Cloning and characterization of the gene (rfc) encoding O-antigen polymerase of Pseudomonas aeruginosa PAO1.
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) AK1401, 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.
Coyne
M J
MJ
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115-5899, USA.
Goldberg
J B
JB
D001482:Base Composition; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003062:Codon; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000472:pathogenicity; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D003062:Codon; D004269:DNA, Bacterial; D006602:Hexosyltransferases; C085652:O-antigen polymerase