Polymorphism and Regulation of the spxB (Pyruvate Oxidase) Virulence Factor Gene in Streptococcus pneumoniae

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, signaling, and oxidative 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 semi-transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency 3x10-5) 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 transcription in response to energy and metabolic state. Finally, microarray analyses of a spxR mutant demonstrated that SpxR positively regulates the strH exoglycosidase gene, which like spxB, has been implicated in colonization. Keywords: bacterial genetic modification Microarray analyses were performed comparing relative transcript amounts for strains D39 (IU1690) with D39(IU1690) spxR6 (Table 2). Total RNA was prepared from bacteria grown exponentially in CDM broth to OD620 = 0.2 as described previously {Lanie, 2007 #58; Ng, 2005 #35}. S. pneumoniae microarrays were purchased from Ocimum Biosolutions). Samples were collected from three independent biological replicates and included one dye swap. Synthesis, labeling, hybridization, scanning, and analysis using the Cyber-T web interface were performed as described previously {Lanie, 2007 #58; Ng, 2005 #35} with the exception of normalization software used. Data were normalized with web-based BASE (BioArray Software Environment, iubase.cgb.indiana.edu/index/phtml) without background subtraction, excluding empty wells and Arabidopsis controls with the plug-in Lowess Normalization