NUCLEOTIDE-SEQUENCE AND MUTATIONAL ANALYSIS OF THE GENE ENCODING KPSD, A PERIPLASMIC PROTEIN INVOLVED IN TRANSPORT OF POLYSIALIC ACID IN ESCHERICHIA-COLI K1

The 17-kb kps gene cluster encodes proteins necessary for the synthesi s, assembly, and translocation of the polysialic acid capsule of Esche richia coli K1. We previously reported that one of these genes, kpsD, encodes a 60-kDa periplasmic protein that is involved in the transloca tion of the polymer to the cell surface. The nucleotide sequence of th e 2.4-kb BamHI-PstI fragment accommodating the kpsD gene was determine d. Sequence analysis showed an open reading frame for a 558-amino-acid protein,vith a typical N-terminal prokaryotic signal sequence corresp onding to the first 20 amino acids. KpsD was overexpressed, partially purified, and used to prepare polyclonal antiserum. A chromosomal inse rtion mutation was generated in the kpsD gene and results in loss of s urface expression of the polysialic acid capsule. Immunodiffusion anal ysis and electron microscopy indicated that polysaccharide accumulates in the periplasmic space of mutant cells. A wild-type copy of kpsD su pplied in trans complemented the chromosomal mutation, restoring extra cellular expression of the K1 capsule. However, a kpsD deletion deriva tive (kpsD Delta C11), which results in production of a truncated KpsD protein lacking its 11 C-terminal amino acids, was nonfunctional. Wes tern 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.