Metabolite profiling and peptidoglycan analysis of transient cell wall‐deficient bacteria in a new Escherichia coli model system

Abstract

Many bacteria are able to assume a transient cell wall-deficient (or L-form) state under favourable osmotic conditions. Cell wall stress such as exposure to β-lactam antibiotics can enforce the transition to and maintenance of this state. L-forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adopted Escherichia coli as a model system for the controlled transition to and reversion from the L-form state, and have studied these dynamics with genetics, cell biology and 'omics' technologies. As such, a transposon mutagenesis screen underscored the requirement for the Rcs phosphorelay and colanic acid synthesis, while proteomics show only little differences between rods and L-forms. In contrast, metabolome comparison reveals the high abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized fatty acids in E. coli L-forms. This increase of membrane lipids associated with increased membrane fluidity may facilitate proliferation through bud formation. Visualization of the residual peptidoglycan with a fluorescently labelled peptidoglycan binding protein indicates de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud constriction. The DD-carboxypeptidases PBP5 and PBP6 are threefold and fourfold upregulated in L-forms, indicating a specific role for regulation of crosslinking during L-form proliferation.

Colanic Acid Intermediates Prevent De Novo Shape Recovery of Escherichia coli Spheroplasts, Calling into Question Biological Roles Previously Attributed to Colanic Acid

“…CA has been deemed to be essential for the continued growth and longterm survival of wall-less E. coli cells (variously described as Lforms or L-form-like cells) (15,16,20). However, we find that, in the complete absence of the entire CA biosynthetic pathway, LI spheroplasts survive, grow, and revert to a wild-type rod shape.…”

“…Cambré et al recently created wall-less E. coli by treating cells with cefsulodin, an antibiotic that inhibits penicillin-binding proteins 1a and 1b (PBPs 1a and 1b) (15). The resulting cells are technically not L-forms but are instead ␤-lactam-induced spheroplasts, similar to the LI spheroplasts that we discuss here.…”

“…Triggering the Rcs system induces expression of this operon, leading to the production of copious amounts of this extracellular capsular carbohydrate (21,32). Because CA was reported to be essential for creating L-forms (15,16,20), it seemed possible that Rcs-deficient spheroplasts would not recover, because they could not synthesize this compound. Consistent with this expectation, spheroplasts lacking wza did not revert to a normal rod shape but instead expanded to become giant spheroids that lysed (Fig.…”

“…In particular, lysozyme-induced (LI) spheroplasts of Escherichia coli can regenerate their walls and morphology when grown in an osmotically protective medium (12,15). However, this recovery process is impeded if the original rodshaped cells lack one of several proteins, including penicillinbinding protein 1b (PBP 1b), LpoB, or Lpp, or if the cells cannot mount an active Rcs stress response (12).…”

“…Though both spheroplasts and L-forms may sometimes regenerate a properly formed cell wall (13), L-forms must be able to divide and survive in the absence of a cell wall, and several genes and proteins are important for this transformation (15)(16)(17). One component reported to be required for L-form survival is the production of colanic acid (CA), a product of the Rcs stress response that regulates about 150 genes (15,16,(18)(19)(20). CA is an extracellular capsular polysaccharide common to members of Enterobacteriaceae but absent from other bacteria (21), and its putative role in L-form survival is undefined.…”