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1.
Commun Biol ; 2: 259, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31312728

RESUMO

Antibiotic killing does not occur at a single, precise time for all cells within a population. Variability in time to death can be caused by stochastic expression of genes, resulting in differences in endogenous stress-resistance levels between individual cells in a population. Here we investigate whether single-cell differences in gene expression prior to antibiotic exposure are related to cell survival times after antibiotic exposure for a range of genes of diverse function. We quantified the time to death of single cells under antibiotic exposure in combination with expression of reporters. For some reporters, including genes involved in stress response and cellular processes like metabolism, the time to cell death had a strong relationship with the initial expression level of the genes. Our results highlight the single-cell level non-uniformity of antibiotic killing and also provide examples of key genes where cell-to-cell variation in expression is strongly linked to extended durations of antibiotic survival.


Assuntos
Antibacterianos/farmacologia , Biologia Computacional , Infecções por Escherichia coli/tratamento farmacológico , Biologia de Sistemas , Fator de Transcrição AraC/metabolismo , Carbenicilina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Processamento de Imagem Assistida por Computador , Regiões Promotoras Genéticas , Processos Estocásticos
2.
Acta Crystallogr D Struct Biol ; 75(Pt 7): 639-646, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31282473

RESUMO

The structure of BgaR, a transcriptional regulator of the lactose operon in Clostridium perfringens, has been solved by SAD phasing using a mercury derivative. BgaR is an exquisite sensor of lactose, with a binding affinity in the low-micromolar range. This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form, and was compared with AraC, another saccharide-binding transcriptional regulator. It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold, but that part of the saccharide is exposed to bulk solvent. This differs from the classical AraC saccharide-binding site, which is mostly sequestered from the bulk solvent. The structures of BgaR bound to lactose and to lactulose highlight how specific and nonspecific interactions lead to a higher binding affinity of BgaR for lactose compared with lactulose. Moreover, solving multiple structures of BgaR in different space groups, both bound to saccharides and unbound, verified that the dimer interface along a C-terminal helix is similar to the dimer interface observed in AraC.


Assuntos
Fator de Transcrição AraC/química , Clostridium perfringens/metabolismo , Lactose/metabolismo , Lactulose/metabolismo , Sítios de Ligação , Cristalização , Escherichia coli/genética , Óperon Lac
3.
Microb Pathog ; 132: 343-354, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31100406

RESUMO

In the present study, an attempt has been made to explore the antibiofilm activity of bioactive compound 1-hydroxy-1-norresistomycin (HNM) derived from coral mucus associated actinomycete Streptomyces variabilis. Initially, different concentration of HNM inhibited the biofilm formation of human clinical pathogens Escherichia coli, Vibrio cholerae and Staphylococcus aureus was determined using crystal-violet staining assay. The light microscopic analysis showed that HNM reduced the biofilm formation and adherence of bacterial cells on the surface of coverslip. HNM also damages the 3D architecture with reduced thickness as well as cell aggregation of biofilm forming bacteria analysed by confocal laser scanning microscopy (CLSM). In addition, HNM also demonstrated the efficiency in inhibiting theoretical adhesion by altering the surface hydrophobicity that can potentially hamper cellular adhesion and prevent biofilm formation. Furthermore, the molecular docking showed the significant interaction between HNM and key biofilm forming proteins proved an excellent antibiofilm activity of HNM. Together, these results suggest that the HNM can serve as potential antibiofilm agent in controlling the infections of E. coli, V. cholerae and S. aureus.


Assuntos
Biofilmes/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Pirenos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Vibrio cholerae/efeitos dos fármacos , Actinobacteria/metabolismo , Animais , Antozoários/microbiologia , Fator de Transcrição AraC/efeitos dos fármacos , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/efeitos dos fármacos , Simulação por Computador , Cisteína Endopeptidases/efeitos dos fármacos , Proteínas de Escherichia coli/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Pirenos/química , Streptomyces/metabolismo , Transativadores/efeitos dos fármacos , Peixe-Zebra
4.
Microbiol Res ; 209: 14-20, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29580618

RESUMO

Corynebacterium glutamicum can degrade phenol by a meta-cleavage pathway, which depends on ncgl2588 (phe) of the phe operon encoding phenol hydroxylase. An additional gene, ncgl2587 (pheR), is located upstream of phe. The pheR encodes an AraC/XylR-type regulator protein with 377 amino acid residues and is transcribed in the same direction as phe. Disruption of pheR by homologous recombination resulted in the accumulation of phenol in C. glutamicum. PheR demonstrates a low type of constitutive expression where phenol induces phe expression. PheR shares 75% sequence identity with AraC-type regulator of Corynebacterium lubricantis and 37 conserved residues, characteristic of AraC family, were located. A constructed pK18mobsacB-Pphe:lacZ transcriptional fusion plasmid was transformed into the wild-type, ΔpheR, and ΔpheR+ strains, and the results indicated that PheR activates the expression of phe encoding phenol hydroxylase. Electrophoretic mobility shift assay (EMSA) demonstrated a direct interaction of PheR with the phe promoter region and binding site of PheR on the Pphe was located 109-bp upstream of phe, as indicated by foot printing analysis. Our research provides deep insight into PheR expression and its regulatory function on Phe in C. glutamicum.


Assuntos
Fator de Transcrição AraC/genética , Corynebacterium glutamicum/genética , Regulação Bacteriana da Expressão Gênica/genética , Oxigenases de Função Mista/genética , Transcrição Genética/genética , Corynebacterium glutamicum/metabolismo , DNA Bacteriano/genética , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Oxigenases de Função Mista/biossíntese
5.
ACS Synth Biol ; 7(1): 107-120, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29113433

RESUMO

Living cells are known for their capacity for versatile signal processing, particularly the ability to respond differently to the same stimuli using biochemical networks that integrate environmental signals and reconfigure their dynamic responses. However, the complexity of natural biological networks confounds the discovery of fundamental mechanisms behind versatile signaling. Here, we study one specific aspect of reconfigurable signal processing in which a minimal biological network integrates two signals, using one to reconfigure the network's transfer function with respect to the other, producing an emergent switch between induction and repression. In contrast to known mechanisms, the new mechanism reconfigures transfer functions through genetic networks without extensive protein-protein interactions. These results provide a novel explanation for the versatility of genetic programs, and suggest a new mechanism of signal integration that may govern flexibility and plasticity of gene expression.


Assuntos
Modelos Biológicos , Transdução de Sinais/genética , Fator de Transcrição AraC/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Isopropiltiogalactosídeo/farmacologia , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Proteínas Virais/genética
6.
PLoS Pathog ; 13(8): e1006545, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28806780

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.


Assuntos
Fator de Transcrição AraC/metabolismo , Escherichia coli Enteropatogênica/metabolismo , Infecções por Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Virulência/fisiologia , 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
7.
BMC Microbiol ; 17(1): 170, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28774286

RESUMO

BACKGROUND: In bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome. RESULTS: We developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication. CONCLUSION: Regions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.


Assuntos
Fator de Transcrição AraC/metabolismo , Escherichia coli K12/genética , Proteínas de Escherichia coli/metabolismo , Regiões Operadoras Genéticas , Fator de Transcrição AraC/genética , Arabinose/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Óperon , Regiões Promotoras Genéticas
8.
Proc Natl Acad Sci U S A ; 114(24): E4822-E4831, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28559336

RESUMO

Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal "RxxxR" motif and a ß-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP-responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP-responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins.


Assuntos
Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Polissacarídeos Bacterianos/biossíntese , Sinorhizobium meliloti/metabolismo , Transativadores/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Fator de Transcrição AraC/química , Fator de Transcrição AraC/genética , Fator de Transcrição AraC/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência Conservada , Cristalografia por Raios X , GMP Cíclico/metabolismo , Modelos Moleculares , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Sinorhizobium meliloti/genética , Transativadores/química , Transativadores/genética
9.
Artigo em Inglês | MEDLINE | ID: mdl-28507116

RESUMO

The multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa However, in so-called nfxC mutants, mutational alteration of the gene mexS results in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA (for chloramphenicol resistance activator). In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprN through MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosa to the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.


Assuntos
Fator de Transcrição AraC/genética , Proteínas da Membrana Bacteriana Externa/genética , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Acroleína/análogos & derivados , Acroleína/farmacologia , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/biossíntese , Cloranfenicol/farmacologia , Glioxal/farmacologia , Proteínas de Membrana Transportadoras/biossíntese , Testes de Sensibilidade Microbiana , Aldeído Pirúvico/farmacologia
10.
Mol Biol Evol ; 34(2): 408-418, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28025272

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.


Assuntos
Proteínas de Bactérias/genética , Aptidão Genética , 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
11.
Antimicrob Agents Chemother ; 60(12): 7200-7205, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27671067

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.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Oxazolidinonas/farmacologia , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , 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
12.
Appl Environ Microbiol ; 82(22): 6715-6727, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27613678

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 lacIq-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.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , 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
13.
J Biotechnol ; 236: 1-9, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27498315

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.


Assuntos
Arabinose/metabolismo , Clonagem Molecular/métodos , Escherichia coli/genética , Regiões Promotoras Genéticas/genética , Engenharia de Proteínas/métodos , Fator de Transcrição AraC/genética , Proteínas de Escherichia coli/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
14.
J Bacteriol ; 198(18): 2503-14, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27381916

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.


Assuntos
Fator de Transcrição AraC/metabolismo , Proteínas de Bactérias/metabolismo , Burkholderia/metabolismo , Colina/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Transaminases/metabolismo , Fator de Transcrição AraC/genética , Proteínas de Bactérias/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas , Transaminases/química
15.
Appl Microbiol Biotechnol ; 100(15): 6725-6737, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27147534

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.


Assuntos
Regiões 5' não Traduzidas/genética , DNA Circular/biossíntese , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Engenharia Genética/métodos , Recombinases/genética , Fator de Transcrição AraC/genética , Proteínas de Escherichia coli/metabolismo , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Recombinases/metabolismo , Recombinação Genética
16.
Biochim Biophys Acta ; 1858(8): 1876-82, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27155568

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.


Assuntos
Gelatinases/química , Proteínas de Membrana/química , Serina Endopeptidases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Fator de Transcrição AraC/genética , Dimerização , Proteínas de Escherichia coli/genética , Gelatinases/genética , Gelatinases/metabolismo , Células HEK293 , 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
17.
Mol Microbiol ; 101(2): 314-32, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27038276

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.


Assuntos
Fator de Transcrição AraC/genética , Genes araC/genética , 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
18.
Protein Sci ; 25(4): 804-14, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26749125

RESUMO

The Escherichia coli regulatory protein AraC regulates expression of ara genes in response to l-arabinose. In efforts to develop genetically encoded molecular reporters, we previously engineered an AraC variant that responds to the compound triacetic acid lactone (TAL). This variant (named "AraC-TAL1") was isolated by screening a library of AraC variants, in which five amino acid positions in the ligand-binding pocket were simultaneously randomized. Screening was carried out through multiple rounds of alternating positive and negative fluorescence-activated cell sorting. Here we show that changing the screening protocol results in the identification of different TAL-responsive variants (nine new variants). Individual substituted residues within these variants were found to primarily act cooperatively toward the gene expression response. Finally, X-ray diffraction was used to solve the crystal structure of the apo AraC-TAL1 ligand-binding domain. The resolved crystal structure confirms that this variant takes on a structure nearly identical to the apo wild-type AraC ligand-binding domain (root-mean-square deviation 0.93 Å), suggesting that AraC-TAL1 behaves similar to wild-type with regard to ligand recognition and gene regulation. Our results provide amino acid sequence-function data sets for training and validating AraC modeling studies, and contribute to our understanding of how to design new biosensors based on AraC.


Assuntos
Substituição de Aminoácidos , Fator de Transcrição AraC/genética , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Pironas/metabolismo , Fator de Transcrição AraC/química , Fator de Transcrição AraC/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Modelos Moleculares , Simulação de Dinâmica Molecular , Distribuição Aleatória
19.
Proteins ; 84(4): 448-60, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26800223

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.


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 , Termodinâmica
20.
Infect Immun ; 84(3): 723-34, 2015 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-26712209

RESUMO

Staphylococcus aureus is an important human pathogen that can cause two categories of severe infections. Acute infections are characterized by pronounced toxin production, while chronic infections often involve biofilm formation. However, it is poorly understood how S. aureus controls the expression of genes associated with acute versus biofilm-associated virulence. We here identified an AraC-type transcriptional regulator, Rsp, that promotes the production of key toxins while repressing major biofilm-associated genes and biofilm formation. Genome-wide transcriptional analysis and modeling of regulatory networks indicated that upregulation of the accessory gene regulator (Agr) and downregulation of the ica operon coding for the biofilm exopolysaccharide polysaccharide intercellular adhesin (PIA) were central to the regulatory impact of Rsp on virulence. Notably, the Rsp protein directly bound to the agrP2 and icaADBC promoters, resulting in strongly increased levels of the Agr-controlled toxins phenol-soluble modulins (PSMs) and alpha-toxin and reduced production of PIA. Accordingly, Rsp was essential for the development of bacteremia and skin infection, representing major types of acute S. aureus infection. Our findings give important insight into how S. aureus adapts the expression of its broad arsenal of virulence genes to promote different types of disease manifestations and identify the Rsp regulator as a potential target for strategies to control acute S. aureus infection.


Assuntos
Fator de Transcrição AraC/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Fator de Transcrição AraC/metabolismo , Proteínas de Bactérias/genética , Humanos , Regiões Promotoras Genéticas , Staphylococcus aureus/genética
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