A Rhizobium Cytochrome bc1 Mutant Elicits Inactive Root Nodules with Abnormal Pigmentation

Eric Rosado
DePaul University
Mentor: Dr. Dale Noel

Bean plants and Rhizobium etli establish a mutualistic symbiosis 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 organic acids and oxygen so that it may conduct cellular respiration to generate ATP for nitrogen fixation. 

One approach to understand the symbiosis is to create bacterial mutations. Tracing the mutation back to the mutated gene and comparing wild-type and mutant phenotypes allow one to understand the functional and developmental factors of the bacteria involved in symbiosis.

This project continues the exploration of a bacterial mutant strain known as CE119. Unlike the wild type, CE119 can not carry out nitrogen fixation (Fix-). Fix- phenotypes are associated with defects in several bacterial genes. However, CE119 is unique in conferring another phenotype, an abnormal bright green pigmentation of the root nodule that occurs about 14-21 days after inoculation of the plant. 

Within the DNA of CE119, there is a transposon insertion (Tn5) that may be the cause of its phenotypes of abnormal pigmentation and lack of nitrogen fixation. A 9kb segment of CE119 DNA surrounding this Tn5 insertion was cloned into a plasmid vector. Determination of its nucleotide sequence revealed that the Tn5 was inserted 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 the mutant was shown to be greatly decreased compared to the wild type. The soluble plant portions of nodules induced by CE119 and wild type bacteria were also 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. 

We hypothesize that the Cytochrome bc1 deficiency disrupts the respiratory generation of ATP, which could explain how CE119 is Fix-. We also suggest that the bright green pigmentation of the nodule may be due to degradation of leghemoglobin as a consequence of the lack of oxygen consumption by the bacteria. Leghemoglobin ferries oxygen to the bacteria at high flux under the normal condition of low free oxygen concentration in the nodule interior. Lack of oxygen consumption by the bacteria may be sensed as an abnormally high relative concentration of oxygenated leghemoglobin, and perhaps high free oxygen as well. A negative feedback response in the plant then might degrade the heme from leghemoglobin to biliverdin, which is a bright green pigment.
 
 

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