||<p>The lipid A component of lipopolysaccharide (LPS) in the nitrogen-fixing plant
endosymbionts <italic>Rhizobium leguminosarum</italic> and <italic>Rhizobium etli</italic>
is strikingly different when compared to that of enteric bacteria such as <italic>Escherichia
coli</italic>. The <italic>Rhizobium</italic> species produce several unique enzymes
that process the lipid A biosynthetic intermediate Kdo<sub>2</sub>-lipid IV<sub>A</sub>.
These enzymes include a 1-phosphatase (LpxE), a 4´-phosphatase (LpxF), a 3-O-deacylase
(PagL), and a lipid A oxidase (LpxQ). The biological functions and enzymological properties
of many of the modification enzymes have remained unconfirmed and/or unknown. The
purpose of these studies was to confirm the activities of these enzymes and to explore
the functional significance of the resulting lipid A modifications.</p>
<p>To confirm the proposed biological functions of the enzymes <italic>in vivo</italic>,
homologs of the lipid A phosphatases, LpxE and LpxF, from <italic>Francisella novicida</italic>
and the lipid A oxidase LpxQ, were expressed heterologously in combination in <italic>E.
coli</italic>. The resulting novel lipid A hybrids were analyzed by thin-layer chromatography
(TLC) and electrospray ionization-mass spectrometry (ESI-MS). </p>
<p>The lipid A oxidase LpxQ, was characterized further biochemically. Two new purification
procedures and a new <italic>in vitro</italic> assay were developed to analyze the
properties of the enzyme. Purified LpxQ was shown to be dependent on oxygen and divalent
cations for activity. Hydrogen peroxide was found to be a product of lipid A oxidation.
A new fluorescence-based assay based on the detection of hydrogen peroxide was developed
to monitor oxidation. LpxQ did not co-purifiy with any discernable cofactors, suggesting
that it may employ a unique mechanism for the oxidation of lipid A.</p>
<p>The biological roles of LpxE and LpxF in plant nodulation were analyzed. Deletion
mutants of the two phosphatases were generated in <italic>R. etli</italic>. The mutant
strains accumulated the expected structures, confirming the specificity of the enzymes.
Single and double phosphatase mutants were able to fix nitrogen <italic>in planta</italic>.
Antimicrobial susceptibility testing indicated that dephosphorylation of lipid A increases
resistance to cationic antimicrobials.</p>
<p>The biological role of the 3-O-deacylase, PagL, was also investigated. The <italic>pagL</italic>
gene was identified using systematic homology searches. PagL was shown to be stimulated
by calcium. A deletion mutant of the enzyme in <italic>R. etli</italic> was constructed
and analyzed. The deletion mutant was found to be viable and unaltered in its ability
to fix nitrogen. In conclusion, these studies have confirmed the roles of LpxE, LpxF,
PagL, and LpxQ in Rhizobium lipid A biosynthesis and contributed new knowledge regarding
the biochemical properties of LpxQ.</p>