Control of Inducer Accumulation Plays a Key Role in Succinate-Mediated Catabolite Repression in Sinorhizobium meliloti

Abstract

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the ␣-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other ␣-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of ␤-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of ␣-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce ␤-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinatemediated catabolite repression. Thus, some raffinose could be transported by the overproduced ␤-galactoside transporters and cause the induction of ␣-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a ␤-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.Bacteria belonging to the genera Sinorhizobium, Rhizobium, and Bradyrhizobium are members of the ␣-proteobacteria, a fascinating group of organisms, many of which are intracellular symbionts or pathogens. Sinorhizobium meliloti can grow in soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodules of alfalfa and a few other plants belonging to the family Leguminosae (3,9,15,19,22,34).Free-living S. meliloti, like many heterotrophic bacteria, utilizes a wide variety of compounds as sources of carbon for growth. S. meliloti can utilize ␣-galactosides in laboratory medium and also when growing in the rhizospheres of host and nonhost plants (4). The utilization of ␣-galactosides requires genes which are part of an operon located on pSymB, a 1.7-Mb plasmid, in S. meliloti (Fig. 1) (7, 12). The agpA gene encodes a 77-kDa periplasmic protein that is required for ␣-galactoside transport and that is similar to periplasmic binding protein components of the oligopeptide family of permeases ( Fig. 1) (12). ...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…In some bacteria, CCR is governed by other preferences and is possibly mediated by additional mechanisms. For instance, ␣-proteobacteria of the genera Sinorhizobium, Rhizobium, and Bradyrhizobium (83,889) and ␥-proteobacteria of the Pseudomonas family (135) prefer acetate or tricarboxylic acid (TCA) cycle intermediates such as succinate over carbon sources such as glucose, fructose, or lactose. The underlying mechanisms have not yet been unraveled, but in pseudomonads, they involve the catabolite repression control (Crc) protein (22,331,332,573,747,982), and rhizobacteria, they involve inducer accumulation (83).…”

Sinorhizobium meliloti pSymB carries genes necessary for arabinose transport and catabolism

“…Succinate is a preferred carbon source for S. meliloti, and has been shown to repress catabolism and/or transport of other carbon sources such as lactose and raffinose (Ucker & Signer, 1978;Gage & Long, 1998;Bringhurst & Gage, 2002). However, glucose has also been shown to repress the expression of catabolic genes in R. leguminosarum (Oresnik et al, 1998;Richardson et al, 2004).…”

HPrK Regulates Succinate-Mediated Catabolite Repression in the Gram-Negative SymbiontSinorhizobium meliloti

“…It has been shown that succinate represses genes needed for utilization of secondary carbon sources such as lactose (31,57) and that it can prevent the intracellular accumulation of secondary carbon sources, lactose and raffinose, through inducer exclusion (6). This is called succinate-mediated catabolite repression (SMCR) and, though well documented in S. meliloti, the molecular mechanisms of its operation remain obscure.…”

“…C 4 -dicarboxylic acids are used to fuel and provide reducing equivalents for nitrogen fixation by bacteroids (20,49). Free-living S. meliloti also utilize succinate and do so in preference to many sugars and other substrates (6,28,31,45,57).…”