Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Capsule Mediates the Cell Shape of pcsB Mutants in Serotype 2 Streptococcus pneumoniae

ABSTRACT: PcsB is a protein of unknown function that plays a critical role in cell division in Streptococcus pneumoniae and other ovococcus species of Streptococcus. We constructed isogenic sets of mutants expressing different amounts of PcsB in laboratory strain R6 and virulent serotype 2 strain D39 to evaluate its cellular roles. Insertion mutagenesis in parent and pcsB+ merodiploid strains indicated that pcsB is essential in serotype 2 S. pneumoniae. Quantitative Western blotting of wild-type and epitope-tagged PcsB showed that all PcsB was processed into cell-associated and secreted forms of the same molecular mass. These analyses showed that PcsB bound to cells is present in relatively low amounts of only â 300 molecules per cell. Controlled expression and complementation experiments indicated that there was a causative relationship between the severity of defects in cell division and decreasing PcsB amount. These experiments also indicated that perturbations of expression of the upstream mreCD genes did not contribute to the cell division defects of pcsB mutants and that mreCD could readily be deleted in these strains. Unexpectedly, the defects in cell division and cell shape in pcsB mutants or other mutants defective in cell wall biosynthesis, such as dacA, were strongly influenced by capsule. Underexpression of PcsB did not result in changes in the amounts or composition of lactoyl-muropeptides, which were markedly different in the R6 and D39 strains, and there was no correlation between decreased PcsB amount and sensitivity to penicillin. Finally, microarray analyses indicated that underexpression of PcsB may generate a signal that increases expression of the VicRK regulon, which includes pcsB. Four independent hybridizations using independent RNA preparations from the strain IU1533 (reference strain) and strain IU1979 (a strain with decreased expression of PcsB protein) were performed. Dye swap was performed with the reference strain labeled with Cy3 in two hybridizations and Cy5 in the other two hybridizations.

ORGANISM(S): Streptococcus pneumoniae

SUBMITTER: Skye Barendt

PROVIDER: E-GEOD-13107 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Influences of capsule on cell shape and chain formation of wild-type and pcsB mutants of serotype 2 Streptococcus pneumoniae.

Barendt Skye M SM Land Adrian D AD Sham Lok-To LT Ng Wai-Leung WL Tsui Ho-Ching T HC Arnold Randy J RJ Winkler Malcolm E ME

Journal of bacteriology 20090306 9

PcsB is a protein of unknown function that plays a critical role in cell division in Streptococcus pneumoniae and other ovococcus species of Streptococcus. We constructed isogenic sets of mutants expressing different amounts of PcsB in laboratory strain R6 and virulent serotype 2 strain D39 to evaluate its cellular roles. Insertion mutagenesis in parent and pcsB(+) merodiploid strains indicated that pcsB is essential in serotype 2 S. pneumoniae. Quantitative Western blotting of wild-type and epi ...[more]

PMID: 19270090

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Project description:The VicRK two-component system of S. pneumoniae (Pneumococcus) is a member of the essential WalRK (YycFG) family found in low G+C gram positive bacteria. Previously, we showed that phosphorylated VicR response regulator (RR) is essential, because of its strong positive regulation of the pcsB gene, which mediates cell division. Little is known about the signals sensed by the VicK or WalK histidine kinases (HK), and it is unknown why pneumococcal VicK is not essential under aerobic growth conditions, whereas other the WalK HKs in other species are essential. VicK contains the only PAS domain in the Pneumococcal genome. PAS domains in other HKs have been shown to sense oxygen or other metabolic signals, such as NAD+, and it has been speculated that VicK may be involved in sensing oxygen in S. pneumoniae. Contrary to previous reports, we found that delta vicK mutants do not grow anaerobically, implying a conditional requirement for the VicK HK. Point mutations and domain deletions in vicK indicated that the autokinase activity of VicK is required for anaerobic growth, whereas the PAS domain and VicK phosphatase activity are not required. These combined results are consistent with the idea that maintenance of VicR~P level is required for anaerobic growth. The inability of delta vicK mutants to grow anaerobically was suppressed by spontaneous high- and low-growth yield mutants. Spontaneous suppressor mutants were still VicR dependent and did not result in bypass of the VicR signaling pathway. The genome of a high yield suppressor was sequenced using the Illumina method, which identified three mutations in the suppressor, two of which are predicted to play roles in phosphate transport (pnpR RR and phosphate ABC transporter). Manual sequencing of regions in additional independently isolated ?vicK suppressor mutants identified similar mutations, and deletion of the pnpR RR gene in the ?vicK background fully suppressed the anaerobic growth requirement for the VicK HK. Ongoing experiments will determine whether this suppression requires the PnpS HK or is mediated through changes in signaling. Finally, Western blotting showed that the level of VicK during anaerobic growth is about 3-fold less than that of aerobic cultures. This result suggests that the level of VicRKX operon expression is lower anaerobically, which may prevent crosstalk that occurs during aerobic culture to phosphorylate VicR. Three independent hybridizations using independent RNA preparations from the strain IU1781 (reference strain) and strain IU1896 (a strain with vicK deletion) were performed. Dye swap was performed with the reference strain labeled with Cy3 in two hybridizations and Cy5 in one hybridization.

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.

Dataset Information

Capsule Mediates the Cell Shape of pcsB Mutants in Serotype 2 Streptococcus pneumoniae

Publications

Influences of capsule on cell shape and chain formation of wild-type and pcsB mutants of serotype 2 Streptococcus pneumoniae.

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