Project name: Streptococcus pneumoniae

Description: 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 modificationOverall design: 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

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Medical

Release date: 2008-01-04

Last updated: 2007-10-10