Changing Substrate Recognition by the S. meliloti DctA Transporter

Washington State University, Pullman WA

Investigators

Abstract

Legume plants like alfalfa, soybean and peanut, can obtain the nitrogen compounds needed for growth through a symbiotic association with "nitrogen-fixing" bacteria. These bacteria are able to take relatively inert dinitrogen gas from the atmosphere and biochemically convert it into a more useful form. This conversion has a cost-the bacteria require plant-derived carbon compounds that can be metabolized to obtain energy for the nitrogen fixation reactions and a microaerobic environment in which the biochemistry can occur. It is thought that the bacteria receive a major fraction of their nutrition in the form of dicarboxylic acids and their ability to import dicarboxylic acids using the bacterial DctA protein has been shown to be essential to bacterial nitrogen-fixation. After traversing a plant membrane, dicarboxylic acids are imported by symbiotic bacteria using the DctA dicarboxylate transporter, a member of a glutamate transporter family of proteins better known for their role in neurotransmitter metabolism. In free-living growth, DctA can transport a number of physiologically important compounds, including malate, fumarate, succinate, aspartate and oxaloacetate. It is unclear how important each of these is in symbiosis. Mutations of the Sinorhizobium meliloti DctA protein will be isolated that have altered specificity or affinity for the different substrates in order to discover more about the transport properties of DctA. Various mutant isolation strategies will be used, including isolation of functional mutants resistant to a toxic DctA substrate, mutants with higher transport rates with dicarboxylic acids for which DctA has low affinity, and mutants able to compete well for limiting amounts of dicarboxylic acids. Transport properties of the mutants will be determined and, in addition, the mutants will be tested on their alfalfa host plants for the ability to support symbiotic nitrogen fixation. In this way, the mutants function as in vivo probes of nodule function. Analysis of the mutants should contribute to the understanding of the relationship of DctA sequence to substrate recognition and thus provide a novel view into one class of this important family of transport proteins.