Comamonas testosteroni antA encodes an antimonite-translocating P-type ATPase

Highlights

• The ant operon is specifically induced by Sb(III).

• AntA specifically confers the resistance to Sb(III).

• AntA is a new subclass of ATPases named as P_1B-8.

• The ant-operon is the first report to specifically detoxify Sb(III) in bacteria.

Abstract

Antimony, like arsenic, is a toxic metalloid widely distributed in the environment. Microbial detoxification of antimony has recently been identified. Here we describe a novel bacterial P_1B-type antimonite (Sb(III))-translocating ATPase from the antimony-mining bacterium Comamonas testosterone JL40 that confers resistance to Sb(III). In a comparative proteomics analysis of strain JL40, an operon (ant operon) was up-regulated by Sb(III). The ant operon includes three genes, antR, antC and antA. AntR belongs to the ArsR/SmtB family of metalloregulatory proteins that regulates expression of the ant operon. AntA belongs to the P_1B family of the P-type cation-translocating ATPases. It has both similarities to and differences from other members of the P_1B-1 subfamily and appears to be the first identified member of a distinct subfamily that we designate P_1B-8. Expression AntA in E. coli AW3110 (Δars) conferred resistance to Sb(III) and reduced the intracellular concentration of Sb(III) but not As(III) or other metals. Everted membrane vesicles from cells expressing antA accumulated Sb(III) but not As(III), where uptake in everted vesicles reflects efflux from cells. AntC is a small protein with a potential Sb(III) binding site, and co-expression of AntC with AntA increased resistance to Sb(III). We propose that AntC functions as an Sb(III) chaperone to AntA, augmenting Sb(III) efflux. The identification of a novel Sb(III)-translocating ATPase enhances our understanding of the biogeochemical cycling of environmental antimony by bacteria.

Introduction

Antimony belongs to the 15th main group in the periodic table, located below arsenic and has similar chemical and toxicological properties to arsenic (Niels and Diethard, 1999). It exists primarily in two valence states, highly toxic trivalent antimonite (Sb(III)) and less toxic pentavalent antimonate (Sb(V)) (Li et al., 2016). Antimony has many industrial uses, including production of alloys, paint pigments and semiconductors, and occupational exposure is the major threat of antimony to human health(Li et al., 2016; Montserrat et al., 2002; Ettler et al., 2007). Chronic exposure to antimony causes respiratory problems, lung damage, cardiovascular effects, gastrointestinal disorders as well as reproductive problems. Thus, in addition to environmental exposure, anthropogenic activities are expanding antimony exposure world-wide (Montserrat et al., 2002; Ettler et al., 2007).

Bacteria are generally sensitive to antimony because Sb(III) enter cells though aquaglyceroporins such as the glycerol facilitator GlpF(Sanders et al., 1997). Many bacteria have evolved arsenic resistance (ars) genes that confer high-level tolerance to both arsenite (As(III)) and Sb(III) by oxidation, methylation or efflux(Shi et al., 2018; Meng et al., 2004; Li et al., 2015). The first identified Sb(III) resistance was the Escherichia coli arsB Sb(III)/H⁺ antiporter(Meng et al., 2004). A second Sb(III) efflux permease, ArsK, was recently identified in ars gene clusters(Shi et al., 2018). While ArsB and ArsK are both secondary efflux permeases, they are unrelated and belong to different transporter families. Secondary carriers that couple to the proton motive force are not nearly as effective in reducing intracellular concentrations of toxic compounds as primary pumps that couple to chemical energy such as ATP (Rosen and Kashket, 1978). For example, ArsB has the ability to form a complex with the ArsA ATPase to become a primary ATP-coupled Sb(III) pump that confers considerably elevated Sb(III) resistance(Meng et al., 2004). E. coli in which the ars operon was deleted is hypersensitive to Sb(III); expression of the arsB Sb(III)/As(III) efflux permease increases resistance 10-fold, while expression of the arsAB efflux ATPase increase resistance an additional 10-fold (Meng et al., 2004).

One of the largest superfamily of primary pumps is the P-type ATPase group (Chan et al., 2010). Members of the subgroup of P_IB-type ATPases are widely distributed in prokaryotes, plants and animals (Arguello et al., 2007). Their substrates include Cu(I), Cu(II), Ag(I), Zn(II), Cd(II), Pb(II) and As(III), and they play key roles in metal ion homeostasis. In archaea and bacteria, P_1B-type ATPases confer tolerance to toxic metals by extrusion from cells (Arguello et al., 2007; Smith et al., 2014). In general, P_IB-type ATPases consist of three conserved domains: (1) a membrane domain with 6–8 transmembrane helices (TMs); (2) an ATP-binding domain (ATP-BD); and (3) an actuator domain (AD), which transmits conformational changes in the ATP-BD to the transmembrane region, driving the catalytic cycle. In addition, some P_IB-type ATPases also have one or more metal binding domains (MBDs) at the N- or C-terminus of the protein that play roles in regulating the rate of transport (Smith et al., 2014; Arguello et al., 2016). TM helices 6, 7 and 8 have highly conserved residues that determine substrate specificity in the metal binding site (TM-MBS) (Smith et al., 2014; Andersson et al., 2014). P_1B-ATPases are divided into seven subfamilies according to substrate specificity and conserved residues at the TM-MBS (Arguello et al., 2007; Smith et al., 2014). To date the substrates of P_1B-ATPases are primarily metals. Only one P_1B-2 type subfamily ATPase has been reported to confer resistance to both the metal Cd(II) and the metalloid As(III) (Antonucci et al., 2017; Antonucci et al., 2018). In this study we identify a novel microbial P_1B-type ATPase, AntA, an Sb(III)-specific efflux ATPase that confers resistance to the metalloid Sb(III). AntA has sufficient sequence differences from other P_1B-type ATPases to suggests it should be considered the first identified member of a new P_1B-8 ATPase subfamily.

The antA gene one of three in the antRCA operon in which antR encodes a member of the ArsR-SmtB family of metalloregulatory proteins (Busenlehner et al., 2003). These transcriptional repressors respond to a variety of metal/metalloid ions such as ArsR (As(III), Sb(III), Bi(III)), SmtB (Zn(II)), CadC (Cd(II)), AztR (Zn(II)) and CmtR (Cd(II)) (Busenlehner et al., 2003; Arunkumar et al., 2009; Chen et al., 2017; Ordonez et al., 2008; Prabaharan et al., 2019). In the absence of metal ions, the homodimeric repressor binds to the promoter, preventing transcription. When substrate binds, the repressor dissociates from the promoter, allowing gene expression (Busenlehner et al., 2003; Arunkumar et al., 2009; Ordonez et al., 2008). AntR is highly selective for Sb(III), sensing environmental antimony to drive transcription of the antA and antC genes. Associated with some P_1B-type ATPases are small metal-binding proteins that serve as chaperones to bind and deliver substrate to the transporter. AntC is a small protein with a potential Sb(III) binding site, and we propose that it is an Sb(III) chaperone for the AntA ATPase. This is the first report of a genetic determinant for detoxification of environmental antimony by an Sb(III)-specific P_1B-type ATPase that sheds light on how life has adapted to the presence of this toxic metalloid.