An Alternative and Conserved Cell Wall Enzyme That Can Substitute for the Lipid II Synthase MurG

Abstract

The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces. CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis. IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.

Reproducible switching between a walled and cell wall-deficient lifestyle of actinomycetes using gradient agar plates

“…We previously showed that K. viridifaciens alpha , an unstable L-form strain obtained after induction with penicillin and lysozyme, can either grow filamentous or wall-deficient depending on the osmolarity of the media (Ramijan et al, 2018; Zhang et al, 2021). Using gradient plates, we indeed found that alpha could readily switch between these distinct morphologies (Fig.…”

“…K. viridifaciens Δ dcw was found to be unable to revert to the walled state, caused by the absence of a large part of the dcw cluster (Zhang et al, 2021). Interestingly, our gradient plates reveal colonies at an LPMA percentage of 20% LPMA, indicating that these L-forms could tolerate a much lower concentration of osmolytes than previously thought.…”

“…Strain K. viridifaciens M1 is a natural L-form cell line, obtained after prolonged exposure of S-cells to hyperosmotic stress (Ramijan et al, 2018). The non-switching L-form strain K. viridifaciens Δ dcw was created by replacing ftsW, murG, ftsQ, ftsZ, ylmD, ylmE, sepG, sepF , and divIVA with the apramycin resistance marker aac(3)IV in alpha (Zhang et al, 2021). To obtain spores, K. viridifaciens DSM40239 and Streptomyces coelicolor A3(2) M145 were grown at 30°C for 4 days on Maltose-Yeast Extract-Malt Extract (MYM) medium, after which spores were harvested as described (Kieser et al, 2000).…”

“…viridifaciens DSM40239, alpha is unable to differentiate and form aerial hyphae (Zhang et al, 2021) (see also Fig. 3).…”

Adrenaline-mediated activation of antibiotic production in Streptomyces highlights catechol as elicitor of specialized metabolism

“…For this, a 20 nt spacer sequence was introduced into the sgRNA scaffold by PCR using forward primers KSβ_TF or KSβ_NT2F together with reverse primer SgTermi_R_B. The PCR products were cloned into pGWS1370 55 via NcoI / BamHI restriction sites. The resulting constructs pGWS1516 (targeting template strand of KSβ, control) and pGWS1517 (targeting non-template strand of KSβ) were then introduced into Streptomyces sp.…”