Microbiology (2000), 146, 573–579 Printed in Great Britain Systematic study of gene expression and transcription organization in the gntZ–ywaA region of the Bacillus subtilis genome Ken-ichi Yoshida, Izumi Ishio, Eishi Nagakawa, Yoshiyuki Yamamoto, Mami Yamamoto and Yasutaro Fujita Author for correspondence : Y. Fujita. Tel : 81 849 36 2111. Fax: 81 849 36 2459. e-mail : yfujitabt.fubt.fukuyama-u.ac.jp Department of Biotechnology, Faculty of Engineering, Fukuyama University, 985 Sanzo, Higashimura-cho, Fukuyama 729-0292, Japan Within the framework of the international project ‘ The functional analysis of the Bacillus subtilis genome ’ in Japan and Europe, the gene expression and transcription organization of the gntZ–ywaA region (160 kb) of the B. subtilis genome has been systematically analysed. First, all unanalysed genes comprising more than 80 amino acids (125 genes) in this region were inactivated through integration of plasmid pMUTIN. No essential gene was found which could not be inactivated. All the integrants grew normally in both nutrient sporulation medium and glucose minimal medium. But an integrant in the yxbG gene exhibited an oligosporogenic phenotype in the nutrient sporulation medium. The synthesis of β-galactosidase was examined, as a reporter for expression of the inactivated genes, during growth and sporulation in the two media. The results indicated that 36 % of the promoters were inactive when cells were grown in at least one of these two media. Furthermore, the transcription of the 119 genes in this region was analysed by Northern blotting, resulting in a transcription map. The results indicate that the gntZ–ywaA region contains at least 24 polycistronic operons, including several published ones. The operons newly found in this work are yxaAB, yxaGH, yxaJKL, yxbBA–yxnB–asnH–yxaM, yxbCD, yxcED, yxdJK, yxeFGH, yxeKLMNOPQ, yxeR–yxxB, hutPHUIGM, bglPH–yxiE, wapA–yxxG, yxiM–deaD, katB–yxiS, yxjCDEF, yxjJI and yxkF–mmsX. Keywords : Bacillus subtilis, genome, transcription, gene inactivation, gene expression INTRODUCTION Recently, tens of genomes of micro-organisms, including Saccharomyces cerevisiae (Goffeau et al., 1997) and Bacillus subtilis (Kunst et al., 1997), have been sequenced. Among the numerous identified genes, nearly half were found to be functionally unknown ones. Determining the functions of the unknown genes of these micro-organisms will be an important step for a more complete and comprehensive understanding of their cellular processes and metabolic pathways. Thus, international projects on functional genomics of genetic- ally well characterized S. cerevisiae and B. subtilis have been started, in which many research groups are ................................................................................................................................................. Abbreviations : β-Gal, β-galactosidase ; DSM, defined nutrient sporulation medium ; MM, glucose minimal medium ; SD, Shine–Dalgarno. participating. Very recently, great progress in the field of functional genomics of S. cerevisiae has been made by research groups in Europe and North America, with the production of deletion strains for more than 2000 unknown genes and the assaying of the phenotypes of 500 of them in parallel (Winzeler et al., 1999). On the other hand, many groups participating in the Japanese and European project on the functional analysis of the B. subtilis genome, which was initiated in 1996, have already inactivated nearly 2000 of the unanalysed genes through plasmid integration and are currently assaying the phenotypes of the integrants constructed, according to the following strategy. The first step in the common strategy adopted in the B. subtilis genome function project is that each partici- pating group is responsible for the inactivation of the unanalysed genes in a certain region of the genome. This 0002-3750  2000 SGM 573