Cell wall morphogenesis in Bacillus subtilis

Abstract

Almost all bacteria are surrounded by a peptidoglycan cell wall. This cell wall imposes the shape of the bacterial cell and enables the cell to resist turgor pressure, localise proteins and coordinate cell division. Despite being ‘essential’, bacteria can grow and divide indefinitely in a wall-less state known as the L-form. Conventionally, L-form bacteria in the laboratory are grown at high concentrations of osmoprotectant such as salt or sucrose due to their sensitivity to turgor. However, L-forms have been isolated from various environments, including from persistent infections where such isotonic conditions may not be relevant. Here I show that L-forms derived from the Gram-positive model organism Bacillus subtilis can be readily adapted to media containing very low concentrations of osmoprotectant (sucrose or salt), lending support to theories regarding the natural role of L-forms. Regeneration of the wall of the adapted strains revealed a raft of phenotypic changes including, but not limited to: cell branching, misplaced septa and increased cell length. Whole genome sequencing of these mutant strains revealed several mutations. Among the mutations identified was a partial deletion of the gene coding for the actin homologue, MreB. In the walled state, MreB directs and coordinates the peptidoglycan synthesis machinery as a means to control the morphology of the cell. Characterisation of the mutation in mreB revealed that loss of this important protein is sufficient to enable the growth of L-forms in low osmolarites. The cell wall of Gram-positive bacteria also contains teichoic acids (TA). TAs linked to the lipids in the cell membrane (Lipoteichoic acids; LTA) are essential for the viability of the pathogen Staphylococcus aureus. In contrast, LTA is dispensable in B. subtilis, where loss of the primary synthase, LtaS, acts as a suppressor mutation in strains that lack the MreB homologue, Mbl. Based on the observation that loss of ltaS improved the growth of a Δmbl mutant in B. subtilis a screening method was previously developed from which 5 candidate actinomycete strains producing potential inhibitors of LtaS were identified. Preliminary experiments narrowed the focus to one strain that reproducibly produced an active compound, which was purified and characterised. Various properties of the compound 4 were consistent with it acting as an inhibitor of LtaS. Mass spectrometry (MS) and MS-MS data suggested a potential identity for the inhibitor that could plausibly represent a substrate analogue. Treatment with the compound impaired the growth of S. aureus.