Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side-chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1

The rfb_kpO1 gene cluster of Klebsiella pneumoniae O1 directs synthesis of the D-galactan I component of the lipopolysaccharide O-antigen. The first two genes in the rfb_kpO1cluster encode Rrfb_kpO1and RfbB_KpO1, with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respectively. RfbB_KpO1 contains a consensus ATP-binding domain and shares homology with several proteins which function as ATP-binding components of cell surface polysaccharide transporters. RfbA_KpO1 is predicted to be an integral membrane protein with five putative membrane-spanning domains and its transmembrane topology was confirmed by TnphoA mutagenesis. The hydropathy plot of RfbA_KpO1 resembles KpsM, the transcytoplasmic membrane component of the capsular polysaccharide transporter from Escherichia coli K-1 and K-5. These relationships suggest that RfbA_KpO1 and RfbB_KpO1 belong to a family of two-component ABC (ATP-binding cassette) transporters. E. coli K-12 containing a plasmid carrying an rfb_KpO1 gene cluster deleted in rfbA_KpO1 and rfbB_KpO1 expresses rough lipopolysaccharide molecules on its surface and accumulates cytoplasmic O-antigen. When RfbA_KpO1 and RfbB_KpO1 are supplied in trans by a compatible plasmid, O-polysaccharide transport is restored and smooth D-galactan l-substituted lipopolysaccharide is produced. RfbA_KpO1 and RfbB_KpO1 are, therefore, proposed to constitute a system required for transport of D-galactan I across the cytoplasmic membrane, where RfbA_KpO1 represents the membrane-spanning translocator and RfbB_KpO1 couples the energy of ATP hydrolysis to the transport process.