RESUMO
Type 6 secretion systems (T6SSs) are specialized multiprotein complexes that inject protein effectors into prokaryotic and/or eukaryotic cells. We previously described the role of the T6SS of the phytopathogen Xanthomonas citri pv. citri as an anti-eukaryotic nanoweapon that confers resistance to predation by the amoeba Dictyostelium discoideum. Transcription of the X. citri T6SS genes is induced by a signaling cascade involving the Ser/Thr kinase PknS and the extracytoplasmic function sigma factor EcfK. Here, we used a strain overexpressing a phosphomimetic constitutively active version of EcfK (EcfKT51E ) to identify the EcfK regulon, which includes a previously uncharacterized transcription factor of the AraC-family (TagK), in addition to T6SS genes and genes encoding protein homeostasis factors. Functional studies demonstrated that TagK acts downstream of EcfK, binding directly to T6SS gene promoters and inducing T6SS expression in response to contact with amoeba cells. TagK controls a small regulon, consisting of the complete T6SS, its accessory genes and additional genes encoded within the T6SS cluster. We conclude that a singular regulatory circuit consisting of a transmembrane kinase (PknS), an alternative sigma factor (EcfK) and an AraC-type transcriptional regulator (TagK) promotes expression of the X. citri T6SS in response to a protozoan predator.
Assuntos
Dictyostelium , Sistemas de Secreção Tipo VI , Xanthomonas , Fator sigma/genética , Fator sigma/metabolismo , Fator de Transcrição AraC/genética , Regulação Bacteriana da Expressão Gênica/genética , Dictyostelium/genética , Dictyostelium/metabolismo , Células Eucarióticas , Eucariotos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Xanthomonas/genética , Xanthomonas/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Genetic experiments with full length AraC and biophysical experiments with its dimerization domain plus linker suggest that arabinose binding to the dimerization domain changes the properties of the inter-domain linker which connects the dimerization domain to the DNA binding domain via interactions that do not depend on the DNA binding domain. Normal AraC function was found to tolerate considerable linker sequence alteration excepting proline substitutions. The proline substitutions partially activate transcription even in the absence of arabinose and hint that a structural shift between helix and coil may be involved. To permit fluorescence anisotropy measurements that could detect arabinose-dependent dynamic differences in the linkers, IAEDANS was conjugated to a cysteine residue substituted at the end of the linker of dimerization domain. Arabinose, but not other sugars, decreased the steady-state anisotropy, indicating either an increase in mobility and/or an increase in the fluorescence lifetime of the IAEDANS. Time-resolved fluorescence measurements showed that the arabinose-induced anisotropy decrease did not result from an increase in the excited-state lifetime. Hence arabinose-induced decreases in anisotropy appear to result from increased tumbling of the fluorophore. Arabinose did not decrease the anisotropy in mutants incapable of binding arabinose nor did it alter the anisotropy when IAEDANS was conjugated elsewhere in the dimerization domain. Experiments with heterodimers of the dimerization domain showed that the binding of arabinose to one subunit of the dimer decreases the fluorescence anisotropy of only a fluorophore on the linker of the other subunit.
Assuntos
Fator de Transcrição AraC/química , Arabinose/química , Cisteína/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Prolina/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Fator de Transcrição AraC/genética , Fator de Transcrição AraC/metabolismo , Arabinose/metabolismo , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Polarização de Fluorescência , Expressão Gênica , Mutação , Naftalenossulfonatos/química , Prolina/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , TermodinâmicaRESUMO
The AraC/XylS family of transcription factors, which include proteins that are involved in the regulation of diverse biological processes, has been of considerable interest recently and has been constantly expanding by means of in silico predictions and experimental analysis. In this work, using a HMM based on the DNA binding domain of 58 experimentally characterized proteins from the AraC/XylS (A/X), 1974 A/X proteins were found in 149 out of 212 bacterial genomes. This domain was used as a template to generate a phylogenetic tree and as a tool to predict the putative regulatory role of the new members of this family based on their proximity to a particular functional cluster in the tree. Based on this approach we assigned a functional regulatory role for 75% of the TFs dataset. Of these, 33.7% regulate genes involved in carbon-source catabolism, 9.6% global metabolism, 8.3% nitrogen metabolism, 2.9% adaptation responses, 8.9% stress responses, and 11.7% virulence. The abundance of TFs involved in the regulation of metabolic processes indicates that bacteria have optimized their regulatory systems to control energy uptake. In contrast, the lower percentage of TFs required for stress, adaptation and virulence regulation reflects the specialization acquired by each subset of TFs associated with those processes. This approach would be useful in assigning regulatory roles to uncharacterized members of other transcriptional factor families and it might facilitate their experimental analysis.