Relationships between rfb gene clusters required for biosynthesis of identical D-galactose-containing O antigens in Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16

Abstract

The lipopolysaccharide O antigens of Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16 both contain a repeating unit disaccharide of [33)-␤-D-Galf-(133)-␣-D-Galp-(13]; the resulting polymer is known as D-galactan I. In K. pneumoniae serotype O1, the genes responsible for the synthesis of D-galactan I are found in the rfb gene cluster (rfb KpO1 ). We report here the cloning and analysis of the rfb cluster from S. marcescens serotype O16 (rfb SmO16 ). This is the first rfb gene cluster examined for the genus Serratia. Synthesis of D-galactan I is an rfe-dependent process for both K. pneumoniae serotype O1 and S. marcescens serotype O16. Hybridization experiments with probes derived from each of the six rfb KpO1 genes indicate that the cloned rfb SmO16 cluster contains homologous genes arranged in the same order. However, the degree of homology at the nucleotide sequence level was sufficiently low that hybridization was detected only under low-stringency conditions. rfbAB SmO16 genes were subcloned and shown to encode an ABC-2 (ATP-binding cassette) transporter which is functionally identical to the one encoded by the corresponding rfb genes from K. pneumoniae serotype O1. The amino acid sequences of the predicted RfbA and RfbB homologs showed identities of 75.7% (87.9% total similarity) and 78.0% (86.5% total similarity), respectively. The last gene of the rfb KpO1 cluster, rfbF KpO1 , encodes a bifunctional galactosyltransferase which initiates the formation of D-galactan I. RfbF KpO1 and RfbF SmO16 are 57.6% identical (with 71.1% total similarity), and both show similarity with RfpB, the galactosyltransferase involved in the synthesis of Shigella dysenteriae type I O-polysaccharide. The G؉C contents of the rfbAB genes from each organism are quite similar, and values are lower than those typical for the species. However, the G؉C content of rfbF SmO16 (47.6%) was much higher than that of rfbF KpO1 (37.3%), despite the fact that the average for each species (52 to 60%) falls within the same range.Lipopolysaccharide (LPS) is located in the outer leaflet of gram-negative bacteria and is responsible for the biological activity of endotoxin. In enteric bacteria, this molecule consists of three regions: lipid A, the core oligosaccharide, and the O polysaccharide. Lipid A forms the outer leaflet of the outer membrane bilayer. The core oligosaccharide region is attached to lipid A, and the O polysaccharide extends outward from the core region. O polysaccharides are composed of repeating oligosaccharide units with variable numbers of sugar residues. There is extensive structural diversity within O polysaccharides (22, 64). The composition, sequence, linkage, and substitution of the individual monomer residues within a repeating unit is characteristic for a given LPS and the parental bacterial strain. Thus, the O polysaccharide gives rise to epitopes (O antigens) which contribute to the immunological specificity of LPS.Klebsiella pneumoniae serotype O1 produces a high-molecular-weight smooth LPS (S-LPS)...

Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa

“…Pairwise comparisons in Tables 2 and 3 reveal that integral membrane proteins and ATP-binding proteins from K. pneumoniae O1 and O8 and S. marcescens O16 show high primary sequence similarity, likely due to a conserved D-galactan I structure in the O-polysaccharide region (8,61). KpsM and KpsT from E. coli K1 and K5 also demonstrate high similarity, possibly due to common capsular polysaccharide structures.…”

“…ABC transporters in general have been found in both prokaryotic and eukaryotic systems and are responsible for the import and export of various proteins, peptides, polysaccharides, and drugs (17). ABC transport systems have recently been identified for the export of LPS O polysaccharides for Yersinia enterocolitica O:3, K. pneumoniae O1, E. coli O9, Serratia marcescens O16, Vibrio cholerae O1, and Myxococcus xanthus (8,20,33,48,61,67). These transport systems are comprised of two components: a hydrophilic protein containing a highly conserved ATP-binding domain and a hydrophobic protein with five to six membrane-spanning domains.…”

A Novel Pathway for O-Polysaccharide Biosynthesis in Salmonella enterica Serovar Borreze

“…The O:54 O-polysaccharide shares a number of characteristics with the O-polysaccharides synthesized by the Rfc-independent O-polysaccharide biosynthetic pathway including the following: 1) it is a homopolymer; and 2) Rfe is required for synthesis although GlcNAc does not form part of the repeating unit. The Rfcindependent pathway has so far only been identified for a few homopolymeric O-polysaccharides in Klebsiella O1 (7,8), Escherichia coli O8 and O9 (9 -11), and Serratia marcescens O16 (12). In this pathway, Rfe initiates synthesis by transferring GlcpNAc-1-P to the carrier lipid to form the acceptor upon which the complete O-polysaccharide chain is subsequently polymerized.…”

ABC Transporters Involved in Export of Cell Surface Glycoconjugates

“…3, NBDs from the K. pneumoniae D-galactan I systems are represented by the homologs from serotype O1 (whose sequence is identical to that of the NBD from O2a) and serotype O8 (which shows some sequence diversity) (67). Serratia marcescens O16 also contains D-galactan I (107), and the genetic locus is well conserved (156). The exception is the glycan synthesized by Rhodopseudomonas palustris, which likely contains L-rhamnose due to the presence of the rml (dTDP-L-rhamnose biosynthesis) genes immediately upstream of wzm and wzt.…”