title: Colanic Acid Intermediates Prevent De Novo Shape Recovery of Escherichia coli Spheroplasts, Calling into Question Biological Roles Previously Attributed to Colanic Acid.
abstract: After losing their protective peptidoglycan, bacterial spheroplasts can resynthesize a cell wall to recreate their normal shape. In Escherichia coli, this process requires the Rcs response. In its absence, spheroplasts do not revert to rod shapes but instead form enlarged spheroids and lyse. Here, we investigated the reason for this Rcs requirement. Rcs-deficient spheroids exhibited breaks and bulges in their periplasmic spaces and failed to synthesize a complete peptidoglycan cell wall, indicating that the bacterial envelope was defective. To determine the Rcs-dependent gene(s) required for shape recovery, we tested spheroplasts lacking selected RcsB-regulated genes and found that colanic acid (CA) biosynthesis appeared to be involved. Surprisingly, though, extracellular CA was not required for recovery. Instead, lysis was caused by mutations that interrupted CA biosynthesis downstream of the initial glycosyl transferase, WcaJ. Deleting wcaJ prevented lysis of spheroplasts lacking ensuing steps in the pathway, and providing WcaJ in trans to a mutant lacking the entire CA operon triggered spheroplast enlargement and lysis. Thus, CA is not required for spheroplast recovery. Instead, CA intermediates accumulate as dead-end products which inhibit recovery of wall-less cells. The results strongly imply that CA may not be required for the survival E. coli L-forms. More broadly, these findings mandate that previous conclusions about the role of colanic acid in biofilm formation or virulence must be reevaluated.
  
  
  OBJECTIVE
  Wall-less bacteria can resynthesize their walls and recreate a normal shape, which in Escherichia coli requires the Rcs response. While attempting to identify the Rcs-dependent gene required for shape recovery, we found that colanic acid (CA) biosynthesis appeared to be involved. Surprisingly, though, cell death was caused by mutations that interrupted CA biosynthesis downstream of the initial step in the pathway, creating dead-end compounds that inhibited recovery of wall-less cells. When testing for the biological role of CA, most previous experiments used mutants that would accumulate these deadly intermediates, meaning that all prior conclusions must be reexamined to determine if the results were caused by these lethal side effects instead of accurately reflecting the biological purpose of CA itself.
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
authors.forename: Dev K
authors.initials: DK
authors.lastname: Ranjit
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA kdyoung@uams.edu.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Young
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011134:Polysaccharides; C004275:colanic acid
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