Cell Envelope Stress Response in Cell Wall-Deficient L-Forms of Bacillus subtilis

Abstract

b L-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-positive model bacterium Bacillus subtilis was constructed that allowed controlled switching between rod-shaped wildtype cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to ␤-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here we investigated the response of B. subtilis to the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS-and BceRS-dependent cell envelope stress biosensors. Our results show that B. subtilis L-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, we established a lacZ-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).

An Acyl-Linked Dimer of Daptomycin Is Strongly Inhibited by the Bacterial Cell Wall

“…On these L-forms, the MIC of the monomer at 5 mM Ca ++ decreased from 0.5 to 0.075 µg/mL; this increase in activity agrees with previous findings (23). With the dimer, however, activity increased dramatically, resulting in an MIC of 0.2 µg/mL which was only slightly (but reproducibly) above that of the monomer.…”

The cell envelope stress response of Bacillus subtilis: from static signaling devices to dynamic regulatory network

“…Spontaneous in-frame deletions in the C-terminus of LiaF revealed a locked-ON phenotype similar to the liaF deletion strain, suggesting that the C-terminus is functionally important for stimulus perception and/or signaling to LiaS (Jordan et al 2006). Although the precise stimulus for the LiaFSR system remains elusive to date, later work suggested that it is unable to sense AMPs directly (Wolf et al 2012). Specifically, the response of the P liaI promoter towards its inducers bacitracin, mersacidin, vancomycin, nisin and daptomycin was completely absent in inducible L-forms of B. subtilis, which were depleted of cell wall precursors by repression of the murE operon (Wolf et al 2012).…”

“…If the bacitracin level is increased further, despite upregulation of BceAB, cell wall synthesis is being affected and first damage to the cell envelope occurs. This induces the Lia system (Wolf et al 2012), which is part of the secondary layer of resistance that protects the cell envelope by a mechanism yet to be identified (Radeck et al 2016). Finally, at even higher bacitracin concentrations, perturbations of the lipid II cycle lead to activation of σ M , which in turn upregulates the expression of cell envelope protecting proteins, amongst them BcrC Helmann 2016).…”

“…Although the precise stimulus for the LiaFSR system remains elusive to date, later work suggested that it is unable to sense AMPs directly (Wolf et al 2012). Specifically, the response of the P liaI promoter towards its inducers bacitracin, mersacidin, vancomycin, nisin and daptomycin was completely absent in inducible L-forms of B. subtilis, which were depleted of cell wall precursors by repression of the murE operon (Wolf et al 2012). This strongly suggested that the LiaFSR system requires the presence After the addition of bacitracin and formation of the UPP-Bac complex, BceB recognizes UPP-Bac and removes bacitracin from its target employing a so far unknown transport mechanism dependent on ATP hydrolysis by BceA.…”

“…But what is the true nature of the substrate bound by the BceB permease? Initial evidence from cell wall-less L-forms of B. subtilis showed that the Bce system is triggered in the absence of cell wall biosynthesis (Wolf et al 2012), arguing for a 'drug-sensing' mechanism of stimulus perception. This view was supported by in vitro results indicating that BceB is able to bind bacitracin directly (Dintner et al 2014).…”