Cloning-independent and counterselectable markerless mutagenesis system in streptococcus mutans

Zhoujie Xie, Toshinori Okinaga, Fengxia Qi, Zhijun Zhang, Justin Merritt

Research output: Contribution to journalArticlepeer-review

86 Scopus citations


Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches.

Original languageEnglish (US)
Pages (from-to)8025-8033
Number of pages9
JournalApplied and Environmental Microbiology
Issue number22
StatePublished - Nov 2011
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology


Dive into the research topics of 'Cloning-independent and counterselectable markerless mutagenesis system in streptococcus mutans'. Together they form a unique fingerprint.

Cite this