Genetic analysis of lipopolysaccharide and purine biosynthesis and their requirement for nodulation in Rhizobium leguminosarum strain CFN42

Joseph Richard Cava, Marquette University


Bacteria of the genus Rhizobium establish a nitrogen-fixing symbiosis with leguminous plants. Rhizobia elicit the formation of root nodules and enter the nodules by way of an infection thread. Wild-type R. leguminosarum biovar phaseoli strain CFN42 nodulates bean. Two classes of symbiotically-defective mutants derived from CFN42 were studied. Symbiosis was blocked at different stages of infection in the two classes. Each class involved chromosomally-located mutations. Mutants of the first class, characterized by eliciting abortive infection threads, were altered in lipopolysaccharide (LPS) synthesis. These mutants had undetectable or greatly diminished amounts of LPS molecules containing the O-antigen polysaccharide portion of LPS. Three genetic regions required for LPS biosynthesis were found to be necessary for complete nodulation. The three regions were represented on eight cloned DNA inserts isolated from a cosmid library of the wild-type strain. When the Lps mutants were complemented to Lps+ by the appropriate cloned DNA, the ability to elicit complete nodules was restored. This restoration showed that the ability to synthesize O-antigen containing LPS was required for infection thread development in bean. Most of the lps mutations clustered in two of the genetic regions; one mutation was located in the third region. Additional mutations were induced in the first two lps regions by transposon Tn5 insertion mutagenesis of cloned DNA. A number of these mutations caused incomplete LPS synthesis. Complementation analysis with strains carrying a plasmid-borne lps mutation has identified at least thirteen of the approximately thirty genes required for complete LPS synthesis. The biochemical and immunological characterization of the LPS from two mutant strains suggested that O-antigen containing LPS, besides being present, must be in sufficient quantities as well for infection to continue. In addition, the O-antigen need not be structurally identical as the wild-type O-antigen in order to serve its function in symbiosis. The second class of symbiotically-defective mutants consists of purine (pur) auxotrophs. These strains do not initiate infection thread formation. Complementation of the purine auxotrophs by cloned DNA has shown that the failure to initiate infection was the direct result of the pur mutations. Complementation and hybridization studies indicated that most of the pur alleles were not closely linked.

Recommended Citation

Cava, Joseph Richard, "Genetic analysis of lipopolysaccharide and purine biosynthesis and their requirement for nodulation in Rhizobium leguminosarum strain CFN42" (1988). Dissertations (1962 - 2010) Access via Proquest Digital Dissertations. AAI8925431.