An Exhaustive Multiple Knockout Approach to Understanding Cell Wall Hydrolase Function inBacillus subtilis

Abstract

Most bacteria are surrounded by their cell wall, a highly crosslinked protective envelope of peptidoglycan. To grow, bacteria must continuously remodel their wall, inserting new material and breaking old bonds. Bond cleavage is performed by cell wall hydrolases, allowing the wall to expand. Understanding the functions of individual hydrolases has been impeded by their redundancy: single knockouts usually present no phenotype. We used an exhaustive multiple-knockout approach to determine the minimal set of hydrolases required for growth in Bacillus subtilis. We identified 42 candidate cell wall hydrolases. Strikingly, we were able to remove all but two of these genes in a single strain; this 'Δ40' strain shows a normal growth rate, indicating that none of the 40 hydrolases are necessary for cell growth. The Δ40 strain does not shed old cell wall, demonstrating that turnover is not essential for growth. The remaining two hydrolases in the Δ40 strain are LytE and CwlO, previously shown to be synthetically lethal. Either can be knocked out in Δ40, indicating that either hydrolase alone is sufficient for cell growth. Environmental screening and zymography revealed that LytE activity is inhibited by Mg2+ and that RlpA-like proteins may stimulate LytE activity. Together, these results demonstrate that the only essential function of cell wall hydrolases in B. subtilis is to enable cell growth by expanding the wall and that LytE or CwlO alone is sufficient for this function. These experiments introduce the Δ40 strain as a tool to study hydrolase activity and regulation in B. subtilis.

Insights into the role of lipoteichoic acids and MprF function in Bacillus subtilis

“…Our analysis showed that magnesium decreased the abundance of LytE significantly in ΔponA and ΔponA ΔmprF (Appendix Fig S13 ) but not as much in ΔponA ΔltaS. Perhaps, the unknown mechanism by which exogenous magnesium helps the growth of some cell wall mutants might partially require LtaS activity to tune LytE (Wilson and Garner, 2021).…”

“…Autolysins or cell wall hydrolases help shape and recycle the peptidoglycan during cell elongation and division. B. subtilis expresses a larger number of them but only the loss of both PG D,L-endopeptidases CwlO and LytE is lethal (Hashimoto et al, 2012, Salzberg et al, 2013, Do et al, 2020, Wilson and Garner, 2021. Our interest in LytE derived from the facts that this major autolysin is secreted and thought to be anchored to the lateral cell wall and the septum (Dominguez-Cuevas et al, 2013).…”

“…The D4 strain is conditionally lethal on PAB agar medium unless supplemented with magnesium (Kawai et al, 2009b). It is thought that this divalent cation alters the structure of the wall by binding to teichoic acids or PG and by doing so, might alter the activity of cell wall hydrolases, the enzymes that modify and sculpt synthesised PG to allow the cell to elongate and divide (Neuhaus and Baddiley, 2003, Do et al, 2020, Wilson and Garner, 2021.…”

WhyD tailors surface polymers to prevent premature bacteriolysis and direct cell elongation in Streptococcus pneumoniae

“…Since the PG layer is a continuous matrix, cell wall biogenesis also requires the activity of enzymes that cut bonds in the network called PG hydrolases or autolysins. These space-making factors are important for breaking bonds in the matrix to allow for its expansion and the insertion of new material into the pre-existing meshwork ( Bisicchia et al, 2007 ; Carballido-López et al, 2006 ; Dobihal et al, 2019 ; Dörr et al, 2013 ; Meisner et al, 2013 ; Singh et al, 2012 ; Sycuro et al, 2010 ; Vollmer et al, 2008 ; Wilson and Garner, 2021 ). They also play important roles in cleaving shared cell wall material connecting daughter cells during cytokinesis ( Hashimoto et al, 2018 ; Heidrich et al, 2002 ; Vollmer et al, 2008 ; Wilson and Garner, 2021 ).…”

Mircubactin B rescues the lethal effect of cell wall biosynthesis mutations in Bacillus subtilis

“…23 Wilson and Garner (2021) identified LytE as a likely target for this inhibition. 24 The TAs are underexplored potential antibiotic targets for Gram-positive pathogens. Compounds active on WTA 25 or LTA 26 have been described but, so far, have not yet entered clinical development.…”