Complex regulation of symbiotic functions is coordinated by MucR and quorum sensing in Sinorhizobium meliloti

Abstract

In Sinorhizobium meliloti, the production of exopolysaccharides such as succinoglycan and exopolysaccharide II (EPS II) enables the bacterium to invade root nodules on Medicago sativa and establish a nitrogen-fixing symbiosis. While extensive research has focused on succinoglycan, less is known concerning the regulation of EPS II or the mechanism by which it mediates entrance into the host plant. Previously, we reported that the ExpR/Sin quorum-sensing system is required to produce the symbiotically active low-molecular-weight fraction of this exopolysaccharide. Here, we show that this system induces EPS II production by increasing expression of the expG-expC operon, encoding both a transcriptional regulator (ExpG) and a glycosyl transferase (ExpC). ExpG derepresses EPS II production at the transcriptional level from MucR, a RosR homolog, while concurrently elevating expression of expC, resulting in the synthesis of the low-molecular-weight form. While the ExpR/Sin system abolishes the role of MucR on EPS II production, it preserves a multitude of other quorum-sensing-independent regulatory functions which promote the establishment of symbiosis. In planktonic S. meliloti, MucR properly coordinates a diverse set of bacterial behaviors by repressing a variety of genes intended for expression during symbiosis and enhancing the bacterial ability to induce root nodule formation. Quorum sensing precisely modulates the functions of MucR to take advantage of both the production of symbiotically active EPS II as well as the proper coordination of bacterial behavior required to promote symbiosis.

FIG. 1.

Expression of expC is not restored by disruption of mucR. Extracted RNA from various strains of S. meliloti grown in MGM low-phosphate medium to an OD₆₀₀ of 1.2 was analyzed by quantitative real-time PCR and compared to expression observed in a sinI quorum-sensing-deficient mutant. Disruption of expG resulted in expression levels of the exp gene family similar to those observed in an sinI mutant incapable of quorum sensing. Introduction of a disrupted mucR in the expG mutant restored expression of all members of the exp gene family with the exception of expG and the downstream expC. WT, wild type.

FIG. 2.

Expression of expC is critical for the wild-type (WT) mucoid phenotype. Mutant strains of S. meliloti streaked on LB-MC agar plates produced phenotypes correlating with gene expression of the exp gene family measured by real-time PCR analysis. Disruption of expG prevented the production of EPS II, resulting in dry colonies. The mucR expG mutant appeared mucoid although the viscosity and volume of the exopolysaccharide were indicative of the absence of the low-molecular-weight fraction. Restoration of expC expression (via pJTpexpC) to wild-type levels restored the fully mucoid phenotype characteristic of strains producing HMW and LMW EPS II.

FIG. 3.

Restored expression of expC results in the biosynthesis of LMW EPS II. (A) Purified EPS II produced from wild-type (WT) and mutant strains was analyzed by HPAEC for the presence of LMW EPS II. Analysis of exopolysaccharide produced by the wild-type S. meliloti confirmed the presence of LMW EPS II based upon distinctive peaks between 24 and 27 min. The mucR expG mutant failed to produce LMW EPS II. Supplementation (by introduction of pJTpexpC) of transcriptional levels of expC in this mutant strain restored the synthesis of the low-molecular-weight fraction. Results were overlaid for comparison. (B) The presence of LMW EPS II produced expected levels of biofilm formation and attachment. The mucR expG mutant strain failed to form biofilm while the strain carrying pJTpexpC attached at equivalent levels to the level of the wild type. (C) In the absence of succinoglycan production, the result of a disruption in exoY, the ability of S. meliloti to invade the host plant and establish a nitrogen-fixing symbiosis, visually discernible by the development of large pink nodules, is a clear indicator of the production of LMW EPS II. The disruption of exoY in the mucR expG mutant abolished the capacity of the strain to invade the plant. Supplementation of expression levels of expC in this strain through the introduction of pJTpexpC resulted in an invasive capacity comparable to that of the exoY strain, confirming the synthesis of LMW EPS II as a result of restored expression of expC.

FIG. 4.

MucR represses the exp gene family prior to quorum. (A) Wild-type S. meliloti was grown in MGM low-phosphate medium to OD₆₀₀ of 0.02, 0.1, and 1.2. Analysis by real-time PCR and comparison to the sinI mutant indicated that expression levels of the exp gene family, in particular, expE, expA, and expD directly responsible for EPS II biosynthesis, were first induced by quorum at a population density between an OD₆₀₀ of 0.02 and an OD₆₀₀ of 1.2. (B) Expression of rem confirmed that low expression levels observed at an OD₆₀₀ of 0.02 were particular to the exp gene family, not a by-product of low global gene expression at this stage of growth. (C) Disruption of mucR resulted in derepression of expE, expA, and expD operons at an OD₆₀₀ of 0.02. However, the repressive effect of an intact mucR attenuated to negligible levels by an OD₆₀₀ of 0.1, suggesting that MucR facilitates the inhibition of EPS II production at a low population density until quorum is reached. At this point, the ExpR/Sin system derepresses the exp gene family. Expression levels of expG and expC are not shown as this operon is not under the transcriptional regulation of MucR.

FIG. 5.

MucR increases nod operon gene expression in response to luteolin. In the absence of induction by luteolin, a disruption in mucR resulted in a negligible change in nodA expression. Addition of luteolin resulted in an approximately 90-fold induction of the nod operon while disruption of mucR attenuated this effect to roughly 10-fold. Complementation of mucR with pJTpmucR restored this expression. Expression of the first open reading frame (nodA) of the nod operon is shown here, as similar transcriptional levels were observed for downstream nodB and nodC (data not shown).

FIG. 6.

The presence of an intact mucR provides an advantage for nodule induction in M. sativa. (A) The ability of wild-type and mutant strains of S. meliloti to develop nodules was examined daily after inoculation of bacteria onto seedlings. Disruption of mucR in strains incapable of invading resulted in a decrease in the ability to induce the formation of nodules on the host plant. Complementation of mucR restored this ability to wild-type levels. All strains were disrupted for expA and exoY in order to prevent invasion of the plant and establishment of symbiosis from interfering with nodule development as an indicator of nod factor production. (B) Direct comparison of the ability of strains to produce nodules (panel A) between those carrying either an intact or disrupted mucR indicates an initial 7-fold advantage in nodule induction associated with the presence of an intact mucR gene sequence. This advantage attenuates to roughly 1.3-fold by day 30.

FIG. 7.

Schematic model for the quorum-sensing (QS)-dependent and -independent roles of MucR. At a low population density (OD₆₀₀ of ≤0.02), MucR prevents the unnecessary production of EPS II prior to the quorum required for attachment and biofilm formation. At a high population density (OD₆₀₀ of ≥0.1), AHL-bound ExpR induces expression of the expG-expC operon encoding both the ExpG transcriptional regulator and the ExpC glycosyl transferase. ExpG derepresses operons expE, expA, and expD from MucR, allowing for the biosynthesis of EPS II (4). Concurrently, increased levels of ExpC result in the production of the symbiotically active low-molecular-weight fraction. Throughout all stages of growth prior to symbiosis, MucR increases production of nod factor as well as the symbiotically active exopolysaccharide succinoglycan while repressing premature expression of bacteroid genes and mitigating motility.