Dataset Information

Negative regulation of MurZ and MurA underlies the essentiality of GpsB- and StkP-mediated protein phosphorylation in Streptococcus pneumoniae D39.

ABSTRACT: GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP(Spn) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress ΔgpsB or ΔstkP. These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overproduction of MurZ caused by ΔkhpAB mutations suppressed ΔgpsB or ΔstkP phenotypes to varying extents. ΔgpsB and ΔstkP were also suppressed by MurZ amino-acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram-positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, ΔgpsB and ΔstkP were not suppressed by ΔclpCP, which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB(Spn), is the only essential requirement for StkP-mediated protein phosphorylation in exponentially growing D39 pneumococcal cells.

SUBMITTER: Tsui HT

PROVIDER: S-EPMC10530524 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Negative regulation of MurZ and MurA underlies the essentiality of GpsB- and StkP-mediated protein phosphorylation in Streptococcus pneumoniae D39.

Tsui Ho-Ching Tiffany HT Joseph Merrin M Zheng Jiaqi J JJ Perez Amilcar J AJ Manzoor Irfan I Rued Britta E BE Richardson John D JD Branny Pavel P Doubravová Linda L Massidda Orietta O Winkler Malcolm E ME

Molecular microbiology 20230714 3

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP(Spn) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress ΔgpsB or ΔstkP. These duplications arose fr ...[more]

PMID: 37452010

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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: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 ΔvicK suppressor mutants identified similar mutations, and deletion of the pnpR RR gene in the ΔvicK 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.

Dataset Information

Negative regulation of MurZ and MurA underlies the essentiality of GpsB- and StkP-mediated protein phosphorylation in Streptococcus pneumoniae D39.

Publications

Negative regulation of MurZ and MurA underlies the essentiality of GpsB- and StkP-mediated protein phosphorylation in Streptococcus pneumoniae D39.

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