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Journal of Bacteriology, February 2006, p. 1039-1048, Vol. 188, No. 3
0021-9193/06/$08.00+0     doi:10.1128/JB.188.3.1039-1048.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Biochemical Analysis of Lpt3, a Protein Responsible for Phosphoethanolamine Addition to Lipooligosaccharide of Pathogenic Neisseria

Ellen T. O'Connor,1 Andrzej Piekarowicz,2 Karen V. Swanson,1,3 J. McLeod Griffiss,3 and Daniel C. Stein1*

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742,1 Institute of Microbiology, University of Warsaw, 02-096 Warsaw, Poland,2 Department of Laboratory Medicine and Veterans Affairs Medical Center, University of California San Francisco, San Francisco, California 941213

Received 28 April 2005/ Accepted 18 October 2005

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 F62{Delta}LgtA, cloned, mutagenized, and inserted into the chromosome of N. gonorrhoeae strain F62{Delta}LgtA, producing strain F62{Delta}LgtAlpt3::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 F62{Delta}LgtA indicated that this strain contained two PEA modifications on its LOS. F62{Delta}LgtAlpt3::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 F62{Delta}LgtAlpt3::Tn5.

* Corresponding author. Mailing address: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742. Phone: (301) 405-5448. Fax: (301) 314-9489. E-mail: dcstein{at}umd.edu.

Journal of Bacteriology, February 2006, p. 1039-1048, Vol. 188, No. 3
0021-9193/06/$08.00+0     doi:10.1128/JB.188.3.1039-1048.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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