An International Project for Systematic Analysis of Gene Function of Bacillus subtilis
Naotake OGASAWARA*
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-01, Japan
The 4,215 kb genome sequence of a spore forming, gram-positive bacterium, B. subtilis, an important source of industrial enzymes, has been completed by an international consortium, consisting of 25 European, 7 Japanese and 1 Korean laboratory together with 2 biotechnology companies. The availability of powerful genetic tools will allow the B. subtilis genome sequence data to be fully exploited within the framework of a systematic functional analysis program, undertaken by a consortium of 18 European and 10 Japanese laboratories. The main approach to assess gene function is the construction of mutants of the target genes and the analysis of the mutant phenotypes. An insertional vector is being used to construct the mutants. Its integration within a target gene (i) inactivates the gene, (ii) places a reporter β-galactosidase gene under the control of the natural expression signals of the target gene, (iii) places the possible downstream genes under the control of an IPTG- dependent promoter. Its integration upstream of a target gene yields conditional, IPTG-dependent, mutants. Over 400 mutants each have already been constructed in Japan and Europe and are undergoing preliminary characterization, including growth properties in a rich and a synthetic medium and the pattern ofβ-galactosidase expression. RNA analysis is also being carried out by Northern hybridization. The mutants will be subjected to three levels of the phenotype tests. The first level tests will have the purpose to assign target genes to broad categories, such as metabolism of small molecules and inorganics, macromolecule metabolism, cell structure and motility, stress response and survival in stationary phase, and various cell processes including sporulation, competence and secretion. The second level tests will be more complex, and will serve to confirm or reject the preliminary assignment. The third level tests should establish the gene function. A systematic analysis of the 213 kb region containing the replication origin of the genome is in progress in our laboratory. Among 197 ORFs identified there, 140 ORFs had not been analyzed experimentally. We have constructed knockout mutant for 117 ORFs. In addition, 70RFs were shown to be essential as mutation of these genes could be obtained only by intercistronic and the growth of the mutants became IPTG dependent. These essential genes include a sensor kinase and response regulator set and ORFs conserved widely in microorganisms. Analysis of expression of the target genes in a sporulation
