Genetic analysis of the Serratia marcescens N28b O4 antigen gene cluster

The Serratia marcescens N28b wbbL gene has been shown to complement the rfb -50 mutation of Escherichia coli K-12 derivatives, and a wbbL mutant has be en shown to be impaired in O4-antigen biosynthesis (X. Rubires, F. Saigi, N . Pique, N, Climent, S, Merino, S. Alberti, J, M, Tomas, and M, Regue, J, B acteriol. 179:7581-7586, 1997), We analyzed a recombinant cosmid containing the wbbL gene by subcloning and determination of O-antigen production phen otype in E. coli DH5 alpha by sodium dodecyl sulfate-polyacrylamide electro phoresis and Western blot experiments with S. 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 an E. coli K-12 strain with a deletion of the w b cluster was used, suggesting that the O4 wb cluster is located in pSUB6. No O4 antigen was produced when plasmid pSUB6 was introduced in a wecA muta nt E. coli strain, suggesting that O4-antigen production is wecA dependent. Nucleotide sequence determination of the whole insert in plasmid pSUB6 sho wed seven open reading frames (ORFs), On the basis of protein similarity an alysis of the ORF-encoded proteins and analysis of the S, marcescens N28b w bbA insertion mutant and wzm-wzt deletion mutant, we suggest that the O4 wb cluster codes for two dTDP-rhamnose biosynthetic enzymes (RmlDC), a rhamno syltransferase (WbbL), a two-component ATP-binding-cassette-type export sys tem (Wzm Wzt), and a putative glycosyltransferase (WbbA), A sequence showin g 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 O4 wb cluster.