Abstract
Studies of the dental caries pathogen Streptococcus mutans have benefitted tremendously from its sophisticated genetic system. As part of our own efforts to further improve upon the S. mutans genetic toolbox, we previously reported the development of the first cloning-independent markerless mutagenesis (CIMM) system for S. mutans and illustrated how this approach could be adapted for use in many other organisms. The CIMM approach only requires overlap extension PCR (OE-PCR) protocols to assemble counterselectable allelic replacement mutagenesis constructs, and thus greatly increased the speed and efficiency with which markerless mutations could be introduced into S. mutans. Despite its utility, the system is still subject to a couple limitations. Firstly, CIMM requires negative selection with the conditionally toxic phenylalanine analog p-chlorophenylalanine (4-CP), which is efficient, but never perfect. Typically, 4-CP negative selection results in a small percentage of naturally resistant background colonies. Secondly, CIMM requires two transformation steps to create markerless mutants. This can be inherently problematic if the transformability of the strain is negatively impacted after the first transformation step, which is used to insert the counterselection cassette at the mutation site on the chromosome. In the current study, we develop a next-generation counterselection cassette that eliminates 4-CP background resistance and combine this with a new direct repeat-mediated cloning-independent markerless mutagenesis (DR-CIMM) system to specifically address the limitations of the prior approach. DR-CIMM is even faster and more efficient than CIMM for the creation of all types of deletions, insertions, and point mutations and is similarly adaptable for use in a wide range of genetically tractable bacteria.
Highlights
A large number of medically significant Streptococcus species are highly amenable to genetic manipulation, which has been a tremendous boon for our mechanistic understanding of their prominent roles as both human commensals and pathogens
While 4-CP negative selection has proven to be a major advance for markerless mutagenesis in numerous species, multiple studies have reported a small percentage of 4-CP resistant background clones arising after negative selection, especially when using complex media (Kristich et al, 2007; Xie et al, 2011; Miyazaki, 2015; Argov et al, 2017)
Since both of the T251A/A294G and T251S/A294G ePheS mutants exhibited stringent negative selection in the presence of 4-CP, we were curious to test whether these mutations would improve 4-CP selection in S. mutans
Summary
A large number of medically significant Streptococcus species are highly amenable to genetic manipulation, which has been a tremendous boon for our mechanistic understanding of their prominent roles as both human commensals and pathogens. Insertion-duplication mutations are unstable and require constant selective pressure to prevent spontaneous plasmid excision and a regeneration of the parental genotype (Liu et al, 2014) These mutations introduce a number of plasmid-encoded genes and regulatory elements onto the bacterial chromosome, which frequently trigger a variety of unanticipated genetic regulatory artifacts within the vicinity of the insertion site (Lee et al, 1998). To circumvent this issue, later generations of S. mutans insertion-duplication constructs integrated both positive and negative selection markers (i.e., counterselection) to create markerless mutations (Merritt et al, 2007). Be adaptable for use in many other species for highly efficient and facile markerless mutagenesis
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