Co-expression of Cyanobacterial Genes for Arsenic Methylation and Demethylation in Escherichia coli Offers Insights into Arsenic Resistance

Abstract

Arsenite [As(III)] and methylarsenite [MAs(III)] are the most toxic inorganic and methylated arsenicals, respectively. As(III) and MAs(III) can be interconverted in the unicellular cyanobacterium Nostoc sp. PCC 7120 (Nostoc), which has both the arsM gene (NsarsM), which is responsible for arsenic methylation, and the arsI gene (NsarsI), which is responsible for MAs(III) demethylation. It is not clear how the cells prevent a futile cycle of methylation and demethylation. To investigate the relationship between arsenic methylation and demethylation, we constructed strains of Escherichia coli AW3110 (ΔarsRBC) expressing NsarsM or/and NsarsI. Expression of NsarsI conferred MAs(III) resistance through MAs(III) demethylation. Compared to NsArsI, NsArsM conferred higher resistance to As(III) and lower resistance to MAs(III) by methylating both As(III) and MAs(III). The major species found in solution was dimethylarsenate [DMAs(V)]. Co-expression of NsarsM and NsarsI conferred As(III) resistance at levels similar to that with NsarsM alone, although the main species found in solution after As(III) biotransformation was methylarsenate [MAs(V)] rather than DMAs(V). Co-expression of NsarsM and NsarsI conferred a higher level of resistance to MAs(III) than found with expression of NsarsM alone but lower than expression of only NsarsI. Cells co-expressing both genes converted MAs(III) to a mixture of As(III) and DMAs(V). In Nostoc NsarsM is constitutively expressed, while NsarsI is inducible by either As(III) or MAs(III). Thus, our results suggest that at low concentrations of arsenic, NsArsM activity predominates, while NsArsI activity predominates at high concentrations. We propose that coexistence of arsM and arsI genes in Nostoc could be advantageous for several reasons. First, it confers a broader spectrum of resistance to both As(III) and MAs(III). Second, at low concentrations of arsenic, the MAs(III) produced by NsArsM will possibly have antibiotic-like properties and give the organism a competitive advantage. Finally, these results shed light on the role of cyanobacteria in the arsenic biogeochemical cycle.

In vitro model insights into the role of human gut microbiota on arsenic bioaccessibility and its speciation in soils

“…The arsenic speciation was determined by high-performance liquid chromatography coupled with inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). A Hamilton PRP-X100 column (250×4.6 mm, 10 μm) (Yan et al, 2017) and a Phoenix C18 column (250×4.6 mm, 10 μm) (Yan et al, 2015) were used. The chromatographic condition details are provided in Table S1.…”

Optimization of arsB (Acr3) as a positive selection marker in the cyanobacterium Synechocystis sp. PCC 6803

“…Finally, the ΔSARSBHC strain will very useful to explore/test the function of the new putative arsenic resistance genes found in cyanobacteria, which have been poorly characterised in vivo (Huertas et al, 2014). Even though some of these genes have been used to complement hypersensitive E. coli strains to characterise their function (Yin et al, 2011; Xue et al, 2017b, 2019; Yan et al, 2017), cyanobacteria possess completely different metabolism and cell organization. For example, when analysing membrane transporters or redox proteins the results might be not clear in E. coli due the lack of proper targeting to the membranes (thylakoid or plasma membrane in cyanobacteria; (Mullineaux and Liu, 2020)) or the lack of correct redox partners which markedly differs between E. coli and cyanobacteria (Florencio et al, 2006).…”

“…The function of these genes in cyanobacteria (with the exception of arsI from Anabaena sp. PCC 7120; (Yan et al, 2015(Yan et al, , 2017) in relation to arsenic resistance remains to be determined, but it is possible that they will work like their characterised homologs. Furthermore, arsK and arsP genes homologs can be also identified, some of which are associated together with other arsenic resistance genes (our unpublished observations).…”

“…More recently it has been shown to be able to oxidise methylarsenite to methylarsenate which is much less toxic (Chen et al, 2015a). arsI codes for an methylarsenite lyase that belongs to nonheme iron-dependent dioxygenase family of proteins and catalyse the demethylation of organic arsenite (Yoshinaga and Rosen, 2014; Pawitwar et al, 2017; Yan et al, 2017). arsS codes for the first committed step in arsenosugar synthesis which uses the methylarsenite species generated by ArsM as substrate (Xue et al, 2019).…”

Arsenite efflux limits microbial arsenic volatilization

“…Since both As(III) and MAs(III) can serve as substrates for ArsM, it is inferred that factors lowering the intracellular As(III) and MAs(III) content inevitably limit As methylation and subsequent As volatilization. More recently, studies have shown that demethylation of As limited the volatilization of As [30,31]. However, it is not known whether other internal factors that reduce intracellular As concentrations would limit microbial As volatilization.…”