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

  • Xuemeng Ji
  • Ping Lu
  • Stijn van der Veen
Methods and protocols


Markerless genetic engineering of bacterial genomes is commonly performed by two-step homologous recombination methods using vectors carrying flanking regions of the target gene for site-specific vector integration and counterselection markers to provide positive selection pressure on the second recombination step resulting in vector excision. Here, we provide the proof-of-principle of a novel counterselection method that exploits antagonistic activities between bactericidal and bacteriostatic antibiotics and which can provide selection pressure on the second recombination step by selective killing of bacteria retaining the antibiotic selection marker. This method was optimized for the bacterial pathogens Listeria monocytogenes and Neisseria meningitidis by screening for antagonistic activities between the bactericidal aminoglycosides kanamycin, streptomycin, and gentamicin in combination with the bacteriostatic antibiotics chloramphenicol and erythromycin. The largest difference in selective killing of both L. monocytogenes and N. meningitidis containing an antibiotic selection marker versus wild-type bacteria was observed for the combination of erythromycin, gentamicin, and ermC as antibiotic selection marker. Therefore, this combination was used to generate two markerless deletion mutants for both L. monocytogenes and N. meningitidis. After applying the dual-antimicrobial selection pressure on cultures during the second recombination step, surviving colonies were replica plated on agar with and without erythromycin. On average, 12–13% of the randomly selected bacterial colonies had lost the selection marker due to a second recombination event and approximately half of these colonies were the desired markerless in-frame deletion mutants. Therefore, this method proved to be easy and fast and should be applicable to a wide variety of bacterial species.


Counterselection Dual-antimicrobial Markerless mutant Listeria monocytogenes Neisseria meningitidis 



This study was funded by Zhejiang University.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9565_MOESM1_ESM.pdf (36 kb)
ESM 1 (PDF 35 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Microbiology and Parasitology, School of MedicineZhejiang UniversityHangzhouChina
  2. 2.Department of Dermatology, Sir Run Run Shaw Hospital, School of MedicineZhejiang UniversityHangzhouChina
  3. 3.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of MedicineZhejiang UniversityHangzhouChina

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