The mdoC gene of Escherichia coli encodes a membrane protein that is required for succinylation of osmoregulated periplasmic glucans

Abstract

Osmoregulated periplasmic glucans (OPGs) of Escherichia coli are anionic oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by phosphoglycerol originating from the membrane phospholipids and by succinyl residues from unknown origin. A phosphoglycerol-transferase-deficient mdoB mutant was subjected to Tn5 transposon mutagenesis, and putative mutant clones were screened for changes in the anionic character of OPGs by thin-layer chromatography. One mutant deficient in succinylation of OPGs was obtained, and the gene inactivated in this mutant was characterized and named mdoC. mdoC, which encodes a membrane-bound protein, is closely linked to the mdoGH operon necessary for the synthesis of the OPG backbone.

FIG. 1

Thin-layer chromatographic analysis of OPGs extracted from NFB732 (mdoB, lanes 1 and 2) or NFB1864 (mdoB mdoC, lanes 3 and 4). Extracts were applied directly to thin-layer chromatography plates (lanes 1 and 3) or first subjected to mild alkali treatment to remove succinyl substituents (lanes 2 and 4).

FIG. 2

DEAE-Sephacel anion exchange column chromatography profiles of [U-¹⁴C] glucose labeled OPGs from strains DF214 (mdo⁺), NFB734 (mdoB), NFB1933 (mdoB mdoC), and NFB1919 (mdoC). Ionic strength was increased by steps of 0.05 M NaCl at fractions indicated by the arrows. Fractions (4 ml) were collected and aliquots were counted. Peak I, neutral glucans; peaks II to V, anionic glucans. The number on the right of each peak refers to the percentage of the total for the corresponding subfraction.

FIG. 3

Genetic organization of the mdoC region of the E. coli chromosome (top) and restriction map of the 8-kb insert of pNF239 and its derivatives (bottom). Only relevant restriction sites are shown; arrows and T indicate transcriptional units and transcriptional terminators, respectively. PstI* and colE1 are part of the insert originating from the colE1 vector (9). Horizontal empty bars indicate the structure of the various inserts of the plasmids.