The transport of group 2 capsular polysaccharides across the periplasmic space in Escherichia coli. Roles for the KpsE and KpsD proteins.
The cell surface expression of group 2 capsular polysaccharides involves the translocation of the polysaccharide from its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the repeat structure of the polysaccharide, and translocation across the periplasm requires the cytoplasmic membrane-anchored protein KpsE and the periplasmic protein KpsD. In this paper we establish the topology of the KpsE protein and demonstrate that the C terminus interacts with the periplasmic face of the cytoplasmic membrane. By chemical cross-linking we show that KpsE is likely to exist as a dimer and that dimerization is independent of the other Kps proteins or the synthesis of capsular polysaccharide. No interaction between KpsD and KpsE could be demonstrated by chemical cross-linking, although in the presence of both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer membrane fractions and that the correct membrane association of KpsD was dependent on KpsE, Lpp, and the secreted polysaccharide molecule. Both KpsD and KpsE showed increased proteinase K sensitivity in the different mutant backgrounds, reflecting conformational changes in the KpsD and KpsE proteins as a result of the disruption of the transport process. Collectively the data suggest that the trans-periplasmic export involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process.
Arrecubieta
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C
University of Manchester, 1.800 Stopford Building, School of Biological Sciences, Oxford Road, Manchester, M13 9PT, United Kingdom.
Hammarton
T C
TC
Barrett
B
B
Chareonsudjai
S
S
Hodson
N
N
Rainey
D
D
Roberts
I S
IS
D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D001692:Biological Transport; D017931:DNA Primers; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D019897:Periplasm / Q000378:metabolism*; D033903:Periplasmic Proteins; D011134:Polysaccharides / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C088016:KpsD protein, E coli; D026901:Membrane Transport Proteins; D033903:Periplasmic Proteins; D011134:Polysaccharides; C084729:kpsE protein, E coli; D019286:Endopeptidase K