Document Detail

Identification of a locus involved in meningococcal lipopolysaccharide biosynthesis by deletion mutagenesis.
MedLine Citation:
PMID:  8830268     Owner:  NLM     Status:  MEDLINE    
A novel method for insertion/deletion mutagenesis in meningococci was devised. This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin-resistance marker (ermC), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76. Southern blotting of a number of the resulting erythromycin-resistant transformants demonstrated that all carried the ermC gene inserted at different positions in the chromosome. Mutants with a specific phenotype were identified by screening with the anti-lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two independent L3-negative mutant strains were isolated. In transformation experiments with chromosomal DNA from these mutants, erythromycin-resistance and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis. On SDS-PAGE, the mutant LPS displayed an electrophoretic mobility intermediate between that produced by the previously isolated galE and rfaF mutant strains. Chemical analysis of the mutant LPS revealed that the structure was probably lipid A-(KDO)2-(Hep)2. Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used as probe for the isolation of the corresponding region of the wild-type strain. From hybridization and polymerase chain reaction (PCR) analysis, it could be concluded that both mutations map to the same locus. The affected gene probably encodes the glycosyltransferase necessary for adding N-acetylglucosamine to heptose.
P van der Ley; M Kramer; L Steeghs; B Kuipers; S R Andersen; M P Jennings; E R Moxon; J T Poolman
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Molecular microbiology     Volume:  19     ISSN:  0950-382X     ISO Abbreviation:  Mol. Microbiol.     Publication Date:  1996 Mar 
Date Detail:
Created Date:  1996-10-17     Completed Date:  1996-10-17     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8712028     Medline TA:  Mol Microbiol     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  1117-25     Citation Subset:  IM    
Laboratory of Vaccine Development and Immune Mechanisms, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
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MeSH Terms
Base Sequence
Carbohydrate Sequence
Cloning, Molecular
DNA Primers
Glycosyltransferases / genetics
Lipopolysaccharides / biosynthesis*
Molecular Sequence Data
Neisseria meningitidis / genetics*,  metabolism
Sequence Deletion
Transformation, Bacterial
Reg. No./Substance:
0/DNA Primers; 0/Lipopolysaccharides; EC 2.4.-/Glycosyltransferases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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