Preliminary Characterization Of A Choline Transporter BetT In Bacterial Osmatic Regulation

In hypertonic environments,Escherichia coli maintains intracellular and extracellular osmotic balance by increasing the concentration of compatible solutes inside the cell.Recent studies have found that glycine betaine is one of the important compatible solutes in osmotic regulation.E.coli can accumulate glycine betaine by directly taking it in from the environment through the glycine betaine transporter protein or by synthesizing it itself.Although direct uptake is more energy efficient than biosynthesis for bacterial growth,biosynthesis becomes particularly important when glycine betaine is not available in the environment.Choline is a precursor for glycine betaine synthesis in E.coli and is transported into the cell by the choline transporter protein Bet T,followed by catalytic synthesis of glycine betaine by choline dehydrogenase and betaine aldehyde dehydrogenase.Although the substrate transport mechanism of the compatible solute transport system in E.coli has been well studied,research on the structure and substrate transport mechanism of the choline transporter protein Bet T is still limited.In this study,Bet T was selected as the research object to obtain a naturally stable protein conformation.The protein structure under several commonly used detergents was characterized by negative stain electron microscopy and cryo-electron microscopy to select the most suitable detergent.Uniformly dispersed and evenly sized protein particles were observed in LMNG buffer under negative stain electron microscopy.The protein features in GDN buffer were more obvious under cryo-electron microscopy.In addition,the substrate binding characteristics of the protein were determined using fluorescence spectroscopy,and the results showed that Bet T had specificity for choline transport and may be coupled with sodium ions.Furthermore,a bet T-deficient strain was constructed,and by measuring the bacterial growth curve,it was found that Bet T protein could transport choline and enable E.coli to tolerate high osmotic environments.The above experimental results have significant implications for the analysis of the Bet T protein structure and the in-depth investigation of its molecular mechanism.Additionally,it provides important references for the characterization of the structures and functions of other transporter proteins in the osmoregulation system of E.coli.