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.
