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TheSerratia marcescens N28bwbbL gene has been shown to complement therfb-50 mutation ofEscherichia coli K-12 derivatives, and awbbL mutant has been shown to be impaired in O4-antigen biosynthesis (X. Rubirés, F. Saigı́, N. Piqué, N. Climent, S. Merino, S. Albertı́, J. M. Tomás, and M. Regué, J. Bacteriol. 179:7581–7586, 1997). We analyzed a recombinant cosmid containing thewbbL gene by subcloning and determination of O-antigen production phenotype inE. coli DH5α by sodium dodecyl sulfate-polyacrylamide electrophoresis and Western blot experiments withS. marcescens O4 antiserum. The results obtained showed that a recombinant plasmid (pSUB6) containing about 10 kb of DNA insert was enough to induce O4-antigen biosynthesis. The same results were obtained when anE. coli K-12 strain with a deletion of thewb cluster was used, suggesting that the O4wb cluster is located in pSUB6. No O4 antigen was produced when plasmid pSUB6 was introduced in awecA mutantE. coli strain, suggesting that O4-antigen production iswecA dependent. Nucleotide sequence determination of the whole insert in plasmid pSUB6 showed seven open reading frames (ORFs). On the basis of protein similarity analysis of the ORF-encoded proteins and analysis of theS. marcescens N28bwbbA insertion mutant andwzm-wzt deletion mutant, we suggest that the O4wb cluster codes for two dTDP-rhamnose biosynthetic enzymes (RmlDC), a rhamnosyltransferase (WbbL), a two-component ATP-binding-cassette-type export system (Wzm Wzt), and a putative glycosyltransferase (WbbA). A sequence showing DNA homology to insertion element IS4 was found downstream from the last gene in the cluster (wbbA ), suggesting that an IS4 -like element could have been involved in the acquisition of the O4wb cluster.

Genetic Analysis of the Serratia marcescens N28b O4 Antigen Gene Cluster