Summary Arsenic, a highly cytotoxic and cancerogenic metalloid, is brought into the biosphere through geochemical sources and anthropogenic activities. A global biogeochemical arsenic biotransformation cycle exists in which inorganic arsenic species are transformed into organoarsenicals, which are subsequently mineralized again into inorganic arsenic compounds. Microorganisms contribute to this biotransformation process greatly and one of the organoarsenicals synthesized and degraded in this cycle is arsenobetaine. Its nitrogen‐containing homologue glycine betaine is probably the most frequently used compatible solute on Earth. Arsenobetaine is found in marine and terrestrial habitats and even in deep‐sea hydrothermal vent ecosystems. Despite its ubiquitous occurrence, the biological function of arsenobetaine has not been comprehensively addressed. Using Bacillus subtilis as a well‐understood platform for the study of microbial osmostress adjustment systems, we ascribe here to arsenobetaine both a protective function against high osmolarity and a cytoprotective role against extremes in low and high growth temperatures. We define a biosynthetic route for arsenobetaine from the precursor arsenocholine that relies on enzymes and genetic regulatory circuits for glycine betaine formation from choline, identify the uptake systems for arsenobetaine and arsenocholine, and describe crystal structures of ligand‐binding proteins from the OpuA and OpuB ABC transporters complexed with either arsenobetaine or arsenocholine.

The accumulation of compatible solutes is a common defense of bacteria against the detrimental effects of high osmolarity. Uptake systems for these compounds are cornerstones in cellular osmostress responses because they allow the energy preserving scavenging of osmostress protectants from environmental sources. is well studied with respect to the import of compatible solutes and its five transport systems (OpuA, OpuB, OpuC, OpuD, OpuE) for these stress protectants have previously been comprehensively studied. Building on this knowledge and taking advantage of the unabated appearance of new genome sequences of members of the genus, we report here the discovery, physiological characterization, and phylogenomics of a new member of the Opu family of transporters, OpuF (OpuFA-OpuFB). OpuF is not present in but it is widely distributed in members of the large genus. OpuF is a representative of a sub-group of ABC transporters in which the substrate-binding protein (SBP) is fused to the trans-membrane domain (TMD). We studied the salient features of the OpuF transporters from and by functional reconstitution in a chassis strain lacking known Opu transporters. A common property of the examined OpuF systems is their substrate profile; OpuF mediates the import of glycine betaine, proline betaine, homobetaine and the marine osmolyte dimethylsulfoniopropionate (DMSP). An model of the SBP domain of the TMD-SPB hybrid protein OpuFB was established. It revealed the presence of an aromatic cage, a structural feature commonly present in ligand-binding sites of compatible solute importers. The high affinity import of compatible solutes from environmental sources is an important aspect of the cellular defense of many and against the harmful effects of high external osmolarity. The accumulation of these osmostress protectants counteracts high osmolarity instigated water efflux, drop in turgor to non-physiological values, and an undue increase in molecular crowding of the cytoplasm; they thereby foster microbial growth under osmotically unfavorable conditions. Importers for compatible solutes allow the energy-preserving scavenging of osmoprotective and physiologically compliant organic solutes from environmental sources. We report here the discovery, exemplary physiological characterization, and phylogenomics of a new compatible solute importer (OpuF) widely found in members of the genus. The OpuF system is a representative of a growing sub-group of ABC transporters in which the substrate-scavenging function of the substrate-binding protein (SBP) and the membrane-embedded substrate trans-locating subunit (TMD) are fused into a single polypeptide chain.

Accumulation of compatible solutes is a common stress response of microorganisms challenged by high osmolarity; it can be achieved either through synthesis or import. These processes have been intensively studied in Bacillus subtilis, where systems for the production of the compatible solutes proline and glycine betaine have been identified, and in which five transporters for osmostress protectants (Opu) have been characterized. Glycine betaine synthesis relies on the import of choline via the substrate-restricted OpuB system and the promiscuous OpuC transporter and its subsequent oxidation by the GbsAB enzymes. Transcription of the opuB and gbsAB operons is under control of the MarR-type regulator GbsR, which acts as an intracellular choline-responsive repressor. Modeling studies using the X-ray structure of the Mj223 protein from Methanocaldococcus jannaschii as the template suggest that GbsR is a homo-dimer with an N-terminal DNA-reading head and C-terminal dimerization domain; a flexible linker connects these two domains. In the vicinity of the linker region, an aromatic cage is predicted as the inducer-binding site, whose envisioned architecture resembles that present in choline and glycine betaine substrate-binding proteins of ABC transporters. We used bioinformatics to assess the phylogenomics of GbsR-type proteins and found that they are widely distributed among Bacteria and Archaea. Alignments of GbsR proteins and analysis of the genetic context of the corresponding structural genes allowed their assignment into four sub-groups. In one of these sub-groups of GbsR-type proteins, gbsR-type genes are associated either with OpuA-, OpuB-, or OpuC-type osmostress protectants uptake systems. We focus here on GbsR-type proteins, named OpuAR by us, that control the expression of opuA-type gene clusters. Using such a system from the marine bacterium Bacillus infantis, we show that OpuAR acts as a repressor of opuA transcription, where several compatible solutes (e.g., choline, glycine betaine, proline betaine) serve as its inducers. Site-directed mutagenesis studies allowed a rational improvement of the putative inducer-binding site in OpuAR with respect to the affinity of choline and glycine betaine binding. Collectively, our data characterize GbsR-/OpuAR-type proteins as an extended sub-group within the MarR-superfamily of transcriptional regulators and identify a novel type of substrate-inducible import system for osmostress protectants.

Bacillus subtilis adjusts to high osmolarity surroundings through the amassing of compatible solutes. It synthesizes the compatible solute glycine betaine from prior imported choline and scavenges many pre-formed osmostress protectants, including glycine betaine, from environmental sources. Choline is imported through the substrate-restricted ABC transporter OpuB and the closely related, but promiscuous, OpuC system, followed by its GbsAB-mediated oxidation to glycine betaine. We have investigated the impact of two MarR-type regulators, GbsR and OpcR, on gbsAB , opuB , and opuC expression. Judging by the position of the previously identified OpcR operator in the regulatory regions of opuB and opuC [ Lee et al. (2013) Microbiology 159, 2087−2096], and that of the GbsR operator identified in the current study, we found that the closely related GbsR and OpcR repressors use different molecular mechanisms to control transcription. OpcR functions by sterically hindering access of RNA-polymerase to the opuB and opuC promoters, while GbsR operates through a roadblock mechanism to control gbsAB and opuB transcription. Loss of GbsR or OpcR de-represses opuB and opuC transcription, respectively. With respect to the osmotic control of opuB and opuC expression, we found that this environmental cue operates independently of the OpcR and GbsR regulators. When assessed over a wide range of salinities, opuB and opuC exhibit a surprisingly different transcriptional profile. Expression of opuB increases monotonously in response to incrementally increase in salinity, while opuC transcription levels decrease after an initial up-regulation at moderate salinities. Transcription of the gbsR and opcR regulatory genes is up-regulated in response to salt stress, and is also affected through auto-regulatory processes. The opuB and opuC operons have evolved through a gene duplication event. However, evolution has shaped their mode of genetic regulation, their osmotic-stress dependent transcriptional profile, and the substrate specificity of the OpuB and OpuC ABC transporters in a distinctive fashion.