Nucleotide sequence and mutational analysis of the gene encoding KpsD, a periplasmic protein involved in transport of polysialic acid in Escherichia coli K1

Abstract

The 17-kb kps gene cluster encodes proteins necessary for the synthesis, assembly, and translocation of the polysialic acid capsule of Escherichia coli K1. We previously reported that one of these genes, kpsD, encodes a 60-kDa periplasmic protein that is involved in the translocation of the polymer to the cell surface. The nucleotide sequence of the 2.4-kb BamHI-PstI fragment accommodating the kpsD gene was determined. Sequence analysis showed an open reading frame for a 558-amino-acid protein with a typical N-terminal prokaryotic signal sequence corresponding to the first 20 amino acids. KpsD was overexpressed, partially purified, and used to prepare polyclonal antiserum. A chromosomal insertion mutation was generated in the kpsD gene and results in loss of surface expression of the polysialic acid capsule. Immunodiffusion analysis and electron microscopy indicated that polysaccharide accumulates in the periplasmic space of mutant cells. A wild-type copy of kpsD supplied in trans complemented the chromosomal mutation, restoring extracellular expression of the K1 capsule. However, a kpsD deletion derivative (kpsD delta C11), which results in production of a truncated KpsD protein lacking its 11 C-terminal amino acids, was nonfunctional. Western blot (immunoblot) data from cell fractions expressing KpsD delta C11 suggest that the truncated protein was inefficiently exported into the periplasm and localized primarily to the cytoplasmic membrane.

Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane

“…For instance, the export of the tightly surface-associated capsular polysaccharides across the outer membrane occurs via a novel type of octameric ␣-helical pore formed by Wza and its otholog KpsD (41)(42)(43), whereas the exopolysaccharide alginate secretin AlgE forms an 18-strand ␤-barrel pore with an arginine-rich highly electropositive pore constriction acting as a selectivity filter for the negatively charged alginate polymer (29). Apart from this, the extracellular loop L2 and a long periplasmic loop T8 of the AlgE ␤-barrel seem to play important roles in regulating alginate secretion ( Fig.…”

The localization of KpsC, S and T, and KfiA, C and D proteins involved in the biosynthesis of the Escherichia coli K5 capsular polysaccharide: evidence for a membrane-bound complex

“…The KpsF protein is not essential for encapsulation, but may play a role in the export of capsular polysaccharide (Simpson et al, 1996;Cieslewicz & Vimr, 1997). The KpsE and KpsD proteins are believed to be involved in translocation of polymer across the periplasm and onto the cell surface (Bronner et al, 1993a, b;Wunder et al, 1994;Rosenow et al, 1995a). The KpsU protein is a capsule-specific cytoplasmic CMP-Kdo synthetase enzyme (Pazzani et al, 1993;Rosenow et al, 1995b) that generates CMP-Kdo for the synthesis of phosphatidylKdo, which is linked to the reducing end of group I1 capsular polysaccharides (Schmidt & Jann, 1982;Jann & Jann, 1992).…”

KpsF Is the Arabinose-5-phosphate Isomerase Required for 3-Deoxy-d-manno-octulosonic Acid Biosynthesis and for Both Lipooligosaccharide Assembly and Capsular Polysaccharide Expression in Neisseria meningitidis

“…KpsU has been shown to encode a second copy of the CMP-Kdo synthetase, KdsB (16). KpsD is a periplasmic protein, and mutation of kpsD results in periplasmic accumulation of capsular polysaccharide (17). The functions of KpsF, NeuD, and NeuE are currently unknown.…”