Cell Growth of Wall-Free L-Form Bacteria Is Limited by Oxidative Damage

Abstract

SummaryThe peptidoglycan (PG) cell wall is a defining feature of the bacterial lineage and an important target for antibiotics, such as β-lactams and glycopeptides. Nevertheless, many bacteria are capable of switching into a cell-wall-deficient state, called the “L-form” [1–3]. These variants have been classically identified as antibiotic-resistant forms in association with a wide range of infectious diseases [4]. L-forms become completely independent of the normally essential FtsZ cell division machinery [3, 5]. Instead, L-form proliferation is driven by a simple biophysical process based on an increased ratio of surface area to cell volume synthesis [6, 7]. We recently showed that only two genetic changes are needed for the L-form transition in Bacillus subtilis [7]. Class 1 mutations work to generate excess membrane synthesis [7]. Until now, the function of the class 2 mutations was unclear. We now show that these mutations work by counteracting an increase in the cellular levels of reactive oxygen species (ROS) originating from the electron transport pathway, which occurs in wall-deficient cells. Consistent with this, addition of a ROS scavenger or anaerobic culture conditions also worked to promote L-form growth without the class 2 mutations in both Gram-positive B. subtilis and Gram-negative Escherichia coli. Our results suggest that physiological compensation for the metabolic imbalance that occurs when cell wall synthesis is blocked is crucial for L-form proliferation in a wide range of bacteria and also provide new insights into the mode of action of antibiotics that target the bacterial cell wall.

Disturbance of the bacterial cell wall specifically interferes with biofilm formation

“…This is consistent with the recently published results of Kawai et al . (), where L‐forms devoid of cell wall avoid an excess of oxidative stress by mutations affecting the ETC. We also observed an overrepresentation of proteins that function in the acquisition and uptake of iron (http://onlinelibrary.wiley.com/doi/10.1111/1758-2229.12346/suppinfo).…”

Induction of the Spx regulon by cell wall stress reveals novel regulatory mechanisms in Bacillus subtilis

“…Induction of the yjbC‐spx operon has been observed in transcriptomic studies of B. subtilis cells treated with vancomycin (Cao et al ., ; Eiamphungporn and Helmann, ), enduracidin and bacitracin (Rukmana et al ., ), and ramoplanin and moenomycin (Salzberg et al ., ). B. subtilis protoplasts also display significant spx upregulation compared to either walled cells or L‐forms (Kawai et al ., ). We here confirmed the upregulation of spx by treatment with early and late PG synthesis inhibitors and further show that the P M1 promoter, which is primarily controlled by σ M , is the major driver of this transcriptional induction (Fig.…”

“…Although the precise molecular signals remain unknown, r M , r W and r X respond to inhibitors of peptidoglycan (PG) synthesis, membrane-active compounds, and antimicrobial cationic peptides respectively (Helmann, 2016). Interestingly, the yjbC-spx operon contains two promoters that appear to be regulated by ECF r factors (Jervis et al, 2007;Eiamphungporn and Helmann, 2008), and cell envelope antibiotics induce spx and Spx-dependent genes (Cao et al, 2002b;Eiamphungporn and Helmann, 2008;Wecke et al, 2011;Kawai et al, 2015).…”

Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes

“…The first class of mutations leads to an increase in membrane synthesis, either directly by increasing fatty acid biosynthesis, or indirectly by reducing cell wall synthesis 20 . The second class of mutations reduces oxidative damage caused by reactive oxygen species, which are detrimental to proliferation of L-forms 21 . Notably, proliferation of L-forms is independent of the FtsZ-based division machinery 15, 22 Instead, their proliferation can be explained solely by biophysical processes, in which an imbalance between the cell surface area to volume ratio leads to spontaneous blebbing and the subsequent generation of progeny cells 20 .…”