The glucose kinase of Bacillus subtilis

Abstract

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.

FIG. 1

Expression of glucose kinase activity during exponential growth and the early stationary phase. Wild-type B. subtilis cells were grown in Luria broth. The corresponding optical density at 600 nm of the culture is indicated. Aliquots were harvested at the indicated times, and glucose kinase activity was determined as described in the text and expressed in nanomoles of NADP reduced minute⁻¹ milligram of protein⁻¹ of cell extracts.

FIG. 2

Map of the glcK (yqgR) locus presenting the protein and RNA coding regions at kilobase position 2570.85 on the B. subtilis genome (17). The locations, orientations, and sizes of glcK and the flanking open reading frames (gray arrows) are indicated by arrows. The 1.1-kb DNA fragment containing the glcK gene whose nucleotide sequence has been determined is presented below. The insertion of a spc cassette between two NaeI restriction sites leading to inactivation of glcK in strain MD186 is depicted below the map. P1 and T1 indicate a potential promoter and terminator, respectively. The corresponding sequences are presented below, showing a potential ς^A-dependent promoter with −35 and −10 regions in front of the ribosome binding site (Shine-Dalgarno sequence [SD]) of yqgP (start codon is underlined) and a palindrome of the suggested terminator T1.

FIG. 3

Overproduction and purification of glucose kinase. (A) Glucose kinase was overproduced as described in the text and analyzed on an SDS-12%PAG. Aliquots were taken from uninduced cells (lane 1) and IPTG-induced cells after 1 h (lane 2), 2 h (lane 3), 3 h (lane 4), 4 h (lane 5). The positions of molecular mass standards (st) are indicated to the left of the gel. (B) Purification of glucose kinase was performed by loading a Ni²⁺ HiTrap chelating column with crude cell extracts from harvested cells after 4 h of IPTG induction as depicted in panel A. The elution profile in panel B was monitored as presented by the protein absorption at 280 nm (in milliabsorption units) correlated with the column volume (1 ml). (Insert) Four selected fractions from the elution fractions as indicated by the gray lines are presented on an SDS-12%PAG, showing purification of GlcK. Molecular size standards (rightmost lane) with the indicated masses (in kilodaltons) are indicated to the right of the inserted gel.

FIG. 4

Phosphorylation of glucose by glucose kinase GlcK. Ten-microliter samples of each reaction mixture were spotted onto thin-layer chromatography plates (see text). In addition to the buffer contents, the mixtures contain glucose (lane 1), glucose-6-phosphate (lane 2), glucose and glucose kinase (lane 3), and glucose, ATP, and glucose kinase (lane 4).