Brian Burghardt 
Marquette University
Milwaukee, WI
Faculty Mentor: Dr. Dale Noel

Rhizobium is a gram-negative bacterial species responsible for nitrogen fixation in legume root nodules. Under particular environmental conditions, there are modifications of the lipopolysaccharide (LPS) structure of the bacteria. These modifications occur in the O-antigen portion of the LPS, whose structure is known to be important in the infection of root nodules. One of the conditions thought to be associated with the root cell surfaces during infection by the bacteria is low pH. Growth of the bacteria at low pH leads to O-antigen modifications, including increased methylation and addition of a galactose residue. After these changes the LPS no longer reacts with a particular monoclonal antibody. 

To study the genetic and cellular basis of these induced changes, mutants unable to make modifications to the O-antigen were sought. Previous students initiated the project by screening for such mutants among bacteria mutated by insertions of a transposon. To identify genuine mutants among the candidates isolated in this prior study, the LPS of these bacterial isolates was analyzed by gel electrophoresis and reaction with a monoclonal antibody. A few of the isolates did not undergo the antigenic modifications at low pH that the wild type 

One overall objective is to determine the sequence of each DNA locus that has been mutated. The first step in doing so is to clone the DNA flanking the site of the transposon mutation. The DNA has been extracted from the cells and digested with restriction enzymes that do not cleave within the transposon. The DNA fragments from the digest will be ligated into a plasmid vector. A recombinant plasmid containing the mutated DNA fragment will be selected by resistance to spectinomycin and ampicillin, which are encoded by the transposon and plasmid, respectively. By using primers from the known sequences of the transposon and the cloning vector, the mutant genetic locus can then be sequenced. 

The genetic regions targeted by the mutations may control one or more steps in the pathway that senses low pH and modifies the O-antigen. The sequences sought in this project could help identify these steps and the protein components involved. Some mutations may be in genes that encode the biosynthetic enzymes that modify the LPS, proteins that sense changes in pH, or proteins that regulate transcription in response to pH. If any of these classes of genes have been targeted by the mutations, there is a high probability of finding matches of their sequences with genes of known function in the sequence databases. Future research would then involve testing the biochemical functions predicted by the sequence matches.