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[PMID]: 28806780 [Au] Autor: Santiago AE; Yan MB; Hazen TH; Sauder B; Meza-Segura M; Rasko DA; Kendall MM; Ruiz-Perez F; Nataro JP [Ad] Endereço: Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia, United States of America. [Ti] Título: The AraC Negative Regulator family modulates the activity of histone-like proteins in pathogenic bacteria. [So] Source: PLoS Pathog;13(8):e1006545, 2017 Aug. [Is] ISSN: 1553-7374 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: The AraC Negative Regulators (ANR) comprise a large family of virulence regulators distributed among diverse clinically important Gram-negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., and pathogenic E. coli strains. We have previously reported broad effects of the ANR members on regulators of the AraC/XylS family. Here, we interrogate possible broader effects of the ANR members on the bacterial transcriptome. Our studies focused on Aar (AggR-activated regulator), an ANR family archetype in enteroaggregative E. coli (EAEC) isolate 042. Transcriptome analysis of EAEC strain 042, 042aar and 042aar(pAar) identified more than 200 genes that were differentially expressed (+/- 1.5 fold, p<0.05). Most of those genes are located on the bacterial chromosome (195 genes, 92.85%), and are associated with regulation, transport, metabolism, and pathogenesis, based on the predicted annotation; a considerable number of Aar-regulated genes encoded for hypothetical proteins (46 genes, 21.9%) and regulatory proteins (25, 11.9%). Notably, the transcriptional expression of three histone-like regulators, H-NS (orf1292), H-NS homolog (orf2834) and StpA, was down-regulated in the absence of aar and may explain some of the effects of Aar on gene expression. By employing a bacterial two-hybrid system, LacZ reporter assays, pull-down and electrophoretic mobility shift assay (EMSA) analysis, we demonstrated that Aar binds directly to H-NS and modulates H-NS-induced gene silencing. Importantly, Aar was highly expressed in the mouse intestinal tract and was found to be necessary for maximal H-NS expression. In conclusion, this work further extends our knowledge of genes under the control of Aar and its biological relevance in vivo. [Mh] Termos MeSH primário: Fator de Transcrição AraC/metabolismo Escherichia coli Enteropatogênica/metabolismo Infecções por Escherichia coli/metabolismo Regulação Bacteriana da Expressão Gênica/fisiologia Virulência/fisiologia [Mh] Termos MeSH secundário: Animais Ensaio de Desvio de Mobilidade Eletroforética Escherichia coli Enteropatogênica/patogenicidade Proteínas de Escherichia coli/metabolismo Histonas/metabolismo Camundongos Camundongos Endogâmicos BALB C Reação em Cadeia da Polimerase [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (AraC Transcription Factor); 0 (Escherichia coli Proteins); 0 (Histones) [Em] Mês de entrada: 1710 [Cu] Atualização por classe: 171002 [Lr] Data última revisão: 171002 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170815 [St] Status: MEDLINE [do] DOI: 10.1371/journal.ppat.1006545

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[PMID]: 28025272 [Au] Autor: Lind PA; Arvidsson L; Berg OG; Andersson DI [Ad] Endereço: Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden. [Ti] Título: Variation in Mutational Robustness between Different Proteins and the Predictability of Fitness Effects. [So] Source: Mol Biol Evol;34(2):408-418, 2017 Feb 01. [Is] ISSN: 1537-1719 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: Random mutations in genes from disparate protein classes may have different distributions of fitness effects (DFEs) depending on different structural, functional, and evolutionary constraints. We measured the fitness effects of 156 single mutations in the genes encoding AraC (transcription factor), AraD (enzyme), and AraE (transporter) used for bacterial growth on l-arabinose. Despite their different molecular functions these genes all had bimodal DFEs with most mutations either being neutral or strongly deleterious, providing a general expectation for the DFE. This contrasts with the unimodal DFEs previously obtained for ribosomal protein genes where most mutations were slightly deleterious. Based on theoretical considerations, we suggest that the 33-fold higher average mutational robustness of ribosomal proteins is due to stronger selection for reduced costs of translational and transcriptional errors. Whereas the large majority of synonymous mutations were deleterious for ribosomal proteins genes, no fitness effects could be detected for the AraCDE genes. Four mutations in AraC and AraE increased fitness, suggesting that slightly advantageous mutations make up a significant fraction of the DFE, but that they often escape detection due to the limited sensitivity of commonly used fitness assays. We show that the fitness effects of amino acid substitutions can be predicted based on evolutionary conservation, but those weakly deleterious mutations are less reliably detected. This suggests that large-effect mutations and the fraction of highly deleterious mutations can be computationally predicted, but that experiments are required to characterize the DFE close to neutrality, where many mutations ultimately fixed in a population will occur. [Mh] Termos MeSH primário: Proteínas de Bactérias/genética Aptidão Genética [Mh] Termos MeSH secundário: Fator de Transcrição AraC/genética Arabinose/genética Evolução Biológica Regulação Bacteriana da Expressão Gênica Variação Genética Modelos Genéticos Proteínas de Transporte de Monossacarídeos/genética Mutação Proteínas Ribossômicas/genética Salmonella typhimurium/genética Fatores de Transcrição/genética [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (AraC Transcription Factor); 0 (AraE protein, bacteria); 0 (Bacterial Proteins); 0 (Monosaccharide Transport Proteins); 0 (Ribosomal Proteins); 0 (Transcription Factors); B40ROO395Z (Arabinose) [Em] Mês de entrada: 1706 [Cu] Atualização por classe: 170608 [Lr] Data última revisão: 170608 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 161228 [St] Status: MEDLINE [do] DOI: 10.1093/molbev/msw239

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[PMID]: 27671067 [Au] Autor: Fan R; Li D; Wang Y; He T; Feßler AT; Schwarz S; Wu C [Ad] Endereço: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, People's Republic of China. [Ti] Título: Presence of the optrA Gene in Methicillin-Resistant Staphylococcus sciuri of Porcine Origin. [So] Source: Antimicrob Agents Chemother;60(12):7200-7205, 2016 Dec. [Is] ISSN: 1098-6596 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: A total of 57 methicillin-resistant Staphylococcus aureus (MRSA) isolates and 475 methicillin-resistant coagulase-negative staphylococci (MRCoNS) collected from pigs in the Guangdong province of China in 2014 were investigated for the presence of the novel oxazolidinone-phenicol resistance gene optrA The optrA gene was detected in 6.9% (n = 33) of the MRCoNS, all of which were Staphylococcus sciuri isolates, but in none of the MRSA isolates. Five optrA-carrying methicillin-resistant (MR) S. sciuri isolates also harbored the multiresistance gene cfr Pulsed-field gel electrophoresis (PFGE) and dru typing of the 33 optrA-carrying MR S. sciuri isolates revealed 25 patterns and 5 sequence types, respectively. S1 nuclease PFGE and Southern blotting confirmed that optrA was located in the chromosomal DNAs of 29 isolates, including 1 cfr-positive isolate. The remaining four isolates harbored a ∼35-kb pWo28-3-like plasmid on which optrA and cfr were located together with other resistance genes, as confirmed by sequence analysis. Six different types of genetic environments (types I to VI) of the chromosome-borne optrA genes were identified; these types had the optrA gene and its transcriptional regulator araC in common. Tn558 was found to be associated with araC-optrA in types II to VI. The optrA gene in types II and III was found in close proximity to the ccr gene complex of the respective staphylococcal cassette chromosome mec element (SCCmec). Since oxazolidinones are last-resort antimicrobial agents for the control of serious infections caused by methicillin-resistant staphylococci in humans, the location of the optrA gene close to the ccr complex is an alarming observation. [Mh] Termos MeSH primário: Antibacterianos/farmacologia Proteínas de Bactérias/genética Farmacorresistência Bacteriana Múltipla/genética Oxazolidinonas/farmacologia Staphylococcus/efeitos dos fármacos Staphylococcus/genética [Mh] Termos MeSH secundário: Animais Fator de Transcrição AraC/genética China Resistência Microbiana a Medicamentos/genética Eletroforese em Gel de Campo Pulsado Resistência a Meticilina/genética Tipagem Molecular Staphylococcus/isolamento & purificação Suínos [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (Anti-Bacterial Agents); 0 (AraC Transcription Factor); 0 (Bacterial Proteins); 0 (CFR protein, Staphylococcus sciuri); 0 (Oxazolidinones) [Em] Mês de entrada: 1710 [Cu] Atualização por classe: 171002 [Lr] Data última revisão: 171002 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 160928 [St] Status: MEDLINE

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[PMID]: 27613678 [Au] Autor: Meisner J; Goldberg JB [Ad] Endereço: Division of Pulmonary, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA. [Ti] Título: The Escherichia coli rhaSR-PrhaBAD Inducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa. [So] Source: Appl Environ Microbiol;82(22):6715-6727, 2016 Nov 15. [Is] ISSN: 1098-5336 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: The araC-ParaBAD inducible promoter system is tightly controlled and allows gene expression to be modulated over a wide range in Escherichia coli, which has led to its widespread use in other bacteria. Although anecdotal evidence suggests that araC-ParaBAD is leaky in Pseudomonas aeruginosa, neither a thorough analysis of this inducible promoter system in P. aeruginosa nor a concerted effort to identify alternatives with improved functionality has been reported. Here, we evaluated the functionality of the araC-ParaBAD system in P. aeruginosa Using transcriptional fusions to a lacZ reporter gene, we determined that the noninduced expression from araC-ParaBAD is high and cannot be reduced by carbon catabolite repression as it can in E. coli Modulating translational initiation by altering ribosome-binding site strength reduced the noninduced activity but also decreased the maximal induced activity and narrowed the induction range. Integrating the inducible promoter system into the posttranscriptional regulatory network that controls catabolite repression in P. aeruginosa significantly decreased the noninduced activity and increased the induction range. In addition to these improvements in the functionality of the araC-ParaBAD system, we found that the lacI -Ptac and rhaSR-PrhaBAD inducible promoter systems had significantly lower noninduced expression and were inducible over a broader range than araC-ParaBAD We demonstrated that noninduced expression from the araC-ParaBAD system supported the function of genes involved in antibiotic resistance and tryptophan biosynthesis in P. aeruginosa, problems that were avoided with rhaSR-PrhaBAD. rhaSR-PrhaBAD is tightly controlled, allows gene expression over a wide range, and represents a significant improvement over araC-ParaBAD in P. aeruginosa IMPORTANCE: We report the shortcomings of the commonly used Escherichia coli araC-ParaBAD inducible promoter system in Pseudomonas aeruginosa, successfully reengineered it to improve its functionality, and show that the E. coli rhaSR-PrhaBAD system is tightly controlled and allows inducible gene expression over a wide range in P. aeruginosa. [Mh] Termos MeSH primário: Proteínas de Bactérias/genética Escherichia coli/genética Regulação Bacteriana da Expressão Gênica Regiões Promotoras Genéticas Pseudomonas aeruginosa/genética [Mh] Termos MeSH secundário: Fator de Transcrição AraC/genética Sítios de Ligação Repressão Catabólica/genética Farmacorresistência Bacteriana/genética Proteínas de Escherichia coli/genética Genes Reporter Engenharia Genética/métodos Óperon Lac Fatores de Transcrição/genética Triptofano/biossíntese [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (AraC Transcription Factor); 0 (AraC protein, E coli); 0 (Bacterial Proteins); 0 (Escherichia coli Proteins); 0 (Transcription Factors); 8DUH1N11BX (Tryptophan) [Em] Mês de entrada: 1710 [Cu] Atualização por classe: 171031 [Lr] Data última revisão: 171031 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 160911 [St] Status: MEDLINE

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[PMID]: 27498315 [Au] Autor: Széliová D; Krahulec J; Safránek M; Lisková V; Turna J [Ad] Endereço: Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia. [Ti] Título: Modulation of heterologous expression from PBAD promoter in Escherichia coli production strains. [So] Source: J Biotechnol;236:1-9, 2016 Oct 20. [Is] ISSN: 1873-4863 [Cp] País de publicação: Netherlands [La] Idioma: eng [Ab] Resumo: Promoter PBAD is frequently used for heterologous gene expression due to several advantages, such as moderately high expression levels, induction by an inexpensive and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly important for expression of toxic proteins. A drawback of this promoter is all-or-none induction that occurs at subsaturating inducer concentrations. Although the overall expression level of the cell culture seems to correlate with increasing arabinose concentrations, the population is a mixture of induced and uninduced cells and with increasing arabinose concentrations, only the fraction of induced cells increases. This phenomenon is caused by autocatalytic gene expression - the expression of the arabinose transporter AraE is induced by the transported molecule. In this work the promoter PE, controlling the expression of araE, was exchanged for the stronger PBAD promoter in two Escherichia coli strains commonly used for heterologous protein production. This modification should increase a basal number of arabinose transporters in the cell wall and reduce the threshold concentration required for induction and thus reduce heterogeneity of cell population. Heterogeneity and level of expression in individual cells were analysed by flow cytometry using gfp as a reporter gene. In the strain BL21ai, the promoter exchange increased the number of induced cells at subsaturating arabinose concentrations as well as a yield of protein at saturating inducer concentration. In contrast, the modification did not improve these characteristics in RV308ai. In both strains it was possible to modulate the expression level in induced cells 3-6-fold even at subsaturating arabinose concentrations. [Mh] Termos MeSH primário: Arabinose/metabolismo Clonagem Molecular/métodos Escherichia coli/genética Regiões Promotoras Genéticas/genética Engenharia de Proteínas/métodos [Mh] Termos MeSH secundário: Fator de Transcrição AraC/genética Proteínas de Escherichia coli/genética Proteínas Recombinantes/genética Proteínas Recombinantes/metabolismo [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (AraC Transcription Factor); 0 (AraC protein, E coli); 0 (Escherichia coli Proteins); 0 (Recombinant Proteins); B40ROO395Z (Arabinose) [Em] Mês de entrada: 1703 [Cu] Atualização por classe: 170309 [Lr] Data última revisão: 170309 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 160808 [St] Status: MEDLINE

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[PMID]: 27381916 [Au] Autor: Nock AM; Wargo MJ [Ad] Endereço: Department of Microbiology and Molecular Genetics, University of Vermont College of Medicine, Burlington, Vermont, USA. [Ti] Título: Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators. [So] Source: J Bacteriol;198(18):2503-14, 2016 Sep 15. [Is] ISSN: 1098-5530 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: UNLABELLED: Burkholderia thailandensis is a soil-dwelling bacterium that shares many metabolic pathways with the ecologically similar, but evolutionarily distant, Pseudomonas aeruginosa Among the diverse nutrients it can utilize is choline, metabolizable to the osmoprotectant glycine betaine and subsequently catabolized as a source of carbon and nitrogen, similar to P. aeruginosa Orthologs of genes in the choline catabolic pathway in these two bacteria showed distinct differences in gene arrangement as well as an additional orthologous transcriptional regulator in B. thailandensis In this study, we showed that multiple glutamine amidotransferase 1 (GATase 1)-containing AraC family transcription regulators (GATRs) are involved in regulation of the B. thailandensis choline catabolic pathway (gbdR1, gbdR2, and souR). Using genetic analyses and sequencing the transcriptome in the presence and absence of choline, we identified the likely regulons of gbdR1 (BTH_II1869) and gbdR2 (BTH_II0968). We also identified a functional ortholog for P. aeruginosa souR, a GATR that regulates the metabolism of sarcosine to glycine. GbdR1 is absolutely required for expression of the choline catabolic locus, similar to P. aeruginosa GbdR, while GbdR2 is important to increase expression of the catabolic locus. Additionally, the B. thailandensis SouR ortholog (BTH_II0994) is required for catabolism of choline and its metabolites as carbon sources, whereas in P. aeruginosa, SouR function can by bypassed by GbdR. The strategy employed by B. thailandensis represents a distinct regulatory solution to control choline catabolism and thus provides both an evolutionary counterpoint and an experimental system to analyze the acquisition and regulation of this pathway during environmental growth and infection. IMPORTANCE: Many proteobacteria that occupy similar environmental niches have horizontally acquired orthologous genes for metabolism of compounds useful in their shared environment. The arrangement and differential regulation of these components can help us understand both the evolution of these systems and the potential roles these pathways have in the biology of each bacterium. Here, we describe the transcriptome response of Burkholderia thailandensis to the eukaryote-enriched molecule choline, identify the regulatory pathway governing choline catabolism, and compare the pathway to that previously described for Pseudomonas aeruginosa These data support a multitiered regulatory network in B. thailandensis, with conserved orthologs in the select agents Burkholderia pseudomallei and Burkholderia mallei, as well as the opportunistic lung pathogens in the Burkholderia cepacia clade. [Mh] Termos MeSH primário: Fator de Transcrição AraC/metabolismo Proteínas de Bactérias/metabolismo Burkholderia/metabolismo Colina/metabolismo Regulação Bacteriana da Expressão Gênica/fisiologia Transaminases/metabolismo [Mh] Termos MeSH secundário: Fator de Transcrição AraC/genética Proteínas de Bactérias/genética Regulação Enzimológica da Expressão Gênica/fisiologia Regiões Promotoras Genéticas Transaminases/química [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (AraC Transcription Factor); 0 (Bacterial Proteins); EC 2.6.1.- (Transaminases); EC 2.6.1.15 (glutamine-pyruvate aminotransferase); N91BDP6H0X (Choline) [Em] Mês de entrada: 1705 [Cu] Atualização por classe: 170525 [Lr] Data última revisão: 170525 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 160707 [St] Status: MEDLINE [do] DOI: 10.1128/JB.00372-16

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[PMID]: 27155568 [Au] Autor: Wonganu B; Berger BW [Ad] Endereço: Program in Bioengineering, Lehigh University, Bethlehem, PA 18015, United States. [Ti] Título: A specific, transmembrane interface regulates fibroblast activation protein (FAP) homodimerization, trafficking and exopeptidase activity. [So] Source: Biochim Biophys Acta;1858(8):1876-82, 2016 08. [Is] ISSN: 0006-3002 [Cp] País de publicação: Netherlands [La] Idioma: eng [Ab] Resumo: Fibroblast activation protein (FAP) is a cell-surface serine protease which promotes invasiveness of certain epithelial cancers and is therefore a potential target for cancer drug development and delivery. Unlike dipeptidyl peptidase IV (DPPIV), FAP exhibits prolyl endopeptidase activity and is active as a homodimer with specificity for type I collagen. The mechanism that regulates FAP homodimerization and its relation to prolyl endopeptidase activity is not completely understood. Here, we investigate key residues in the FAP TM domain that may be significant for FAP homodimerization. Mutations to predicted TM interfacial residues (G10L, S14L, and A18L) comprising a small-X3-small motif reduced FAP TM-CYTO dimerization relative to wild type as measured using the AraTM assay, whereas predicted off-interface residues showed no significant change from wild type. The results implied that the predicted small-X3-small dimer interface affect stabilization of FAP TM-CYTO homodimerization. Compared with FAPwild-type, the interfacial TM residue G10L significantly decreased FAP endopeptidase activity more than 25%, and also reduced cell-surface versus intracellular expression relative to other interfacial residues S14L and A18L. Thus, our results suggest FAP dimerization is important for both trafficking and protease activity, and is dependent on a specific TM interface. [Mh] Termos MeSH primário: Gelatinases/química Proteínas de Membrana/química Serina Endopeptidases/química [Mh] Termos MeSH secundário: Motivos de Aminoácidos Sequência de Aminoácidos Fator de Transcrição AraC/genética Dimerização Proteínas de Escherichia coli/genética Gelatinases/genética Gelatinases/metabolismo Células HEK293 Seres Humanos Proteínas de Membrana/genética Proteínas de Membrana/metabolismo Mutagênese Sítio-Dirigida Domínios Proteicos Transporte Proteico Proteólise Proteínas Recombinantes de Fusão/química Proteínas Recombinantes de Fusão/metabolismo Alinhamento de Sequência Homologia de Sequência de Aminoácidos Serina Endopeptidases/genética Serina Endopeptidases/metabolismo Frações Subcelulares/química [Pt] Tipo de publicação: COMPARATIVE STUDY; JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, U.S. GOV'T, NON-P.H.S. [Nm] Nome de substância: 0 (AraC Transcription Factor); 0 (AraC protein, E coli); 0 (Escherichia coli Proteins); 0 (Membrane Proteins); 0 (Recombinant Fusion Proteins); EC 3.4.21.- (Serine Endopeptidases); EC 3.4.21.- (fibroblast activation protein alpha); EC 3.4.24.- (Gelatinases) [Em] Mês de entrada: 1710 [Cu] Atualização por classe: 171025 [Lr] Data última revisão: 171025 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 160508 [St] Status: MEDLINE

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[PMID]: 27147534 [Au] Autor: Simcíková M; Alves CP; Brito L; Prather KL; Prazeres DM; Monteiro GA [Ad] Endereço: iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal. [Ti] Título: Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase. [So] Source: Appl Microbiol Biotechnol;100(15):6725-37, 2016 Aug. [Is] ISSN: 1432-0614 [Cp] País de publicação: Germany [La] Idioma: eng [Ab] Resumo: The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5' untranslated region (5'-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5'-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6-14.9 kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48-817-fold) translation initiation rate. No effect of the 5'-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5'-UTR. Interestingly, the amount of this transcript was 2.6-3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5'-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5'-UTR (∼1.8 ± 0.7-fold). Overall, these results show that the improvements made in the 5'-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation. [Mh] Termos MeSH primário: Regiões 5´ não Traduzidas/genética DNA Circular/biossíntese Proteínas de Escherichia coli/genética Escherichia coli/genética Engenharia Genética/métodos Recombinases/genética [Mh] Termos MeSH secundário: Fator de Transcrição AraC/genética Proteínas de Escherichia coli/metabolismo Regiões Promotoras Genéticas/genética Biossíntese de Proteínas RNA Mensageiro/genética Recombinases/metabolismo Recombinação Genética [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (5' Untranslated Regions); 0 (AraC Transcription Factor); 0 (AraC protein, E coli); 0 (DNA, Circular); 0 (Escherichia coli Proteins); 0 (ParA protein, E coli); 0 (RNA, Messenger); 0 (Recombinases) [Em] Mês de entrada: 1702 [Cu] Atualização por classe: 170202 [Lr] Data última revisão: 170202 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 160506 [St] Status: MEDLINE [do] DOI: 10.1007/s00253-016-7565-x

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[PMID]: 27038276 [Au] Autor: Santiago AE; Yan MB; Tran M; Wright N; Luzader DH; Kendall MM; Ruiz-Perez F; Nataro JP [Ad] Endereço: Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA, USA. [Ti] Título: A large family of anti-activators accompanying XylS/AraC family regulatory proteins. [So] Source: Mol Microbiol;101(2):314-32, 2016 Jul. [Is] ISSN: 1365-2958 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners. [Mh] Termos MeSH primário: Fator de Transcrição AraC/genética Genes araC/genética [Mh] Termos MeSH secundário: Fator de Transcrição AraC/metabolismo Proteínas de Bactérias/metabolismo Proteínas de Ligação a DNA/metabolismo Escherichia coli/genética Proteínas de Escherichia coli/metabolismo Regulação Bacteriana da Expressão Gênica/genética Genes araC/fisiologia Bactérias Gram-Negativas/genética Mutagênese Sítio-Dirigida Filogenia Relação Estrutura-Atividade Transativadores/metabolismo Fatores de Transcrição/metabolismo Virulência/genética [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (AraC Transcription Factor); 0 (Bacterial Proteins); 0 (DNA-Binding Proteins); 0 (Escherichia coli Proteins); 0 (Trans-Activators); 0 (Transcription Factors) [Em] Mês de entrada: 1707 [Cu] Atualização por classe: 170718 [Lr] Data última revisão: 170718 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 160403 [St] Status: MEDLINE [do] DOI: 10.1111/mmi.13392

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[PMID]: 26800223 [Au] Autor: Malaga F; Mayberry O; Park DJ; Rodgers ME; Toptygin D; Schleif RF [Ad] Endereço: Biology Department, UPCH, Lima, San Martín De Porres, Peru. [Ti] Título: A genetic and physical study of the interdomain linker of E. Coli AraC protein--a trans-subunit communication pathway. [So] Source: Proteins;84(4):448-60, 2016 Apr. [Is] ISSN: 1097-0134 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: 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. [Mh] Termos MeSH primário: Fator de Transcrição AraC/química Arabinose/química Cisteína/química Proteínas de Escherichia coli/química Escherichia coli/química Prolina/química Subunidades Proteicas/química [Mh] Termos MeSH secundário: 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 Termodinâmica [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (AraC Transcription Factor); 0 (AraC protein, E coli); 0 (Escherichia coli Proteins); 0 (Naphthalenesulfonates); 0 (Protein Subunits); 0 (Recombinant Proteins); 6V7G304M5M (1,5-I-AEDANS); 9DLQ4CIU6V (Proline); B40ROO395Z (Arabinose); K848JZ4886 (Cysteine) [Em] Mês de entrada: 1612 [Cu] Atualização por classe: 161230 [Lr] Data última revisão: 161230 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 160123 [St] Status: MEDLINE [do] DOI: 10.1002/prot.24990

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