Dataset Information

Identification of the promoter region for the colanic acid polysaccharide biosynthetic genes in Escherichia coli K-12.

ABSTRACT: The colanic acid polysaccharide capsule biosynthetic genes (cps genes) are primarily clustered at one site located at about 45 min on the Escherichia coli chromosome. The network of proteins involved in regulating the expression of these genes includes the two positive regulators RcsA and RcsB. This work describes the site of action of these two activator proteins and the promoter of the cps genes. It is likely that the cps genes are arranged in a single long operon that is at least 13.5 kb. The promoter region was identified with fusions to lacZ that resulted in regulated expression by the Rcs network of regulatory proteins, and the start site of transcription was identified by primer extension. The operator region was cloned from Kohara phage to multicopy plasmids and identified by titrating RcsA or RcsB. Sequence analysis of the promoter and operator region revealed homology to the JUMPstart element found in the untranslated region of many exopolysaccharide biosynthetic operons. In addition, the deduced amino acid sequence of the amino terminus of the first open reading frame of the cps operon was found to be homologous to proteins encoded by the exopolysaccharide biosynthetic operons of Klebsiella pneumoniae and Erwinia amylovora.

SUBMITTER: Stout V

PROVIDER: S-EPMC178186 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Identification of the promoter region for the colanic acid polysaccharide biosynthetic genes in Escherichia coli K-12.

Stout V V

Journal of bacteriology 19960701 14

The colanic acid polysaccharide capsule biosynthetic genes (cps genes) are primarily clustered at one site located at about 45 min on the Escherichia coli chromosome. The network of proteins involved in regulating the expression of these genes includes the two positive regulators RcsA and RcsB. This work describes the site of action of these two activator proteins and the promoter of the cps genes. It is likely that the cps genes are arranged in a single long operon that is at least 13.5 kb. The ...[more]

PMID: 8763957

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Project description:UnlabelledAfter 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.ImportanceWall-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.

Dataset Information

Identification of the promoter region for the colanic acid polysaccharide biosynthetic genes in Escherichia coli K-12.

Publications

Identification of the promoter region for the colanic acid polysaccharide biosynthetic genes in Escherichia coli K-12.

Similar Datasets

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