Abstract
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 105-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.
Highlights
Genetic manipulations of Streptococcus pneumoniae have been a powerful approach to understand virulence factors and drug targets in this important human pathogen
SpnYL003) or Sweet Janus (SJ) (SpnYL001 and SpnYL004) were grown to midlog phase in THY and subjected to titer determination on SBA plates supplemented with Km (500 mg/ml), Sm (200 mg/ml), sucrose sensitivity (Sus) (10% w/v), or a mix of Sm and Su (Sm+Su, final concentrations 200 mg/ml and 10% w/v, respectively)
The SJ-carrying strains (KmrSmsSus) were subjected to titer determination on SBA plates supplemented with Km, Sm, Su, or a mix of Sm and Su (Sm+Su) to determine sensitivity to selective agents
Summary
Genetic manipulations of Streptococcus pneumoniae (pneumococcus) have been a powerful approach to understand virulence factors and drug targets in this important human pathogen. The Janus cassette [1] permits marker-free allelic replacement or knockout in Streptococcus pneumoniae through sequential positive and negative selection [2,3,4]. The first transformation tags the locus of interest with the Janus cassette by homologous recombination while conferring a Kmr phenotype that can be selected (positive selection) and Sms. The resulting Janus-carrying strain (KmrSms) is transformed by donor DNA to replace Janus with an allele of interest. To identify true allelic replacement, Smr clones resulted from the negative selection need to be duplicated on Km and Sm plates to screen for Kms ones. When the transformation frequency is around 1027, as reported for several pneumococcal strains [5,6], more than 99% of the Smr clones would be false-positives, making the post-selection screening a time-consuming process
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