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 synthes is of the D-galactan I component of the lipopolysaccharide O-antigen. The first two genes in the rfb(KpO1) cluster encode RfbA(KpO1) and Rfb B(KpO1), with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respec tively. RfbB(KpO1) contains a consensus ATP-binding domain and shares homology with several proteins which function as ATP-binding component s of cell surface polysaccharide transporters. RfbA(KpO1) is predicted to be an integral membrane protein with five putative membrane-spanni ng domains and its transmembrane topology was confirmed by TnphoA muta genesis. The hydropathy plot of RfbA(KpO1) resembles KpsM, the transcy toplasmic membrane component of the capsular polysaccharide transporte r 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 car rying an rfb(KpO1) gene cluster deleted in rfbA(KpO1) and rfbB(KpO1) e xpresses rough lipopolysaccharide molecules on its surface and accumul ates cytoplasmic O-antigen. When RfbA(Kpo1) and RfbB(KpO1) are supplie d in trans by a compatible plasmid, O-polysaccharide transport is rest ored and smooth D-galactan I-substituted lipopolysaccharide is produce d. RfbA(KpO1) and RfbA(KpO1) are, therefore, proposed to constitute a system required for transport of D-galactan I across the cytoplasmic m embrane, where RfbA(KpO1) represents the membrane-spanning translocato r and RfbB(KpO1) couples the energy of ATP hydrolysis to the transport process.