RESUMO
acs encodes acetyl-coenzyme A synthetase, a high-affinity enzyme that allows cells to scavenge for acetate during carbon starvation. CRP activates acs transcription by binding tandem DNA sites located upstream of the major promoter, acsP2. Here, we used electrophoretic mobility shift assays and DNase I footprint analyses to demonstrate that the nucleoid proteins FIS and IHF each bind multiple sites within the acs regulatory region, that FIS competes successfully with CRP for binding to their overlapping and neighbouring sites and that IHF binds independently of either FIS or CRP. Using in vitro transcription assays, we demonstrated that FIS and IHF independently reduce CRP-dependent acs transcription. Using in vivo reporter assays, we showed that disruption of DNA sites for FIS or deletion of DNA sites for IHF increases acs transcription. We propose that FIS and IHF each function directly as anti-activators of CRP, each working independently at different times during growth to set the levels of CRP-dependent acs transcription.
Assuntos
Acetato-CoA Ligase/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Receptores de Superfície Celular/genética , Fatores de Transcrição , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Proteína Receptora de AMP Cíclico , Primers do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Nucleoproteínas/genética , Receptores de Superfície Celular/metabolismo , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
Spores produced by bacilli are encased in a proteinaceous multilayered coat and, in some species (including Bacillus anthracis), further surrounded by a glycoprotein-containing exosporium. To characterize bacillus spore surface morphology and to identify proteins that direct formation of coat surface features, we used atomic-force microscopy (AFM) to image the surfaces of wild-type and mutant spores of Bacillus subtilis, as well as the spore surfaces of Bacillus cereus 569 and the Sterne strain of Bacillus anthracis. This analysis revealed that the coat surfaces in these strains are populated by a series of bumps ranging between 7 and 40 nm in diameter, depending on the species. Furthermore, a series of ridges encircled the spore, most of which were oriented along the long axis of the spore. The structures of these ridges differ sufficiently between species to permit species-specific identification. We propose that ridges are formed early in spore formation, when the spore volume likely decreases, and that when the spore swells during germination the ridges unfold. AFM analysis of a set of B. subtilis coat protein gene mutants revealed three coat proteins with roles in coat surface morphology: CotA, CotB, and CotE. Our data indicate novel roles for CotA and CotB in ridge pattern formation. Taken together, these results are consistent with the view that the coat is not inert. Rather, the coat is a dynamic structure that accommodates changes in spore volume.
