Date of Award
8-1989
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Dale Noel
Second Advisor
Mary Lynne Collins
Third Advisor
James B. Courtright
Fourth Advisor
Reinhold D. Mueller
Fifth Advisor
Gail L. Waring
Abstract
The lipopolysaccharide (LPS) of R. leguminosarum contains two polysaccharide domains attached to lipid A: a conserved core oligosaccharide and a longer O-antigen polysaccharide whose composition varies from strain to strain. Being a major cell-surface molecule, LPS is likely to play a role in early symbiotic events. To examine the requirement of LPS in nodulation of clover by R. leguminosarum strain ANU843, two Lps mutants, TB104 and TB112, were isolated following Tn5 mutagenesis of strain ANU843. These mutants were found to lack O-antigen and a portion of the core. When inoculated onto clover, they elicited poorly developed nodules with much lower than normal bacterial populations. Two genetic regions, $\alpha$ and $\beta$, required for LPS synthesis in R. leguminosarum strain CFN42 are represented by the cosmid clones pCOS109.11 and pCOS126, respectively. Based on the arrangement of lps genes in Salmonella spp., in which genetic region rfa directs LPS core synthesis and region rfb directs O-antigen synthesis, it was hypothesized that pCOS126 and pCOS109.11 may represent similar regions in R. leguminosarum. This was tested by transferring pCOS109.11 and pCOS126 to mutants TB104 and TB112. Mutant TB104 was restored to apparently wild-type LPS by the lps $\beta$ DNA of strain CFN42, suggesting that this region encodes a conserved function(s). Mutant TB112, carrying lps region $\alpha$ DNA of strain CFN42, produced a unique LPS with some determinants of CFN42 LPS. It was thus concluded that lps region $\alpha$ encodes enzymes required for O-antigen synthesis. In both cases, nodulation proficiency was restored to that of the wild-type. Therefore, considerable latitude in LPS structure is tolerated in the symbiosis with clover. lps Regions $\alpha$ and $\beta$ were used as probes in Southern hybridizations to study the conservation of lps genes among R. leguminosarum wild isolates. The DNA of lps region $\beta$ was found to be conserved among the R. leguminosarum strains tested. Interestingly, lps region $\beta$ was found to be plasmid encoded in some cases. The DNA of lps region $\alpha$, encoding O-antigen structure, appeared to be fairly specific to the strain from which it was cloned.