Environmental Microbiology (2018) 20(1), 305–323 doi:10.1111/1462-2920.13999 Arsenobetaine: an ecophysiologically important organoarsenical confers cytoprotection against osmotic stress and growth temperature extremes Tamara Hoffmann,1† Bianca Warmbold,1† transformed into organoarsenicals, which are subse- Sander H. J. Smits,2† Britta Tschapek,2 quently mineralized again into inorganic arsenic Stefanie Ronzheimer,1 Abdallah Bashir,1,3,4 compounds. Microorganisms contribute to this bio- Chiliang Chen,1,5 Anne Rolbetzki,1 transformation process greatly and one of the Marco Pittelkow,1 Mohamed Jebbar,6 organoarsenicals synthesized and degraded in this Andreas Seubert,7 Lutz Schmitt2* and cycle is arsenobetaine. Its nitrogen-containing homo- Erhard Bremer1,5** logue glycine betaine is probably the most frequently 1Laboratory for Microbiology, Department of Biology, used compatible solute on Earth. Arsenobetaine is Philipps-University Marburg, Karl-von-Frisch Str. 8, found in marine and terrestrial habitats and even in Marburg D-35043, Germany. deep-sea hydrothermal vent ecosystems. Despite its 2Institute of Biochemistry, Heinrich Heine University ubiquitous occurrence, the biological function of Dusseldorf,€ Universitats€ Str. 1, Dusseldorf€ D-402325, arsenobetaine has not been comprehensively Germany. addressed. Using Bacillus subtilis as a well- 3Faculty of Science Biology Department, Al-Azhar understood platform for the study of microbial osmo- stress adjustment systems, we ascribe here to University-Gaza, Gaza, P.O. Box 1277, Palestine. arsenobetaine both a protective function against 4Emeritus Group of R.K. Thauer, Max Planck Institute high osmolarity and a cytoprotective role against for Terrestrial Microbiology, Karl-von-Frisch Str. 10, extremes in low and high growth temperatures. We Marburg D-35043, Germany. define a biosynthetic route for arsenobetaine from 5LOEWE-Center for Synthetic Microbiology, the precursor arsenocholine that relies on enzymes Philipps-University Marburg, Hans-Meerwein Str. 6, and genetic regulatory circuits for glycine betaine for- Marburg D-35043, Germany. mation from choline, identify the uptake systems for 6 European Institute of Marine Studies, Technopole arsenobetaine and arsenocholine, and describe crys- Brest-Iroise, Laboratory of Extreme Environments, tal structures of ligand-binding proteins from the Microbiology, University of West Brittany (Brest), OpuA and OpuB ABC transporters complexed with Plouzane F-29280, France. either arsenobetaine or arsenocholine. 7Faculty of Chemistry, Analytical Chemistry, Philipps-University Marburg, Hans-Meerwein Str. 4, Marburg D-35043, Germany. Introduction Arsenic is an abundant constituent of the Earth crust and Summary is also an important constituent of the biosphere (Mukho- Arsenic, a highly cytotoxic and cancerogenic metal- padhyay et al., 2002; Oremland and Stolz, 2003; Li et al., loid, is brought into the biosphere through 2016; Zhu et al., 2017). Millions of humans suffer from its geochemical sources and anthropogenic activities. A cytotoxic and cancerogenic effects through their exposure global biogeochemical arsenic biotransformation to contaminated water sources (Oremland and Stolz, cycle exists in which inorganic arsenic species are 2005). This situation is exacerbated by the widespread use of man-made organoarsenicals for disease-prevention and Received 24 September, 2017; revised 9 November, 2017; growth promotion of animals (Yoshinaga and Rosen, accepted 16 November, 2017. For correspondence. *E-mail Lutz. 2014). As a result of its wide distribution and abundance in [email protected]; Tel. (149)-211-81-10773; Fax (149)-211-81- the environment, microorganisms have been exposed to 15310. **E-mail [email protected]; Tel. (149)-6421- 2821529; Fax (149)-6421-2828979. †These authors contributed arsenic essentially since the origin of life. A global arsenic equally to this work. biotransformation cycle exists in nature, to which microbial VC 2017 Society for Applied Microbiology and John Wiley & Sons Ltd 306 T. Hoffmann et al. Fig. 1. Osmoprotectant uptake (Opu) systems in B. subtilis and the pathway used for the synthesis of glycine betaine and arsenobetaine from their precursor molecules. The transcription of the operons encoding the choline/arsenocholine- specific ABC transporter (OpuB) and the enzymes for glycine betaine/ arsenobetaine synthesis (GbsB/GbsA) is regulated via the GbsR repressor protein. The promoters and terminators of the corresponding operons are indicated by arrows and lollipops respectively. Choline and arsenocholine bound to GbsR are represented by a black sphere. metabolic activities contribute greatly (Oremland and Stolz, for its formation rely mostly on the detection of potential 2003; Slyemi and Bonnefoy, 2012; Li et al., 2016; Zhu biosynthetic precursors and intermediates. Two proposed et al., 2017). Microorganisms have not only developed an biosynthetic pathways envision the formation of arsenobe- impressive array of systems to resist and detoxify arsenic, taine from di- or tri-methylated arsenosugars that are but they have also learned to exploit it as a means for primarily produced by eukaryotic organisms at the bottom energy-generation through oxidative and reductive bio- of the aquatic food chain. The breakdown of these organo- chemical transformations (Slyemi and Bonnefoy, 2012; Li arsenicals lead either to the formation of arsenocholine as et al., 2016; Zhao, 2016; Chen et al., 2017; Edwardson an intermediate that then could be further oxidized to and Hollibaugh, 2017; Zhang et al., 2017; Zhu et al., arsenobetaine, or to the synthesis of dimethylarsinoyl- 2017). ethanol, which could serve through several biotransforma- There is one notable exception to the severe cytotoxicity tion reactions as precursor for arsenobetaine production. of inorganic and organic arsenic-containing compounds, An alternative route for its synthesis proposes dimethylar- the organoarsenical arsenobetaine (Fig. 1); it has an esti- senite as the starting compound (Caumette et al., 2012; mated median lethal dose (LD50)inmiceofabout10 Foster and Maher, 2016; Taylor et al., 2017; Zhu et al., gkg21 body weight (Kaise et al., 1985). As arsenobetaine 2017). is widely found in marine ecosystems, humans ingest Despite the fact that arsenobetaine is an environmen- arsenobetaine primarily through the consumption of sea- tally ubiquitous organoarsenical (Ciulla et al., 1997; food and they excrete it again through their urine (Molin Caumette et al., 2012; Molin et al., 2015; Foster and et al., 2015; Thomas and Bradham, 2016; Taylor et al., Maher, 2016; Popowich et al., 2016; Taylor et al., 2017) its 2017). Since its discovery 40 years ago (Edmonds et al., potential biological function has not yet been addressed 1977), studies monitoring arsenic species in environmental systematically. The chemical relatedness of arsenobetaine and food samples consistently find arsenobetaine, often in to the nitrogen-containing and environmentally abundant substantial quantities (Caumette et al., 2012; Molin et al., glycine betaine molecule (Yancey, 2005) (Fig. 1) fostered 2015; Popowich et al., 2016; Thomas and Bradham, 2016; speculations about its possible function as an osmostress Taylor et al., 2017). Evidence for its production by microor- protectant (Peddie et al., 1994; Ciulla et al., 1997; Devesa ganisms (Ritchie et al., 2004), terrestrial fungi (Nearing et al., 2005; Nearing et al., 2015), as glycine betaine prom- et al., 2015) and phytoplankton (Caumette et al., 2012; inently plays such a role in many Prokarya and Eukarya Foster and Maher, 2016), has been presented, but the (Csonka and Hanson, 1991; da Costa et al., 1998; Kempf details of arsenobetaine synthesis are still incompletely and Bremer, 1998; Wood et al., 2001; Yancey, 2005; Burg understood (Popowich et al., 2016). The current models and Ferraris, 2008). However, the available evidence for VC 2017 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 20, 305–323 Stress protection by uptake and synthesis of glycine betaine 307 such a biological role of arsenobetaine is mostly indirect physiological roles played by the ubiquitously distributed (Popowich et al., 2016). arsenobetaine molecule, and its biosynthetic precursor Increases in the external osmolarity triggers water efflux arsenocholine, in terrestrial and marine ecosystems. from microbial cells, and as a result, vital turgor and growth will be impaired (Csonka and Hanson, 1991; Kempf and Results Bremer, 1998; Wood et al., 2001; Wood, 2011). Many Osmostress protection by arsenobetaine and its members of the Bacteria and Archaea either import or syn- dependence on the OpuA, OpuC and OpuD osmolyte thesize specific organic osmolytes, the so-called transporters compatible solutes, under these stressful conditions to bal- ance the osmotic gradient across their cytoplasmic To assess the osmostress protective potential of arsenobe- membrane and to optimize the solvent properties of the taine for the B. subtilis wild-type strain JH642, we cytoplasm for biochemical reactions (Csonka and Hanson, challenged cultures grown in a chemically defined medium 1991; da Costa et al., 1998; Kempf and Bremer, 1998; (SMM) with sustained high salinity (1.2 M NaCl) and then Roesser and Muller,€ 2001; Wood et al., 2001; Wood, monitored the influence of both glycine betaine and arsen- 2011). Glycine betaine is such a compatible solute and obetaine on cell growth. The addition of 1.2 M NaCl to the can be synthesized under