The Genetic Basis of Delayed Bean Root Nodulation by a Bacterial Mutant

Rachel Kowalski
Marquette University
Mentor:  Dr. Dale Noel

Rhizobium etli bacteria are able to form a symbiotic relationship with Phaseolus vulgaris bean plants through root nodule formation.  Within the nodule, nitrogen is fixed into ammonia by the bacteria and then released to the plant.   Nodule formation is a developmental process that only occurs when bacteria are present. Bacterial mutations that affect nodule development allow for determination of bacterial components required for the symbiotic relationship.  Mutant bacterial strain CE123 has a phenotype that affects the period in nodule development that is least understood.  It carries a mutation from random Tn5 mutagenesis, which may be the cause of this phenotype.

Wild-type nodulation gives clumps of pink nodules located close to the top of the root system, which start to develop about 8 days after inoculation (dai).  The pink color indicates mature nodules that fix nitrogen.  These nodules have a large amount of nitrogen-fixing activity at 14 dai and the activity continues for several weeks after inoculation.  The nodules elicited by mutant CE123 at 14 dai were small, light in color, sparsely dispersed throughout the roots, and unable to fix nitrogen.  At 21 dai some CE123 nodules were large and pink but others were not. These developed nodules were able to fix a small amount of nitrogen compared to the wild-type. 

A genetic investigation of CE123 involved cloning the fragment of DNA carrying the Tn5 insertion for sequencing and reconstructing the mutant.  Sequencing revealed the general location of the Tn5 insert but not the specific gene.  Further analysis allowed for localization of the Tn5 within 100 base pairs of the genomic DNA sequence.  This location places the insertion within or in between the genes greA and lpcC.  Little is known about greA, but previous studies suggest that, due to its location in the genome, it may affect lipopolysacchride (LPS), which is a bacterial component necessary for developing the symbiotic relationship.  Gene lpcC encodes a mannosyl transferase, which is necessary to form LPS.  In as much as both genes suggest a connection with LPS; it may be relevant that the mutant CE123 LPS appears to be antigenically altered. 

Further genetic investigation of CE123 is aimed at reconstructing the mutant.  The mutant CE123 DNA fragment was subcloned so that it could be transferred into the wild type bacteria through a double recombination event.  The reconstructed bacteria will be inoculated onto plants.  If the mutant phenotype is again observed, then the Tn5 insertion is the cause of the deficient nodule formation.  If not, then some other mutation or perhaps multiple mutations may be the cause of the symbiotic deficiency.
 
 

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