Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans

• DOI: 10.1128/aem.06362-11
• Publication Date: 2011-09-24T05:25:36Z
• Authors: Zhoujie Xie; Toshinori Okinaga; Fengxia Qi; Zhijun Zhang; Justin Merritt
• Keywords: Cloning (programming)

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