CLONALLY DIVERSE RFB GENE CLUSTERS ARE INVOLVED IN EXPRESSION OF A FAMILY OF RELATED D-GALACTAN O-ANTIGEN IN KLEBSIELLA SPECIES

Klebsiella species express a family of structurally related lipopolysa ccharide O antigens which share a common backbone known as D-galactan I. Serotype specificity results from modification of D-galactan I by a ddition of domains of altered structure or by substitution with O-acet yl and/or alpha-D-Galp side groups with various linkages and stoichiom etries. In the prototype, Klebsiella serotype O1, the his-linked rfb g ene cluster is required for synthesis of D-galactan I, but genes confe rring serotype specificity are unlinked. The D-galactan I part of the O polysaccharide is O acetylated in Klebsiella serotype O8. By cloning the rfb region from Klebsiella serotype O8 and analyzing the O polysa ccharide synthesized in Escherichia coil K-12 hosts, we show that, lik e rfb(O1), the rf(O8) region directs formation of unmodified D-galacta n I. The rfbAB genes encode an ATP-binding cassette transporter requir ed for export of polymeric D-galactan I across the plasma membrane pri or to completion of the lipopolysaccharide molecule by ligation of the O polysaccharide to lipid A-core. Complementation experiments show th at the rfbAB gene products in serotypes O1 and O8 are functionally equ ivalent and interchangeable. Hybridization experiments and physical ma pping of the rfb regions in related Klebsiella serotypes suggest the e xistence of shared rfb genes with a common organization. However, desp ite the functional equivalence of these rfb gene clusters, at least th ree distinct clonal groups were detected in different Klebsiella speci es and subspecies, on the basis of Southern hybridization experiments carried out under high-stringency conditions. The clonal groups cannot be predicted by features of the O-antigen structure. To examine the r elationships in more detail, the complete nucleotide sequence of the s erotype O8 rfb fluster was determined and compared with that of the se rotype O1 prototype. The nucleotide sequences for the six rfb genes sh owed variations in moles percent G+C values and in the values for nucl eotide sequence identity, which ranged from 66.9 to 79.7%. The predict ed polypeptides ranged from 63.3% identity (78.4% total similarity) to 94.3% identity (98.0% similarity). The results presented here are not consistent with dissemination of the Klebsiella D-galactan I rfb gene s through recent lateral transfer events.