The principles of a markerless mutagenesis system, originally designed for use in S. mutans (19), were adapted to construct a S. gordonii–specific mutagenesis cassette, JHMD1. All bacterial strains are listed in table S5. Briefly, the cassette uses erythromycin resistance for the first allelic exchange event. A mutated S. gordonii pheS sensitizes cells to 4-Cl-Phe providing identification of the final allelic exchange and resolution of the in-frame mutation. The S. gordonii ldh promoter in the JHMD1 cassette drives expression of the selection markers. The cassette was constructed in silico and produced by GenScript, USA, producing plasmid pJHMD1. To construct the single in-frame markerless gene deletions, the JHMD1 cassette and regions of homology [~300 to 500 base pairs (bp)] upstream and downstream of the region to be mutated were PCR-amplified from pJHMD1 and DL1 genomic DNA, respectively. The PCR primers were designed to delete >98% of the structural gene but maintain the native transcription regulatory regions and start and stop codon sequences. The upstream and downstream regions were spliced to the JHMD1 cassette using splice overlap extension PCR (SOE-PCR) (19). The PCR product was transformed into wild-type DL1 (10) and plated on selective THerm agar. The same regions of chromosomal homology were then spliced to each other using SOE-PCR, transformed into the erythromycin-resistant intermediary strain, and plated on selective TH4-Cl-Phe. The mutations were confirmed by diagnostic PCR and sequencing.

Double deletions were constructed progressively using DNA from a verified ErmR intermediary. Next, the genomic DNA of a resolved, sequence-verified mutant was used as a template for PCR amplification. Each PCR product was transformed sequentially through positive and negative selection until the second mutation was achieved. Chromosomal nucleotide changes were constructed as above. The mutation of interest was incorporated into the upstream reverse oligonucleotide PCR primer and carried through until resolution of the mutation.

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