IADR Abstract Archives
Next-Generation Counterselection Cassettes for Markerless Genome Editing in Streptococcus Species
Objectives: Markerless mutagenesis is currently only available in a very limited number of organisms due to the dearth of widely applicable negative selection markers and their high background during selection. Our goal was to develop a new negative selection system that could circumvent these limitations using xylose-inducible toxin genes.
Methods: The pZX9 plasmid and various toxin genes were used as templates for overlap extension PCR reactions generating candidate counterselection cassettes each containing a xylose-inducible toxin and spectinomycin resistance gene. The candidate cassettes were then integrated into the genomes of Streptococcus species, such as S. mutans UA159, UA140, JF243, and CL1, S. sanguinis SK36, and S. gordonii DL1. Confirmed transformants were finally transformed with a second construct to remove the counterselection cassettes by plating on media supplemented with xylose to induce negative selection. This system was used to engineer markerless gene deletions, gene insertions, and point mutations in S. mutans, S. sanguinis, and S. gordonii.
Results: Candidate toxins were derived from both endogenous S. mutans genes as well as from exogenous sources. Of these, two toxins exhibited robust xylose-inducible negative selection, the toxin subunit of the chromosomally-encoded Fst-sm toxin-antitoxin module and the ParE toxin encoded by the addiction module found on the cryptic S. mutans plasmid pUA140.
Conclusions: Our results demonstrate a markerless mutagenesis strategy that could be easily adapted for use in many other organisms. Potential negative selection markers can be derived from the toxin genes that are naturally present in nearly all bacterial genomes as well as from naturally occurring cryptic plasmids that encode addiction modules. Given the variable levels of toxicity encoded by these genes, several candidates may need to be compared to identify those with the appropriate level of toxicity for its paired toxin gene expression system.
Methods: The pZX9 plasmid and various toxin genes were used as templates for overlap extension PCR reactions generating candidate counterselection cassettes each containing a xylose-inducible toxin and spectinomycin resistance gene. The candidate cassettes were then integrated into the genomes of Streptococcus species, such as S. mutans UA159, UA140, JF243, and CL1, S. sanguinis SK36, and S. gordonii DL1. Confirmed transformants were finally transformed with a second construct to remove the counterselection cassettes by plating on media supplemented with xylose to induce negative selection. This system was used to engineer markerless gene deletions, gene insertions, and point mutations in S. mutans, S. sanguinis, and S. gordonii.
Results: Candidate toxins were derived from both endogenous S. mutans genes as well as from exogenous sources. Of these, two toxins exhibited robust xylose-inducible negative selection, the toxin subunit of the chromosomally-encoded Fst-sm toxin-antitoxin module and the ParE toxin encoded by the addiction module found on the cryptic S. mutans plasmid pUA140.
Conclusions: Our results demonstrate a markerless mutagenesis strategy that could be easily adapted for use in many other organisms. Potential negative selection markers can be derived from the toxin genes that are naturally present in nearly all bacterial genomes as well as from naturally occurring cryptic plasmids that encode addiction modules. Given the variable levels of toxicity encoded by these genes, several candidates may need to be compared to identify those with the appropriate level of toxicity for its paired toxin gene expression system.