NDLI: Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans ▿

Content Provider	PubMed Central
Author	Xie, Zhoujie Okinaga, Toshinori Qi, Fengxia Zhang, Zhijun Merritt, Justin
Copyright Year	2011
Abstract	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.
Related Links	http://dx.doi.org/10.1128/aem.06362-11
Ending Page	8033
Page Count	9
Starting Page	8025
File Format	PDF
ISSN	00992240
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	22
Volume Number	77
Language	English
Publisher	American Society for Microbiology
Publisher Date	2011-11-15
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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