| Mutation
of Three Genes in Order to Probe O-antigen Biosynthesis and the Features
of Its Structure Required in Symbiosis
Amber
Lorge
Marian
College
Summer
Mentor: Dr. Dale Noel
Rhizobium
etli are gram-negative bacteria that form a symbiotic relationship
with Phaseolus vulgaris. They undergo a controlled invasion of the
plant roots, leading to the formation of root nodules which fix nitrogen
into a form that the plant is able to use. A major molecule required for
this infection is lipopolysaccharide I, which is a part of the bacterial
outer membrane. Without the O-antigen polysaccharide region of LPS I, proper
infection does not occur, leading to incompletely developed nodules. Past
research has suggested that specific regions of the O-antigen are required
for its role in symbiosis. The bacterium is able to make specific changes
to its O-antigen structure when in the presence of the plant. Other evidence
comes from mutations that eliminate specific features of the O-antigen
and result in greatly slowed infection and nodule development. The goal
of this project was to generate mutations that should affect different
structural features of the LPS that previously were not targeted by mutation.
The resulting mutants may then provide a way to test the importance of
the affected structures in symbiosis.
The
large stretch of DNA known as the lps-? region is thought to include all
the genes necessary for O-antigen biosynthesis. The complete nucleotide
sequence of this genetic region has been determined. Over half of the genes
located in this region have been mutated and studied. Three contiguous
genes were targeted for mutagenesis in this study: wbpS, tesA,
and dtsE. Sequence comparisons indicate the following putative function
for each gene: amidotransferase (wbpS), acyl hydrolase (tesA),
and epimerase (dtsE). The exact function of these genes in the formation
of the O-antigen has not been determined, and it is possible that they
may not play a role in O-antigen biosynthesis at all.
The
approach taken to study gene function was in vitro mutagenesis by
inserting DNA that encodes antibiotic resistance into each gene. This multi-step
process involves amplifying each individual gene by polymerase chain reaction
and sub-cloning the mutated region into a plasmid that can be transferred
into Rhizobium. During this process a unique restriction site, located
within the gene, is used to insert the antibiotic resistant cassette. The
mutant allele is then passed into Rhizobium, and double recombination
is selected to replace the endogenous wild-type gene with the mutant allele.
The mutants thereby obtained can be studied to indicate the role of each
gene in the formation of the O-antigen and its impact in symbiosis. O-antigen
content will be studied by gel electrophoresis, immunoblots with antibodies
that bind in the O-antigen region, and sugar analysis if gel electrophoresis
indicates the presence of an O-antigen. The effect of the mutation on symbiosis
would also be tested if the O-antigen is present in normal amounts but
appears to be altered in structure.
Genes
wbpS
and dtsE have been mutated and the mutant alleles are currently
being transferred into R. etli CE3. The formation of the mutant
tesA gene has not reached this phase. However, it has been determined
during this project that R. etli CE346, a previously isolated mutant
strain, has a Tn5 mutation in the tesA region. Analysis of this
mutant has shown that there is an effect on lipopolysaccharide structure,
but not a significant defect in symbiosis.
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