The glk gene from Bacillus megaterium, which encodes glucose kinase, was isolated and analyzed. Disruption by a transcriptional glk-luxAB fusion indicated that glk is the only glucose kinase gene in that strain but did not affect growth of that mutant on glucose. Determination of luciferase activity under various growth conditions revealed constitutive transcription of glk. Expression of a xylA-lacZ fusion was repressed by glucose in the strain with the glk disruption about twofold less efficiently than in the wild type. The potential contribution of glk expression to glucose repression is discussed.Glucose kinase catalyzes the ATP-dependent conversion of glucose into glucose-6-phosphate, the entry compound of glycolysis. In some bacteria, like Escherichia coli, glucose kinase seems to be important for metabolic pathways only because it phosphorylates intracellular glucose originating, e.g., from disaccharide hydrolysis. In contrast, in Streptomyces coelicolor glucose kinase is essential for catabolite repression (2). Even in Staphylococcus xylosus, in which catabolite repression is mainly exerted by the CcpA-dependent pathway (5), inactivation of glkA, which encodes glucose kinase, leads to reduced catabolite repression of several genes (17).We have investigated catabolite repression of xylose utilization in Bacillus megaterium (9) and report here the isolation and inactivation of glk, which encodes glucose kinase. The resulting strain, although deficient in ATP-dependent phosphorylation of glucose, grows like the wild type (wt) on glucose or trehalose as the sole carbon source but shows reduced glucose repression of xylose utilization.The strategy for cloning the glucose kinase gene from B. megaterium involved complementation of the E. coli glk mutant UE26 (3). E. coli UE26 (ptsG ptsM glk) is unable to utilize glucose as a carbon source and, therefore, forms white colonies on glucose-containing MacConkey plates, while the wt forms red colonies. Since E. coli UE26 cannot transport glucose via the phosphotransferase system, we supplemented the plates with 100 mM fucose. This leads to the induction of galactose permease, which can also transport glucose (8). The unphosphorylated glucose can be metabolized only if it is converted to glucose-6-phosphate by glucose kinase. We transformed E. coli UE26 (3) with a gene library from B. megaterium consisting of a partial AluI digest of total DNA inserted into the EcoRV site of pWH1509K (13) and screened the transformants on MacConkey agar supplemented with 100 mM fucose and 50 mM glucose. One candidate turned red on this agar and remained white on MacConkey agar supplemented with either 50 mM glucose or 50 mM mannose, indicating that the insert did not complement ptsG or ptsM. The plasmid prepared from that candidate contained an insert of 6.45 kbp and was called pWH665.Glucose kinase activity was quantified in a crude protein extract supplemented with glucose dehydrogenase, ATP, and NADP (15). Glucose-6-phosphate is converted to gluconate-6-phosphate, thereby producing ...