OpuF, a New Bacillus Compatible Solute ABC Transporter with a Substrate-Binding Protein Fused to the Transmembrane Domain

Abstract

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.

The GbsR Family of Transcriptional Regulators: Functional Characterization of the OpuAR Repressor

“…The B. subtilis OpuA transporter consists of an ATPase (OpuAA), the trans-membrane component OpuAB, and the substrate-binding protein OpuAC ( Kempf and Bremer, 1995 ; Horn et al, 2006 ; Smits et al, 2008 ), a lipoprotein tethered to the outer face of the cytoplasmic membrane ( Kempf et al, 1997 ). Variants of the OpuA system exist in which the substrate-binding protein is fused to the trans -membrane domain of the ABC transporter ( van der Heide and Poolman, 2002 ); e.g., the OpuA system from Lactococcus lactis ( Obis et al, 1999 ; Mahmood et al, 2006 ; Wolters et al, 2010 ) and various representatives of the Bacillus genus ( Teichmann et al, 2018 ). We found both types of OpuA transporters in our dataset, and these are primarily present in Paenibacillius (Figure 2 ).…”

“…The genome sequence of B. infantis NRRL B-14911 predicts in addition to OpuA, the presence of several other types of osmostress protectant uptake systems (e.g., OpuF, OpuD, OpuE) ( Teichmann et al, 2018 ), a feature that precludes an assignment of a defined substrate spectrum to the OpuA ABC transporter. We therefore cloned the opuA gene cluster ( opuAR/opuAA-opuAB-opuAC ) (Figure 4B ) and inserted it as a single copy into the chromosomal amyE gene of a B. subtilis chassis strain ( Teichmann et al, 2017 ) with defective OpuA, OpuB, OpuC, and OpuD systems (this strain possesses the L -proline transporter OpuE).…”

“…By inspecting the Mj223-derived in silico model of the B. subtilis GbsR protein, we noticed a striking clustering of aromatic amino acids whose side-chains could potentially form an aromatic cage-like structure that is positioned near the flexible linker region ( Nau-Wagner et al, 2012 ; Figures 1A – C ). Aromatic cage-like structures are characteristic features of many substrate-binding proteins mediating the high-affinity capturing of osmostress protectants with fully methylated head-groups for their import into the cytoplasm via ABC transport systems ( Schiefner et al, 2004a , b ; Horn et al, 2006 ; Oswald et al, 2008 ; Smits et al, 2008 ; Wolters et al, 2010 ; Du et al, 2011 ; Pittelkow et al, 2011 ; Lang et al, 2015 ; Teichmann et al, 2018 ). In these aromatic cages, the positively charged head-group of the substrate (e.g., choline, glycine betaine, proline betaine) is coordinated via cation–π interactions ( Schiefner et al, 2004a , b ; Mahadevi and Sastry, 2013 ).…”

Management of Osmoprotectant Uptake Hierarchy in Bacillus subtilis via a SigB-Dependent Antisense RNA

“…The OpuB and OpuC transporters consist of a homo-dimeric ATPase (OpuBA/OpuCA), hetero-dimeric trans-membrane components (OpuBB/OpuBD; OpuCB/OpuCD), and an extracellular substrate binding protein (OpuBC/OpuCC) that is tethered to the outer face of the cytoplasmic membrane via a lipid modification at its N-terminus (Kappes et al, 1999;Hoffmann and Bremer, 2017). The entire substrate spectrum of the OpuB system is shown, while only some of the 15 known substrates of the OpuC transporter are indicated (Hoffmann and Bremer, 2017;Teichmann et al, 2018). (B) Genetic organization of the opuB and opuC operons and their flanking regions.…”

Evidence from Mutational Analysis for a Single Transmembrane Substrate Binding Site in the Histidine ATP-Binding Cassette Transporter of Salmonella enterica Serovar Typhimurium

“…In other archaea, SBPs are tethered to the membrane by an N-terminal hydrophobic alpha-helix. Thus far, in a few cases, solute binding domains were found that are fused to the TMDs of their cognate transporters, resulting in one, two, or four substrate binding sites (2,(14)(15)(16)(17).…”