Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28

The trimethylammonium compound glycine betaine (N,N,N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced o smo- and cryotolerance upon Listeria monocytogenes. We report the identific ation of betL, a gene encoding a glycine betaine uptake system in L. monocy togenes, isolated by functional complementation of the betaine uptake mutan t Escherichia coli MKH13, The betL gene is preceded by a consensus sigma(B) -dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions, BetL exhibits significant seq uence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, who se substrates possess a fully methylated quaternary ammonium group, The obs erved K-m value of 7.9 mu M for glycine betaine uptake after heterologous e xpression of betL in E. coli MKH13 is consistent with values obtained for L . monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. T his mutant is also unable to withstand concentrations of salt as high as ca n the BetL(+) parent, signifying the role of the transporter in Listeria os motolerance.