Identification of lipopolysaccharide O antigen synthesis genes required for attachment of the S-layer of Caulobacter crescentus

The outer surface of Caulobacter crescentus consists of a two-dimensional crystalline protein lattice layer (S-layer), A fraction of the LPS has an O antigen polymer attached to the core to form a 'smooth' LPS (S-LPS). which is required for attachment of the protein S-layer to the outer-membrane surface. A method to screen for strains defective in LPS production, based on loss of S-layer attachment, was developed and applied to libraries of transposon-generated mutants. Eighteen distinct insertions were found with transposon interruptions in genes affecting S-LPS production. 12 of which were located near the S-layer subunit protein gene, rsaA, and its transporter genes. Sequence adjacent to transposon insertion points was determined and used to search a C. crescentus genome database. Twelve ORFs likely to be involved in S-LPS synthesis were identified. Seven of the predicted ORFs were linked to rsaA, Six of the putative genes had identity with proteins involved in synthesis of sugar residues. including five predicted to make perosamine. The remaining six ORFs were similar to glycosyltransferases involved in forming linkages between sugar residues in the O antigen, while one may be a transcription repressor. Other chemical and preliminary proton NMR studies of the S-LPS O antigen indicate that it contains an N-acetylated 4,6-dideoxy-4-aminohexose, but is not assembled as a simple, uniform homopolymer. consisting of several different linkages between sugar residues. The ORFs described here include homologues of all the enzymes involved in the synthesis of N-acetylperosamine, a 4,6-dideoxy-4-aminohexose. Overall, the data are consistent with the hypothesis that the O antigen of C. crescentus S-LPS consists primarily of N-acetylperosamine residues polymerized with multiple anomeric linkages.