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(KpO1) cluster encode RfbA(KpO1) and 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 I-substituted lipopolysaccharide is produced. RfbA(KpO1) and RfbA(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.