Direct Observation of Conversion From Walled Cells to Wall-Deficient L-Form and Vice Versa in Escherichia coli Indicates the Essentiality of the Outer Membrane for Proliferation of L-Form Cells

Abstract

Gram-negative bacteria such as Escherichia coli are surrounded by an outer membrane, which encloses a peptidoglycan layer. Even if thinner than in many Gram-positive bacteria, the peptidoglycan in E. coli allows cells to withstand turgor pressure in hypotonic medium. In hypertonic medium, E. coli treated with a cell wall synthesis inhibitor such as penicillin G form wall-deficient cells. These so-called L-form cells grow well under anaerobic conditions (i.e., in the absence of oxidative stress), becoming deformed and dividing as L-form. Upon removal of the inhibitor, they return to the walled rod-shaped state. Recently, the outer membrane was reported to provide rigidity to Gram-negative bacteria and to strengthen wall-deficient cells. However, it remains unclear why L-form cells need the outer membrane for growth. Using a microfluidic system, we found that, upon treatment with the outer membrane-disrupting drugs polymyxin B and polymyxin B nonapeptide or with the outer membrane synthesis inhibitor CHIR-090, the cells lysed during cell deformation and division, indicating that the outer membrane was important even in hypertonic medium. L-form cells could return to rod-shaped when trapped in a narrow space, but not in a wide space, likely due to insufficient physical force. Outer membrane rigidity could be compromised by lack of outer membrane proteins; Lpp, OmpA, or Pal. Deletion of lpp caused cells to lyse during cell deformation and cell division. In contrast, ompA and pal mutants could be deformed and return to small oval cells even when less physical force was exerted. These results strongly suggest that wall-deficient E. coli cells require a rigid outer membrane to survive, but not too rigid to prevent them from changing cell shape.

Understanding Beta-Lactam-Induced Lysis at the Single-Cell Level

“…Rather, both turgor pressure and cell wall synthesis may be modulated to generate cells of different widths, and our results provide evidence that the mechanical stresses in the cell envelope are regulated during the normal growth of cells. Indeed, the response of cells to osmotic stress, both hypoosmotic and hyperosmotic, has been appreciated as physiologically relevant in other contexts (Sleator and Hill, 2002 ), including the conversion of walled cells to wall-less L-forms (Ramijan et al, 2018 ; Claessen and Errington, 2019 ; Mickiewicz et al, 2019 ; Osawa and Erickson, 2019 ; Chikada et al, 2021 ). We anticipate future experiments, for instance those involving osmotic shocks, to further validate the hypothesis that the mechanical stresses in cells of different widths are approximately constant.…”

Characterization of the L-form switch in the Gram-negative pathogen Streptobacillus moniliformis

“…Although this result suggested a critical importance of OM growth for proliferation of S. moniliformis L-forms, which was also recently described for E. coli L-forms (Osawa and Erickson 2019 ; Chikada et al . 2021 ), separation of progeny cells through OM scission was not evident under the conditions of our time-lapse experiments with liquid medium. This may reflect a physiological barrier to the proliferation of L-forms in liquid media.…”

“… 2018 ; Chikada et al . 2021 ). In several bacteria, L-form proliferation has been shown to be completely independent of the normally essential FtsZ-based bacterial division machinery or cell wall synthetic enzymes (Leaver et al .…”

“… 2020 ; Chikada et al . 2021 ). This work has started to uncover central conserved properties of the unusual L-form state, as well as the genetic and other mechanisms involved in the process of L-form formation and proliferation.…”

“… 2015 , 2019 ; Chikada et al . 2021 ). Crucially, these effects do not occur in a Gram-positive bacterium, Enterococcus faecium , which lacks the RC pathway (Kawai et al .…”