The open reading frame yqgR (now termed glcK), which had been sequenced as part of the genome project, encodes a glucose kinase of Bacillus subtilis. A 1.1-kb DNA fragment containing glcK complemented an Escherichia coli strain deficient in glucose kinase activity. Insertional mutagenesis of glcK resulted in a complete inactivation of glucose kinase activity in crude protein extracts, indicating that B. subtilis contains one major glucose kinase. The glcK gene encodes a 321-residue protein with a molecular mass of 33.5 kDa. The glucose kinase was overexpressed as a fusion protein to a six-His affinity tag and purified to homogeneity. The enzyme had K(m) values for ATP and glucose of 0.77 and 0.24 mM, respectively, and a Vmax of 93 mumol min-1 mg-1. A B. subtilis strain deficient for glucose kinase grew at the same rate on different carbon sources tested, including disaccharides such as maltose, trehalose, and sucrose.