To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among Ϸ4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

Previous determination of the nucleotide sequence of the iol region of the Bacillus subtilis genome allowed us to predict the structure of the iol operon for myo-inositol catabolism, consisting of 10 iol genes (iolA to iolJ); iolG corresponds to idh, encoding myo-inositol 2-dehydrogenase (Idh). Primer extension analysis suggested that an inositol-inducible promoter for the iol operon (iol promoter) might be a promoter-like sequence in the 5 region of iolA, which is probably recognized by A . S1 nuclease analysis implied that a -independent terminator-like structure in the 3 region of iolJ might be a terminator for iol transcription. Disruption of the iol promoter prevented synthesis of the iol transcript as well as that of Idh, implying that the iol operon is most probably transcribed as an 11.5-kb mRNA containing the 10 iol genes. Immediately upstream of the iol operon, two genes (iolR and iolS) with divergent orientations to the iol operon were found. Disruption of iolR (but not iolS) caused constitutive synthesis of the iol transcript and Idh, indicating that the iolR gene encodes a transcriptionnegative regulator (presumably a repressor) for the iol operon. Northern and S1 nuclease analyses revealed that the iolRS genes were cotranscribed from another inositol-inducible promoter, which is probably recognized by A . The promoter assignments of the iol and iolRS operons were confirmed in vivo with a lacZ fusion integrated into the amyE locus.myo-Inositol is abundant in nature, especially in soil. Various microorganisms including soil bacteria are able to grow on inositol as the sole carbon source. The pathway of inositol catabolism in Klebsiella aerogenes has been extensively studied (1, 2, 4, 5). myo-Inositol 2-dehydrogenase (Idh; EC 1.1.1.18) is the first enzyme responsible for inositol catabolism in this organism (4). Other microorganisms, such as Bacillus subtilis (17), Rhizobium leguminosarum bv. viciae (16), and Cryptococcus melibiosum (20), probably possess similar pathways involving Idh. These findings suggested that inositol catabolism might be conserved among these microorganisms. However, the molecular genetics of the genes involved in inositol catabolism have not been well studied.B. subtilis Idh has been purified and characterized (17). Idh synthesis is induced upon the addition of inositol and is also under catabolite repression (13,14,17). We succeeded in the cloning and expression of the idh gene of B. subtilis in Escherichia coli (11). The regions surrounding the idh gene were cloned by chromosome walking (23, 24). The nucleotide sequences of these regions implied a putative operon structure consisting of 10 genes, which are probably involved in inositol catabolism (iol) (23-25).As a first step toward elucidating the pathway and regulation of inositol catabolism of B. subtilis, we analyzed the iol transcript. We present here the results regarding the organization and transcription of the iol operon. Moreover, we identified a transcription-negative regulator gene (iolR) for this operon. MATERIA...