OusB, a Broad-Specificity ABC-Type Transporter fromErwinia chrysanthemi, Mediates Uptake of Glycine Betaine and Choline with a High Affinity

Abstract

The ability of Erwinia chrysanthemi to cope with environments of elevated osmolality is due in part to the transport and accumulation of osmoprotectants. In this study we have identified a high-affinity glycine betaine and choline transport system in E. chrysanthemi. By using a pool of Tn5-B21 ousA mutants, we isolated a mutant that could grow in the presence of a toxic analogue of glycine betaine (benzyl-glycine betaine) at high osmolalities. This mutant was impaired in its ability to transport all effective osmoprotectants in E. chrysanthemi. The DNA sequence of the regions flanking the transposon insertion site revealed three chromosomal genes (ousVWX) that encode components of an ABC-type transporter (OusB): OusV (ATPase), OusW (permease), and OusX (periplasmic binding protein). The OusB components showed a significant degree of sequence identity to components of ProU from Salmonella enterica serovar Typhimurium and Escherichia coli. OusB was found to restore the uptake of glycine betaine and choline through functional complementation of an E. coli mutant defective in both ProU and ProP osmoprotectant uptake systems. Competition experiments demonstrated that choline, dimethylsulfoniacetate, dimethylsulfoniopropionate, and ectoine were effective competitors for OusB-mediated betaine transport but that carnitine, pipecolate, and proline were not effective. In addition, the analysis of single and double mutants showed that OusA and OusB were the only osmoprotectant transporters operating in E. chrysanthemi.

TheErwinia chrysanthemi3937 PhoQ Sensor Kinase Regulates Several Virulence Determinants

“…4B) in the presence of 1 M NaCl. ProW is the ABC transporter component of OusB that is involved in adaptation to osmotic stress in Erwinia chrysanthemi (11). Consistent with the increased transcript levels of sodC (ID 17092), the phoQ mutant showed slightly higher survivability in the presence of hydrogen peroxide (Fig.…”

Characterization of the Osmoprotectant Transporter OpuC from Pseudomonas syringae and Demonstration that Cystathionine-β-Synthase Domains Are Required for Its Osmoregulatory Function

“…For competition experiments, unlabeled compounds were used at final concentrations of 100 M and 1 mM, whereas [methyl- 14 C]glycine betaine and [methyl- 14 C]choline were used at a final concentration of 10 M (with specific activity of 0.55 mCi/mmol). The protein content of cell suspensions was determined using the Bio-Rad Bradford assay (Bio-Rad, Hercules, CA) following incubation of a subsample of cells in 1 M of NaOH at 95°C for 5 min.…”

“…These include OpuA, OpuB, and OpuC from Bacillus subtilis (30), Gbu and OpuC from Listeria monocytogenes (3), and OpuA (BusA) from Lactococcus lactis (52). The Erwinia chrysanthemi OusB transporter is among the few identified in a gram-negative species and is similar to ProU in sequence, substrate range, expression, and activation profile (14). S. meliloti ABC transporters that function in the uptake of osmoprotectant compounds have also been identified (1,8,19,28), but the primary or sole function of each appears to be for catabolism rather than osmoprotection.…”

The Compatible-Solute-Binding Protein OpuAC from Bacillus subtilis : Ligand Binding, Site-Directed Mutagenesis, and Crystallographic Studies

“…DMSA also serves as an osmoprotectant for Pseudomonas aeruginosa PAO1 (10) and the lactic acid bacterium Tetragenococcus halophila (2). Furthermore, DMSA is a substrate for the periplasmic binding protein from the glycine betaine and choline transporter OusB from Erwinia chrysanthemi (7). Interestingly, uptake of DMSA in Sinorhizobium meliloti is toxic and it becomes only osmoprotective in mutants that are unable to dimethylate this sulfobetaine (38).…”