Bacterial Respiration and metabolic diversity.
Relevant publications
D.J. Westenberg, 2007 B. japonicum, Agriculture and the Bacterium, BIOforum Europe, 11:16
Fumeaux, C., Bakkou, N, Kopciñska, J, Golinowski, W., Westenberg, D.J., Müller, P., and X. Perret. 2011 Functional analysis of the nifQdctA1y4vGHIJ operon of Sinorhizobium fredii strain NGR234 using a transposon with a NifA-dependent read-out promoter. Microbiology 157:2745-58.
*Gulley, V. and Westenberg, D.J. 2002 Regulation of the Bradyrhizobium japonicum sdh operon encoding succinate dehydrogenase. In Finan et al (eds.), Nitrogen Fixation: Global Perspectives. CABI Publishing, Oxford University Press, Cary, NC
Westenberg. D. J. and M. L. Guerinot. 1999. Succinate dehydrogenase (Sdh) from Bradyrhizobium japonicum is closely related to mitochondrial Sdh and is sensitive to the agricultural fungicide carboxin. J. Bacteriol. 181:4676-4679
Westenberg, D. J. and M. L. Guerinot. 1997. The role of metals in bacterial gene regulation. Adv. Genetics. 36:187-238.
In the microbial world, diversity is most apparent in the area of metabolic diversity. Bacteria have myriad ways to get energy for growth. The most efficient method for obtaining energy from available nutrients is through electron transport phosphorylation, aka respiration. Besides oxygen, bacteria can use an amazing variety of molecules as terminal electron acceptors and an even greater variety of molecules can serve as electron donors. Both electron donors and electron acceptors can range from organic compounds such as fumarate, to inorganic material such as metals. Frequently, a single species of bacteria can utilize multiple electron donors or terminal electron acceptors or use multiple pathways toward the same terminal electron acceptor. Our lab is interested in how organisms deal will varying availability of terminal electron donors and acceptors and how those responses relate to important physiological process. Some of the questions we wish to address include, how these bacteria control the composition of their respiratory chain to respond to the presence or absence of specific electron donors or acceptors, the varying concentration of those materials or choose which donor or acceptor to use when multiple material are available. In addition, we want to determine what impact those options have on key physiological processes. The model organism currently studied in our lab is the nitrogen-fixing soybean symbiont, Bradyrhizobium japonicum.
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 enzymes of the B. japonicum respiratory chain. With respect to terminal oxidases, B. japonicum can utilize a number of terminal oxidases that are differentially expressed depending upon oxygen availability and/or association with the symbiotic partner. Association with soybean has a dramatic impact on gene expression in B. japonicum as it undergoes conversion to a pleomorphic form referred to as a bacteroid. In addition to the expression of alternative terminal oxidases, the bacteroids must also express a number of other enzymes necessary for utilization of the nutrients available inside the nodule. For example, the dicarboxylic acids succinate and malate serve as energy rich substrates for the bacteroids. These compounds can be used to provide electrons to the respiratory chain of the bacterium. Therefore, these changes in the respiratory chain of the bacteroid are reflected in the amounts and types of cytochromes present.
From a practical point of view, 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.
Work in our lab has focused on the enzymes involved in transferring 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 enzymes take 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 directly to ubiquinone.
| The majority of the work in our lab involves Sdh. We are looking at regulation of the sdhCDAB operon in B. japonicum using fusions to the beta-galactosidase gene from E. coli. Regulation of the B. japonicum sdhCDAB operon is of particular interest because expression appears to increase under reduced oxygen conditions. This is in contrast to the regulation seen in other model organisms such as E. coli. We are also constructing a strain of B. japonicum that is deleted for the sdhCDAB operon that will allow us to introduce modified versions of the B. japonicum sdhCDAB operon (either regulatory or catalytic modifications) as well as heterologous sdhCDAB operons into B. japonicum. |
Other projects are looking at interaction of B. japonicum Sdh with inhibitors. We have shown that B. japonicum Sdh is inhibited by carboxins and are currently screening for carboxin resistant mutants and determining the inhibitory effectiveness of modified carboxins.
The main goal of these projects is to increase the nitrogen fixing capacity of B. japonicum but the results of this work should also have implications with respect to a number of other organism. This is due to the observation that the amino acid sequences of Nuo and Sdh from B. japonicum are quite similar with the same enzymes from Paracoccus denitrificans and mitochondria . In fact, the B. japonicum sequences are more similar to mitochondrial sequences than to other bacterial sequences. This sequence similarity indicates that these studies will provide information relevant to people interested in the function of the mitochondrial enzyme. We can then use the relatively simple bacterial system to address fundamental questions about respiratory complex structure and function.