Unknown,Transcriptomics,Genomics,Proteomics

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Streptococcus pneumoniae D39 spxB- mutant vs. D39 wild-type

ABSTRACT: Comparison of the Streptococcus pneumoniae D39 spxB- mutant vs D39. For DNA microarray analysis, D39 wild-type and two independently obtained D39spxB- mutants were grown as four, two and two biological replicates, respectively, in Glc-M17 under semi-aerobic conditions and harvested at an OD595 of approximately 0.25 (mid-exponential). The RNA of both D39spxB- strains was compared to D39 and the combined data was analyzed. All other procedures regarding microarray analyses were done as described before. A gene was considered differentially expressed when the fold change was M-bM-^IM-% 2, M-bM-^IM-$ 0.5, with a Bayes p M-bM-^IM-$ 0.00001 and when at least 7 measurements were available. One condition design comparision of two strains including a dye swap.

ORGANISM(S): Streptococcus pneumoniae D39

SUBMITTER: Jetta Bijlsma

PROVIDER: E-GEOD-45971 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Previous studies have indicated that PsaR of Streptococcus pneumoniae is a manganese-dependent regulator, negatively affecting the expression of at least seven genes. Here, we extended these observations by transcriptome and proteome analysis of psaR mutants in strains D39 and TIGR4. The microarray analysis identified three shared PsaR targets: the psa-operon, pcpA, and prtA. Additionally, we found 31 genes to be regulated by PsaR in D39 only, most strikingly a cellobiose-specific PTS and a putative bacteriocin operon (sp0141-sp0146). In TIGR4, 14 PsaR gene targets were detected, with the rlrA pathogenicity islet being the most pronounced. Proteomics confirmed most of the shared gene targets. To examine the contribution of PsaR to pneumococcal virulence, we compared D39 and TIGR4 wild-type (wt) and psaR-mutants in three murine infection models. During colonization, no clear effect was observed of the psaR mutation in either D39 or TIGR4. In the pneumonia model, small but significant differences were observed in the lungs of mice infected with either D39wt or âpsaR: D39âpsaR had an initial advantage in survival in the lungs. Conversely, TIGR4âpsaR-infected mice had significantly lower bacterial loads at 24h only. Finally, during experimental bacteremia, D39âpsaR-infected mice had significantly lower bacterial loads in the blood stream than wt-infected mice for the first 24h of infection. TIGR4âpsaR showed attenuation at 36h only. In conclusion, our results show that PsaR of D39 and TIGR4 has a strain-specific role in global gene expression and in the development of bacteremia in mice. Microarray analysis was performed essentially as described (Hendriksen et al., 2007; Hendriksen et al., 2008a). In short, 500 ml of CDM was inoculated with 10-20 colonies from agar plates, and these cultures were statically grown at 37Â°C. Samples for RNA isolation were taken when the cultures reached an optical density (OD600) of 0.2 (mid-log growth). RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Hendriksen et al., 2007; Hendriksen et al., 2008a). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures. Synthesis, subsequent labeling of cDNA, and microarray hybridization was performed as described (Hendriksen et al., 2007; Kloosterman et al., 2006). In all cases, dye-swapping was performed with one of the three biological replicates. Microarrays used in this study were constructed as described (Hendriksen et al., 2007; Kloosterman et al., 2006) and contain amplicons representing 2,087 ORFs of S. pneumoniae TIGR4 and 184 ORFs unique for S. pneumoniae R6, all spotted in duplicate.

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Streptococcus pneumoniae D39 spxB- mutant vs. D39 wild-type

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