Cloning and characterization of the gene ( rfc) encoding O-antigen polymerase of Pseudomonas aeruginosa PAO1

Abstract

The lipopolysaccharide (LPS) O-antigen polymerase is the product of the rfc gene. Loss of O-antigen polymerase activity due to mutation in rfc gives rise to a characteristic LPS phenotype known as core-plus-one or semi-rough, wherein the LPS core is capped with a single oligosaccharide unit. Pseudomonas aeruginosa (Pa) AK 1401, a derivative of strain PAO1 (serogroup O5), expresses a semi-rough LPS; this mutant phenotype was complemented by a 2.2-kb NsiI- SacI fragment of Pa PAO1 DNA. Sequence analysis of this fragment revealed a 1317-bp open reading frame (ORF) potentially encoding a 438-amino-acid (aa) protein of 48 849 Da. This DNA sequence and the inferred aa sequence contain many of the features of other O-antigen polymerases, including an aberrantly low G+C content (particularly apparent in the high-G+C background of Pa), an unusual codon usage pattern, and a hydrophobicity profile indicative of a membrane protein. A 345-bp fragment internal to the ORF hybridized to genomic DNA from two of ten Pa serogroup strains examined by Southern blot; these two strains express O antigens structurally related to that of strain PAO1.