Bacillus subtilis cell diameter is determined by the opposing actions of two distinct cell wall synthetic systems

Abstract

Introductory Paragraph Rod-shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: The Rod complex moves around the cell circumference, while class A penicillin-binding proteins do not. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each affects the growth and width of Bacillus subtilis, as well as the mechanical anisotropy and orientation of material within their sacculi. Rod width is not determined by MreB, rather it depends on the balance between the systems: The Rod complex reduces diameter, while aPBPs increase it. Increased Rod complex activity correlates with an increased density of directional MreB filaments and a greater fraction of directional PBP2a enzymes. This increased circumferential synthesis increases the relative amount of oriented material within the sacculi, making them more resistant to stretching across their width, thereby reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems builds and maintains rods of different widths. Escherichia coli Rod mutants also show the same correlation between width and directional MreB filament density, suggesting this model may be generalizable to bacteria that elongate via the Rod complex.

Cell-wall synthases contribute to bacterial cell-envelope integrity by actively repairing defects

“…While cell shape was hardly affected by PBP1ab repression, we found a mild but significant positive correlation between cell diameter PBP1ab levels, consistent with a previous study of a ΔPBP1a mutant ( Banzhaf et al, 2012 ). A much stronger correlation between a class-A PBP levels and cell diameter of same sign was recently also observed in B. subtilis ( Dion et al, 2019 ). In both species, it is conceivable that increased aPBP activity depletes a common pool of lipid II precursors and thus indirectly reduces the capacity of the Rod complex to maintain a narrower cell diameter.…”

“…In contrast, a 10-fold decrease in the level of Rod-complex-related operons PBP2-RodA or MreBCD increased diameter by about 800 nm (Figure 1D), as previously demonstrated ( Vigouroux et al, 2018 ). Our observations are also in stark contrast to B. subtilis , where a similar change of the level of the major class A PBP PBP1 leads to a 600 nm increase in diameter ( Dion et al, 2019 ). As a control, we used an alternative setup based on the inducible P Bad promoter (strains AV100, AV101) and checked the lack of major shape phenotype at low PBP1ab induction (Figure 1 - Supplement 2BC).…”

“…We then used combinations of different CRISPR guides targeting GFP and RFP with a variable number of mismatches ( Vigouroux et al, 2018 ). To extend the range of possible repression levels, CRISPR guides were expressed in two different forms: i) as a CRISPR RNA (crRNA) co-expressed with the tracrRNA, on the pCRRNAcos vector ( Vigouroux et al, 2018 ) or ii) as a single-guide (sgRNA) with fused crRNA and tracrRNA, on the pAV20 vector ( Dion et al, 2019 ) (Figure 1A). The CRISPR guides are named according to their complementarity to GFP (G) or RFP (R), Ø designating a control guide.…”

“…Therefore, Rod complex and aPBPs might serve different functions despite catalyzing the same chemical reactions ( Zhao et al, 2017 ; Pazos et al, 2017 ). In agreement with this viewpoint, recent work in the gram-positive Bacillus subtilis showed that the two machineries have opposing actions on cell diameter and lead to either circumferentially organized or disordered cell-wall deposition ( Dion et al, 2019 ).…”

“…The CRISPR plasmids are either from the pcrRNA collection described in ( Vigouroux et al, 2018 ), or were assembled using the pAV20 double-sgRNA vector ( Dion et al, 2019 ). In the later case, complementary oligonucleotide pairs (table 8) were phosphorylated with T4 PNK in the presence of T4 ligase buffer (New England Biolabs) and then annealed.…”