Limits...
Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

Koprowski P, Grajkowski W, Balcerzak M, Filipiuk I, Fabczak H, Kubalski A - PLoS ONE (2015)

Bottom Line: MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel.Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein.Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Pasteur 3, Warsaw, Poland.

ABSTRACT
Bacterial mechano-sensitive (MS) channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS) family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

No MeSH data available.


Related in: MedlinePlus

ABDOM binds FtsZ but does not interfere with its polymerization neither in vitro nor in vivo.A. Purified MBP-ABDOM bound assembled FtsZ in vitro. The MBP-ABDOM fusion protein, but not MBP alone, co-sedimented with polymerized FtsZ. Fixed concentration of FtsZ (16 μM) was co-sedimented by addition of 1mM GTP in the presence of increasing concentration of MBP or MBP-ABDOM. For reference, equal amounts of MBP-ABDOM and MBP supernatant without FtsZ are shown in rightmost lane. Proteins were detected with anti-MBP antibody. B. MBP-ABDOM had no effect on FtsZ polymerization/depolymerization in vitro, as detected by 90° angle light scattering. FtsZ was polymerized after addition of 1 mM GTP (arrow) in the presence of either MBP-ABDOM (black line) or MBP (red line) C. Filaments produced by MscSΔ266–286 expression contain multiple nonfunctional Z-rings (upper row), which resemble Z-rings arrested by the cephalexin inhibition of PBP3 (middle row), control cells (lower row). FtsZ-YFP was expressed to visualize Z-rings. Scale bar represents 10 μm. D. Peptidoglycan synthesis detected by immunofluorescent D-cysteine labeling. Overexpression of MscSΔ266–286 resulted in cell filamentation, with clear dark rings of newly synthesized murein in septal areas (left). A similar pattern of murein segregation was observed in cells treated with 1μg/ml aztreonam, a selective inhibitor of PBP3 whose septal localization depends on functional Z-rings (right). Experiments were carried out over one cell cycle in D-cysteine, followed by 30 min. chase. The labeled, older murein is seen as bright spots, while newly made murein is seen as dark areas. Scale bar represents 1 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4440785&req=5

pone.0127029.g002: ABDOM binds FtsZ but does not interfere with its polymerization neither in vitro nor in vivo.A. Purified MBP-ABDOM bound assembled FtsZ in vitro. The MBP-ABDOM fusion protein, but not MBP alone, co-sedimented with polymerized FtsZ. Fixed concentration of FtsZ (16 μM) was co-sedimented by addition of 1mM GTP in the presence of increasing concentration of MBP or MBP-ABDOM. For reference, equal amounts of MBP-ABDOM and MBP supernatant without FtsZ are shown in rightmost lane. Proteins were detected with anti-MBP antibody. B. MBP-ABDOM had no effect on FtsZ polymerization/depolymerization in vitro, as detected by 90° angle light scattering. FtsZ was polymerized after addition of 1 mM GTP (arrow) in the presence of either MBP-ABDOM (black line) or MBP (red line) C. Filaments produced by MscSΔ266–286 expression contain multiple nonfunctional Z-rings (upper row), which resemble Z-rings arrested by the cephalexin inhibition of PBP3 (middle row), control cells (lower row). FtsZ-YFP was expressed to visualize Z-rings. Scale bar represents 10 μm. D. Peptidoglycan synthesis detected by immunofluorescent D-cysteine labeling. Overexpression of MscSΔ266–286 resulted in cell filamentation, with clear dark rings of newly synthesized murein in septal areas (left). A similar pattern of murein segregation was observed in cells treated with 1μg/ml aztreonam, a selective inhibitor of PBP3 whose septal localization depends on functional Z-rings (right). Experiments were carried out over one cell cycle in D-cysteine, followed by 30 min. chase. The labeled, older murein is seen as bright spots, while newly made murein is seen as dark areas. Scale bar represents 1 μm.

Mentions: To determine if FtsZ interacts with ABDOM directly, we assayed co-sedimentation of FtsZ with a fusion of ABDOM to maltose binding protein (MBP). In contrast to dimeric GST, MBP is monomeric, therefore advantageous in co-sedimentation experiments. MBP-ABDOM, MBP and FtsZ were each purified (S2 Fig), combined in a pair-wise fashion and tested in vitro. We found that MBP-ABDOM, but not MBP alone, co-sedimented with GTP-polymerized FtsZ (Fig 2, panel A), indicating that ABDOM interacts directly with a polymerized form of FtsZ. No co-sedimention of MBP-ABDOM was observed in the absence of either GTP (S3 Fig, panel B) or FtsZ (S3 Fig, panels A and C).


Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

Koprowski P, Grajkowski W, Balcerzak M, Filipiuk I, Fabczak H, Kubalski A - PLoS ONE (2015)

ABDOM binds FtsZ but does not interfere with its polymerization neither in vitro nor in vivo.A. Purified MBP-ABDOM bound assembled FtsZ in vitro. The MBP-ABDOM fusion protein, but not MBP alone, co-sedimented with polymerized FtsZ. Fixed concentration of FtsZ (16 μM) was co-sedimented by addition of 1mM GTP in the presence of increasing concentration of MBP or MBP-ABDOM. For reference, equal amounts of MBP-ABDOM and MBP supernatant without FtsZ are shown in rightmost lane. Proteins were detected with anti-MBP antibody. B. MBP-ABDOM had no effect on FtsZ polymerization/depolymerization in vitro, as detected by 90° angle light scattering. FtsZ was polymerized after addition of 1 mM GTP (arrow) in the presence of either MBP-ABDOM (black line) or MBP (red line) C. Filaments produced by MscSΔ266–286 expression contain multiple nonfunctional Z-rings (upper row), which resemble Z-rings arrested by the cephalexin inhibition of PBP3 (middle row), control cells (lower row). FtsZ-YFP was expressed to visualize Z-rings. Scale bar represents 10 μm. D. Peptidoglycan synthesis detected by immunofluorescent D-cysteine labeling. Overexpression of MscSΔ266–286 resulted in cell filamentation, with clear dark rings of newly synthesized murein in septal areas (left). A similar pattern of murein segregation was observed in cells treated with 1μg/ml aztreonam, a selective inhibitor of PBP3 whose septal localization depends on functional Z-rings (right). Experiments were carried out over one cell cycle in D-cysteine, followed by 30 min. chase. The labeled, older murein is seen as bright spots, while newly made murein is seen as dark areas. Scale bar represents 1 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440785&req=5

pone.0127029.g002: ABDOM binds FtsZ but does not interfere with its polymerization neither in vitro nor in vivo.A. Purified MBP-ABDOM bound assembled FtsZ in vitro. The MBP-ABDOM fusion protein, but not MBP alone, co-sedimented with polymerized FtsZ. Fixed concentration of FtsZ (16 μM) was co-sedimented by addition of 1mM GTP in the presence of increasing concentration of MBP or MBP-ABDOM. For reference, equal amounts of MBP-ABDOM and MBP supernatant without FtsZ are shown in rightmost lane. Proteins were detected with anti-MBP antibody. B. MBP-ABDOM had no effect on FtsZ polymerization/depolymerization in vitro, as detected by 90° angle light scattering. FtsZ was polymerized after addition of 1 mM GTP (arrow) in the presence of either MBP-ABDOM (black line) or MBP (red line) C. Filaments produced by MscSΔ266–286 expression contain multiple nonfunctional Z-rings (upper row), which resemble Z-rings arrested by the cephalexin inhibition of PBP3 (middle row), control cells (lower row). FtsZ-YFP was expressed to visualize Z-rings. Scale bar represents 10 μm. D. Peptidoglycan synthesis detected by immunofluorescent D-cysteine labeling. Overexpression of MscSΔ266–286 resulted in cell filamentation, with clear dark rings of newly synthesized murein in septal areas (left). A similar pattern of murein segregation was observed in cells treated with 1μg/ml aztreonam, a selective inhibitor of PBP3 whose septal localization depends on functional Z-rings (right). Experiments were carried out over one cell cycle in D-cysteine, followed by 30 min. chase. The labeled, older murein is seen as bright spots, while newly made murein is seen as dark areas. Scale bar represents 1 μm.
Mentions: To determine if FtsZ interacts with ABDOM directly, we assayed co-sedimentation of FtsZ with a fusion of ABDOM to maltose binding protein (MBP). In contrast to dimeric GST, MBP is monomeric, therefore advantageous in co-sedimentation experiments. MBP-ABDOM, MBP and FtsZ were each purified (S2 Fig), combined in a pair-wise fashion and tested in vitro. We found that MBP-ABDOM, but not MBP alone, co-sedimented with GTP-polymerized FtsZ (Fig 2, panel A), indicating that ABDOM interacts directly with a polymerized form of FtsZ. No co-sedimention of MBP-ABDOM was observed in the absence of either GTP (S3 Fig, panel B) or FtsZ (S3 Fig, panels A and C).

Bottom Line: MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel.Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein.Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Pasteur 3, Warsaw, Poland.

ABSTRACT
Bacterial mechano-sensitive (MS) channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS) family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

No MeSH data available.


Related in: MedlinePlus