A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli DH5␣, and clones were screened for serum resistance. One clone was found resistant to serum, to bacteriocin 28b, and to bacteriophages TuIa and TuIb. This clone also showed O antigen in its lipopolysaccharide. Subcloning and sequencing experiments showed that a 2,124-bp DNA fragment containing the rmlD and wbbL genes was responsible for the observed phenotypes. On the basis of amino acid similarity, we suggest that the 288-residue RmlD protein is a dTDP-L-rhamnose synthase. Plasmid pJT102, containing only the wbbL gene, was able to induce O16-antigen production and serum resistance in E. coli DH5␣. These results suggest that the 282-residue WbbL protein is a rhamnosyltransferase able to complement the rfb-50 mutation in E. coli K-12 derivatives, despite the low level of amino acid identity between WbbL and the E. coli rhamnosyltransferase (24.80%). S. marcescens N28b rmlD and wbbL mutants were constructed by mobilization of suicide plasmids containing a portion of rmlD or wbbL. These insertion mutants were unable to produce O antigen; since strain N28b produces O4 antigen, these results suggest that both genes are involved in O4-antigen biosynthesis.Serratia marcescens N28b is able to produce bacteriocin 28b (6,35). In studies aimed at identifying a putative bacteriocin 28b immunity system, a gene coding for a 17-kDa outer membrane protein (Omp4) has been previously characterized by us (11). The Omp4 protein was similar to a family of small outer membrane proteins of Enterobacteriaceae. Two members of this protein family, Ail from Yersinia enterocolitica and Rck from Salmonella typhimurium, have been associated with complement resistance, and when the genes encoding these proteins were expressed in Escherichia coli HB101, both conferred serum resistance (3, 13). Despite the high level of similarity among Omp4, Ail, and Rck proteins, E. coli HB101 or DH5␣ producing Omp4 protein remained serum sensitive but became bacteriocin 28b resistant (11).S. marcescens strains have been classified as serum sensitive, delayed serum sensitive, and serum resistant (32). As a general rule in gram-negative bacteria, with many exceptions, smooth strains (O ϩ ) are serum resistant while rough strains (O Ϫ ) are serum sensitive (30). S. marcescens N28b is a serum-resistant strain, and probably this phenotype is related to O-antigen production. We screened a previously constructed cosmidbased genomic library of this strain (11) introduced into E. coli DH5␣ (which is O Ϫ and serum sensitive) for serum resistance. In this work we report the characterization of a gene from S. marcescens N28b that confers the ability to produce E. coli O16-antigen lipopolysaccharide (LPS) and serum resistance on E. coli DH5␣ and other K-12-derived strains.S. marcescens N28b (7) and E. coli strains used in this study were grown in Luria-Bertani (LB)-Miller broth and LB-Miller agar (18) supplemented with ampicillin (50 g/ml), chloramphenicol (50 g/ml), kanamycin...