CbsT2 from Lactobacillus johnsonii 100-100 is a transport protein of the major facilitator superfamily that facilitates bile acid antiport.
We previously identified two conjugated bile acid transporters, CbsT1 and CbsT2, in Lactobacillus johnsonii 100-100 and Lactobacillus acidophilus KS-13 that are gene duplicates encoded in tandem with a conjugated bile salt hydrolase (BSH) [Elkins and Savage, J. Bacteriol. 180:4344-4349, 1998; Elkins et al., Microbiology 147: 3403-3412, 2001]. CbsT2 from 100-100 was shown to increase taurocholic acid (TCA) uptake in Escherichia coli; however, higher levels were achieved when an extracellular factor (EF) from 100-100 was present in the assay medium (spent medium from 100-100, pH 4.2). We continued this study here to determine the role of EF in this transport system. Kinetic studies revealed that the previously observed CbsT2- and EF-mediated TCA accumulation is rapid (< 15 s) but not saturable, suggesting that EF is limiting. In addition, uptake of TCA by E. coli expressing CbsT2 was insensitive to ionophores, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, and thus, is independent of the proton motive force. Since BSH converts [24-(14)C]TCA to [24-(14)C]cholic acid (CA), we measured net radiolabel uptake in E. coli cells expressing transporter(s) and BSH. Interestingly, such cells accumulated less 14C radiolabel (by approximately half) than cells expressing CbsT2 alone. These data can be explained if CA diffuses out of E. coli through the transporter(s). We, therefore, added exogenous, unlabeled CA to EF-spent media, which under our assay conditions, performed similarly to EF+ culture supernatant in TCA and CA uptake assays. Thus, unlabeled CA (a protonated, neutral lipophile) can partition directly into E. coli cells especially at low pH. These findings were validated in uptake assays with [3H]TCA, which yields [3H]taurine (a hydrophilic moiety) upon hydrolysis by the BSH. Amounts of cell-associated 3H radiolabel remained similar in cells expressing CbsT2 and BSH versus cells expressing only CbsT2, both of which were higher than in cells expressing BSH alone. Our data support a hypothesis that these transporters, which comprise a new subfamily of the major facilitator superfamily, facilitate antiport of TCA and CA.
Elkins
Christopher A
CA
Department of Microbiology, University of Tennessee, Knoxville, Tenn., USA. Caelkins@nctr.fda.gov
Savage
Dwayne C
DC
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001647:Bile Acids and Salts / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D017077:Culture Media, Conditioned / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D017136:Ion Transport / Q000502:physiology; D007700:Kinetics; D007778:Lactobacillus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D013656:Taurocholic Acid / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001647:Bile Acids and Salts; D002352:Carrier Proteins; C444423:CbsT2 protein, Lactobacillus johnsonii; D003470:Culture Media; D017077:Culture Media, Conditioned; D026901:Membrane Transport Proteins; D013656:Taurocholic Acid