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.
Highlights
Most bacteria are surrounded by a cell wall or peptidoglycan, a large molecule consisted glycan strands cross-linked by short peptides
In a hypotonic medium, when peptidoglycan synthesis is inhibited by antibiotics such as penicillin, or peptidoglycan is degraded by lysozyme, bacterial cells are lysed by turgor pressure (Vollmer and Seligman, 2010; Egan et al, 2020)
Consistent with previous reports that show the importance of the outer membrane for growth of the L-form (Rojas et al, 2018; Osawa and Erickson, 2019), we found that in a medium containing a low concentration of Mg2+ or outer membrane synthesis inhibitors, walled E. coli cells converted to the L-form, but lysed and failed to grow
Summary
Most bacteria are surrounded by a cell wall or peptidoglycan, a large molecule consisted glycan strands cross-linked by short peptides. Many bacteria, following inhibition of peptidoglycan synthesis, can switch to a state, the L-form, in which they proliferate without the cell wall These cells cope well with decreased turgor pressure under hypertonic conditions and can revert to walled rod-shaped cells by restoring cell wall synthesis (Ranjit and Young, 2013; Billings et al, 2014; Kawai et al, 2014; Mercier et al, 2014). Cell division of walled E. coli and B. subtilis is regulated by FtsZ, a tubulin homolog; whereas division of the B. subtilis L-form appears to be unregulated (Leaver et al, 2009) These findings can be extrapolated to other Grampositive or Gram-negative bacteria, including E. coli (Mercier et al, 2016). Contrasting evidence may be explained by different experimental conditions employed, such as aeration and the antibiotics used to induce L-form conversion
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