Periodic Reporting for period 1 - BACTOSHAPE (Single cell biophysics of bacterial cell shape)
Reporting period: 2015-04-01 to 2017-03-31
Little is known about the mechanisms determining cell shape, and the main question concerning morphogenesis is the same in prokaryotic and eukaryotic systems. How cell shape is determined and maintain? In bacteria, the tough external cell wall (CW), a 3D polymer network that is one of the most prominent targets for antibiotics, is traditionally known to be a primary determinant of cell shape. However, the complex CW ultrastructure and the molecular mechanisms that control CW morphogenesis remain unknown. The discovery about a decade ago of a bacterial actin-like cytoskeleton changed our understanding of bacterial cell morphogenesis. Since then, MreB homologues have been shown to serve as organizers for the movement and assembly of molecular complexes involved in CW biogenesis in bacteria with complex shapes (non-spherical). Thus MreB homologues play a critical role in cell shape determination and maintenance. However, the mechanistic details used by the MreB cytoskeleton to fulfill this role remain to be elucidated.
The main objectives of this project are to understand the role of cell wall organization and of the actin like proteins in cell shape determination and maintenance and to describe the mechanical aspect of cell shape maintenance.
The main objectives of this project are to understand the role of cell wall organization and of the actin like proteins in cell shape determination and maintenance and to describe the mechanical aspect of cell shape maintenance.
Cell wall structure and mechanical properties of ∆mreB
Bacillus subtilis is known to tightly control its cell width, however, when mreB is deleted, in absence of excess Mg2+, cells deform and eventually lyse. In order to understand the mechanism by which cell width increases and bulges form in ∆mreB mutant we first used Atomic Force Microscopy (AFM) to study cell surface structure and mechanical properties. When ∆mreB cells were grown without excess Mg for few generations, cells deform and present a lower rigidity, and a rougher cell wall surface. In cross section pieces of the cell wall are visible peeling off the surface of ∆mreB mutant grown without added Mg.
Dynamic of cell wall synthesis and degradation
In order to determine which aspect of cell wall homeostasis is affected by mreB deletion, we visualized PG insertion with and without excess Mg2+ using a fluorescent dye. In ∆mreB, no differential staining was observed at sites of deformation indicating that the PG is regularly inserted along the sidewall. To investigate the dynamic of PG degradation during cell elongation, we carried out pulse chase experiments with the PG fluorescent dye. In wild type and ∆mreB grown in presence of excess Mg2+, staining pattern becomes punctate due to insertion of new PG. In contrast, in the ∆mreB mutant grown without added Mg2+, large gaps without fluorescence are observed at the sites of bulging. Swelling cells showed a contrasting staining pattern, parts of the cells were very bright while other parts presented large gaps in fluorescence, suggesting that PG degradation was non uniform in the cell.
Effect of mreB deletion on cell wall composition
We analyzed muropeptide composition of wild type and ∆mreB mutant grown in presence or in absence of excess Mg2+. Results revealed a significant increase of muropeptides resulting from endopeptidase activities in ∆mreB mutant. Additionally, amidation of dimeric muropeptides is higher in ∆mreB grown without added Mg compare to the wild type in the same culture conditions.
Surface analysis revealed that ∆mreB mutant present a higher concentration of nitrogen compare to the wild type while phosphorus concentration vary very slightly. This finding shows that amount of teichoic acid at the cell surface does not change in response to mreB deletion or change of Mg concentration in the medium. Changes in nitrogen concentration are most probably due to an increase of peptidoglycan amidation or teichoic acids alanylation in ∆mreB mutant.
Effect of magnesium on the cell wall of Bacillus subtilis
Magnesium can compensate the absence of a number of otherwise essential cell wall related genes in B. subtilis. We have tested the effect of Mg2+ on the rigidity of the cell wall of wild-type B. subtilis cells using AFM and have found no effect. However, we identified a significant decrease in the degree of amidation of mDAP in cells grown in the presence of high concentrations of Mg2+. In B. subtilis and other species where it has been reported, mDAP amidation seems to slightly differ in different growth condition, suggesting a possible regulatory role in cell wall homeostasis.
We aimed to understand the interaction of Mg with the cell wall and the equilibrium of charges at the cell surface. Overall, gram positive bacteria are negatively charged and thus bind cations. Analyses reveal that Mg2+ concentration in LB is not sufficient to saturate binding site of the cell wall and that Mg compete with Na. Moreover, when Mg is in excess in the growth medium it binds not only to phosphate but also to carboxyl groups.
Bacillus subtilis is known to tightly control its cell width, however, when mreB is deleted, in absence of excess Mg2+, cells deform and eventually lyse. In order to understand the mechanism by which cell width increases and bulges form in ∆mreB mutant we first used Atomic Force Microscopy (AFM) to study cell surface structure and mechanical properties. When ∆mreB cells were grown without excess Mg for few generations, cells deform and present a lower rigidity, and a rougher cell wall surface. In cross section pieces of the cell wall are visible peeling off the surface of ∆mreB mutant grown without added Mg.
Dynamic of cell wall synthesis and degradation
In order to determine which aspect of cell wall homeostasis is affected by mreB deletion, we visualized PG insertion with and without excess Mg2+ using a fluorescent dye. In ∆mreB, no differential staining was observed at sites of deformation indicating that the PG is regularly inserted along the sidewall. To investigate the dynamic of PG degradation during cell elongation, we carried out pulse chase experiments with the PG fluorescent dye. In wild type and ∆mreB grown in presence of excess Mg2+, staining pattern becomes punctate due to insertion of new PG. In contrast, in the ∆mreB mutant grown without added Mg2+, large gaps without fluorescence are observed at the sites of bulging. Swelling cells showed a contrasting staining pattern, parts of the cells were very bright while other parts presented large gaps in fluorescence, suggesting that PG degradation was non uniform in the cell.
Effect of mreB deletion on cell wall composition
We analyzed muropeptide composition of wild type and ∆mreB mutant grown in presence or in absence of excess Mg2+. Results revealed a significant increase of muropeptides resulting from endopeptidase activities in ∆mreB mutant. Additionally, amidation of dimeric muropeptides is higher in ∆mreB grown without added Mg compare to the wild type in the same culture conditions.
Surface analysis revealed that ∆mreB mutant present a higher concentration of nitrogen compare to the wild type while phosphorus concentration vary very slightly. This finding shows that amount of teichoic acid at the cell surface does not change in response to mreB deletion or change of Mg concentration in the medium. Changes in nitrogen concentration are most probably due to an increase of peptidoglycan amidation or teichoic acids alanylation in ∆mreB mutant.
Effect of magnesium on the cell wall of Bacillus subtilis
Magnesium can compensate the absence of a number of otherwise essential cell wall related genes in B. subtilis. We have tested the effect of Mg2+ on the rigidity of the cell wall of wild-type B. subtilis cells using AFM and have found no effect. However, we identified a significant decrease in the degree of amidation of mDAP in cells grown in the presence of high concentrations of Mg2+. In B. subtilis and other species where it has been reported, mDAP amidation seems to slightly differ in different growth condition, suggesting a possible regulatory role in cell wall homeostasis.
We aimed to understand the interaction of Mg with the cell wall and the equilibrium of charges at the cell surface. Overall, gram positive bacteria are negatively charged and thus bind cations. Analyses reveal that Mg2+ concentration in LB is not sufficient to saturate binding site of the cell wall and that Mg compete with Na. Moreover, when Mg is in excess in the growth medium it binds not only to phosphate but also to carboxyl groups.
MreB proteins including mreb and mbl are known to be essential proteins in normal culture condition. Depleting mreB and mbl lead to cell deformation and eventually lysis.
Our results suggest that cell deformation in ∆mreB results from a weakened cell wall due to an increase of endopeptidase products which explain lower rigidity of the cell wall. Weakened cell wall due to misregulated hydrolases activity is unable to bear the stress from osmotic pressure leading to more stretching of PG structure at the cell surface as imaged by AFM and TEM and thus to increase cell width and/or bulging. Observation of wider cells as well as cells with bulges suggests that both localization and level of activity of hydrolases is affected. MreB proteins form a large complex at the membrane with morphogenetic proteins and cell wall synthesizing/degrading enzymes that rotate around the short axis of the cells and inserts new PG. Our results reveal that mreB deletion affects both PG synthesis and degradation by altering localization and/or activity of enzymes such as transpeptidases, carboxypeptidases (DacA) and endopeptidases. However, cell deformation is mainly the result of uncontrolled activity of hydrolytic enzymes.
When cultivated in a medium containing an excess of Mg, ∆mreB mutants are viable and present an almost wild type phenotype. Mg has also been shown to rescue other cell wall related mutants including mbl, mreC, mreD and ponA. The mechanism by which Mg rescue viability and/or cell shape in these mutants is not known. In this study we found that excess Mg does not change cell rigidity but induces changes in cell wall chemistry by decreasing the level of mDAP amidation, suggesting that Mg has a more complex role. We have shown that in presence of excess Mg, cell wall roughness and mechanical properties of ∆mreB mutant are restored to the wild type level. In term of muropeptides composition, Mg was able to reduce endopeptidases products almost to the wild type level. These results suggest that Mg restore rod shape in ∆mreB mutant by reducing the activity of endopeptidase. This hypothesis is supported by the ability of Mg to reduce autolysis rate and protect the cells against cell wall degrading enzymes such as the endopeptidase lytE. Bacterial cell wall is negatively charged because of its high phosphate content and the presence of carboxyl groups. Mg could directly act on the capacity of enzymes to interact with the cell wall. Alternatively, it could induce changes in cell wall organization altering capacity of cell wall enzymes to diffuse through the cell wall.
Our results suggest that cell deformation in ∆mreB results from a weakened cell wall due to an increase of endopeptidase products which explain lower rigidity of the cell wall. Weakened cell wall due to misregulated hydrolases activity is unable to bear the stress from osmotic pressure leading to more stretching of PG structure at the cell surface as imaged by AFM and TEM and thus to increase cell width and/or bulging. Observation of wider cells as well as cells with bulges suggests that both localization and level of activity of hydrolases is affected. MreB proteins form a large complex at the membrane with morphogenetic proteins and cell wall synthesizing/degrading enzymes that rotate around the short axis of the cells and inserts new PG. Our results reveal that mreB deletion affects both PG synthesis and degradation by altering localization and/or activity of enzymes such as transpeptidases, carboxypeptidases (DacA) and endopeptidases. However, cell deformation is mainly the result of uncontrolled activity of hydrolytic enzymes.
When cultivated in a medium containing an excess of Mg, ∆mreB mutants are viable and present an almost wild type phenotype. Mg has also been shown to rescue other cell wall related mutants including mbl, mreC, mreD and ponA. The mechanism by which Mg rescue viability and/or cell shape in these mutants is not known. In this study we found that excess Mg does not change cell rigidity but induces changes in cell wall chemistry by decreasing the level of mDAP amidation, suggesting that Mg has a more complex role. We have shown that in presence of excess Mg, cell wall roughness and mechanical properties of ∆mreB mutant are restored to the wild type level. In term of muropeptides composition, Mg was able to reduce endopeptidases products almost to the wild type level. These results suggest that Mg restore rod shape in ∆mreB mutant by reducing the activity of endopeptidase. This hypothesis is supported by the ability of Mg to reduce autolysis rate and protect the cells against cell wall degrading enzymes such as the endopeptidase lytE. Bacterial cell wall is negatively charged because of its high phosphate content and the presence of carboxyl groups. Mg could directly act on the capacity of enzymes to interact with the cell wall. Alternatively, it could induce changes in cell wall organization altering capacity of cell wall enzymes to diffuse through the cell wall.