Development of a dual-antimicrobial counterselection method for markerless genetic engineering of bacterial genomes

Ji, Xiaofu; Lü, Ping; Veen, Stijn van der

doi:10.1007/s00253-018-9565-5

Cited by 11 publications

(4 citation statements)

References 33 publications

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“…We demonstrate, however, that the high 2-DOG concentration leads to a high success rate for obtaining unmarked mutants. The average success rate of our method is >85 %, considerably higher than previously employed methods for generating markerless mutations in N. gonorrhoeae [11,39], and comparable to other counter-selection markers in different species [38,40,41].…”

Section: Discussionmentioning

confidence: 54%

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Markerless gene editing in Neisseria gonorrhoeae

et al. 2022

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Neisseria gonorrhoeae , the gonococcus, is a pathogen of major public health concern, but sophisticated approaches to gene manipulation are limited for this species. For example, there are few methods for generating markerless mutations, which allow the generation of precise point mutations and deletions without introducing additional DNA sequence. Markerless mutations are central to studying pathogenesis, the spread of antimicrobial resistance (AMR) and for vaccine development. Here we describe the use of galK as a counter-selectable marker that can be used for markerless mutagenesis in N. gonorrhoeae . galK encodes galactokinase, an enzyme that metabolizes galactose in bacteria that can utilize it as a sole carbon source. GalK can also phosphorylate a galactose analogue, 2-deoxy-galactose (2-DOG), into a toxic, non-metabolisable intermediate, 2-deoxy-galactose-1-phosphate. We utilized this property of GalK to develop a markerless approach for mutagenesis in N. gonorrhoeae . We successfully deleted both chromosomally and plasmid-encoded genes, that are important for gonococcal vaccine development and studies of AMR spread. We designed a positive-negative selection cassette, based on an antibiotic resistance marker and galK, that efficiently rendered N. gonorrhoeae susceptible to growth on 2-DOG. We then adapted the galK-based counter-selection and the use of 2-DOG for markerless mutagenesis, and applied biochemical and phenotypic analyses to confirm the absence of target genes. We show that our markerless mutagenesis method for N. gonorrhoeae has a high success rate, and should be a valuable gene editing tool in the future.

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Section: Discussionmentioning

confidence: 54%

“…The average success rate of our method is >85 %, considerably higher than previously employed methods for generating markerless mutations in N. gonorrhoeae [11, 39], and comparable to other counter-selection markers in different species [38, 40, 41].…”

Section: Discussionmentioning

confidence: 69%

Markerless gene editing in Neisseria gonorrhoeae

et al. 2022

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“…A diverse range of genetic tools has been developed only to prevent the negative impacts of genetically modified organisms on the field, including antibiotic gene-free genetic engineering tools (Ji et al 2019 ) and suicide genetic systems (Honjo et al 2019 ; Marguet et al 2010 ; Scott et al 2017 ). By using synthetic biology and metabolic engineering, an attempt has been already made to engineer microorganisms, which can be efficiently used as self-eliminate microbial scavengers for the bioremediation of various toxic environmental pollutants (Moog et al 2019 ; French et al 2020 ).…”

Section: Modern Biotechnological Methods To Enhance Microplastic Degr...mentioning

confidence: 99%

Biotechnological methods to remove microplastics: a review

et al. 2023

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Microplastics pollution is major threat to ecosystems and is impacting abiotic and biotic components. Microplastics are diverse and highly complex contaminants that transport other contaminants and microbes. Current methods to remove microplastics include biodegradation, incineration, landfilling, and recycling. Here we review microplastics with focus on sources, toxicity, and biodegradation. We discuss the role of algae, fungi, bacteria in the biodegradation, and we present biotechnological methods to enhance degradation, e.g., gene editing tools and bioinformatics.

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“…To cure pGW1 from the strain C. sakazakii GZcsf-1, the specific fragment that was amplified from the plasmid to be removed using the primer pair pGW1-F and pGW1-R was mixed with plasmid pCVD442 that was linearized using the primer pair pCVD442-F1 and pCVD442-R1 and the ermC fragment amplified from pUC57-ermC (Ji et al, 2019) using the primer pair ermC-F and ermC-R. The following procedure was identical as the protocols described for the removal of pESA3, but 300 mg/L erythromycin was used to select strains harboring the hybrid plasmid.…”

Section: Curing Of Endogenous Plasmidsmentioning

confidence: 99%

Function Characterization of Endogenous Plasmids in Cronobacter sakazakii and Identification of p-Coumaric Acid as Plasmid-Curing Agent

Lü

et al. 2021

Front. Microbiol.

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Virulence traits and antibiotic resistance are frequently provided by genes located on plasmids. However, experimental verification of the functions of these genes is often lacking due to a lack of related experimental technology. In the present study, an integrated suicide vector was used to efficiently and specifically delete a bacterial endogenous plasmid in Cronobacter sakazakii. The pESA3 plasmid was removed from C. sakazakii BAA-894, and we confirmed that this plasmid contributes to the invasion and virulence of this strain. In addition, the pGW1 plasmid was expunged from C. sakazakii GZcsf-1, and we confirmed that this plasmid confers multidrug resistance. We further screened plasmid-curing agents and found that p-coumaric acid had a remarkable effect on the curing of pESA3 and pGW1 at sub-inhibitory concentrations. Our study investigated the contribution of endogenous plasmids pESA3 and pGW1 by constructing plasmid-cured strains using suicide vectors and suggested that p-coumaric acid can be a safe and effective plasmid-curing agent for C. sakazakii.

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Development of a dual-antimicrobial counterselection method for markerless genetic engineering of bacterial genomes

Cited by 11 publications

References 33 publications

Markerless gene editing in Neisseria gonorrhoeae

Markerless gene editing in Neisseria gonorrhoeae

Biotechnological methods to remove microplastics: a review

Function Characterization of Endogenous Plasmids in Cronobacter sakazakii and Identification of p-Coumaric Acid as Plasmid-Curing Agent

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