Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in Enterococcus faecalis

ABSTRACT Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication. IMPORTANCE Enterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

Download Full-text

Multiplex CRISPRi-Cas9 silencing of planktonic and stage-specific biofilm genes in Enterococcus faecalis

10.1101/2020.04.30.071571 ◽

2020 ◽

Author(s):

Irina Afonina ◽

June Ong ◽

Jerome Chua ◽

Timothy Lu ◽

Kimberly A. Kline

Keyword(s):

Enterococcus Faecalis ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Expression System ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Essential Genes ◽

Dual Vector ◽

Wide Range ◽

Life Threatening

ABSTRACTEnterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multi-level regulation. Compared to established knockout methodologies, CRISPRi approaches enable rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both non-template and template strand targeting. Since nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust, and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacteria interactions, and inter-species communication.IMPORTANCEEnterococcus faecalis causes multidrug resistant life-threatening infections, and is often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labour-intensive allelic exchange methods. Built upon streptococcal dCas9, we developed an easily-modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

Download Full-text

CRISPRi enables studies of enterococcal biofilm initiation, maturation and maintenance.

10.5194/biofilms9-40 ◽

2020 ◽

Author(s):

Irina Afonina ◽

Timothy Lu ◽

Kimberly Kline

Keyword(s):

Patient Outcomes ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Multidrug Resistant ◽

Allelic Exchange ◽

Complex Interactions ◽

Interference System ◽

Wide Range ◽

Life Threatening ◽

Human Infections

Among the Enterococci, Enterococcus faecalis is most frequently associated with human infections ranging from the urinary tract and wound infection to endocarditis and bacteraemia. These infections are often multidrug-resistant and, hence, life-threatening. Moreover, E. faecalis are often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections contributing to disease progression and poorer patient outcomes. Genetic tools to dissect complex interactions in biofilms and mixed microbial communities are largely limited to transposon mutagenesis and traditional allelic exchange methods requiring time- and labour-intensive two-step integration and excision screening that can take a week or more to make a single mutant. We built upon the well-characterized CRISPR interference system using streptococcal dCas9 to develop an easily-modifiable, inducible system for E. faecalis that can efficiently silence single and multiple genes in a matter of hours. We show that this system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This inducible CRISPRi system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal regulation networks within the biofilms, including polymicrobial biofilms.&#160;

Download Full-text

Characterization of φEf-vB1 prophage infecting oral Enterococcus faecalis and enhancing bacterial biofilm formation

Journal of Medical Microbiology ◽

10.1099/jmm.0.001246 ◽

2020 ◽

Vol 69 (9) ◽

pp. 1151-1168

Author(s):

Ahmed Askora ◽

Mohamed El-Telbany ◽

Gamal El-Didamony ◽

Eman Ariny ◽

Momen Askoura

Keyword(s):

Enterococcus Faecalis ◽

Biofilm Formation ◽

Type Species ◽

Sequence Similarity ◽

Bacterial Biofilm ◽

Phenotypic Traits ◽

High Sequence Similarity ◽

Content Type ◽

Link Type ◽

Wide Range

Introduction. Enterococcus faecalis is a facultative, anaerobic, opportunistic pathogen associated with medical and dental diseases. Bacterial phenotypic traits and pathogenesis are often influenced by lysogeny. Aim. The aim of this study was to characterize both the morphology and complete genome sequences of induced prophages puriﬁed from E. faecalis clinical isolates. Methodology. E. faecalis isolates were recovered from the roots of teeth of patients attending an endodontic clinic. The morphological features of isolated phage were characterized using transmission electron microscopy (TEM). DNA sequencing was performed using the Illumina MiSeq platform. Results. TEM indicated that the isolated φEf-vB1 prophage belongs to the family Siphoviridae. The φEf-vB1 prophage was stable over a wide range of temperatures and pH. Sequencing of φEf-vB1 DNA revealed that the phage genome is 37 561 bp in length with a G+C content of 37.6mol% and contained 53 ORFs. Comparison with previously predicted prophage genomes using blast revealed that φEf-vB1 has a high sequence similarity to previously characterized phage genomes. The lysogenic E. faecalis strain exhibited a higher biofilm formation capacity relative to the non-lysogenic strain. Conclusion. The current findings highlight the role of lysogeny in modification of E. faecalis properties and reveal the potential importance of prophages in E. faecalis biology and pathogenesis.

Download Full-text

Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance

Journal of Bacteriology ◽

10.1128/jb.00813-15 ◽

2016 ◽

Vol 198 (7) ◽

pp. 1087-1100 ◽

Cited By ~ 14

Author(s):

Gursonika Binepal ◽

Kamal Gill ◽

Paula Crowley ◽

Martha Cordova ◽

L. Jeannine Brady ◽

...

Keyword(s):

Stress Tolerance ◽

Streptococcus Mutans ◽

Transport System ◽

Biofilm Formation ◽

Cellular Response ◽

Pathogenic Bacteria ◽

Transport Systems ◽

Content Type ◽

Growth And Survival ◽

Dental Biofilm

ABSTRACTPotassium (K+) is the most abundant cation in the fluids of dental biofilm. The biochemical and biophysical functions of K+and a variety of K+transport systems have been studied for most pathogenic bacteria but not for oral pathogens. In this study, we establish the modes of K+acquisition inStreptococcus mutansand the importance of K+homeostasis for its virulence attributes. TheS. mutansgenome harbors four putative K+transport systems that included two Trk-like transporters (designated Trk1 and Trk2), one glutamate/K+cotransporter (GlnQHMP), and a channel-like K+transport system (Kch). Mutants lacking Trk2 had significantly impaired growth, acidogenicity, aciduricity, and biofilm formation. [K+] less than 5 mM eliminated biofilm formation inS. mutans. The functionality of the Trk2 system was confirmed by complementing anEscherichia coliTK2420 mutant strain, which resulted in significant K+accumulation, improved growth, and survival under stress. Taken together, these results suggest that Trk2 is the main facet of the K+-dependent cellular response ofS. mutansto environment stresses.IMPORTANCEBiofilm formation and stress tolerance are important virulence properties of caries-causingStreptococcus mutans. To limit these properties of this bacterium, it is imperative to understand its survival mechanisms. Potassium is the most abundant cation in dental plaque, the natural environment ofS. mutans. K+is known to function in stress tolerance, and bacteria have specialized mechanisms for its uptake. However, there are no reports to identify or characterize specific K+transporters inS. mutans. We identified the most important system for K+homeostasis and its role in the biofilm formation, stress tolerance, and growth. We also show the requirement of environmental K+for the activity of biofilm-forming enzymes, which explains why such high levels of K+would favor biofilm formation.

Download Full-text

Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

mBio ◽

10.1128/mbio.00183-10 ◽

2010 ◽

Vol 1 (4) ◽

Cited By ~ 106

Author(s):

Judith H. Merritt ◽

Dae-Gon Ha ◽

Kimberly N. Cowles ◽

Wenyun Lu ◽

Diana K. Morales ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Extracellular Polysaccharide ◽

Diguanylate Cyclase ◽

Flagellar Motility ◽

Critical Question ◽

Cyclic Diguanylate ◽

Critical Signal ◽

Content Type ◽

Wide Range

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

Download Full-text

CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

mSphere ◽

10.1128/msphere.00064-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 43

Author(s):

Valerie J. Price ◽

Wenwen Huo ◽

Ardalan Sharifi ◽

Kelli L. Palmer

Keyword(s):

Antibiotic Resistance ◽

High Risk ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Treatment Options ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Content Type ◽

New Genes ◽

Restriction Modification

ABSTRACT Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics. Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.

Download Full-text

Pumilacidin-Like Lipopeptides Derived from Marine Bacterium Bacillus sp. Strain 176 Suppress the Motility of Vibrio alginolyticus

Applied and Environmental Microbiology ◽

10.1128/aem.00450-17 ◽

2017 ◽

Vol 83 (12) ◽

Cited By ~ 21

Author(s):

Pengyuan Xiu ◽

Rui Liu ◽

Dechao Zhang ◽

Chaomin Sun

Keyword(s):

Cell Death ◽

Antibiotic Resistance ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Marine Bacterium ◽

Vibrio Alginolyticus ◽

Bacterial Motility ◽

Great Promise ◽

Content Type ◽

Cyclic Lipopeptides

ABSTRACT Bacterial motility is a crucial factor during the invasion and colonization processes of pathogens, which makes it an attractive therapeutic drug target. Here, we isolated a marine bacterium (Vibrio alginolyticus strain 178) from a seamount in the tropical West Pacific that exhibits vigorous motility on agar plates and severe pathogenicity to zebrafish. We found that V. alginolyticus 178 motility was significantly suppressed by another marine bacterium, Bacillus sp. strain 176, isolated from the same niche. We isolated, purified, and characterized two different cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The two related CLPs have a pumilacidin-like structure and were both effective inhibitors of V. alginolyticus 178 motility. The CLPs differ by only one methylene group in their fatty acid chains. In addition to motility suppression, the CLPs also induced cell aggregation in the medium and reduced adherence of V. alginolyticus 178 to glass substrates. Notably, upon CLP treatment, the expression levels of two V. alginolyticus flagellar assembly genes (flgA and flgP) dropped dramatically. Moreover, the CLPs inhibited biofilm formation in several other strains of pathogenic bacteria without inducing cell death. This study indicates that CLPs from Bacillus sp. 176 show promise as antimicrobial lead compounds targeting bacterial motility and biofilm formation with a low potential for eliciting antibiotic resistance. IMPORTANCE Pathogenic bacteria often require motility to establish infections and subsequently spread within host organisms. Thus, motility is an attractive therapeutic target for the development of novel antibiotics. We found that cyclic lipopeptides (CLPs) produced by marine bacterium Bacillus sp. strain 176 dramatically suppress the motility of the pathogenic bacterium Vibrio alginolyticus strain 178, reduce biofilm formation, and promote cellular aggregation without inducing cell death. These findings suggest that CLPs hold great promise as potential drug candidates targeting bacterial motility and biofilm formation with a low overall potential for triggering antibiotic resistance.

Download Full-text

Interactions between Candida albicans and Enterococcus faecalis in an Organotypic Oral Epithelial Model

Microorganisms ◽

10.3390/microorganisms8111771 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1771

Author(s):

Akshaya Lakshmi Krishnamoorthy ◽

Alex A. Lemus ◽

Adline Princy Solomon ◽

Alex M. Valm ◽

Prasanna Neelakantan

Keyword(s):

Candida Albicans ◽

Enterococcus Faecalis ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Surface Erosion ◽

Host Immune System ◽

Microbial Invasion ◽

Mucosal Surfaces ◽

Life Threatening ◽

Hyphal Formation

Candida albicans as an opportunistic pathogen exploits the host immune system and causes a variety of life-threatening infections. The polymorphic nature of this fungus gives it tremendous advantage to breach mucosal barriers and cause oral and disseminated infections. Similar to C. albicans, Enterococcus faecalis is a major opportunistic pathogen, which is of critical concern in immunocompromised patients. There is increasing evidence that E. faecalis co-exists with C. albicans in the human body in disease samples. While the interactive profiles between these two organisms have been studied on abiotic substrates and mouse models, studies on their interactions on human oral mucosal surfaces are non-existent. Here, for the first time, we comprehensively characterized the interactive profiles between laboratory and clinical isolates of C. albicans (SC5314 and BF1) and E. faecalis (OG1RF and P52S) on an organotypic oral mucosal model. Our results demonstrated that the dual species biofilms resulted in profound surface erosion and significantly increased microbial invasion into mucosal compartments, compared to either species alone. Notably, several genes of C. albicans involved in tissue adhesion, hyphal formation, fungal invasion, and biofilm formation were significantly upregulated in the presence of E. faecalis. By contrast, E. faecalis genes involved in quorum sensing, biofilm formation, virulence, and mammalian cell invasion were downregulated. This study highlights the synergistic cross-kingdom interactions between E. faecalis and C. albicans in mucosal tissue invasion.

Download Full-text

Bioinformatic and Molecular Analysis of Inverse Autotransporters from Escherichia coli

mSphere ◽

10.1128/msphere.00572-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 3

Author(s):

Kelvin G. K. Goh ◽

Danilo G. Moriel ◽

Steven J. Hancock ◽

Minh-Duy Phan ◽

Mark A. Schembri

Keyword(s):

Escherichia Coli ◽

Cell Surface ◽

Biofilm Formation ◽

Secretion System ◽

Content Type ◽

E Coli ◽

Wide Range ◽

Type V Secretion ◽

K 12 ◽

Type V

ABSTRACT Proteins secreted by the type V secretion system possess multiple functions, including the capacity to mediate adhesion, aggregation, and biolfilm formation. The type V secretion system can be divided into five subclasses, one of which is the type Ve system. Proteins of the type Ve secretion system are also referred to as inverse autotransporters (IATs). In this study, we performed an in silico analysis of 126 completely sequenced Escherichia coli genomes available in the NCBI database and identified several distinct IAT-encoding gene families whose distribution varied throughout the E. coli phylogeny. The genes included three characterized IATs (intimin, fdeC, and yeeJ) and four uncharacterized IATs (here named iatA, iatB, iatC, and iatD). The four iat genes were cloned from the completely sequenced environmental E. coli strain SMS-3-5 and characterized. Three of these IAT proteins (IatB, IatC, and IatD) were expressed at the cell surface and possessed the capacity to mediate biofilm formation in a recombinant E. coli K-12 strain. Further analysis of the iatB gene, which showed a unique association with extraintestinal E. coli strains, suggested that its regulation is controlled by the LeuO global regulator. Overall, this study provides new data describing the prevalence, sequence variation, domain structure, function, and regulation of IATs found in E. coli. IMPORTANCE Escherichia coli is one of the most prevalent facultative anaerobes of the human gut. E. coli normally exists as a harmless commensal but can also cause disease following the acquisition of genes that enhance its pathogenicity. Adhesion is an important first step in colonization of the host and is mediated by an array of cell surface components. In E. coli, these include a family of adhesins secreted by the type V secretion system. Here, we identified and characterized new proteins from an emerging subclass of the type V secretion system known as the inverse autotransporters (IATs). We found that IAT-encoding genes are present in a wide range of strains and showed that three novel IATs were localized on the E. coli cell surface and mediated biofilm formation. Overall, this study provides new insight into the prevalence, function, and regulation of IATs in E. coli.

Download Full-text

Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria

Applied and Environmental Microbiology ◽

10.1128/aem.02296-13 ◽

2013 ◽

Vol 79 (21) ◽

pp. 6795-6802 ◽

Cited By ~ 40

Author(s):

Andreas Kaczmarczyk ◽

Julia A. Vorholt ◽

Anne Francez-Charlot

Keyword(s):

Gene Expression ◽

Caulobacter Crescentus ◽

Paracoccus Denitrificans ◽

Expression System ◽

Model Organisms ◽

Inducible Gene Expression ◽

Gene Expression System ◽

Content Type ◽

Wide Range ◽

Inducible Gene

ABSTRACTTunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of thePseudomonas putidaF1cym/cmtsystem. The resulting promoter, termed PQ5, responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in otherAlphaproteobacteria, such as the model organismsCaulobacter crescentus,Paracoccus denitrificans, andMethylobacterium extorquens. In the noninduced state, expression from PQ5is low enough to allow gene depletion analysis, as demonstrated with the essential genephyPofSphingomonassp. strain Fr1. A set of PQ5-based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins.

Download Full-text