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 ...

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The transcriptional regulatory gene spoOH encodes an RNA polymerase sigma factor called sigma H that directs gene expression at an early stage of sporulation in the Gram-positive bacterium Bacillus subtilis. We now report that conditions that induce sporulation cause a rapid increase in the cellular concentration of sigma H. This increase could account for the stimulated transcription of certain sigma H-controlled genes at the onset of sporulation. Experiments in which the expression of spoOH was monitored by use of a spoOH-lacZ fusion and in which expression of spoOH was artificially manipulated by use of an isopropyl-beta-D-thiogalacto-side-inducible promoter indicate that sporulation-induced increases in the amount of sigma H are not controlled at the level of the transcription of its structural gene. Rather, we infer the existence of post-transcriptional control mechanisms that govern sigma H levels, and we present evidence suggesting that increases in the amount of sigma H at the start of sporulation are due to increased translation or stability of the spoOH mRNA and, to a lesser extent, decreased turnover of spoOH protein.

Rhodococcus sp. strain RHA1 is a gram-positive polychlorinated biphenyl (PCB) degrader which can degrade 10 ppm of PCB48 (equivalent to Aroclor1248), including tri-, tetra-, and pentachlorobiphenyls, in a few days. We isolated the 7.6-kb EcoRI-BamHI fragment carrying the biphenyl catabolic genes of RHA1 and determined their nucleotide sequence. On the basis of deduced amino acid sequence homology, we identified six bph genes, bphA1A2A3A4, bphB, and bphC, that are responsible for the initial three steps of biphenyl degradation. The order of bph genes in RHA1 is bphA1A2A3A4-bphC-bphB. This gene order differs from that of other PCB degraders reported previously. The amino acid sequences deduced from the RHA1 bph genes have a higher degree of homology with the tod genes from Pseudomonas putida F1 (49 to 79%) than with the bph genes of Pseudomonas sp. strains KF707 and KKS102 (30 to 65%). In Escherichia coli, bphA gene activity was not observed even when expression vectors were used. The activities of bphB and bphC, however, were confirmed by observing the transformation of biphenyl to a meta-cleavage compound with the aid of benzene dioxygenase activity that complemented the bphA gene activity (S. Irie, S. Doi, T. Yorifuji, M. Takagi, and K. Yano, J. Bacteriol. 169:5174-5179, 1987). The expected products of the cloned bph genes, except bphA3, were observed in E. coli in an in vitro transcription-translation system. Insertion mutations of bphA1 and bphC of Rhodococcus sp. strain RHA1 were constructed by gene replacement with cloned gene fragments. The bphA1 and bphC insertion mutants lost the ability to grow on biphenyl, demonstrating that the cloned bph genes are essential for biphenyl catabolism in this strain.