RESUMO
The gut microbiota is implicated in numerous aspects of health and disease, but dissecting these connections is challenging because genetic tools for gut anaerobes are limited. Inducible promoters are particularly valuable tools because these platforms allow real-time analysis of the contribution of microbiome gene products to community assembly, host physiology, and disease. We developed a panel of tunable expression platforms for the prominent genus Bacteroides in which gene expression is controlled by a synthetic inducer. In the absence of inducer, promoter activity is fully repressed; addition of inducer rapidly increases gene expression by four to five orders of magnitude. Because the inducer is absent in mice and their diets, Bacteroides gene expression inside the gut can be modulated by providing the inducer in drinking water. We use this system to measure the dynamic relationship between commensal sialidase activity and liberation of mucosal sialic acid, a receptor and nutrient for pathogens. VIDEO ABSTRACT.
Assuntos
Bacteroides/genética , Microbioma Gastrointestinal , Engenharia Genética/métodos , Animais , Bacteroides/classificação , Expressão Gênica , Humanos , Camundongos , Neuraminidase/metabolismo , Regiões Promotoras GenéticasRESUMO
An important toxin-antitoxin (TA) system hok/sok, encoded by R1 plasmid of Escherichia coli, is involved in the post segregation killing of cells that have lost the plasmid. The lethal properties of hok protein have been utilized for the environmental containment of microbes and the development of potential vaccine candidates. This study aimed to demonstrate the potent anti-microbial property of a 19 amino acid (AA) long N-terminal fragment of hok peptide. This was accomplished by designing a conditional suicide system based on hok gene expression cloned in an anhydrotetracycline (aTc) inducible vector - pASK75. Heat shock and electroporation were utilized for the transformation of Escherichia coli and Vibrio cholerae cells, respectively. The minimal induction concentration (MId C) of aTc, determined by analyzing the expression of green fluorescent protein cloned separately into pASK75 vector, was 30 ng/mL. As hok gene was synthesized de novo (using recombinant polymerase chain reaction) in our study, various random sized hok fragments were generated (as a result of the error-prone nature of Taq polymerase). The smallest hok fragment able to bring about effective antimicrobial killing was a 19 AA long N-terminal fragment of hok having the wild type sequence, except for the carboxy terminus AA residue. The MId C of aTc in our experiments was 6-fold lower than previously reported, making our bacterial clones suitable for use in mammalian systems as potential vaccine candidates. Based on our experiments, we hypothesize the 19 AA long N-terminal fragment of hok peptide to be the smallest possible hok fragment sufficient to bring about effective antimicrobial killing.
Assuntos
Aminoácidos/metabolismo , Antibacterianos/farmacologia , Clonagem Molecular , Vetores Genéticos/genética , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Tetraciclinas/farmacologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Plasmídeos/genética , Proteínas Recombinantes , Vibrio choleraeRESUMO
We report the construction of a tetracycline inducible expression vector that allows regulated gene expression in the enteric pathogen Vibrio cholerae. The expression vector, named pXB300, contains the tetracycline regulatory elements from Tn10, a multiple cloning site downstream of the tetA promoter and operator sequences, a ColE1 origin of replication, a ß-lactamase resistance gene for positive selection, and the hok/sok addiction system for selection in the absence of antibiotic. The function of the tetracycline expression system was demonstrated by cloning lacZ under control of the tetA promoter and quantifying ß-galactosidase expression in Escherichia coli and V. cholerae. The utility for pXB300 was documented by complementation of V. cholerae virulence mutants during growth under virulence inducing conditions. The results showed that pXB300 allowed high-level expression of recombinant genes with linear induction in response to the exogenous concentration of the inducer anhydrotetracycline. We further show that pXB300 was reliably maintained in V. cholerae during growth in the absence of antibiotic selection.
Assuntos
Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/efeitos dos fármacos , Tetraciclina/farmacologia , Vibrio cholerae/genética , Antiporters/genética , Proteínas de Bactérias/genética , Toxina da Cólera/genética , Toxina da Cólera/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Teste de Complementação Genética , Vetores Genéticos/genética , Mutação , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Regiões Promotoras Genéticas , Tetraciclinas/farmacologia , Fatores de Transcrição/genética , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The need for early-on diagnostic tools to assess the folding and solubility of expressed protein constructs in vivo is of great interest when dealing with recalcitrant proteins. In this paper, we took advantage of the picomolar sensitivity of the bipartite GFP1-10/GFP11 system to investigate the solubility of the Mycobacterium tuberculosis 4'-phosphopantetheinyl transferase PptT, an enzyme essential for the viability of the tubercle bacillus. In vivo and in vitro complementation assays clearly showed the improved solubility of the full-length PptT compared to its N- and C-terminally truncated counterparts. However, initial attempts to purify the full-length enzyme overexpressed in Escherichia coli cells were hampered by aggregation issues overtime that caused the protein to precipitate within hours. The fact that the naturally occurring Coenzyme A and Mg(2+), essentials for PptT to carry out its function, could play a role in stabilizing the enzyme was confirmed using DSF experiments. In vitro activity assays were performed using the ACP substrate from the type I polyketide synthase PpsC from M. tuberculosis, a 2188 amino-acid enzyme that plays a major role in the virulence and pathogenicity of this microbial pathogen. We selected the most soluble and compact ACP fragment (2042-2188), identified by genetic selection of in-frame fragments from random library experiments, to monitor the transfer of the P-pant moiety from Coenzyme A onto a conserved serine residue of this ACP domain.
Assuntos
Proteínas de Bactérias/biossíntese , Mycobacterium tuberculosis/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/biossíntese , Proteína de Transporte de Acila/química , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Coenzima A/química , Estabilidade Enzimática , Escherichia coli , Expressão Gênica , Proteínas de Fluorescência Verde/biossíntese , Magnésio/química , Dobramento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/isolamento & purificaçãoRESUMO
IMPORTANCE: Mycobacterium abscessus represents the most common rapidly growing mycobacterial pathogen in cystic fibrosis and is extremely difficult to eradicate. Essential genes are required for growth, often participate in pathogenesis, and encode valid drug targets for further chemotherapeutic developments. However, assessing the function of essential genes in M. abscessus remains challenging due to the limited spectrum of efficient genetic tools. Herein, we generated a Tet-OFF-based system allowing to knock down the expression of mmpL3, encoding the mycolic acid transporter in mycobacteria. Using this conditional mutant, we confirm the essentiality of mmpL3 in planktonic cultures, in biofilms, and during infection in zebrafish embryos. Thus, in this study, we developed a robust and reliable method to silence the expression of any M. abscessus gene during host infection.
Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Mycobacterium , Animais , Mycobacterium abscessus/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , Peixe-Zebra , Expressão GênicaRESUMO
Antibiotic resistance is a growing global health threat, demanding urgent responses. Tetracyclines, a widely used antibiotic class, are increasingly succumbing to antibiotic resistance; generating novel analogues is therefore a top priority for public health. Fungal tetracyclines provide structural and enzymatic diversity for novel tetracycline analogue production in tractable heterologous hosts, like yeasts, to combat antibiotic-resistant pathogens. Here, we successfully engineered Saccharomyces cerevisiae (baker's yeast) and Saccharomyces boulardii (probiotic yeast) to produce the nonantibiotic fungal anhydrotetracycline, TAN-1612, in synthetic defined mediaânecessary for clean purificationsâthrough heterologously expressing TAN-1612 genes mined from the fungus, Aspergillus niger ATCC 1015. This was accomplished via (i) a promoter library-based combinatorial pathway optimization of the biosynthetic TAN-1612 genes coexpressed with a putative TAN-1612 efflux pump, reducing TAN-1612 toxicity in yeasts while simultaneously increasing supernatant titers and (ii) the development of a medium-throughput UV-visible spectrophotometric assay that facilitates TAN-1612 combinatorial library screening. Through this multipronged approach, we optimized TAN-1612 production, yielding an over 450-fold increase compared to previously reported S. cerevisiae yields. TAN-1612 is an important tetracycline analogue precursor, and we thus present the first step toward generating novel tetracycline analogue therapeutics to combat current and emerging antibiotic resistance. We also report the first heterologous production of a fungal polyketide, like TAN-1612, in the probiotic S. boulardii. This highlights that engineered S. boulardii can biosynthesize complex natural products like tetracyclines, setting the stage to equip probiotic yeasts with synthetic therapeutic functionalities to generate living therapeutics or biocontrol agents for clinical and agricultural applications.
Assuntos
Saccharomyces cerevisiae , Tetraciclinas , Antibacterianos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tetraciclinas/farmacologia , LevedurasRESUMO
Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.
RESUMO
In this study we report the design, construction and validation of a novel transposon aimed to systematically screen for protein localization and expression patterns in prokaryotes using fluorescence microscopy. Upon random insertion in an open reading frame in the proper frame and orientation, the transposon creates an N-terminal fluorescent protein fusion to the msfGFP reporter. Moreover, in order to examine the localization of fusion proteins whose native expression might be too low or absent, the transposon was fitted with a PLtetO-1 promoter that makes the expression of the generated fluorescent protein fusions controllable by anhydrotetracycline. Importantly, upon flipping out the PLtetO-1 promoter and neighboring antibiotic resistance marker, an in-frame "sandwich" msfGFP fusion is created in which the N- and C-terminal portions of the targeted protein are again controlled by its native promoter.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis/genética , Proteômica/métodos , Mapeamento Cromossômico , Conjugação Genética , Marcadores Genéticos , Proteínas de Fluorescência Verde/genética , Óperon Lac , Mutagênese Insercional , Fases de Leitura Aberta , Regiões Promotoras GenéticasRESUMO
The synthesis and biological evaluation of semisynthetic anhydrotetracycline analogues as small molecule inhibitors of tetracycline-inactivating enzymes are reported. Inhibitor potency was found to vary as a function of enzyme (major) and substrate-inhibitor pair (minor), and anhydrotetracycline analogue stability to enzymatic and nonenzymatic degradation in solution contributes to their ability to rescue tetracycline activity in whole cell Escherichia coli expressing tetracycline destructase enzymes. Taken collectively, these results provide the framework for the rational design of next-generation inhibitor libraries en route to a viable and proactive adjuvant approach to combat the enzymatic degradation of tetracycline antibiotics.
Assuntos
Antibacterianos/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/enzimologia , Tetraciclina/metabolismo , Tetraciclinas/química , Tetraciclinas/farmacologia , Antibacterianos/síntese química , Antibacterianos/farmacologia , Inibidores Enzimáticos/síntese química , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Tetraciclinas/síntese químicaRESUMO
Clostridium difficile remains a leading nosocomial pathogen, putting considerable strain on the healthcare system. The ability to form endospores, highly resistant to environmental insults, is key to its persistence and transmission. However, important differences exist between the sporulation pathways of C. difficile and the model Gram-positive organism Bacillus subtilis. Amongst the challenges in studying sporulation in C. difficile is the relatively poor levels of sporulation and high heterogeneity in the sporulation process. To overcome these limitations we placed Ptet regulatory elements upstream of the master regulator of sporulation, spo0A, generating a new strain that can be artificially induced to sporulate by addition of anhydrotetracycline (ATc). We demonstrate that this strain is asporogenous in the absence of ATc, and that ATc can be used to drive faster and more efficient sporulation. Induction of Spo0A is titratable and this can be used in the study of the spo0A regulon both in vitro and in vivo, as demonstrated using a mouse model of C. difficile infection (CDI). Insights into differences between the sporulation pathways in B. subtilis and C. difficile gained by study of the inducible strain are discussed, further highlighting the universal interest of this tool. The Ptet-spo0A strain provides a useful background in which to generate mutations in genes involved in sporulation, therefore providing an exciting new tool to unravel key aspects of sporulation in C. difficile.
RESUMO
Synthetic biology is the design and construction of new biological parts, devices and circuits not existing in nature. It provides a novel solution to imminent challenges in a wide variety of fields, including the discovery of new drugs, production chemicals, renewable biofuels, value-added products and cellular reprogramming. Many efforts have been made to design and characterize synthetic genetic parts, including promoter, transcription factors, RBS, degradation tags and transcriptional terminators, among others. These genetic parts have been assembled for construction of a number of synthetic devices and circuits like oscillators, toggle switches, amplifiers and biologic gates; they play a vital role in cell reprogramming for better understanding of cellular mechanisms and control of biological process. They are also useful for the periodic and tunable production of drugs, fine chemicals, vaccines and much more. It is the goal of this review to aid and accelerate future research in synthetic biology.
Assuntos
Biologia Sintética , Sítios de Ligação , Proteínas/metabolismo , Ribossomos/metabolismo , Regiões Terminadoras Genéticas , Fatores de Transcrição/metabolismoRESUMO
The lack of a versatile system to control gene expression in Helicobacter pylori has hampered efforts to study H. pylori physiology and pathogenesis. To overcome these limitations, we evaluated the utility of an inducible system based on the well-characterized Tet repressor (TetR) and Tet operator (tetO). As validation of this system, we introduced three copies of tetO into the promoter region upstream of the cagUT operon (encoding two virulence factors required for function of the H. pylori Cag type IV secretion system) and expressed tetR by introducing a codon-optimized gene into the chromosomal ureA locus. Introduction of the tetO copies upstream of cagUT did not disrupt promoter activity, as determined by immunoblotting for CagT. The subsequent introduction of tetR, however, did repress CagT synthesis. Production of CagT was restored when strains were cultured in the presence of the inducer, anhydrotetracycline. To demonstrate one potential application of this new tool, we analyzed the function of the Cag type IV secretion system. When the modified H. pylori strains were co-cultured with AGS cells, activity of the Cag type IV secretion system was dependent on the presence of anhydrotetracycline as evidenced by inducer-dependent induction of IL-8 secretion, CagA translocation, and appearance of type IV secretion system pili at the bacteria-host interface. These studies demonstrate the effectiveness of the tetR-tetO system to control gene expression in H. pylori and provide an improved system for studying H. pylori physiology and pathogenesis.