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.

Importance: 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.

FIG 1

Organization of the periplasm and peptidoglycan in recovering LI spheroplasts. Wild-type E. coli strain MG1655 (A to H) and the isogenic ΔrcsB mutant DR5 (I to P) were visualized immediately before being transformed into LI spheroplasts (Before; n = 8 to 10), immediately after being transformed into LI spheroplasts (0 h; n = 5 to 6), and 1 h into the recovery period (1 h; n = 5 to 6). Cells expressing the periplasmic fluorescent protein DsbA-ss-sfGFP were visualized by phase (A to C, I to K) and fluorescence (E to G, M to O) microscopy. Newly synthesized peptidoglycan in recovering spheroplasts was labeled with the fluorescent d-alanine derivative HADA (D, H, L, P). The images are representative.

FIG 2

Colanic acid genes and the CA biosynthetic pathway. (A) Genes of the CA operon. The promoter region contains a binding sequence that induces gene expression when occupied by the RcsAB heterodimer. (B) Selected substrates and enzymes in the CA biosynthesis and transport pathway (29, 39, 58). (Adapted from reference with permission of the publisher.) Enzymes are devoted to precursor synthesis (CpsG, CpsG, Gmd, WcaG, and WcaH) or to CA assembly and transport (WcaJ, WcaA, WcaC, WcaE, WcaI, WcaL, WcaB, WcaF, WzxC, WcaD, Wzc, Wzb, and Wza). Undecaprenyl pyrophosphate synthase (UppS) catalyzes the condensation reaction of isopentenyl diphosphate and farnesyl diphosphate to generate undecaprenyl pyrophosphate (Und-PP). Undecaprenyl pyrophosphate phosphatase (UppP) converts Und-PP to undecaprenyl phosphate (Und-P). WcaJ, a glycosyl transferase, initiates the first dedicated step of CA assembly by adding glucose-1-phosphate from UDP-glucose (UDP-Glu) to Und-P, generating Und-PP-Glu. Additional sugar residues are added stepwise to Und-PP-Glu: GDP-fucose (GDP-Fuc), UDP-galactose (UDP-Gal), and UDP-glucaronic acid (UDP-GlcA). WzxC, a polysaccharide transporter, flips the lipid-linked CA repeat unit to the periplasmic face of the inner membrane (IM). WcaD polymerizes the repeat units to create CA polymers, and the Wzc-Wza complex transports mature CA through the outer membrane (OM) and out of the cell. OAc, O-acetyl; PG, peptidoglycan; Pyr, pyruvate.

FIG 3

Extracellular colanic acid is not required for spheroplast recovery. Spheroplasts lacking Wza were incubated on recovery medium, and the recovery process was monitored by time-lapse phase-contrast microscopy. Time after plating (hour:minute) is displayed in each panel. The images are representative. (A) E. coli DR36 Δwza (n = 16); (B) E. coli DR37 Δwza, complemented with the pWza plasmid (n = 9); (C) E. coli DR38 Δ(wza to wcaM) (n = 16).

FIG 4

Blocking colanic acid transport inhibits spheroplast recovery. Spheroplasts unable to assemble or transport CA were grown on osmotically protected sucrose recovery medium, and the recovery process was monitored by time-lapse phase-contrast microscopy. Time after plating (hour:minute) is displayed in each panel. The images are representative. (A) E. coli DR39 ΔwcaD (n = 9); (B) E. coli DR40 ΔwzxC (n = 22); (C) E. coli DR44 Δugd (n = 5); (D) E. coli DR45, in which ugd is complemented with the pUgd plasmid (n = 4); (E) E. coli DR41 ΔcpsB (n = 7); (F) E. coli DR42 ΔcpsG (n = 4); (G) E. coli DR43, in which ΔcpsG is complemented with the pCpsG plasmid (n = 8).

FIG 5

Deleting wcaJ restores recovery to spheroplasts lacking components of the colanic acid biosynthesis pathway but not to spheroplasts lacking rcsB. Spheroplasts from the indicated mutants were grown on osmotically protected sucrose recovery medium, and the recovery process was monitored by time-lapse phase-contrast microscopy. Time after plating (hour:minute) is displayed in each panel. The images are representative. (A) E. coli DR46 ΔwcaJ (n = 13); (B) E. coli DR47 ΔwzxC ΔwcaJ (n = 15); (C) E. coli DR48 ΔcpsG ΔwcaJ (n = 14); (D) E. coli DR49 Δugd ΔwcaJ (n = 18); (E) E. coli MG1655 carrying the pWcaJ plasmid (n = 9); (F) E. coli DR51 Δ(wza to wcaM) carrying the pWcaJ plasmid (n = 15); (G) E. coli DR57 Δ(wza to wcaM) ΔrcsB (n = 12).