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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

MscS protects cells from β-lactam antibiotics.Experiments were conducted on plasmid transformed MJF429 strain in two different concentrations of ampicillin: 1.6 μg/ml (A, C, E) and 4.1μg/ml ampicillin (B, D, F). A, B. Overexpression of MscS but not MscS-K258A/R259A or MscS-YFP supports the cell growth in the presence of subMIC of ampicillin. Inset in B: growth of ampicillin treated cells shown using expanded Y axis scale. C, D. The number of elongated cells estimated from forward scatter (FSC) was reduced for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample FSC histograms are presented in insets, for complete FSC analysis see S8 Fig, panels A-C. E, F. The number of cells with compromised membrane integrity estimated using PI staining was decreased for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample scatter plots are presented for complete FC analysis see S8 Fig, panels D-F. Cells grown in A, B were then used in experiments presented in C, D, E, F. For statistically different results p-values < 0.05 of paired Student`s t-test are shown above the graphs (n = 3).
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pone.0127029.g004: MscS protects cells from β-lactam antibiotics.Experiments were conducted on plasmid transformed MJF429 strain in two different concentrations of ampicillin: 1.6 μg/ml (A, C, E) and 4.1μg/ml ampicillin (B, D, F). A, B. Overexpression of MscS but not MscS-K258A/R259A or MscS-YFP supports the cell growth in the presence of subMIC of ampicillin. Inset in B: growth of ampicillin treated cells shown using expanded Y axis scale. C, D. The number of elongated cells estimated from forward scatter (FSC) was reduced for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample FSC histograms are presented in insets, for complete FSC analysis see S8 Fig, panels A-C. E, F. The number of cells with compromised membrane integrity estimated using PI staining was decreased for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample scatter plots are presented for complete FC analysis see S8 Fig, panels D-F. Cells grown in A, B were then used in experiments presented in C, D, E, F. For statistically different results p-values < 0.05 of paired Student`s t-test are shown above the graphs (n = 3).

Mentions: FtsZ is known to be responsible for cell division but also directs cell wall synthesis [36]. We wondered, whether MscS could be responsible for protection against cell wall damage and whether this function requires interactions with FtsZ. Ampicilin is a broad spectrum ß-lactam penicillin that binds to all penicillin binding proteins [37] and by inhibiting PG cross-linking induces general lesions of peptidoglycan [38]. Penicillin treatment often results in membrane protrusions emanating from midcell before they rupture [39, 40]. An accepted hypothesis to explain this is that penicillin causes a defect in the splitting of septal PG during division. Consistent with this idea, blocking of cell division has been shown to have protective effects when E. coli cells are treated with ß–lactams [41]. FtsZ is a central division protein that directs the septal as well as the lateral wall PG synthesis [34, 36]. We expected that if indeed MscS functionally interacts with FtsZ, it could also have protective effect via this interaction against ampicillin treatment. To induce cell wall lesions without cell lysis we used of ampicillin in concentrations below minimal inhibitory concentration (subMIC) in which we did not observe any apparent cell morphology changes. We transformed mscS- strain MJF429 with pWG1 plasmids (kanR derivatives of pTRC99A) carrying mscS alleles. We found that overexpression of MscS, but not MscS-K258A/R259A nor MscS-YFP, increases the growth rate of cells cultivated in the presence of subMIC of ampicillin (lower—1.6 μg/ml or higher—4.2 μg/ml) (Fig 4, panels A and B). These results suggest that MscS protects cells against ampicillin and the lack of this effect in MscS-K258A/R259A or in MscS-YFP suggests that the mechanism of protection is associated with the MscS binding of FtsZ. To get more insight into this mechanism we investigated the ampicillin treated cells by flow cytometry (FC). We measured forward scatter (FSC) which is proportional to the cell length [42]. We noticed that ampicillin treatment resulted in a new population of cells with increased length in all strains tested (Fig 4, panels C and D, see insets above the plots). Fractions of elongated cells in the presence or in the absence of ampicillin are shown as bars on X axes in Fig 4, panels C and D. Larger number of the elongated cells was observed in higher concentration of ampicillin (Fig 4, panel D). It was demonstrated previously that subMIC of ampicillin arrested E. coli cell division by inducing SOS system [41] what in turn resulted in cell elongation [43]. Curiously, in our experiments expression of wt-MscS partially relieved this effect (Fig 4, panels C andD; S8 Fig, panels A-C). In cells expressing MscS, both the percent of elongated cells and the median cell scatter (S2 Table) were lower compared to those in control, while the protective effect of expression of MscS-K258A/R259A was weaker (Fig 4, panels C and D; S8 Fig, panels A-C). In the presence of 1.6 μg/ml ampicillin expression of MscS-YFP resulted in the largest number of elongated cells (Fig 4, panel C) as a consequence of almost complete cell division arrest (Fig 4, panel A). Similar phenomenon was seen for all constructs in 4.2 μg/ml ampicillin (cell elongation is shown in Fig 4, panel D and cell division arrest is shown in Fig 4, panel B).


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)

MscS protects cells from β-lactam antibiotics.Experiments were conducted on plasmid transformed MJF429 strain in two different concentrations of ampicillin: 1.6 μg/ml (A, C, E) and 4.1μg/ml ampicillin (B, D, F). A, B. Overexpression of MscS but not MscS-K258A/R259A or MscS-YFP supports the cell growth in the presence of subMIC of ampicillin. Inset in B: growth of ampicillin treated cells shown using expanded Y axis scale. C, D. The number of elongated cells estimated from forward scatter (FSC) was reduced for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample FSC histograms are presented in insets, for complete FSC analysis see S8 Fig, panels A-C. E, F. The number of cells with compromised membrane integrity estimated using PI staining was decreased for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample scatter plots are presented for complete FC analysis see S8 Fig, panels D-F. Cells grown in A, B were then used in experiments presented in C, D, E, F. For statistically different results p-values < 0.05 of paired Student`s t-test are shown above the graphs (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127029.g004: MscS protects cells from β-lactam antibiotics.Experiments were conducted on plasmid transformed MJF429 strain in two different concentrations of ampicillin: 1.6 μg/ml (A, C, E) and 4.1μg/ml ampicillin (B, D, F). A, B. Overexpression of MscS but not MscS-K258A/R259A or MscS-YFP supports the cell growth in the presence of subMIC of ampicillin. Inset in B: growth of ampicillin treated cells shown using expanded Y axis scale. C, D. The number of elongated cells estimated from forward scatter (FSC) was reduced for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample FSC histograms are presented in insets, for complete FSC analysis see S8 Fig, panels A-C. E, F. The number of cells with compromised membrane integrity estimated using PI staining was decreased for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample scatter plots are presented for complete FC analysis see S8 Fig, panels D-F. Cells grown in A, B were then used in experiments presented in C, D, E, F. For statistically different results p-values < 0.05 of paired Student`s t-test are shown above the graphs (n = 3).
Mentions: FtsZ is known to be responsible for cell division but also directs cell wall synthesis [36]. We wondered, whether MscS could be responsible for protection against cell wall damage and whether this function requires interactions with FtsZ. Ampicilin is a broad spectrum ß-lactam penicillin that binds to all penicillin binding proteins [37] and by inhibiting PG cross-linking induces general lesions of peptidoglycan [38]. Penicillin treatment often results in membrane protrusions emanating from midcell before they rupture [39, 40]. An accepted hypothesis to explain this is that penicillin causes a defect in the splitting of septal PG during division. Consistent with this idea, blocking of cell division has been shown to have protective effects when E. coli cells are treated with ß–lactams [41]. FtsZ is a central division protein that directs the septal as well as the lateral wall PG synthesis [34, 36]. We expected that if indeed MscS functionally interacts with FtsZ, it could also have protective effect via this interaction against ampicillin treatment. To induce cell wall lesions without cell lysis we used of ampicillin in concentrations below minimal inhibitory concentration (subMIC) in which we did not observe any apparent cell morphology changes. We transformed mscS- strain MJF429 with pWG1 plasmids (kanR derivatives of pTRC99A) carrying mscS alleles. We found that overexpression of MscS, but not MscS-K258A/R259A nor MscS-YFP, increases the growth rate of cells cultivated in the presence of subMIC of ampicillin (lower—1.6 μg/ml or higher—4.2 μg/ml) (Fig 4, panels A and B). These results suggest that MscS protects cells against ampicillin and the lack of this effect in MscS-K258A/R259A or in MscS-YFP suggests that the mechanism of protection is associated with the MscS binding of FtsZ. To get more insight into this mechanism we investigated the ampicillin treated cells by flow cytometry (FC). We measured forward scatter (FSC) which is proportional to the cell length [42]. We noticed that ampicillin treatment resulted in a new population of cells with increased length in all strains tested (Fig 4, panels C and D, see insets above the plots). Fractions of elongated cells in the presence or in the absence of ampicillin are shown as bars on X axes in Fig 4, panels C and D. Larger number of the elongated cells was observed in higher concentration of ampicillin (Fig 4, panel D). It was demonstrated previously that subMIC of ampicillin arrested E. coli cell division by inducing SOS system [41] what in turn resulted in cell elongation [43]. Curiously, in our experiments expression of wt-MscS partially relieved this effect (Fig 4, panels C andD; S8 Fig, panels A-C). In cells expressing MscS, both the percent of elongated cells and the median cell scatter (S2 Table) were lower compared to those in control, while the protective effect of expression of MscS-K258A/R259A was weaker (Fig 4, panels C and D; S8 Fig, panels A-C). In the presence of 1.6 μg/ml ampicillin expression of MscS-YFP resulted in the largest number of elongated cells (Fig 4, panel C) as a consequence of almost complete cell division arrest (Fig 4, panel A). Similar phenomenon was seen for all constructs in 4.2 μg/ml ampicillin (cell elongation is shown in Fig 4, panel D and cell division arrest is shown in Fig 4, panel B).

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