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Use of lacZ gene fusions to determine the dependence pattern of the sporulation gene spoIID in spo mutants of Bacillus subtilis 总被引:9,自引:0,他引:9
The spoIID gene, which is involved in Bacillus subtilis sporulation, was fused to the beta-galactosidase gene, lacZ, of Escherichia coli so that the expression of beta-galactosidase would be under the control of the spoIID locus. When the fused product was inserted into the B. subtilis chromosome, production of beta-galactosidase indicated that the spoIID gene was expressed 1.5 h after the start of sporulation. When the spoIID::lacZ fusion was inserted into the chromosome of sporulation mutants, all strains carrying spo0 lesions and those with mutations in spoIIA, spoIIE and spoIIG loci failed to make beta-galactosidase. The proposed provisional order of expression of operons governing stage II is spoIIA----[spoIIG, spoIIE]----[spoIID, spoIIB, spoIIF]. 相似文献
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By rewiring the sporulation gene-regulatory network of Bacillus subtilis, we generated a novel expression system relying on derepression. The gene of interest is placed under the control of the abrB promoter, which is active only when Spo0A is absent, and Spo0A is controlled via an IPTG (isopropyl-beta-d-thiogalactopyranoside)-inducible promoter. 相似文献
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Muchová K Lewis RJ Perecko D Brannigan JA Ladds JC Leech A Wilkinson AJ Barák I 《Molecular microbiology》2004,53(3):829-842
Spo0A, the response regulator protein controlling the initiation of sporulation in Bacillus, has two distinct domains, an N-terminal phosphoacceptor (or receiver) domain and a C-terminal DNA-binding (or effector) domain. The phosphoacceptor domain mediates dimerization of Spo0A on phosphorylation. A comparison of the crystal structures of phosphorylated and unphosphorylated response regulators suggests a mechanism of activation in which structural changes originating at the phosphorylatable aspartate extend to the alpha4beta5alpha5 surface of the protein. In particular, the data show an important role in downstream signalling for a conserved aromatic residue (Phe-105 in Spo0A), the conformation of which alters upon phosphorylation. In this study, we have prepared a Phe-105 to Ala mutant to probe the contribution of this residue to Spo0A function. We have also made an alanine substitution of the neighbouring residue Tyr-104 that is absolutely conserved in the Spo0As of spore-forming Bacilli. The spo0A(Y104A) and spo0A(F105A) alleles severely impair sporulation in vivo. In vitro phosphorylation of the purified proteins by phosphoramidate is unaffected, but dimerization and DNA binding are abolished by the mutations. We have identified intragenic suppressor mutations of spo0A(F105A) and shown that these second-site mutations in the purified proteins restore phosphorylation-dependent dimer formation. Our data support a model in which dimerization and signal transduction between the two domains of Spo0A are mediated principally by the alpha4beta5alpha5 signalling surface in the receiver domain. 相似文献
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Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis. 总被引:48,自引:33,他引:15
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The kinA (spoIIJ) locus contains a single gene which codes for a protein of 69,170 daltons showing strong homology to the transmitter kinases of two component regulatory systems. The purified kinase autophosphorylates in the presence of ATP and mediates the transfer of phosphate to the Spo0A and Spo0F sporulation regulatory proteins. Spo0F protein was a much better phosphoreceptor for this kinase than Spo0A protein in vitro. Mutants with deletion mutations in the kinA gene were delayed in their sporulation. They produced about a third as many spores as the wild type in 24 h, but after 72 h on solid medium, the level of spores approximated that found for the wild-type strain. Such mutations had no effect on the regulation of the abrB gene or on the timing of subtilisin expression and therefore did not impair the repression function of the Spo0A protein. Placement of the kinA locus on a multicopy vector suppressed the sporulation-defective phenotype of spo0B, spo0E, and spo0F mutations but not of spo0A mutations. The results suggest that the spo0B-, spo0E-, and spo0F-dependent pathway of activation (phosphorylation) of the Spo0A regulator may be by-passed through the kinA gene product if it is present at sufficiently high intracellular concentration. The results suggest that multiple kinases exist for the Spo0A protein. 相似文献
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The Bacillus subtilis SinR protein is a repressor of the key sporulation gene spo0A. 总被引:4,自引:3,他引:1
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SinR is a pleiotropic DNA binding protein that is essential for the late-growth processes of competence and motility in Bacillus subtilis and is also a repressor of others, e.g., sporulation and subtilisin synthesis. In this report, we show that SinR, in addition to being an inhibitor of sporulation stage II gene expression, is a repressor of the key early sporulation gene spo0A. The sporulation-specific rise in spo0A expression at time zero is absent in a SinR-overproducing strain and is much higher than normal in strains with a disrupted sinR gene. This effect is direct, since SinR binds specifically to spo0A in vitro, in a region overlapping the -10 region of the sporulation-specific Ps promoter that is recognized by E-sigma H polymerase. Methyl interference and site-directed mutagenesis studies have identified guanine residues that are important for SinR recognition of this DNA sequence. Finally, we present evidence that SinR controls sporulation through several independent genes, i.e., sp0A, spoIIA, and possibly spoIIG and spoIIE. 相似文献