken egg-size rock of orpiment’), but the patient’s‘physical examination was unremarkable, and diagnostictests included a normal electrolyte panel, a normal serumlactate and a normal electrocardiogram.’ A urine sampletaken 12 h post-admission registered 1490 ppb total arse-nic, which is clearly well outside normal (<100 ppb As)(Agency for Toxic Substances and Disease Registry, n.d.),but given the large bolus of As consumed it should beviewed as exceptionally low. Clearly, a small portion of theAs became bioavailable, however, if the ingested As hadbeen a readily bioavailable form (e.g., As(III)), the patientwould not have survived (acute dose ~70–180 mg As(III),depending on body mass) (Agency for Toxic Substancesand Disease Registry, n.d.). This clinical case is consistentwith the view that mineralization of ingested As results inreduced bioavailability during GIT transit and thus reducedtoxicity. Depending on the form ingested, As bioavailabilitywill vary along the different phases of the GIT (Yin et al.,2016), and levels and rates of SO4

2− and As(V) reductionsmay have varying biochemical endpoints that vary intoxicity.

Arsenic adsorption to solid phases will also limit As bio-accessibility, again with the expectation being reduced over-all exposure and toxicity in the GIT. Various studies suggestthis may be important, although with caveats. As(V) bindsstrongly to iron, manganese and aluminium oxides(Hingston et al., 1971; Gupta and Chen, 1978; Manninget al., 2002; Dixit and Hering, 2003). As(III) binding occurswith iron (hydr)oxides (Gupta and Chen, 1978), but thissorption to iron solid phases is relatively weak (Zobrist et al.,2000), readily desorbing at circumneutral pH that occursthroughout most of the human colon. Environmental condi-tions that favour microbial reduction of eitherAs(V) (particularly dissimilatory) and or Fe3+ do not favourAs(V) adsorption (Ahmann et al., 1994; Zobrist et al., 2000;Tufano et al., 2008) suggesting the anaerobic GIT environ-ment might not be conducive to this type of (biogeo)chemis-try. In a SHIME study where FeCl3 was variously added(0-3mg/l) with 600 μg/l As(III), all added arsenic remained insolution in the simulated stomach (pH 2.0), but thendecreased significantly in the small intestine (pH ~ 7),ascending colon (5.5 < pH < 5.9), transverse colon(6.0 < pH < 6.4) and descending colon (6.6 < pH < 6.9)chambers (Yu et al., 2016). Relative to zero Fe controls, ironamendments significantly reduced soluble As in the smallintestine but not in the other chambers. A human lower GITexperimental flow-through system examined the fate offerrihydrite-adsorbed As(V) with transit through the systemin order to model bio-accessible As(V) as it is released(by desorption) from the Fe(III) (Beak et al., 2006). Bio-accessibility of the As was governed by whether the arsenic

sorption capacity of the iron solid phase (ferrihydrite) wasexceeded; if the systemwas spikedwithAs:ferrihydrite sam-ples where the As sorption capacity was not exceeded, thenbio-accessible As was below the detection limit of theirassay. While this was basically a risk assessment study, it isalso informative in the context of host exposure if ingestedAs undergoes sorption reactions while transiting theGIT.

Future directions and concluding remarks

Looking forward, there is much to be done to understandthe response of our microbiomes to the presence of Asoxyanions or any environmental toxicant. SHIME studieshave been valuable in demonstrating the potential for dif-ferent microbial As transformations, but this approachessentially involves As enrichments that select for micro-biome members capable of metabolisms of interest. Thetime lag typically observed suggests significant selectionis occurring, implying that the particular functions/trans-formations being observed are not dominant and do notrepresent equilibrium conditions of the sampled material.We suggest future work should shift to in situ orientedstudies.

Much can be learned from work with germ-free micewherein microbiomes can be customized to focus on spe-cific arsenic transformations. The use of germ-free micewill also avoid the vendor-based variability in microbiomecomposition (Velazquez et al., 2019) that will likelybecome very important as efforts begin to focus on spe-cific members of the GIT microbiome and for systemati-cally comparing results between studies. Mousegenotype can also be manipulated in the germ-free set-ting, allowing for gene-for-gene strategies. To be sure,such approaches are reductionist, but nevertheless allowfor genetically clean comparisons that will provide impor-tant baseline answers to modest questions such as:‘What microbial activities are harmful to the host?’ At thisjuncture, the answer to even this simple query involvessignificant conjecture. To the extent possible, futurecommunity-wide efforts would benefit from experimentalmice having standardized GIT microbiomes, perhapsinvolving a sponsored centralized service lab(s).

The omics-level work that dominates the recent litera-ture has been very important for demonstrating significant(dramatic in some cases) microbiome community shiftsupon As exposure. However, there is much to be done atmore refined taxonomic levels. We predict that autecolog-ical strategies and approaches will be important, if notessential, complements to the community-level efforts,and that some of the most important work will take placeat the strain level. Much effort is required to better under-stand how ingested arsenic is perceived by the microbesalong the different phases of the GIT. Clearly, as hasbeen demonstrated in the above-reviewed studies, a



significant bolus brings about changes in the microbiome.But as an example of the fundamental work that isneeded, we are unaware of any studies that haveassessed the minimal As(V) or As(III) levels required toelicit induction of the ars genes in vivo. What proportionof the ingested arsenic actually makes its way throughthe entire GIT? There is a broad bandwidth of possibleinteractions predicted or possible (Figs 2 and 3), butcharacterizing them in reasonable detail will require con-siderable effort. What type of microbe–microbe interac-tions occur or are important, both in a positive as well asa negative sense? Microbiome responses and microbialtransformation notwithstanding, abiotic chemical reac-tions occurring in the GIT are also an important topic(particularly thiolation reactions).One final point bears mentioning. Microbiome investi-

gations often emphasize gross population shifts(e.g., 16 S rRNA genes) with respect to changing envi-ronmental parameters. But steadily introduced low micro-molar quantities of As(V) into the GIT are less likely tocause notable shifts in sub-populations that can exploitthis oxyanion for growth. Thus, it is important that suchefforts focus on functional arsenic genes (e.g., arsC,arrA, aioA, arxA) in addition to phylogenetic composi-tional parameters, and include transcriptomic approachesthat would delineate whether such genes are expressedwhen the resident sub-population is exposed to arsenicoxyanions. Moreover, because not all such genes havebeen identified in enteric genomes despite the capacityof some species to respire arsenate (e.g., Citrobacterstrain TSA-1), there is ample room for discovery of ‘novel’biochemical mechanisms and their encoding genes tocarry out such reactions (Switzer Blum et al., 2018).While this type of research will be conducted in the con-text of human health and welfare, undoubtedly our under-standing of how and why microbes react to arsenic willgreatly expand.

Acknowledgements

T.R.M. acknowledges primary support for this review fromthe National Institutes of General Medical Sciences and theNational Cancer Institute (R01CA215784), and additionalsupport from the National Science Foundation Systems andSynthetic Biology Program (MCB-1714556), and MontanaAgricultural Experiment Station (Project 911310). J.F.S. andR.S.O. acknowledge support from NASA NNX09AW41G.We also thank Alyssa Veliz in assisting with the upper GITmetagenome searches.

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Supporting Information

Additional Supporting Information may be found in the onlineversion of this article at the publisher’s web-site:

Table S1. List of metagenomes searched via Blastp at theJoint Genome Institute Metagenome Host-Associated data-base. All metagenomes are from the same study: Humanmicrobial communities from the National Institute of Health,USA, HMP



arsenic and the gastrointestinal tract microbiome

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