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The Janus cassette permits marker-free allelic replacement or knockout in streptomycin-resistant  Streptococcus pneumoniae  (pneumococcus) through sequential positive and negative selection. Spontaneous revertants of Janus can lead to high level of false-positives during negative selection, which necessitate a time-consuming post-selection screening process. We hypothesized that an additional counter-selectable marker in Janus would decrease the revertant frequency and reduce false-positives, since simultaneous reversion of both counter-selectable makers is much less likely. Here we report a modified cassette, Sweet Janus (SJ), in which the  sacB  gene from  Bacillus subtilis  conferring sucrose sensitivity is added to Janus. By using streptomycin and sucrose simultaneously as selective agents, the frequency of SJ double revertants was about 10 5 -fold lower than the frequency of Janus revertants. Accordingly, the frequency of false-positives in the SJ-mediated negative selection was about 100-fold lower than what was seen for Janus. Thus, SJ enhances negative selection stringency and can accelerate allelic replacement in pneumococcus, especially when transformation frequency is low due to strain background or suboptimal transformation conditions. Results also suggested the  sacB  gene alone can function as a counter-selectable marker in the Gram-positive pneumococcus, which will have the advantage of not requiring a streptomycin-resistant strain for allelic replacement.

A Modified Janus Cassette (Sweet Janus) to Improve Allelic Replacement Efficiency by High-Stringency Negative Selection in Streptococcus pneumoniae