Roles of relSpn in Stringent Response, Global Regulation, and Virulence of Serotype 2 Streptococcus pneumoniae D39
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ABSTRACT:
ORGANISM(S): Streptococcus pneumoniae
SUBMITTER: Kyle Wayne
PROVIDER: E-GEOD-14750 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Copper is essential for both innate and adaptive immune function and copper resistance has emerged as an important determinant of virulence of microbial pathogens. In the human pathogen Streptococcus pneumoniae (Spn), cytoplasmic copper resistance is mediated by an operon encoding the copper-responsive repressor CopY, CupA, of unknown function, and CopA, a copper effluxing P1B-type ATPase. We show that CupA is a novel cell membrane-anchored Cu(I) chaperone for CopA, and that a Cu(I)-binding competent, membrane-localized CupA, like CopA, is obligatory for copper resistance. Bacteria were grown statically in Brain Heart Infusion media (Bacto BHI, Becton Dickinson) at 37C in an atmosphere of 5% CO2 to a culture density of OD620~0.2 and were processed as described in the related Sample record. Strain IU1781 (D39 rpsL1) served as the reference for the strain comparison. The experiment was repeated one time, and results are consistent with those observed by Shafeeq et al (Mol Microbiol. 2011). Data normalization was performed using the BioArray Software Environment (BASE 1.2; Saal et al, Genome Biol. 2002) using the Lowess (subgrid) method. Median spot intensities were normalized without background subtraction and used to calculate expression ratios. The cut-off for statistical significance of differential expression was set at an average up or down fold change of at least 1.8 fold.
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Project description:Pyruvate oxidase encoded by spxB is a major virulence factor in the human respiratory pathogen Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes large amounts of H2O2 and acetyl phosphate, which can serve as a phosphoryl group donor to response regulators and be converted to ATP. SpxB is the main source of the millimolar concentrations of H2O2 produced and tolerated by pneumococcus, despite its lack of a catalase. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen, similar to those used previously for phase variants, for spontaneous mutations that caused colonies of virulent serotype 2 strain D39 to change from a transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency of 3 x 10-5) were impaired for SpxB function. Modeling suggested that two mutations changed amino acids in SpxB required for FAD cofactor or subunit binding. One mutation deleted a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three independent mutations created the same frameshift near the start of a trans-acting regulatory gene designated as spxR. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in DNA, adenosine, and CoA compound binding, respectively, consistent with the idea that SpxR positively regulates spxB transcript amount in response to energy and metabolic state rather than oxidative state. Finally, microarray analyses of a null spxB or a spxR mutant revealed the presence of a new oxidative stress response in pneumococcus and unexpectedly demonstrated that SpxR strongly positively regulates the transcript amount of the strH exoglycosidase gene, which like spxB, has been implicated in host colonization. Keywords: genetic modification Bacterial strains were grown exponentially in rich (BHI) media at 37C and an atmosphere of 5% CO2, and were processed as described in the related Sample records. Samples were collected from three independent biological replicates and included one dye swap. Data were normalized using the Lowess (subgrid) method without background subtraction.
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