Open AccessBiochemical Analysis of Lpt3, a Protein Responsible forPhosphoethanolamine Addition to Lipooligosaccharide ofPathogenic Neisseria
Author(s) -
Ellen T. O'Connor,
Andrzej Piekarowicz,
Karen V. Swanson,
J. McLeod Griffiss,
Daniel C. Stein
Publication year - 2006
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.188.3.1039-1048.2006
Subject(s) - biology , heptose , complementation , transposable element , neisseria gonorrhoeae , genomic dna , microbiology and biotechnology , neisseria , plasmid , sequence analysis , neisseria meningitidis , strain (injury) , dna , genetics , gene , mutant , bacteria , anatomy
Theinner core of neisserial lipooligosaccharide (LOS) contains heptoseresidues that can be decorated by phosphoethanolamine (PEA). PEAmodification of heptose II (HepII) can occur at the 3, 6, or 7position(s). We used a genomic DNA sequence oflpt3 , derivedfromNeisseria meningitidis MC58, to search the genomicsequence ofN. gonorrhoeae FA1090 and identified a homolog oflpt3 inN. gonorrhoeae . A PCR amplicon containinglpt3 was amplified from F62ΔLgtA, cloned, mutagenized,and inserted into the chromosome ofN. gonorrhoeae strainF62ΔLgtA, producing strainF62ΔLgtAlpt3::Tn5 . LOS isolatedfrom this strain lost the ability to bind monoclonal antibody (MAb)2-1-L8. Complementation of this mutation by genetic removal of thetransposon insertion restored MAb 2-1-L8 binding. Mass spectrometryanalysis of LOS isolated from the F62ΔLgtA indicated that thisstrain contained two PEA modifications on its LOS.F62ΔLgtAlpt3::Tn5 lacked a PEAmodification on its LOS, a finding consistent with the hypothesis thatlpt3 encodes a protein mediating PEA addition onto gonococcalLOS. The DNA encodinglpt3 was cloned into an expressionvector and Lpt3 was purified. Purified Lpt3 was able to mediate theaddition of PEA to LOS isolated fromF62ΔLgtAlpt3::Tn5 .