Control of alpha-galactoside utilization in Sinorhizobium meliloti by the transcriptional activator AgpT and succinate-mediated catabolite repression

The nodulating bacterium Sinorhizobium meliloti and its symbiotic partner alfalfa have been well studied to understand symbiosis and nitrogen fixation. S. meliloti can exist as a free-living organism or as a nitrogen-fixing symbiont. This focus of this work is to better understand the regulation of α-galactoside utilization, including succinate-mediated catabolite repression, in the free-living bacterium. ^ As a free-living organism, S. meliloti can utilize a wide range of substrates. C4-dicarboxylic acids are used preferentially and support the highest growth rate. These organic acids repress growth on secondary carbon sources, including α-galactosides. There are two parts to the work described in this thesis. First, we describe experiments to understand the genetic regulation of the agp operon, which is necessary for uptake and initial cleavage of α-galactosides. Second, we have used the agp operon as a model to study the mechanisms by which the C4-dicarboxylic acid succinate represses use of secondary carbon sources. ^ We have demonstrated that α-galactoside utilization requires an AraC-like transcriptional activator, AgpT. When agpT was inactivated, S. meliloti could not grow on α-galactosides or induce expression of the agp operon. We have also shown that AgpT is a DNA-binding protein that binds to the promoter region of the agp operon. ^ To begin studying the mechanism by which C4-dicarboxylic acids are preferentially used over secondary carbon sources, we performed a random transposon mutagenesis and looked for mutants in which succinate was unable to repress the expression of genes of the agp operon. One mutant isolated from this screen led us to investigate the possibility that inducer exclusion plays a role in succinate-mediated catabolite repression. We demonstrated the presence of an inducer exclusion mechanism using radiolabeled raffinose uptake assays. ^ We used a second genetic screen to identify a gene, SMa0113, which appears to be required for catabolite repression. Early work showed loss of catabolite repression in media containing succinate and raffinose, lactose, or maltose. We have also begun to characterize S. meliloti genes similar to Escherichia coli genes implicated in catabolite repression.