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Altered expression of Group A Streptococcus (GAS) virulence factors, including the M protein, can result as a consequence of spontaneous genetic changes that occur during laboratory and animal passage. Occurrence of such secondary mutations during targeted gene deletion could confound the interpretation of effects attributable to the function of the gene being investigated. Contradicting reports on whether the sagA/pel locus regulates the M protein-encoding emm might be due to inconsistent occurrence of mutations unrelated with sagA. This study examined the possibility that altered emm expression observed in association with sagA/pel deletion mutants is artifactual. sagA deletion mutants (MGAS2221ΔsagA) of M1T1 isolate MGAS2221 obtained using liquid broth for GAS growth during the deletion process had diminished emm transcription and no detectable M protein production. In contrast, a ΔsagA …