Genetic Analysis of a Rhizobium Mutant That Elicits
Nodules with Abnormal Pigmentation

Eric Rosado
DePaul University
Mentor:  Dr. Dale Noel

Bean plants (Phaeolus vulgaris) and Rhizobium etli establish a symbiotic mutualism in which the bacteria stimulate the plant to create an organ called a nodule. In this symbiosis, the bacteria fix nitrogen into ammonia for the benefit of the plant, while the plant provides the bacteria fixed carbon and oxygen so that it may conduct cellular respiration to produce ATP for nitrogen fixation. This research investigated a bacterial mutant strain known as CE119. Unlike the wild type, CE119 cannot fix nitrogen (Fix-). In addition, nodules carrying this mutant change from the normal red pigmentation to a bright green color at about 14 to 21 days after inoculation of the plant. The DNA of CE119 carries an insertion of transposon Tn5. As well as investigating the nodules’ strange coloration, other objectives were to identify the gene mutated by Tn5 and to determine whether this insert is the cause for these phenotypes. 

The red color of normal nodules is due to heme in leghemoglobin, which is the transporter of oxygen to the nitrogen-fixing bacteria. It was suspected that the switch to a green color in the mutant’s nodules was due to the degradation of heme and leghemoglobin. To test this hypothesis, the cytosols of nodules induced by mutant and wild type bacteria were compared. The visible spectra of CE119 nodule cytosol lacked features that normally are attributed to the heme in leghemoglobin. Gel electrophoresis revealed that CE119 nodules lacked leghemoglobin proteins. 

The strategy to determine the genetic location of the Tn5 insert and whether it causes the symbiotic abnormalities of CE119 involved two overall steps of genetic construction. The first step was to clone the DNA fragment of mutant CE119 that contains Tn5 and to determine portions of its nucleotide sequence. The second step was to return this cloned fragment to the wild-type bacteria and replace the wild type’s DNA with the mutated DNA. The first step has been accomplished. The sequence revealed that the Tn5 insertion was within the gene (fbcF) that encodes the iron sulfur protein of cytochrome bc1. Cytochrome bc1 is part of the electron transport chain of the bacteria inside nodules and in free-living cultures. Cytochrome bc1 activity in free-living cultures was assayed by measuring NADH reduction of cytochrome c. This activity in the mutant was greatly decreased compared to the wild type.

 This deficiency could explain how CE119 is fix- and how the green color arises. Although cytochrome bc1 is not essential ex planta, studies of other rhizobial species have suggested that it is essential for electron transport in the bacteria in nodules. Its mutation would prevent synthesis of ATP, which is necessary for nitrogen fixation. It also would prevent oxygen consumption by the bacteria. The lack of nitrogen fixation or oxygen consumption might trigger a negative feedback response in the plant that leads to leghemoglobin degradation and heme conversion to biliverdin, which is a green pigment.

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