Metabolite profiling and peptidoglycan analysis of 3 transient cell wall-deficient bacteria 4 in a new Escherichia coli model system 1 5 6

28 Many bacteria are able to assume a transient cell wall-deficient (or L-form) state 29 under favorable osmotic conditions. Cell wall stress such as exposure to β-lactam antibiotics 30 can enforce the transition to and maintenance of this state. L-forms actively proliferate and 31 can return to the walled state upon removal of the inducing agent. We have adopted 32 Escherichia coli as a model system for the controlled transition to and reversion from the L33 form state, and have studied these dynamics with genetics, cell biology and ‘omics’ 34 technologies. As such, a transposon mutagenesis screen underscored the requirement for 35 the Rcs phosphorelay and colanic acid synthesis, while proteomics show only little 36 differences between rods and L-forms. In contrast, metabolome comparison reveals the high 37 abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized 38 fatty acids in E. coli L-forms. This increase of membrane lipids associated with increased 39 membrane fluidity may facilitate proliferation through bud formation. Visualization of the 40 residual peptidoglycan with a fluorescently labeled peptidoglycan binding protein indicates 41 de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud 42 constriction. The DD-carboxypeptidases PBP5 and PBP6 are threeand four-fold upregulated 43 in L-forms, indicating a specific role for regulation of crosslinking during L-form proliferation. 44 45 A cc ep te d A rti cl e