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Abstract
Escherichia coli K1 is a pathogen commonly associated with neonatal meningitis and childhood pyelonephritis. The K1 capsular polysaccharide is an essential virulence factor for invasive E. coli and contributes significantly to the ability of this organism to survive in host extra-intestinal environments. The 17-kb kps gene cluster of E. coli K1 is divided into three functional regions and encodes the gene products necessary for synthesis and translocation of the K1 capsular polysaccharide. A previously described region 1 gene, kpsD, encodes a 60-kD periplasmic protein required for the expression of K1 polymer on the cell surface. In this study the nucleotide sequence of 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. The KpsD protein was overexpressed and partially purified for preparation of polyclonal antiserum. Preliminary mutational analysis of kpsD was carried out by insertional inactivation and in-frame deletion mutagenesis in chromosomal copies of the kpsD gene. These strains do not elaborate the K1 capsule and accumulate polysaccharide in the periplasm. Plasmid encoded, wild-type kpsD effectively restored K1 capsule expression in these strains. Subsequent mutational analysis resulted in the generation of two interesting deletion derivatives of kpsD. The first kpsD deletion derivative $(kpsD\Delta C$11, corresponding to a loss of 11 amino acids from the C-terminal end of the protein) was unable to complement the chromosomal mutation and resulted in inefficient export of the protein to the periplasm. The second deletion derivative, $kpsD\Delta C$123, was isolated as a dominant negative mutation, as it inhibited K1 capsule expression when overexpressed in the K1/K-12 hybrid, EV36. The dominant negative phenotype was dependent upon the active biosynthesis of the K1 polymer. Other mutations in the kpsD gene did not, however, show any dominant negative effect in EV36. Lastly, the finding that KpsD overexpressed under the lac promoter was lethal in EV36 mutant derivatives and in a number of E coli K-12 backgrounds, but not in the EV36 itself, suggests an interaction of KpsD with some non-kps gene product(s). This lethality requires the presence of KpsD in the periplasm, providing evidence that this non-kps gene product may be a porin. This hypothesis is consistent with data previously showing the requirement of porins for expression of the K1 polymer. The results from this study provide the first evidence that, KpsD not only is required for K1 capsule transport, but is also intimately associated with the synthetic and translocation apparatus required for the cell surface expression of the K1 capsule.