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A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives.

ABSTRACT: A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli DH5alpha, 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 DH5alpha. These results suggest that the 282-residue WbbL protein is a rhamnosyltransferase able to complement the rJb-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.

SUBMITTER: Rubires X

PROVIDER: S-EPMC179713 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives.

Rubirés X X Saigi F F Piqué N N Climent N N Merino S S Albertí S S Tomás J M JM Regué M M

Journal of bacteriology 19971201 23

A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli DH5alpha, 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 similarit ...[more]

PMID: 9393727

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Project description:The gene cluster (waa) involved in Serratia marcescens N28b core lipopolysaccharide (LPS) biosynthesis was identified, cloned, and sequenced. Complementation analysis of known waa mutants from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae led to the identification of five genes coding for products involved in the biosynthesis of a shared inner core structure: [L,D-HeppIIIalpha(1-->7)-L,D-HeppIIalpha(1-->3)-L,D-HeppIalpha(1-->5)-KdopI(4<--2)alphaKdopII] (L,D-Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid). Complementation and/or chemical analysis of several nonpolar mutants within the S. marcescens waa gene cluster suggested that in addition, three waa genes were shared by S. marcescens and K. pneumoniae, indicating that the core region of the LPS of S. marcescens and K. pneumoniae possesses additional common features. Chemical and structural analysis of the major oligosaccharide from the core region of LPS of an O-antigen-deficient mutant of S. marcescens N28b as well as complementation analysis led to the following proposed structure: beta-Glc-(1-->6)-alpha-Glc-(1-->4))-alpha-D-GlcN-(1-->4)-alpha-D-GalA-[(2<--1)-alpha-D,D-Hep-(2<--1)-alpha-Hep]-(1-->3)-alpha-L,D-Hep[(7<--1)-alpha-L,D-Hep]-(1-->3)-alpha-L,D-Hep-[(4<--1)-beta-D-Glc]-(1-->5)-Kdo. The D configuration of the beta-Glc, alpha-GclN, and alpha-GalA residues was deduced from genetic data and thus is tentative. Furthermore, other oligosaccharides were identified by ion cyclotron resonance-Fourier-transformed electrospray ionization mass spectrometry, which presumably contained in addition one residue of D-glycero-D-talo-oct-2-ulosonic acid (Ko) or of a hexuronic acid. Several ions were identified that differed from others by a mass of +80 Da, suggesting a nonstoichiometric substitution by a monophosphate residue. However, none of these molecular species could be isolated in substantial amounts and structurally analyzed. On the basis of the structure shown above and the analysis of nonpolar mutants, functions are suggested for the genes involved in core biosynthesis.

Dataset Information

A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives.

Publications

A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives.

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