RESUMO
Promoter-specific activation of transcript initiation provides an important regulatory device in Escherichia coli and Salmonella. Here, we describe the different mechanisms that operate, focusing on how they have evolved to manage the "housekeeping" bacterial transcription machinery. Some mechanisms involve assisting the bacterial DNA-dependent RNA polymerase or replacing or remodeling one of its subunits. Others are directed to chromosomal DNA, improving promoter function, or relieving repression. We discuss how different activators work together at promoters and how the present complex network of transcription factors evolved.
RESUMO
Escherichia coli K-12 was originally isolated 100 years ago and since then it has become an invaluable model organism and a cornerstone of molecular biology research. However, despite its pedigree, since its initial isolation E. coli K-12 has been repeatedly cultured, passaged and mutagenized, resulting in an organism that carries many genetic changes. To understand more about this important model organism, we have sequenced the genomes of two ancestral K-12 strains, WG1 and EMG2, considered to be the progenitors of many key laboratory strains. Our analysis confirms that these strains still carry genetic elements such as bacteriophage lambda (λ) and the F plasmid, but also indicates that they have undergone extensive laboratory-based evolution. Thus, scrutinizing the genomes of ancestral E. coli K-12 strains leads us to examine whether E. coli K-12 is a sufficiently robust model organism for 21st century microbiology.
Assuntos
Escherichia coli K12 , Escherichia coli , Escherichia coli/genética , Escherichia coli K12/genética , Bacteriófago lambda , Sequência de BasesRESUMO
BACKGROUND: Resistance nodulation division (RND) family efflux pumps, including the major pump AcrAB-TolC, are important mediators of intrinsic and evolved antibiotic resistance. Expression of these pumps is carefully controlled by a network of regulators that respond to different environmental cues. EnvR is a TetR family transcriptional regulator encoded upstream of the RND efflux pump acrEF. METHODS: Binding of EnvR protein upstream of acrAB was determined by electrophoretic mobility shift assays and the phenotypic consequence of envR overexpression on antimicrobial susceptibility, biofilm motility and invasion of eukaryotic cells in vitro was measured. Additionally, the global transcriptome of clinical Salmonella isolates overexpressing envR was determined by RNA-Seq. RESULTS: EnvR bound to the promoter region upstream of the genes coding for the major efflux pump AcrAB in Salmonella, inhibiting transcription and preventing production of AcrAB protein. The phenotype conferred by overexpression of envR mimicked deletion of acrB as it conferred multidrug susceptibility, decreased motility and decreased invasion into intestinal cells in vitro. Importantly, we demonstrate the clinical relevance of this regulatory mechanism because RNA-Seq revealed that a drug-susceptible clinical isolate of Salmonella had low acrB expression even though expression of its major regulator RamA was very high; this was caused by very high EnvR expression. CONCLUSIONS: In summary, we show that EnvR is a potent repressor of acrAB transcription in Salmonella, and can override binding by RamA so preventing MDR to clinically useful drugs. Finding novel tools to increase EnvR expression may form the basis of a new way to prevent or treat MDR infections.
Assuntos
Proteínas de Bactérias , Salmonella typhimurium , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Resistência Microbiana a Medicamentos , Salmonella typhimurium/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
We have developed a novel urea-inducible recombinant protein production system by exploiting the Proteus mirabilis urease ureR-ureD promoter region and the ureR AraC-family transcriptional regulator. Experiments using the expression of ß-galactosidase and green fluorescent protein (GFP) showed that promoter activity is tightly regulated and that varying the concentration of urea can give up to 100-fold induction. Production of proteins of biopharmaceutical interest has been demonstrated, including human growth hormone (hGH), a single chain antibody fragment (scFv) against interleukin-1ß and a potential Neisserial vaccine candidate (BamAENm). Expression levels can be fine-tuned by temperature and different urea concentrations, and can be induced with readily available garden fertilisers and even urine. As urea is an inexpensive, stable inducer, a urea-induced expression system has the potential to considerably reduce the costs of large-scale recombinant protein production.
Assuntos
Proteínas de Escherichia coli , Ureia , Humanos , Ureia/farmacologia , Ureia/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteus mirabilis/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
Encapsulation of a selected DNA molecule in a cell has important implications for bionanotechnology. Non-viral proteins that can be used as nucleic acid containers include proteinaceous subcellular bacterial microcompartments (MCPs) that self-assemble into a selectively permeable protein shell containing an enzymatic core. Here, we adapted a propanediol utilization (Pdu) MCP into a synthetic protein cage to package a specified DNA segment in vivo, thereby enabling subsequent affinity purification. To this end, we engineered the LacI transcription repressor to be routed, together with target DNA, into the lumen of a Strep-tagged Pdu shell. Sequencing of extracted DNA from the affinity-isolated MCPs shows that our strategy results in packaging of a DNA segment carrying multiple LacI binding sites, but not the flanking regions. Furthermore, we used LacI to drive the encapsulation of a DNA segment containing operators for LacI and for a second transcription factor.
Assuntos
Bactérias , Proteínas de Bactérias , Proteínas de Bactérias/metabolismo , Bactérias/genética , Propilenoglicol/química , Propilenoglicol/metabolismo , DNA/genéticaRESUMO
The serine protease autotransporters of the Enterobacteriaceae (SPATEs) are a large family of virulence factors commonly found in enteric bacteria. These secreted virulence factors have diverse functions during bacterial infection, including adhesion, aggregation and cell toxicity. One such SPATE, the Pic mucinase (protein involved in colonisation) cleaves mucin, allowing enteric bacterial cells to utilise mucin as a carbon source and to penetrate the gut mucus lining, thereby increasing mucosal colonisation. The pic gene is widely distributed within the Enterobacteriaceae, being found in human pathogens, such as enteroaggregative Escherichia coli (EAEC), uropathogenic E. coli (UPEC) and Shigella flexneri 2a. As the pic promoter regions from EAEC strain 042 and UPEC strain CFT073 differ, we have investigated the regulation of each promoter. Here, using in vivo and in vitro techniques, we show that both promoters are activated by the global transcription factor, CRP (cyclic AMP receptor protein), but the architectures of the EAEC and the UPEC pic promoter differ. Expression from both pic promoters is repressed by the nucleoid-associated factor, Fis, and maximal promoter activity occurs when cells are grown in minimal medium. As CRP activates transcription in conditions of nutrient depletion, whilst Fis levels are maximal in nutrient-rich environments, the regulation of the EAEC and UPEC pic promoters is consistent with Pic's nutritional role in scavenging mucin as a suitable carbon source during colonisation and infection.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli , Serina Endopeptidases , Escherichia coli Uropatogênica , Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mucinas/metabolismo , Regiões Promotoras Genéticas , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Most Escherichia coli overexpression vectors used for recombinant protein production (RPP) depend on organic inducers, for example, sugars or simple conjugates. However, these can be expensive and, sometimes, chemically unstable. To simplify this and to cut the cost of RPP, we have developed vectors controlled by the Escherichia coli nitrate-responsive NarL transcription activator protein, which use nitrate, a cheap, stable, and abundant inorganic ion, to induce high-level controlled RPP. We show that target proteins, such as green fluorescent protein, human growth hormone, and single-chain variable region antibody fragments can be expressed to high levels using our promoter systems. As nitrate levels are high in many commercial fertilizers, we demonstrate that controlled RPP can be achieved using readily available and inexpensive garden products.
Assuntos
Proteínas de Escherichia coli , Sequência de Bases , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Nitratos/metabolismo , Óperon , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Klebsiella pneumoniae is an important human pathogen in both developing and industrialised countries that can causes a variety of human infections, such as pneumonia, urinary tract infections and bacteremia. Like many Gram-negative bacteria, it is becoming resistant to many frontline antibiotics, such as carbapenem and cephalosporin antibiotics. In Egypt, K. pneumoniae is increasingly recognised as an emerging pathogen, with high levels of antibiotic resistance. However, few Egyptian K. pneumoniae strains have been sequenced and characterised. Hence, here, we present the genome sequence of a multidrug resistant K. pneumoniae strain, KPE16, which was isolated from a child in Assiut, Egypt. We report that it carries multiple antimicrobial resistance genes, including a blaNDM-1 carbapenemase and extended spectrum ß-lactamase genes (i.e., blaSHV-40, blaTEM-1B, blaOXA-9 and blaCTX-M-15). By comparing this strain with other Egyptian isolates, we identified common plasmids, resistance genes and virulence determinants. Our analysis suggests that some of the resistance plasmids that we have identified are circulating in K. pneumoniae strains in Egypt, and are likely a source of antibiotic resistance throughout the world.
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Many commonly used bacterial promoters employed for recombinant protein production (RPP) in Escherichia coli are capable of high-level protein expression. However, such promoter systems are often too strong, being ill suited for expressing proteins that are difficult to fold, targeted to the membrane or secreted out of the cytoplasm. To circumvent this problem, a suite of bacterial promoters has been constructed with a range of different promoter strengths, assigning them specific "promoter activity ratings" (PARs). Selecting three of these PAR promoters, with low, intermediate and high strengths, it is demonstrated that the expression of target proteins, such as green fluorescent protein (GFP), human growth hormone (hGH) and single chain variable region antibody fragments (scFvs), can be set to three levels when expressed in E. coli. It is shown that the PAR promoter system is extremely flexible, operating in a variety of E. coli strains and under various different culture regimes. Furthermore, due to its tight regulation, it is shown that this system can also express a toxic outer membrane protein, at levels which do not affect bacterial growth. Thus, the PAR promoter system can be used to tailor the expression levels of target proteins in E. coli and maximize RPP.
Assuntos
Escherichia coli , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Hormônio do Crescimento Humano/biossíntese , Anticorpos de Cadeia Única/biossínteseRESUMO
Enteroaggregative Escherichia coli (EAEC) is a common diarrhoeagenic human pathogen, isolated from patients in both developing and industrialized countries, that is becoming increasingly resistant to many frontline antibiotics. In this study, we screened 50 E. coli strains from children presenting with diarrhea at the outpatients clinic of Assiut University Children's Hospital, Egypt. We show that all of these isolates were resistant to multiple classes of antibiotics and identified two as being typical EAEC strains. Using whole genome sequencing, we determined that both isolates carried, amongst others, blaCTX-M and blaTEM antibiotic resistance genes, as well as many classical EAEC virulence determinants, including the transcriptional regulator, AggR. We demonstrate that the expression of these virulence determinants is dependent on AggR, including aar, which encodes for a repressor of AggR, Aar. Since biofilm formation is the hallmark of EAEC infection, we examined the effect of Aar overexpression on both biofilm formation and AggR-dependent gene expression. We show that whilst Aar has a minimal effect on AggR-dependent transcription it is able to completely disrupt biofilm formation, suggesting that Aar affects these two processes differently. Taken together, our results suggest a model for the induction of virulence gene expression in EAEC that may explain the ubiquity of EAEC in both sick and healthy individuals.
Assuntos
Antibacterianos/farmacologia , Diarreia/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Biofilmes , Pré-Escolar , Egito , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Genes Bacterianos , Genoma Bacteriano , Humanos , Lactente , Virulência , Fatores de Virulência/genética , Sequenciamento Completo do GenomaRESUMO
Despite enormous progress in understanding the fundamentals of bacterial gene regulation, our knowledge remains limited when compared with the number of bacterial genomes and regulatory systems to be discovered. Derived from a small number of initial studies, classic definitions for concepts of gene regulation have evolved as the number of characterized promoters has increased. Together with discoveries made using new technologies, this knowledge has led to revised generalizations and principles. In this Expert Recommendation, we suggest precise, updated definitions that support a logical, consistent conceptual framework of bacterial gene regulation, focusing on transcription initiation. The resulting concepts can be formalized by ontologies for computational modelling, laying the foundation for improved bioinformatics tools, knowledge-based resources and scientific communication. Thus, this work will help researchers construct better predictive models, with different formalisms, that will be useful in engineering, synthetic biology, microbiology and genetics.
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Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Iniciação da Transcrição Genética , Óperon , Regiões Promotoras Genéticas , Regulon , Fatores de Transcrição/fisiologiaRESUMO
The Escherichia coli NarX/NarL two-component response-regulator system regulates gene expression in response to nitrate ions and the NarL protein is a global transcription factor, which activates transcript initiation at many target promoters. One such target, the E. coli ogt promoter, which controls the expression of an O6-alkylguanine-DNA-alkyltransferase, is dependent on NarL binding to two DNA targets centred at positions -44.5 and -77.5 upstream from the transcript start. Here, we describe ogt promoter derivatives that can be activated solely by NarL binding either at position -44.5 or position -77.5. We show that NarL can also activate the ogt promoter when located at position -67.5. We present data to argue that NarL-dependent activation of transcript initiation at the ogt promoter results from a direct interaction between NarL and a determinant in the C-terminal domain of the RNA polymerase α subunit. Footprinting experiments show that, at the -44.5 promoter, NarL and the C-terminal domain of the RNA polymerase α subunit bind to opposite faces of promoter DNA, suggesting an unusual mechanism of transcription activation. Our work suggests new organisations for activator-dependent transcription at promoters and future applications for biotechnology.
Assuntos
Proteínas de Ligação a DNA/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Metiltransferases/genética , Regiões Promotoras Genéticas , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Nitratos/farmacologia , Iniciação da Transcrição GenéticaRESUMO
Escherichia coli is a heavily used platform for the production of biotherapeutic and other high-value proteins, and a favored strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed-batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 "TatExpress" strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N-terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed-batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an enzyme-linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately two-fold lower.
Assuntos
Escherichia coli/metabolismo , Hormônio do Crescimento Humano/biossíntese , Periplasma/metabolismo , Dobramento de Proteína , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Escherichia coli/genética , Hormônio do Crescimento Humano/genética , Humanos , Periplasma/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
The activity of any bacterial promoter is generally supposed to be set by its base sequence and the different transcription factors that bind in the local vicinity. Here, we review recent data indicating that the activity of the Escherichia coli lac operon promoter also depends upon its chromosomal location. Factors that affect promoter activity include the binding of nucleoid-associated proteins to neighbouring sequences, supercoiling and the activity of neighbouring promoters. We suggest that many bacterial promoters might be susceptible to similar position-dependent effects and we review recent data showing that the expression of mobile genes encoding antibiotic-resistance determinants is also location-dependent, both when carried on a bacterial chromosome or a conjugative plasmid.
Assuntos
Efeitos da Posição Cromossômica , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Regiões Promotoras Genéticas , Cromossomos Bacterianos , Elementos de DNA Transponíveis , Óperon Lac , Plasmídeos , Transcrição GênicaRESUMO
The Escherichia coli lac operon promoter is widely used as a tool to control recombinant protein production in bacteria. Here, we give a brief review of how it functions, how it is regulated, and how, based on this knowledge, a suite of lac promoter derivatives has been developed to give a controlled expression that is suitable for diverse biotechnology applications.
Assuntos
Escherichia coli/metabolismo , Óperon Lac/genética , Regiões Promotoras Genéticas/genética , Escherichia coli/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição Gênica/genéticaRESUMO
Transcription in most bacteria is tightly regulated in order to facilitate bacterial adaptation to different environments, and transcription factors play a key role in this. Here we give a brief overview of the essential features of bacterial transcription factors and how they affect transcript initiation at target promoters. We focus on complex promoters that are regulated by combinations of activators and repressors, combinations of repressors only, or combinations of activators. At some promoters, transcript initiation is regulated by nucleoid-associated proteins, which often work together with transcription factors. We argue that the distinction between nucleoid-associated proteins and transcription factors is blurred and that they likely share common origins.
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Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Iniciação da Transcrição GenéticaRESUMO
We report an approach for visualizing DNA sequence and using these 'DNA barcodes' to search complex mixtures of genomic material for DNA molecules of interest. We demonstrate three applications of this methodology; identifying specific molecules of interest from a dataset containing gigabasepairs of genome; identification of a bacterium from such a dataset and, finally, by locating infecting virus molecules in a background of human genomic material. As a result of the dense fluorescent labelling of the DNA, individual barcodes of the order 40 kb pairs in length can be reliably identified. This means DNA can be prepared for imaging using standard handling and purification techniques. The recorded dataset provides stable physical and electronic records of the total genomic content of a sample that can be readily searched for a molecule or region of interest.
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DNA/química , Genômica/métodos , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Bacteriófago lambda/genética , Sequência de Bases , Sistemas CRISPR-Cas , Simulação por Computador , DNA Bacteriano/química , DNA Viral/química , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Corantes Fluorescentes , Humanos , Klebsiella pneumoniae/genéticaRESUMO
Regulatory interactions at the lac promoter.Activation of the transcription of genes is central to many processes of adaptation and differentiation in bacteria. Here, I review the molecular mechanisms by which transcription factors can activate the initiation of specific transcripts at bacterial promoters. The story is presented in the context of Marjory Stephenson's pioneering work on enzymatic adaptation in bacteria, and sets the different mechanisms in the greater context of how transcription regulatory mechanisms evolved.
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Bactérias/genética , Proteínas de Bactérias/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fator sigma/genética , Fator sigma/metabolismo , Transativadores/genética , Iniciação da Transcrição GenéticaRESUMO
Enteroaggregative Escherichia coli (EAEC) are important intestinal pathogens causing acute and persistent diarrhoeal illness worldwide. Although many putative EAEC virulence factors have been identified, their association with pathogenesis remains unclear. As environmental cues can modulate bacterial virulence, we investigated the effect of oxygen and human intestinal epithelium on EAEC virulence gene expression to determine the involvement of respective gene products in intestinal colonisation and pathogenesis. Using in vitro organ culture of human intestinal biopsies, we established the colonic epithelium as the major colonisation site of EAEC strains 042 and 17-2. We subsequently optimised a vertical diffusion chamber system with polarised T84 colon carcinoma cells for EAEC infection and showed that oxygen induced expression of the global regulator AggR, aggregative adherence fimbriae, E. coli common pilus, EAST-1 toxin, and dispersin in EAEC strain 042 but not in 17-2. Furthermore, the presence of T84 epithelia stimulated additional expression of the mucinase Pic and the toxins HlyE and Pet. This induction was dependent on physical host cell contact and did not require AggR. Overall, these findings suggest that EAEC virulence in the human gut is modulated by environmental signals including oxygen and the intestinal epithelium.