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LocZ is a new cell division protein involved in proper septum placement in Streptococcus pneumoniae.

Holečková N, Doubravová L, Massidda O, Molle V, Buriánková K, Benada O, Kofroňová O, Ulrych A, Branny P - MBio (2014)

Bottom Line: Here, we characterized one of the StkP substrates, Spr0334, which we named LocZ.Consistently, LocZ supports proper Z-ring positioning at midcell.LocZ is conserved only among streptococci, lactococci, and enterococci, which lack homologues of the Min and nucleoid occlusion effectors, indicating that these bacteria adapted a unique mechanism to find their middle, reflecting their specific shape and symmetry.

View Article: PubMed Central - PubMed

Affiliation: Cell and Molecular Microbiology Division, Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic.

No MeSH data available.


Related in: MedlinePlus

Phosphorylation of LocZ. (A) In vitro phosphorylation. WT and mutated derivatives of the recombinant His6-LocZ protein were subjected to phosphorylation by the recombinant StkP-kinase domain in the presence of [γ-32P]ATP. Samples were resolved by SDS-PAGE, stained with Coomassie blue (CB), and exposed to a sensitive screen ([γ-32P]ATP). The arrows on the right indicate positions of radioactively labeled proteins (upper panel) and Coomassie blue-stained proteins (lower panel). (B) In vivo phosphorylation. Phosphorylation of amino acids T67 and T78 was tested in whole-cell lysates of the WT (Sp208) and the mutant expressing unphosphorylatable LocZ-T67A/T78A (Sp234). Arrows indicate the positions of proteins. Note the difference in the LocZ migration, due to differential phosphorylation.
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fig7: Phosphorylation of LocZ. (A) In vitro phosphorylation. WT and mutated derivatives of the recombinant His6-LocZ protein were subjected to phosphorylation by the recombinant StkP-kinase domain in the presence of [γ-32P]ATP. Samples were resolved by SDS-PAGE, stained with Coomassie blue (CB), and exposed to a sensitive screen ([γ-32P]ATP). The arrows on the right indicate positions of radioactively labeled proteins (upper panel) and Coomassie blue-stained proteins (lower panel). (B) In vivo phosphorylation. Phosphorylation of amino acids T67 and T78 was tested in whole-cell lysates of the WT (Sp208) and the mutant expressing unphosphorylatable LocZ-T67A/T78A (Sp234). Arrows indicate the positions of proteins. Note the difference in the LocZ migration, due to differential phosphorylation.

Mentions: To confirm phosphorylation of both residues, we replaced them with unphosphorylatable alanine (A) by site-directed mutagenesis to introduce single or double mutations (Thr to Ala). The corresponding LocZ derivatives were expressed and purified as His-tagged proteins in E. coli. Following incubation with StkP and [γ-32P]ATP, SDS-PAGE–autoradiography revealed that phosphorylation was partially inhibited in the LocZ-T67A and LocZ-T78A mutants and that this inhibition was almost complete in the doubly mutated LocZ-T67A/T78A protein (Fig. 7A). However, a residual phosphorylation signal of LocZ-T67A/T78A suggested that the doubly mutated protein was still weakly phosphorylated. Since no other phosphoresidues were detected, we mutated also S80, previously identified together with T78 as a phosphoacceptor residue in a global study (50). Nevertheless, phosphorylation of the triply mutated LocZ-T67A/T78A/S80A protein was comparable to that of the double mutant (Fig. 7A), indicating that there may be a yet-unidentified phosphoacceptor amino acid in LocZ in vitro, though its phosphorylation is minimal.


LocZ is a new cell division protein involved in proper septum placement in Streptococcus pneumoniae.

Holečková N, Doubravová L, Massidda O, Molle V, Buriánková K, Benada O, Kofroňová O, Ulrych A, Branny P - MBio (2014)

Phosphorylation of LocZ. (A) In vitro phosphorylation. WT and mutated derivatives of the recombinant His6-LocZ protein were subjected to phosphorylation by the recombinant StkP-kinase domain in the presence of [γ-32P]ATP. Samples were resolved by SDS-PAGE, stained with Coomassie blue (CB), and exposed to a sensitive screen ([γ-32P]ATP). The arrows on the right indicate positions of radioactively labeled proteins (upper panel) and Coomassie blue-stained proteins (lower panel). (B) In vivo phosphorylation. Phosphorylation of amino acids T67 and T78 was tested in whole-cell lysates of the WT (Sp208) and the mutant expressing unphosphorylatable LocZ-T67A/T78A (Sp234). Arrows indicate the positions of proteins. Note the difference in the LocZ migration, due to differential phosphorylation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4281919&req=5

fig7: Phosphorylation of LocZ. (A) In vitro phosphorylation. WT and mutated derivatives of the recombinant His6-LocZ protein were subjected to phosphorylation by the recombinant StkP-kinase domain in the presence of [γ-32P]ATP. Samples were resolved by SDS-PAGE, stained with Coomassie blue (CB), and exposed to a sensitive screen ([γ-32P]ATP). The arrows on the right indicate positions of radioactively labeled proteins (upper panel) and Coomassie blue-stained proteins (lower panel). (B) In vivo phosphorylation. Phosphorylation of amino acids T67 and T78 was tested in whole-cell lysates of the WT (Sp208) and the mutant expressing unphosphorylatable LocZ-T67A/T78A (Sp234). Arrows indicate the positions of proteins. Note the difference in the LocZ migration, due to differential phosphorylation.
Mentions: To confirm phosphorylation of both residues, we replaced them with unphosphorylatable alanine (A) by site-directed mutagenesis to introduce single or double mutations (Thr to Ala). The corresponding LocZ derivatives were expressed and purified as His-tagged proteins in E. coli. Following incubation with StkP and [γ-32P]ATP, SDS-PAGE–autoradiography revealed that phosphorylation was partially inhibited in the LocZ-T67A and LocZ-T78A mutants and that this inhibition was almost complete in the doubly mutated LocZ-T67A/T78A protein (Fig. 7A). However, a residual phosphorylation signal of LocZ-T67A/T78A suggested that the doubly mutated protein was still weakly phosphorylated. Since no other phosphoresidues were detected, we mutated also S80, previously identified together with T78 as a phosphoacceptor residue in a global study (50). Nevertheless, phosphorylation of the triply mutated LocZ-T67A/T78A/S80A protein was comparable to that of the double mutant (Fig. 7A), indicating that there may be a yet-unidentified phosphoacceptor amino acid in LocZ in vitro, though its phosphorylation is minimal.

Bottom Line: Here, we characterized one of the StkP substrates, Spr0334, which we named LocZ.Consistently, LocZ supports proper Z-ring positioning at midcell.LocZ is conserved only among streptococci, lactococci, and enterococci, which lack homologues of the Min and nucleoid occlusion effectors, indicating that these bacteria adapted a unique mechanism to find their middle, reflecting their specific shape and symmetry.

View Article: PubMed Central - PubMed

Affiliation: Cell and Molecular Microbiology Division, Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic.

No MeSH data available.


Related in: MedlinePlus