scholarly journals A broad-host-range CRISPRi toolkit for silencing gene expression in Burkholderia

2019 ◽  
Author(s):  
Andrew M. Hogan ◽  
A. S. M. Zisanur Rahman ◽  
Tasia J. Lightly ◽  
Silvia T. Cardona
Keyword(s):  
Gene Expression ◽  
Host Range ◽  
Phenylacetic Acid ◽  
Inducible Promoter ◽  
Broad Host Range ◽  
Proof Of Concept ◽  
Genetic Tools ◽  
Cellular Processes ◽  
Inhibition Of Growth ◽  
Silence Gene

AbstractGenetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus Burkholderia have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit Burkholderia GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in Burkholderia, testing it in B. cenocepacia, B. multivorans and B. thailandensis. Tunable expression was provided by placing a codon-optimized dcas9 from Streptococcus pyogenes under control of a rhamnose-inducible promoter. As a proof of concept, the paaABCDE operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne sgRNAs, resulting in near complete inhibition of growth on phenylacetic acid as the sole carbon source. This was supported by reductions in paaA mRNA expression. The utility of CRISPRi to probe other functions at the single cell level was demonstrated by knocking down phbC and fliF, which dramatically reduces polyhydroxybutyrate granule accumulation and motility, respectively. As a hallmark of the mini-CTX system is the broad host-range of integration, we putatively identified 67 genera of Proteobacteria that might be amenable to modification with our CRISPRi toolkit. Our CRISPRi tool kit provides a simple and rapid way to silence gene expression to produce an observable phenotype. Linking genes to functions with CRISPRi will facilitate genome editing with the goal of enhancing biotechnological capabilities while reducing Burkholderia’s pathogenic arsenal.Author contributionsSTC conceived the idea and design of the research; AMH designed and cloned the dCas9 constructs; AMH and ASMZ designed the sgRNAs, assessed knockdown phenotypes, processed data, and wrote and edited the manuscript; TJL performed RT-qPCR analysis and edited the manuscript; STC supervised the work and provided financial support.Graphical Abstract

scholarly journals Selection and characterization of a glucokinase-deficient mutant of Tetrahymena thermophila.

1982 ◽  
Vol 2 (4) ◽  
pp. 378-385 ◽  
Author(s):  
C T Roberts ◽  
J E Lavine ◽  
D E Morse
Keyword(s):  
Gene Expression ◽  
Mutant Allele ◽  
Tetrahymena Thermophila ◽  
Molecular Defect ◽  
Deficient Mutant ◽  
Cellular Processes ◽  
Inhibition Of Growth ◽  
Selection Of ◽  
Enzymatic Defect

We have isolated a mutant of Tetrahymena thermophila that is resistant to inhibition of growth by the glucose analog 2-deoxyglucose. The mutant exhibits a deficiency in a cytoplasmic glucokinase. This enzymatic defect and the attendant inability to convert 2-deoxyglucose to toxic phosphorylated derivatives is apparently the sole basis for the mutant phenotype since transport of glucose and 2-deoxyglucose is unimpaired; there is no elevation of glucose-6-phosphatase activity, which could decrease the level of toxic 2-deoxyglucose metabolites. Genetic analyses have shown that the mutant allele is recessive and inherited as a single Mendelian mutation. The glucokinase-deficient strain described here is useful for the selection of other mutants in this organism and for the investigation of various cellular processes initiated or modulated by glucose and its analogs. We have exploited the molecular defect in this strain to investigate the initial steps in the cyclic AMP-mediated repression of galactokinase gene expression which is caused by glucose.

scholarly journals Construction of a Broad-Host-Range Tn7-Based Vector for Single-Copy PBAD-Controlled Gene Expression in Gram-Negative Bacteria

2012 ◽  
Vol 79 (2) ◽  
pp. 718-721 ◽  
Author(s):  
F. Heath Damron ◽  
Elizabeth S. McKenney ◽  
Herbert P. Schweizer ◽  
Joanna B. Goldberg
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Host Range ◽  
Single Copy ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Delivery Vector ◽  
Inducible Gene

ABSTRACTWe describe a mini-Tn7-based broad-host-range expression cassette for arabinose-inducible gene expression from the PBADpromoter. This delivery vector, pTJ1, can integrate a single copy of a gene into the chromosome of Gram-negative bacteria for diverse genetic applications, of which several are discussed, usingPseudomonas aeruginosaas the model host.

scholarly journals Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron (“Targetron”) Expressed from a Broad-Host-Range Vector

2007 ◽  
Vol 73 (8) ◽  
pp. 2735-2743 ◽  
Author(s):  
Jun Yao ◽  
Alan M. Lambowitz
Keyword(s):  
Host Range ◽  
Gene Targeting ◽  
Inducible Promoter ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Efficient Gene ◽  
Range Vector ◽  
Group Ii ◽  
Broad Host Range Vector

ABSTRACT Mobile group II introns (“targetrons”) can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acid-inducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.

scholarly journals Broad-Host-Range Shuttle Vectors for Screening of Regulated Promoter Activity in Viridans Group Streptococci: Isolation of a pH-Regulated Promoter

2000 ◽  
Vol 66 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Aldwin J. M. Vriesema ◽  
René Brinkman ◽  
Jan Kok ◽  
Jacob Dankert ◽  
Sebastian A. J. Zaat
Keyword(s):  
Gene Expression ◽  
Host Range ◽  
Pathogenic Bacteria ◽  
Genomic Library ◽  
Regulation Of Gene Expression ◽  
Broad Host Range ◽  
Oral Flora ◽  
Specific Stimulus ◽  
Bacterial Gene ◽  
Viridans Group Streptococci

ABSTRACT Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of thehydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.

scholarly journals Broad-Host-Range Plasmids for Constitutive and Inducible Gene Expression in the Absence of Antibiotic Selection

2019 ◽  
Vol 8 (36) ◽  
Author(s):  
Lindsey Burbank ◽  
Wei Wei
Keyword(s):  
Gene Expression ◽  
Host Range ◽  
Gene Function ◽  
Bacterial Species ◽  
Limited Range ◽  
Broad Host Range ◽  
Bacterial Gene ◽  
Antibiotic Selection ◽  
Broad Host Range Plasmids ◽  
Inducible Gene

Plasmid vectors are a valuable research tool for characterizing bacterial gene function, but there is a limited range of plasmids that are functional in nonmodel bacterial species. Described here is a set of broad-host-range plasmids modified for stability in the absence of antibiotic selection and for gene expression manipulation.

Analysis of the DRR230 family of pea defensins: gene expression pattern and evidence of broad host-range antifungal activity

Plant Science ◽  
2002 ◽  
Vol 163 (4) ◽  
pp. 855-864 ◽  
Author(s):  
Fang-Ming Lai ◽  
Catherine DeLong ◽  
Kangfeng Mei ◽  
Tracy Wignes ◽  
Pierre R. Fobert
Keyword(s):  
Gene Expression ◽  
Antifungal Activity ◽  
Host Range ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Broad Host Range

scholarly journals Development of a CRISPR/Cas9 System forMethylococcus capsulatusIn VivoGene Editing

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Timothy Tapscott ◽  
Michael T. Guarnieri ◽  
Calvin A. Henard
Keyword(s):  
Natural Gas ◽  
Host Range ◽  
Gene Editing ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Broad Host Range ◽  
Methanotrophic Bacteria ◽  
Methylococcus Capsulatus ◽  
Content Type ◽  
Genetic Tools

ABSTRACTMethanotrophic bacteria play a crucial role in the Earth’s biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatableclusteredregularlyinterspacedshortpalindromicrepeats (CRISPR)/Cas9 gene-editing system inMethylococcus capsulatus(Bath). Heterologous coexpression of theStreptococcus pyogenesCas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effectivein vivoediting of plasmid DNA using both Cas9 and Cas9D10Anickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encodingmmoXgene with the Cas9D10Anickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing inM. capsulatus. Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in other methanotrophs and a wide array of Gram-negative microorganisms.IMPORTANCEIn this study, we targeted the development and evaluation of broad-host-range CRISPR/Cas9 gene-editing tools in order to enhance the genetic-engineering capabilities of an industrially relevant methanotrophic biocatalyst. The CRISPR/Cas9 system developed in this study expands the genetic tools available to define molecular mechanisms in methanotrophic bacteria and has the potential to foster advances in the generation of novel biocatalysts to produce biofuels, platform chemicals, and high-value products from natural gas- and biogas-derived methane. Further, due to the broad-host-range applicability, these genetic tools may also enable innovative approaches to overcome the barriers associated with genetically engineering diverse, industrially promising nonmodel microorganisms.

Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression

Plasmid ◽  
1985 ◽  
Vol 13 (2) ◽  
pp. 149-153 ◽  
Author(s):  
Gary Ditta ◽  
Thomas Schmidhauser ◽  
Emanuel Yakobson ◽  
Peter Lu ◽  
Xiao-Wu Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Cloning ◽  
Host Range ◽  
Broad Host Range ◽  
Range Vector ◽  
Monitoring Gene Expression ◽  
Broad Host Range Vector
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