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Journal of Bacteriology, February 1999, p. 973-980, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Effect of wzx (rfbX)
Mutations on A-Band and B-Band Lipopolysaccharide Biosynthesis
in Pseudomonas aeruginosa O5
Lori L.
Burrows and
Joseph S.
Lam*
Department of Microbiology, University of
Guelph, Guelph, Ontario, Canada N1G 2W1
Received 15 July 1998/Accepted 17 November 1998
The wbp cluster of Pseudomonas aeruginosa
O5 encodes a number of proteins involved in biosynthesis of the
heteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the
O-antigen polymerase, and Wzz, the regulator of O-antigen chain length. A gene (formerly wbpF), contiguous with wzy in
the wbp cluster, is predicted to encode a highly
hydrophobic protein with multiple membrane-spanning domains. This
secondary structure is consistent with that of Wzx (RfbX), the putative
O-antigen unit translocase or "flippase." Insertion of a
Gmr cassette at two separate sites within the putative
wzx gene led in both cases to the loss of B-band
lipopolysaccharide (LPS) O-antigen production. To our knowledge, this
is the first report of the successful generation of chromosomal
wzx gene replacement mutations. Surprisingly, inactivation
of wzx also led to a marked delay in production of the
ATP-binding cassette-transporter-dependent, D-rhamnose
homopolymer, A-band LPS. This effect on A-band LPS synthesis was
alleviated by supplying multiple copies of WbpL in trans.
WbpL, a WecA (Rfe) homologue, was shown recently to be essential for
the initiation of both A-band and B-band LPS synthesis in P. aeruginosa O5 (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, Mol. Microbiol. 28:1103-1119, 1998). These results suggest that the delay in A-band LPS production may arise
from insufficient access to WbpL when the completed B-band O unit is
not successfully translocated to the periplasm. Without adequate WbpL,
A-band LPS synthesis is delayed. A subset of wzx mutants
appeared to have accumulated second-site mutations which either
restored the normal expression of A-band LPS or abolished A-band
expression completely. Complementation studies showed that all of the
additional mutations affecting LPS synthesis that were characterized in
this study were located within the B-band LPS genes.
*
Corresponding author. Mailing address: Department of
Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Phone: (519) 824-4120, ext. 3823. Fax: (519) 837-1802. E-mail: jlam{at}uoguelph.ca.
Journal of Bacteriology, February 1999, p. 973-980, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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