Polymorphism and regulation of the spxB (pyruvate oxidase) virulence factor gene by a CBS‐HotDog domain protein (SpxR) in serotype 2 Streptococcus pneumoniae

Summary spxB-encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H2O2 and acetyl phosphate, which play roles in metabolism, signalling, and oxi- dative stress. We report here the first cis- and trans- acting regulatory elements for spxB transcription. These elements were identified in a genetic screen for spontaneous mutations that caused colonies of strain D39 to change from a semitransparent to an opaque appearance. Six of the seven opaque colonies recov- ered (frequency a 3 ¥ 10 -5 ) were impaired for SpxB function or expression. Two mutations changed amino acids in SpxB likely required for cofactor or subunit binding. One mutation defined a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three spontaneous mutations created the same frameshift near the start of the trans-acting spxR regulatory gene. The SpxR protein contains helix-turn-helix, CBS and HotDog domains implicated in binding DNA, adenosyl compounds, and CoA-containing compounds respectively, and suggest that SpxR positively regulates spxB tran- scription in response to energy and metabolic state. Microarray analyses unexpectedly demonstrated that SpxR also positively regulates the strH exoglycosi- dase gene, which, like spxB, has been implicated in colonization. Finally, SpxR is required for full viru- lence in a murine model of infection.