Kristylea Thompson
Marquette University
Milwaukee, WI
Faculty Mentor: Dr. Dale Noel

The symbiotic relationship between the bacterium Rhizobium etli CE3 and the bean plant Phaseolus vulgaris allows the plant access to atmospheric nitrogen that has been fixed into a utilizable form by the bacterium. The relationship is established when the bacterium induces the plant root to form nodules and invades the developing nodule through what is known as an infection thread. This alliance involves several essential interactions and signals from both the plant and the bacterium. One set of molecules that are crucial in these interactions is the bacterial lipopolysaccharides (LPSs). LPSs are the most abundant constituents of the surface layer of the bacteria. Mutant strains of Rhizobium etli that lack the outer-most portion of the LPS structure, known as the O-antigen, do not complete the infection process.

The bacteria alter the structure of the LPS in the presence of the plant. Some changes in the LPS structure are initiated by flavonoid compounds that are present in the plant seed or root exudate. In particular, flavonoids known as anthocyanins, which are released from seeds as they germinate, induce the bacteria to synthesize LPSs that lack the terminal residue of the O-antigen. While this effect is well established, the exact pathway by which induction occurs is uncertain.

Rhizobium etli strains that are mutated in the lpeB gene lack the terminal residue of the O-antigen under all growth conditions. Therefore, at the very least, lpeB is necessary for the synthesis of LPS molecules possessing the terminal residue. The sequence of this gene has the greatest homology to genes encoding known glycosyltransferases, which are enzymes that catalyze the additions of sugars during polysaccharide synthesis. We hypothesize that lpeB encodes the transferase that adds the terminal sugar to the O-antigen. Mutations in lpeB result in an LPS structure that matches that of the LPS structure induced by the plant in that the terminal O-antigen residue is missing. Hence, a further hypothesis is that anthocyanins repress the lpeB gene or inhibit the glycosyltransferase putatively encoded by this gene.

My work has been mainly focused on the possible repression of the lpeB gene. In order to test this hypothesis, Rhizobium etli CE3 cells are exposed to anthocyanins. Through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and immunoblot analysis of LPS from bacteria grown in seed exudate, the loss of the terminal residue can be verified. Once induction is established, the presence of RNA complementary to lpeB is assayed by RT-PCR (reverse transcription-polymerize chain reaction). The RT-PCR product then indicates whether the lpeB is being transcribed. If anthocyanins lead to the repression of lpeB, this RT-PCR product should be absent from cultures that exhibit altered LPS.