Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314 degrees) and sacXY (333 degrees) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety-two open reading frames were predicted to encode putative proteins among which only forty-two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.

The adk gene from Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli under the control of the lac promoter. The primary structure of B. stearothermophilus adenylate kinase exhibited 76% identity with the enzyme from Bacillus subtilis, 60% identity with the enzyme from Lactococcus lactis, and 42% identity with the enzyme from E. coli. The most striking property of the adenylate kinase from B. stearothermophilus is the presence of a structural zinc atom bound to four cysteines in a zinc finger-like fashion. The ability to coordinate zinc is predicted also for a number of other isoforms of bacterial adenylate kinases. Furthermore, the tightly bound metal ion contributes to the high thermodynamic stability of adenylate kinase from B. stearothermophilus.

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The nucleosidediphosphate kinase phosphorylation reaction led to the incorporation of 0.95 i 0.1 phosphate groups per enzyme subunit. The equilibrium constant of the phosphorylation reaction was 0.26. The inhibition of the nucleosidediphosphate kinase activity by Cibacron bluc 3GA was competitive with respect to ATP, the donor nucleotide (apparent Ki = 0.28 pM) and uncompetitive with respect to 8-bromoinosine 5'-diphosphate, the acceptor nucleotide (apparent Ki = 0.31 pM). By difference spectroscopy it was shown that each enzyme subunit bound one Cibacron blue 3GA molecule, whereas the phosphorylated enzyme had no affinity for the dye. ATP was an effective competitor, being able to displace the dye from its bound state. The complex behaviour noted was taken as evidence for cooperative interaction between the enzyme subunits. The data obtained using polarographic techniques agreed with these results.The nucleosidediphosphate (NDP) kinase is a key enzyme in the rephosphorylation of non-adenine nucleoside diphosphates to the corresponding nucleoside triphosphates. The enzyme is built up of six subunits of relatively small size [I -31. The NDP kinases of various origins have a pingpong mechanism. The covalent binding of a phosphate group to a histidine side-chain was noted in all cases [4,5]. Fast kinetic studies [6] and the retention of transfered phosphate group configuration [7] indicate that the phosphorylated enzyme is the true intermediate during the catalysis. The data on the stoichiometry of phosphorylation are conflicting. On the other hand, many structural and mechanistic details are still unknown, such as the location and number of nucleotide binding sites, the interactions among subunits, the role of the conformational transitions, etc.The polysulfonated anthraquinonic dye Cibacron blue 3GA (Reactive Blue 2, Colour Index constitution number 61 21 1) is extensively used as a probe for nucleotide binding sites in enzymes [8 -101. The investigation of the interaction of this dye with N D P kinase is interesting for sevcral reasons. First of all the enzyme binds strongly to blue-Sepharose [1,1 I] and to blue-dextran-Sepharose [12], a property which enabled its purification. Next, given the existence of the phosphorylated enzyme, the use of Cibacron blue 3GA allows the probing (or mapping) of the nucleotide binding site. Finally, if it is true that in spite of known limitations [I31 the dye binds preferentially to the supersecondary structure, called 'dinucleotide fold' [I 4,151, the interaction between Cibacron blue 3GA and N D P kinase might supply information on this functional domain before the three-dimensional structure of the enzyme is elucidated by X-ray diffraction methods.In this study we investigated the interaction of Cibacron blue 3GA with the unphosphorylated and phosphorylated NDP kinase, by steady-state kinetics, difference spectroscopy and polarographic methods. MATERIALS A N D METHODS ChemicalsAll natural nucleotides, substrates and coupling enzymes were from Boehringer (Mannheim). 8-Bromoi...