The gene cluster involved in the choline-glycine betaine conversion pathway was cloned from chromosomal DNA of the Gram-positive moderate halophile Halobacillus dabanensis D-8(T). Nucleotide sequence analysis revealed four genes, designated gbsT, gbsI, gbsA, and gbsB, which are clustered in a 5.1-kb fragment. After heterologous expression of gbsAB in the Escherichia coli mutant strain PD141, the transformed cells were able to grow in a selective M63 medium containing 0.7 M NaCl and 1 mM choline, in contrast to the mutant strain. Glycine betaine biosynthesis was restored and its accumulation was confirmed by using C-13 nuclear magnetic resonance spectroscopy.