We describe a general strategy for the identification of genes that are controlled by a specific regulatory factor in vivo and the use of this strategy to identify genes in Bacillus subtilis that are controlled by spoOH, a regulatory gene required for the initiation of sporulation. The general strategy makes use of a cloned regulatory gene fused to an inducible promoter to control expression of the regulatory gene and random gene fusions to a reporter gene to monitor expression in the presence and absence of the regulatory gene product. spoOH encodes a sigma factor of RNA polymerase, orH, and is required for the extensive reprograming of gene expression during the transition from growth to stationary phase and during the initiation of sporulation. We identified 18 genes that are controlled by (rH (csh genes) in vivo by monitoring expression of random gene fusions to lacZ, made by insertion mutagenesis with the transposon Tn9171ac, in the presence and absence of iFH. These genes had lower levels of expression in the absence of ifH than in the presence of cfH. Patterns of expression of the csh genes during growth and sporulation in wild-type and spoOH mutant cells indicated that other regulatory factors are probably involved in controlling expression of some of these genes. Three of the csh::Tn9l7lac insertion mutations caused noticeable phenotypes. One caused a defect in vegetative growth, but only in combination with a spoOH mutation. Two others caused a partial defect in sporulation. One of these also caused a defect in the development of genetic competence. Detailed characterization of some of the csh genes and their regulatory regions should help define the role of spoOH in the regulation of gene expression during the transition from growth to stationary phase and during the initiation of sporulation.Endospore formation in the gram-positive bacterium Bacillus subtilis involves extensive reprograming of gene expression and a series of complex morphological and physiological changes (14,25,32,37). Rapidly growing cells can be induced to differentiate upon nutrient deprivation, and all conditions of nutrient deprivation that cause efficient sporulation have been shown to cause a drop in the intracellular concentration of GDP and GTP (23,24). Furthermore, conditions that cause a drop in these nucleotides (for example, addition of the drug decoyinine [29]), even in the presence of excess nutrients, can cause efficient sporulation (15), provided that the cells are at relatively high densities (17,38). Thus, it is thought that a drop in the intracellular level of GDP and GTP is necessary and sufficient for efficient sporulation (14, 16).At least seven regulatory genes, called spoO genes, are required for the initiation of sporulation. Mutations in these genes prevent the earliest morphological change associated with sporulation, the formation of an asymmetric division septum following nutrient deprivation (26,32,33 (2,3,(45)(46)(47)(48). spoVG is normally expressed during vegetative growth, and expression increases ...

We present a collection of 182 isogenic strains containing genetically linked antibiotic resistance elements located at approximately 1-min intervals around the Escherichia coli chromosome. At most positions both Tn10 (Tetr) and TN10kan (Kanr) elements are available, so that the collection contains a linked set of alternating antibiotic resistance markers. The map position of each insertion has been aligned to the E. coli genetic map as well as to the Kohara ordered clone bank. These strains are designed to be used in a rapid two-step mapping system in E. coli. In the first step, the mutation is localized to a 5- to 15-min region of the chromosome by Hfr mapping with a set of Hfr strains containing either Tn10 or Tn10kan elements located 20 min from their respective origins of transfer. In the second step, the mutation is localized to a 1-min region by P1 transduction, with a collection of isogenic insertion strains as donors. We discuss the uses of this collection of strains to map and eventually to clone a variety of mutations in E. coli.