Research Teaching Personal People Pastimes
    
Bradyrhizobium japonicum  
This is a micrograph of Bradyrhizobium japonicum. B. japonicum is a gram negative, rod-shaped, nitrogen-fixing bacterium which develops a symbiosis with the soybean plant Glycine max. B. japonicum belongs to the family, Rhizobiaceae which includes other nitrogen-fixing bacteria that develop symbiosis with legumes. This photo shows an individual cell and groups of cells in a characteristic aster formation.

 
Source: D.J. Westenberg
The bacterium "communicates" with the host plant and begins a process of plant and bacterial development that leads to a symbiotic partnership. The bacterium will attach to root hairs and release compounds that cause the roothairs to curl. Coordination of bacterial multiplication and inward directed growth of the root hair results in formation of an infection thread (a tube derived from plant membranes). It is through this infection thread that the bacteria enter the cortical cells of the root and begin to colonize the developing root nodule.
Source: F. Dazzo, Michigan State University
 
Within the developing root nodule, bacteria divide and begin to differentiate into a bacteroid (a term used to refer to the bacterium existing in a symbiotic relationship to distinguish it from the free-living bacterium), that is capable of fixing nitrogen. The bacteroids are located inside a structure refered to as a symbiosome that is derived from plant membrane. One to several bacteroids can be found in a single symbiosome. Therefore, nutrients must traverse multiple membranes to reach the bacteroids and fixed nitrogen must follow a similar complex path to reach the plant tissue.

 
Source: M.L. Guerinot, Dartmouth College
The nodule which results from this process is a highly specialized structure. It provides a physical barrier which keeps the free oxygen concentration low.
Source: Unknown
The plant cells within the nodule produce leghemoglobin which serves as an oxygen carrier to the bacteria within the nodule. This enables the bacteria to obtain enough oxygen for respiration but ensures that the oxygen is in a bound form so that it cannot harm nitrogen fixing enzymes inside the bacteria. Cutting open a nodule reveals the deep red color typical of leghemoglobin when it binds oxygen.
Source: M.L. Guerinot, Dartmouth College
What do the individual partners in this symbiosis get from each other? The plant provides the bacterium with a "safe" environment and a steady supply of carbon for energy and growth. This carbon source is referred to as photosynthate as it is derived from the product of photosynthesis. In most rhizobium/legume symbiosis, photosynthate refers to the dicarboxylic acids, succinate, fumarate and malate. In return, the bacteria provide the plant with fixed nitrogen - nitrogen gas which has been reduced and converted into a form readily utilized by the plant. The result of this symbiosis is a dramatic increase in plant production without the need for adding external fertilizer.
Source: M.L. Guerinot, Dartmouth College
Soybeans are an important crop throughout the world and strategies to improve crop production by manipulating the nitrogen fixing partner are desirable.

Nitrogen fixation requires a large input of energy and reducing potential. As a strictly respiratory organism B. japonicum must get that energy from the respiratory chain by oxidizing energy rich substrates and reducing oxygen. One strategy to improve crop production is to increase the nitrogen fixing capacity of B. japonicum through manipulation of the respiratory chain.

B. japonicum presents an excellent model organism for studying respiratory enzymes. There is a large body of information describing the respiratory chains in B. japonicum. However, other than the terminal oxidases, little information is available on the structure, function and regulation of the enzymes of the B. japonicum respiratory chain. B. japonicum can express a number of terminal oxidases. Specific terminal oxidases are expressed depending upon oxygen availability and/or association with the symbiotic partner. In association with soybean B. japonicum undergoes conversion to a pleomorphic form referred to as a bacteroid. Changes in the respiratory chain of the bacteroid are reflected in the amounts and types of cytochromes present. In addition to the expression of alternative terminal oxidases, the bacteroids must also express a number of other cytochromes necessary for utilization of the nutrients available insside the nodule. The dicarboxylic acids succinate and malate serve as energy rich substrates for the bacteroids. These compounds can be used to provide reducing potential to the respiratory chain of the bacterium and the enzymes involved in conserving this reducing potential contain cytochromes..

Part of the work in our lab focuses on the enzymes involved in transfering electrons from the dicarboxylic acids to the respiratory chain. We are looking at two respiratory complexes, NADH dehydrogenase (NADH ubiquinone oxidoreductase - Nuo) and succinate dehydrogenase (Sdh) (referred to as complexes I and II, respectively, in mitochondria). NADH dehydrogenase takes electrons from NADH and transfers them to ubiquinone, a lipid soluble electron carrier. Possible major sources of NADH in bacteroids are one of the malic enzymes and malate dehydrogenase. Both of these ezymes get electrons from malate one of the most abundant dicarboxylic acids in the soybean nodule. Succinate is another dicarboxylic acid that is very abundant in nodules and the enzyme succinate dehydrogenase takes electrons from succinate and transfers them to ubiquinone.

An additional project in our lab is directed at determining factors that may influence the ability of inoculant strains to compete with native strains for infection of the roothair. One factor would be acyl homoserine lactone (AHL) molecules that are involved in density dependent gene expression - also known as "quorum sensing". Another factor would be inhibitors that would block the growth of native strains but not inoculants. To accomplish this, we are using an inhibitor of succinate dehydrogenase (carboxin - Vitavax) and creating carboxin resistant inoculant strains.

Yet another project in our lab is investigating factors that influence expression of the respiratory enzymes. Factors that we are examining are iron, heme, oxygen and the dicarboxylic acids. Projects include studies of the expression of the hemA, sdhCDAB and nuo genes.

Check out my research interests to find out more about my current research involving B. japonicum.


 
Return to DJW's Homepage
Return to the Biology Dept. Homepage