Date of Award


Document Type

Dissertation - Restricted

Degree Name

Doctor of Philosophy (PhD)


Biological Sciences

First Advisor

Dale Noel

Second Advisor

Walter W. Fredricks

Third Advisor

William F. Millington


Purine auxotrophs of Rhizobium leguminosarum strain CFN42 (biovar phaseoli) induce bean nodules lacking infection threads. Only purine and pyrimidine auxotrophs had this particular symbiotic phenotype on bean. Transfer of mutant alleles affecting purine or pyrimidine synthesis into R. leguminosarum strains which nodulate pea (bv. viciae) or clover (bv. trifolii) had a similar effect on pea and clover nodulation. Several experiments showed that the mutants grew in the rhizosphere as well as the wild-type strain. The addition of 5-aminoimidazole-4-carboxamide riboside (AICAR) enhanced nodule development of the auxotrophs on all of the respective host plants. It is possible that AICAR or the subsequent precursor of inosine, or a derivative of one of them, is an essential factor in promoting nodule development. Ten exopolysaccharide (Exo-) mutants of strain CFN42 were isolated after Tn5 mutagenesis and were grouped into three classes (Class A1, A2 and B). The Class A1 and A2 strains induced normal nitrogen-fixing nodules on beans. The A1 strains contained Tn5 inserts on the same chromosomal EcoRI fragment and could be complemented to Exo+ with cosmids of the CFN42 library carrying this fragment. The A2 strains harbored Tn5 insertions on another chromosomal EcoRI fragment but could not be complemented to Exo+ with cosmids of the CFN42 library. Both classes of mutants did not produce any acidic exopolysaccharide (EPS) but, unlike the wild-type strain excreted lipopolysaccharide (LPS) into the media. The Class B strains induced underdeveloped nodules (Ndv-) on bean. They lacked acidic EPS and were deficient in lipopolysaccharide synthesis (Exo- Lps- Ndv-). These mutants were complemented to Exo+ Lps+ Ndv+ by either of two overlapping cosmids and the Tn5 inserts were found on adjacent chromosomal EcoRI fragments. The exo::Tn5 alleles were transferred to bv. viciae and bv. trifolii strains. The resulting transconjugants were Exo- and failed to induce normal nodules on pea and clover. When these Exo- mutants were complemented to Exo+ by cosmids from the CFN42 library, normal nodulation on pea and clover was restored. Thus, exo mutations which did not affect bean nodulation prevented normal nodule development on pea and clover. Acidic EPS appears to be a compatibility factor on pea and clover, but is unnecessary for symbiosis with bean.



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