Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 285
Filtrar
Mais filtros










Filtros aplicados
Base de dados
Intervalo de ano de publicação
1.
ACS Synth Biol ; 13(1): 206-219, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38113125

RESUMO

In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For AraC family regulators (AFRs) represented in these data, we posited AFR/promoter/inducer groupings. Twelve promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in Escherichia coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled the further diversification of these inducible plasmids.


Assuntos
Proteínas de Escherichia coli , Pseudomonas putida , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regiões Promotoras Genéticas/genética , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Plasmídeos/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Escherichia coli/genética , Fator de Transcrição AraC/genética
2.
J Bacteriol ; 205(4): e0001523, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-36920216

RESUMO

A novel approach to treat the highly virulent and infectious enteric pathogen Shigella flexneri, with the potential for reduced resistance development, is to target virulence pathways. One promising such target is the AraC-family transcription factor VirF, which activates downstream virulence factors. VirF harbors a conserved C-terminal DNA-binding domain (DBD) and an N-terminal dimerization domain (NTD). Previously, we studied the wild type (WT) and seven alanine DBD mutants of VirF binding to the virB promoter (N. J. Ragazzone, G. T. Dow, and A. Garcia, J Bacteriol 204:e00143-22, 2022, https://doi.org/10.1128/jb.00143-22). Here, we report studies of VirF binding to the icsA and rnaG promoters. Gel shift assays (electrophoretic mobility shift assays [EMSAs]) of WT VirF binding to these promoters revealed multiple bands at higher apparent molecular weights, indicating the likelihood of VirF dimerization when bound to DNA. For three of the mutants, we observed consistent effects on binding to the three promoters. For the four other mutants, we observed differential effects on promoter binding. Results of a cell-based, LexA monohybrid ß-galactosidase reporter assay [D. A. Daines, M. Granger-Schnarr, M. Dimitrova, and R. P. Silver, Methods Enzymol 358:153-161, 2002, https://doi.org/10.1016/s0076-6879(02)58087-3] indicated that WT VirF dimerizes in vivo and that alanine mutations to Y132, L137, and L147 significantly reduced dimerization. However, these mutations negatively impacted protein stability. We did purify enough of the Y132A mutant to determine that it binds in vitro to the virB and rnaG promoters, albeit with weaker affinities. Full-length VirF model structures, generated with I-TASSER, predict that these three amino acids are in a "dimerization" helix in the NTD, consistent with our results. IMPORTANCE Antimicrobial-resistant (AMR) infections continue to rise dramatically, and the lack of new approved antibiotics is not ameliorating this crisis. A promising route to reduce AMR is by targeting virulence. The Shigella flexneri virulence pathway is a valuable source for potential therapeutic targets useful to treat this infection. VirF, an AraC-family virulence transcription factor, is responsible for activating necessary downstream virulence genes that allow the bacteria to invade and spread within the human colon. Previous studies have identified how VirF interacts with the virB promoter and have even developed a lead DNA-binding inhibitor, but not much is known about VirF dimerization or binding to the icsA and rnaG promoters. Fully characterizing VirF can be a valuable resource for inhibitor discovery/design.


Assuntos
Proteínas de Ligação a DNA , Shigella flexneri , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Shigella flexneri/genética , Transcrição Gênica , Proteínas de Bactérias/metabolismo , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/farmacologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Fator de Transcrição AraC/genética , DNA/metabolismo , Regulação Bacteriana da Expressão Gênica
3.
J Mol Recognit ; 36(1): e2993, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36112092

RESUMO

Atomic force microscopy (AFM) was used to conduct single-molecule imaging of protein/DNA complexes involved in the regulation of the arabinose operon of Escherichia coli. In the presence of arabinose, the transcription regulatory protein AraC binds to a 38 bp region consisting of the araI1 and araI2 half-sites. The domain positioning of full-length AraC, when bound to DNA, was not previously known. In this study, AraC was combined with 302 and 560 bp DNA and arabinose, deposited on a mica substrate, and imaged with AFM in air. High resolution images of 560 bp DNA, where bound protein was visible, showed that AraC induces a bend in the DNA with an angle 60° ± 12° with a median of 55°. These results are consistent with earlier gel electrophoresis measurements that measured the DNA bend angle based on migration rates. By using known domain structures of AraC, geometric constraints, and contacts determined from biochemical experiments, we developed a model of the tertiary and quaternary structure of DNA-bound AraC in the presence of arabinose. The DNA bend angle predicted by the model is in agreement with the measurement values. We discuss the results in view of other regulatory proteins that cause DNA bending and formation of the open complex to initiate transcription.


Assuntos
Fator de Transcrição AraC , Proteínas de Escherichia coli , Fator de Transcrição AraC/genética , Fator de Transcrição AraC/química , Fator de Transcrição AraC/metabolismo , Proteínas de Escherichia coli/metabolismo , Microscopia de Força Atômica , Citarabina/metabolismo , Proteínas Repressoras/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Bactérias/metabolismo , Arabinose/química , Arabinose/metabolismo , Arabinose/farmacologia , Fatores de Transcrição/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , DNA/metabolismo , Ligação Proteica
4.
Mol Microbiol ; 118(5): 552-569, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36164821

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/metabolismo
5.
Nucleic Acids Res ; 50(10): 5974-5987, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35641097

RESUMO

Rob, which serves as a paradigm of the large AraC/XylS family transcription activators, regulates diverse subsets of genes involved in multidrug resistance and stress response. However, the underlying mechanism of how it engages bacterial RNA polymerase and promoter DNA to finely respond to environmental stimuli is still elusive. Here, we present two cryo-EM structures of Rob-dependent transcription activation complex (Rob-TAC) comprising of Escherichia coli RNA polymerase (RNAP), Rob-regulated promoter and Rob in alternative conformations. The structures show that a single Rob engages RNAP by interacting with RNAP αCTD and σ70R4, revealing their generally important regulatory roles. Notably, by occluding σ70R4 from binding to -35 element, Rob specifically binds to the conserved Rob binding box through its consensus HTH motifs, and retains DNA bending by aid of the accessory acidic loop. More strikingly, our ligand docking and biochemical analysis demonstrate that the large Rob C-terminal domain (Rob CTD) shares great structural similarity with the global Gyrl-like domains in effector binding and allosteric regulation, and coordinately promotes formation of competent Rob-TAC. Altogether, our structural and biochemical data highlight the detailed molecular mechanism of Rob-dependent transcription activation, and provide favorable evidences for understanding the physiological roles of the other AraC/XylS-family transcription factors.


Assuntos
Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Fator de Transcrição AraC/genética , Fator de Transcrição AraC/metabolismo , Proteínas de Bactérias/metabolismo , Citarabina/metabolismo , DNA/química , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ativação Transcricional
6.
Biochem J ; 479(4): 479-501, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35089310

RESUMO

A genetic selection system for activity of HIV protease is described that is based on a synthetic substrate constructed as a modified AraC regulatory protein that when cleaved stimulate l-arabinose metabolism in an Escherichia coli araC strain. Growth stimulation on selective plates was shown to depend on active HIV protease and the scissile bond in the substrate. In addition, the growth of cells correlated well with the established cleavage efficiency of the sites in the viral polyprotein, Gag, when these sites were individually introduced into the synthetic substrate of the selection system. Plasmids encoding protease variants selected based on stimulation of cell growth in the presence of saquinavir or cleavage of a site not cleaved by wild-type protease, were indistinguishable with respect to both phenotypes. Also, both groups of selected plasmids encoded side chain substitutions known from clinical isolates or displayed different side chain substitutions but at identical positions. One highly frequent side chain substitution, E34V, not regarded as a major drug resistance substitution was found in variants obtained under both selective conditions and is suggested to improve protease processing of the synthetic substrate. This substitution is away from the substrate-binding cavity and together with other substitutions in the selected reading frames supports the previous suggestion of a substrate-binding site extended from the active site binding pocket itself.


Assuntos
Fármacos Anti-HIV/farmacocinética , Farmacorresistência Viral/genética , Protease de HIV/genética , Substituição de Aminoácidos , Fator de Transcrição AraC/genética , Arabinose/metabolismo , Quimosina/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Fusão gag-pol/metabolismo , Produtos do Gene gag/metabolismo , Genes araC , Protease de HIV/química , Protease de HIV/isolamento & purificação , Protease de HIV/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Mutação Puntual , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saquinavir/antagonistas & inibidores , Saquinavir/farmacologia , Seleção Genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por Substrato
7.
Proteins ; 90(1): 186-199, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34369028

RESUMO

To create bacterial transcription "circuits" for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coli AraC transcriptional activator, for which binding to DNA is modulated by binding L-arabinose. Here, we engineered chimeric AraC/XylS transcription activators that recognized ara DNA binding sites and responded to varied effector ligands. The first step, identifying domain boundaries in the natural homologs, was challenging because (i) no full-length, dimeric structures were available and (ii) extremely low sequence identities (≤10%) among homologs precluded traditional assemblies of sequence alignments. Thus, to identify domains, we built and aligned structural models of the natural proteins. The designed chimeric activators were assessed for function, which was then further improved by random mutagenesis. Several mutational variants were identified for an XylS•AraC chimera that responded to benzoate; two enhanced activation to near that of wild-type AraC. For an RhaR•AraC chimera, a variant with five additional substitutions enabled transcriptional activation in response to rhamnose. These five changes were dispersed across the protein structure, and combinatorial experiments testing subsets of substitutions showed significant non-additivity. Combined, the structure modeling and epistasis suggest that the common AraC/XylS structural scaffold is highly interconnected, with complex intra-protein and inter-domain communication pathways enabling allosteric regulation. At the same time, the observed epistasis and the low sequence identities of the natural homologs suggest that the structural scaffold and function of transcriptional regulation are nevertheless highly accommodating of amino acid changes.


Assuntos
Fator de Transcrição AraC , Proteínas de Bactérias , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Transativadores , Regulação Alostérica , Aminoácidos/química , Aminoácidos/genética , Fator de Transcrição AraC/química , Fator de Transcrição AraC/genética , Fator de Transcrição AraC/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Mutação/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/química , Transativadores/genética , Transativadores/metabolismo
8.
J Bacteriol ; 203(23): e0018521, 2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34543107

RESUMO

Francisella tularensis is a Gram-negative bacterium that causes a fatal human disease known as tularemia. The Centers for Disease Control and Prevention have classified F. tularensis as a category A tier 1 select agent. The virulence mechanisms of Francisella are not entirely understood. Francisella possesses very few transcription regulators, and most of these regulate the expression of genes involved in intracellular survival and virulence. The F. tularensis genome sequence analysis reveals an AraC (FTL_0689) transcriptional regulator homologous to the AraC/XylS family of transcriptional regulators. In Gram-negative bacteria, AraC activates genes required for l-arabinose utilization and catabolism. The role of the FTL_0689 regulator in F. tularensis is not known. In this study, we characterized the role of FTL_0689 in the gene regulation of F. tularensis and investigated its contribution to intracellular survival and virulence. The results demonstrate that FTL_0689 in Francisella is not required for l-arabinose utilization. Instead, FTL_0689 specifically regulates the expression of the oxidative and global stress response, virulence, metabolism, and other key pathways genes required by Francisella when exposed to oxidative stress. The FTL_0689 mutant is attenuated for intramacrophage growth and virulence in mice. Based on the deletion mutant phenotype, FTL_0689 was termed osrR (oxidative stress response regulator). Altogether, this study elucidates the role of the osrR transcriptional regulator in tularemia pathogenesis. IMPORTANCE The virulence mechanisms of category A select agent Francisella tularensis, the causative agent of a fatal human disease known as tularemia, remain largely undefined. The present study investigated the role of a transcriptional regulator and its overall contribution to the oxidative stress resistance of F. tularensis. The results provide an insight into a novel gene regulatory mechanism, especially when Francisella is exposed to oxidative stress conditions. Understanding such Francisella- specific regulatory mechanisms will help identify potential targets for developing effective therapies and vaccines to prevent tularemia.


Assuntos
Fator de Transcrição AraC/metabolismo , Francisella tularensis/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Estresse Oxidativo/fisiologia , Animais , Fator de Transcrição AraC/genética , Regulação para Baixo , Francisella tularensis/patogenicidade , Deleção de Genes , Teste de Complementação Genética , Camundongos , Camundongos Endogâmicos C57BL , Tularemia/microbiologia , Virulência
9.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064685

RESUMO

Pseudomonas aeruginosa encodes a large set of transcriptional regulators (TRs) that modulate and manage cellular metabolism to survive in variable environmental conditions including that of the human body. The AraC family regulators are an abundant group of TRs in bacteria, mostly acting as gene expression activators, controlling diverse cellular functions (e.g., carbon metabolism, stress response, and virulence). The PA3027 protein from P. aeruginosa has been classified in silico as a putative AraC-type TR. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3027 revealed a spectacular increase in the mRNA levels of PA3026-PA3024 (divergent to PA3027), PA3464, and PA3342 genes encoding proteins potentially involved in glycerolipid metabolism. Concomitantly, chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that at least 22 regions are bound by PA3027 in the PAO1161 genome. These encompass promoter regions of PA3026, PA3464, and PA3342, showing the major increase in expression in response to PA3027 excess. In Vitro DNA binding assay confirmed interactions of PA3027 with these regions. Furthermore, promoter-reporter assays in a heterologous host showed the PA3027-dependent activation of the promoter of the PA3026-PA3024 operon. Two motifs representing the preferred binding sites for PA3027, one localized upstream and one overlapping with the -35 promoter sequence, were identified in PA3026p and our data indicate that both motifs are required for full activation of this promoter by PA3027. Overall, the presented data show that PA3027 acts as a transcriptional regulator in P. aeruginosa, activating genes likely engaged in glycerolipid metabolism. The GliR name, from a glycerolipid metabolism regulator, is proposed for PA3027 of P. aeruginosa.


Assuntos
Fator de Transcrição AraC/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/metabolismo , Óperon , Pseudomonas aeruginosa/metabolismo , Fator de Transcrição AraC/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Humanos , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Transativadores
10.
Nat Chem Biol ; 17(7): 817-827, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33903769

RESUMO

The L-arabinose-responsive AraC and its cognate PBAD promoter underlie one of the most often used chemically inducible prokaryotic gene expression systems in microbiology and synthetic biology. Here, we change the sensing capability of AraC from L-arabinose to blue light, making its dimerization and the resulting PBAD activation light-inducible. We engineer an entire family of blue light-inducible AraC dimers in Escherichia coli (BLADE) to control gene expression in space and time. We show that BLADE can be used with pre-existing L-arabinose-responsive plasmids and strains, enabling optogenetic experiments without the need to clone. Furthermore, we apply BLADE to control, with light, the catabolism of L-arabinose, thus externally steering bacterial growth with a simple transformation step. Our work establishes BLADE as a highly practical and effective optogenetic tool with plug-and-play functionality-features that we hope will accelerate the broader adoption of optogenetics and the realization of its vast potential in microbiology, synthetic biology and biotechnology.


Assuntos
Fator de Transcrição AraC/genética , Arabinose/genética , Proteínas de Escherichia coli/genética , Engenharia Genética , Luz , Fator de Transcrição AraC/metabolismo , Arabinose/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo
11.
ACS Synth Biol ; 10(5): 1227-1236, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33915046

RESUMO

Growth feedback, the inherent coupling between the synthetic gene circuit and the host cell growth, could significantly change the circuit behaviors. Previously, a diverse array of emergent behaviors, such as growth bistability, enhanced ultrasensitivity, and topology-dependent memory loss, were reported to be induced by growth feedback. However, the influence of the growth feedback on the circuit functions remains underexplored. Here, we reported an unexpected damped oscillatory behavior of a self-activation gene circuit induced by nutrient-modulating growth feedback. Specifically, after dilution of the activated self-activation switch into the fresh medium with moderate nutrients, its gene expression first decreases as the cell grows and then shows a significant overshoot before it reaches the steady state, leading to damped oscillation dynamics. Fitting the data with a coarse-grained model suggests a nonmonotonic growth-rate regulation on gene production rate. The underlying mechanism of the oscillation was demonstrated by a molecular mathematical model, which includes the ribosome allocation toward gene production, cell growth, and cell maintenance. Interestingly, the model predicted a counterintuitive dependence of oscillation amplitude on the nutrition level, where the highest peak was found in the medium with moderate nutrients, but was not observed in rich nutrients. We experimentally verified this prediction by tuning the nutrient level in the culture medium. We did not observe significant oscillatory behavior for the toggle switch, suggesting that the emergence of damped oscillatory behavior depends on circuit network topology. Our results demonstrated a new nonlinear emergent behavior mediated by growth feedback, which depends on the ribosome allocation between gene circuit and cell growth.


Assuntos
Proliferação de Células/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Retroalimentação Fisiológica/fisiologia , Engenharia Genética/métodos , Nutrientes , Fator de Transcrição AraC/genética , Fator de Transcrição AraC/metabolismo , Meios de Cultura/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Genes Bacterianos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Microrganismos Geneticamente Modificados , Modelos Genéticos , Modelos Moleculares , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Ribossomos/metabolismo
12.
FEMS Microbiol Rev ; 45(5)2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-33837749

RESUMO

Transcriptional factors play an important role in gene regulation in all organisms, especially in Bacteria. Here special emphasis is placed in the AraC/XylS family of transcriptional regulators. This is one of the most abundant as many predicted members have been identified and more members are added because more bacterial genomes are sequenced. Given the way more experimental evidence has mounded in the past decades, we decided to update the information about this captivating family of proteins. Using bioinformatics tools on all the data available for experimentally characterized members of this family, we found that many members that display a similar functional classification can be clustered together and in some cases they have a similar regulatory scheme. A proposal for grouping these proteins is also discussed. Additionally, an analysis of surveyed proteins in bacterial genomes is presented. Altogether, the current review presents a panoramic view into this family and we hope it helps to stimulate future research in the field.


Assuntos
Transativadores , Fator de Transcrição AraC , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética
13.
Nucleic Acids Res ; 49(5): e25, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33290521

RESUMO

Ligand-inducible genetic systems are the mainstay of synthetic biology, allowing gene expression to be controlled by the presence of a small molecule. However, 'leaky' gene expression in the absence of inducer remains a persistent problem. We developed a leak dampener tool that drastically reduces the leak of inducible genetic systems while retaining signal in Escherichia coli. Our system relies on a coherent feedforward loop featuring a suppressor tRNA that enables conditional readthrough of silent non-sense mutations in a regulated gene, and this approach can be applied to any ligand-inducible transcription factor. We demonstrate proof-of-principle of our system with the lactate biosensor LldR and the arabinose biosensor AraC, which displayed a 70-fold and 630-fold change in output after induction of a fluorescence reporter, respectively, without any background subtraction. Application of the tool to an arabinose-inducible mutagenesis plasmid led to a 540-fold change in its output after induction, with leak decreasing to the level of background mutagenesis. This study provides a modular tool for reducing leak and improving the fold-induction within genetic circuits, demonstrated here using two types of biosensors relevant to cancer detection and genetic engineering.


Assuntos
Regulação Bacteriana da Expressão Gênica , RNA de Transferência/metabolismo , Fator de Transcrição AraC/metabolismo , Arabinose/metabolismo , Códon de Terminação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácido Láctico/metabolismo , Mutagênese , Plasmídeos/genética , Biossíntese de Proteínas , RNA Catalítico , RNA de Transferência/química , Fatores de Transcrição/metabolismo
14.
Protein Eng Des Sel ; 332020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-33215672

RESUMO

We previously described the design of triacetic acid lactone (TAL) biosensor 'AraC-TAL1', based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a 'decoy' ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.


Assuntos
Fator de Transcrição AraC , Técnicas Biossensoriais , Proteínas de Escherichia coli , Escherichia coli , Engenharia de Proteínas , Pironas/análise , Resorcinóis/análise , Fator de Transcrição AraC/química , Fator de Transcrição AraC/genética , Fator de Transcrição AraC/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Sensibilidade e Especificidade
15.
PLoS Pathog ; 16(8): e1008776, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845938

RESUMO

Enteroaggregative Escherichia coli (EAEC) is a diarrheagenic pathotype associated with traveler's diarrhea, foodborne outbreaks and sporadic diarrhea in industrialized and developing countries. Regulation of virulence in EAEC is mediated by AggR and its negative regulator Aar. Together, they control the expression of at least 210 genes. On the other hand, we observed that about one third of Aar-regulated genes are related to metabolism and transport. In this study we show the AggR/Aar duo controls the metabolism of lipids. Accordingly, we show that AatD, encoded in the AggR-regulated aat operon (aatPABCD) is an N-acyltransferase structurally similar to the essential Apolipoprotein N-acyltransferase Lnt and is required for the acylation of Aap (anti-aggregation protein). Deletion of aatD impairs post-translational modification of Aap and causes its accumulation in the bacterial periplasm. trans-complementation of 042aatD mutant with the AatD homolog of ETEC or with the N-acyltransferase Lnt reestablished translocation of Aap. Site-directed mutagenesis of the E207 residue in the putative acyltransferase catalytic triad disrupted the activity of AatD and caused accumulation of Aap in the periplasm due to reduced translocation of Aap at the bacterial surface. Furthermore, Mass spectroscopy revealed that Aap is acylated in a putative lipobox at the N-terminal of the mature protein, implying that Aap is a lipoprotein. Lastly, deletion of aatD impairs bacterial colonization of the streptomycin-treated mouse model. Our findings unveiled a novel N-acyltransferase family associated with bacterial virulence, and that is tightly regulated by AraC/XylS regulators in the order Enterobacterales.


Assuntos
Acetiltransferases/metabolismo , Fator de Transcrição AraC/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica , Acetiltransferases/genética , Acilação , Animais , Fator de Transcrição AraC/química , Fator de Transcrição AraC/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Filogenia , Conformação Proteica , Virulência
16.
Mol Microbiol ; 114(5): 870-886, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32757400

RESUMO

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in crucifers. Our previous findings revealed that Xcc can degrade 4-hydroxybenzoic acid (4-HBA) via the ß-ketoadipate pathway. This present study expands on this knowledge in several ways. First, we show that infective Xcc cells induce in situ biosynthesis of 4-HBA in host plants, and Xcc can efficiently degrade 4-HBA via the pobA/pobR locus, which encodes a 4-hydroxybenzoate hydroxylase and an AraC-family transcription factor respectively. Next, the transcription of pobA is specifically induced by 4-HBA and is positively regulated by PobR, which is constitutively expressed in Xcc. 4-HBA directly binds to PobR dimers, resulting in activation of pobA expression. Point mutation and subsequent isothermal titration calorimetry and size exclusion chromatography analysis identified nine key conserved residues required for 4-HBA binding and/or dimerization of PobR. Furthermore, overlapping promoters harboring fully overlapping -35 elements were identified between the divergently transcribed pobA and pobR. The 4-HBA/PobR dimer complex specifically binds to a 25-bp site, which encompasses the -35 elements shared by the overlapping promoters. Finally, GUS histochemical staining and subsequent quantitative assay showed that both pobA and pobR genes are transcribed during Xcc infection of Chinese radish, and the strain ΔpobR exhibited compromised virulence in Chinese radish. These findings suggest that the ability of Xcc to survive the 4-HBA stress might be important for its successful colonization of host plants.


Assuntos
Parabenos/metabolismo , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismo , Fator de Transcrição AraC/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Parabenos/química , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Virulência/genética , Xanthomonas campestris/patogenicidade
17.
Int J Med Microbiol ; 310(5): 151436, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32654771

RESUMO

Staphylococcus aureus is a gram-positive pathogenic bacterium and is capable of secreting numerous toxins interfering directly with the host to cause acute infections. Rbf, a transcriptional regulator of AraC/XylS family, has been reported to promote biofilm formation in polysaccharide intercellular adhesion (PIA) mediated manner to cause chronic infections. In this study, we revealed the new virulence-mediated role of Rbf that can negatively regulate the hemolytic activity. Furthermore, Rbf can specifically bind to the hla and psmα promoters to repress their expression, resulting in significantly decreased production of phenol-soluble modulins α (PSMα) and alpha-toxin. Accordingly, the rbf mutant strain exhibited the increased pathogenicity compared to the wild-type (WT) strain in a mouse subcutaneous abscess model, representing a type of acute infection by S. aureus. Collectively, our results provide a novel insight into the virulence regulation and acute infections mediated by Rbf in S. aureus.


Assuntos
Fator de Transcrição AraC/metabolismo , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus/genética , Fatores de Virulência/metabolismo , Animais , Fator de Transcrição AraC/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Regulação para Baixo , Feminino , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Hemólise , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Regiões Promotoras Genéticas , Staphylococcus aureus/metabolismo , Virulência , Fatores de Virulência/genética
18.
Appl Environ Microbiol ; 86(19)2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32709725

RESUMO

Rhizobium tropici CIAT 899 is a broad-host-range rhizobial strain that establishes symbiotic interactions with legumes and tolerates different environmental stresses such as heat, acidity, or salinity. This rhizobial strain produces a wide variety of symbiotically active nodulation factors (NF) induced not only by the presence of plant-released flavonoids but also under osmotic stress conditions through the LysR-type transcriptional regulators NodD1 (flavonoids) and NodD2 (osmotic stress). However, the activation of NodD2 under high-osmotic-stress conditions remains elusive. Here, we have studied the role of a new AraC-type regulator (named as OnfD) in the symbiotic interaction of R. tropici CIAT 899 with Phaseolus vulgaris and Lotus plants. We determined that OnfD is required under salt stress conditions for the transcriptional activation of the nodulation genes and therefore the synthesis and export of NF, which are required for a successful symbiosis with P. vulgaris Moreover, using bacterial two-hybrid analysis, we demonstrated that the OnfD and NodD2 proteins form homodimers and OnfD/NodD2 form heterodimers, which could be involved in the production of NF in the presence of osmotic stress conditions since both regulators are required for NF synthesis in the presence of salt. A structural model of OnfD is presented and discussed.IMPORTANCE The synthesis and export of rhizobial NF are mediated by a conserved group of LysR-type regulators, the NodD proteins. Here, we have demonstrated that a non-LysR-type regulator, an AraC-type protein, is required for the transcriptional activation of symbiotic genes and for the synthesis of symbiotically active NF under salt stress conditions.


Assuntos
Fator de Transcrição AraC/genética , Proteínas de Bactérias/genética , Lotus/microbiologia , Phaseolus/microbiologia , Rhizobium tropici/genética , Simbiose/genética , Fator de Transcrição AraC/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Rhizobium tropici/metabolismo , Estresse Salino/genética , Ativação Transcricional/genética
19.
Infect Immun ; 88(10)2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32690633

RESUMO

Successful colonization by enteric pathogens is contingent upon effective interactions with the host and the resident microbiota. These pathogens thus respond to and integrate myriad signals to control virulence. Long-chain fatty acids repress the virulence of the important enteric pathogens Salmonella enterica and Vibrio cholerae by repressing AraC-type transcriptional regulators in pathogenicity islands. While several fatty acids are known to be repressive, we show here that cis-2-unsaturated fatty acids, a rare chemical class used as diffusible signal factors (DSFs), are highly potent inhibitors of virulence functions. We found that DSFs repressed virulence gene expression of enteric pathogens by interacting with transcriptional regulators of the AraC family. In Salmonella enterica serovar Typhimurium, DSFs repress the activity of HilD, an AraC-type activator essential to the induction of epithelial cell invasion, by both preventing its interaction with target DNA and inducing its rapid degradation by Lon protease. cis-2-Hexadecenoic acid (c2-HDA), a DSF produced by Xylella fastidiosa, was the most potent among those tested, repressing the HilD-dependent transcriptional regulator hilA and the type III secretion effector sopB >200- and 68-fold, respectively. Further, c2-HDA attenuated the transcription of the ToxT-dependent cholera toxin synthesis genes of V. cholerae c2-HDA significantly repressed invasion gene expression by Salmonella in the murine colitis model, indicating that the HilD-dependent signaling pathway functions within the complex milieu of the animal intestine. These data argue that enteric pathogens respond to DSFs as interspecies signals to identify appropriate niches in the gut for virulence activation, which could be exploited to control the virulence of enteric pathogens.


Assuntos
Fator de Transcrição AraC/metabolismo , Intestinos/microbiologia , Ácidos Palmíticos/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Animais , Fator de Transcrição AraC/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas/genética , Camundongos , Ácidos Palmíticos/química , Ligação Proteica , Estabilidade Proteica , Salmonella typhimurium/genética , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genética
20.
ACS Synth Biol ; 9(7): 1581-1590, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32525658

RESUMO

Robustness to temperature variation is an important specification in biomolecular circuit design. While the cancellation of parametric temperature dependencies has been shown to improve the temperature robustness of the period in a synthetic oscillator design, the performance of other biomolecular circuit designs in different temperature conditions is relatively unclear. Using a combination of experimental measurements and mathematical models, we assessed the temperature robustness of two biomolecular circuit motifs-a negative feedback loop and a feedforward loop. We found that the measured responses of both the circuits changed with temperature, both in the amplitude and in the transient response. We also found that, in addition to the cancellation of parametric temperature dependencies, certain parameter regimes could facilitate the temperature robustness of the negative feedback loop, although at a performance cost. We discuss these parameter regimes in the context of the measured data for the negative feedback loop. These results should help develop a framework for assessing and designing temperature robustness in biomolecular circuits.


Assuntos
Retroalimentação Fisiológica , Modelos Biológicos , Fator de Transcrição AraC/genética , Escherichia coli/metabolismo , Expressão Gênica , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Temperatura
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...