ABSTRACT
The
inner core of neisserial lipooligosaccharide (LOS) contains heptose
residues that can be decorated by phosphoethanolamine (PEA). PEA
modification of heptose II (HepII) can occur at the 3, 6, or 7
position(s). We used a genomic DNA sequence of lpt3, derived
from Neisseria meningitidis MC58, to search the genomic
sequence of N. gonorrhoeae FA1090 and identified a homolog of
lpt3 in N. gonorrhoeae. A PCR amplicon containing
lpt3 was amplified from F62ΔLgtA, cloned, mutagenized,
and inserted into the chromosome of N. gonorrhoeae strain
F62ΔLgtA, producing strain
F62ΔLgtAlpt3::Tn5. LOS isolated
from this strain lost the ability to bind monoclonal antibody (MAb)
2-1-L8. Complementation of this mutation by genetic removal of the
transposon insertion restored MAb 2-1-L8 binding. Mass spectrometry
analysis of LOS isolated from the F62ΔLgtA indicated that this
strain contained two PEA modifications on its LOS.
F62ΔLgtAlpt3::Tn5 lacked a PEA
modification on its LOS, a finding consistent with the hypothesis that
lpt3 encodes a protein mediating PEA addition onto gonococcal
LOS. The DNA encoding lpt3 was cloned into an expression
vector and Lpt3 was purified. Purified Lpt3 was able to mediate the
addition of PEA to LOS isolated from
F62ΔLgtAlpt3::Tn5.
Molecular Cloning of a Genomic DNA Encoding Yam Class IV Chitinase
