Water-Soluble Humic Materials Regulate Quorum Sensing in Sinorhizobium meliloti Through a Novel Repressor of expR

Abstract

Quorum sensing (QS) plays an important role in the growth, nodulation, and nitrogen fixation of rhizobia. In this study, we show that water-soluble humic materials (WSHM) repress the expression of the QS related genes sinI, sinR, and expR in Sinorhizobium meliloti. This decreased the production of N-acetyl homoserine lactones (AHL) and exopolysaccharides (EPS), and ultimately increased S. meliloti cell density. We also identified a novel regulator, SMc03890 (renamed QsrR), which binds directly to the expR promoter. Deletion of qsrR increased expR expression. WSHM repressed the expression of expR by augmenting the interaction between QsrR and the expR promoter; this was determined by a bacterial-one-hybrid assay. These effects of WSHM on the QS system in S. meliloti may be the underlying mechanism by which WSHM increase the symbiotic nitrogen fixation of Medicago sativa inoculated with S. meliloti. This study provides the first evidence that humic acids regulate the QS of rhizobia and suggests that WSHM could be used as fertilizers to improve the efficiency of symbiotic nitrogen fixation.

Keywords: ExpR regulator; Sinorhizobium meliloti; bacterial communication; humic materials; quorum sensing.

FIGURE 1

Regulatory diagram of the S. meliloti quorum sensing system. The transcription of sinI, which encodes AHL synthase, is induced by SinR. sinI transcription can also be induced by the AHL-ExpR complex at low AHL concentrations (5∼10 nM). The AHL-ExpR complex represses sinR expression at high AHL concentrations (100∼200 nM). Furthermore, the AHL-ExpR complex induces the expression of genes related to EPS biosynthesis, and represses the expression of genes important for motility, nodulation, and nitrogen fixation.

FIGURE 2

Effects of WSHM on the growth (A), AHL production (B), exopolysaccharide production (C), and exoY and expE gene expression (D) in S. meliloti 8530. All the data are expressed as average ± standard deviation (SD) (n = 3). Statistical significance was assessed by Student’s t-tests (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001). For exopolysaccharides production (C), S. meliloti 1021 (ΔexpR) was also included. (A) YM-CN broth: the nitrogen (yeast extract) and carbon (mannitol) source in YM broth were removed.

FIGURE 3

Effects of WSHM on the expression of genes involved in the QS system in S. meliloti 8530 (A), S. meliloti 1021 (B), S. melilotiΔsinR (C), and S. melilotiΔsinI (D). Data are expressed as average ± SD (n = 3). Statistical significance was determined using Student’s t-tests (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001).

FIGURE 4

Expression of P_expR-lacZ in S. meliloti 8530 (WT) and S. melilotiΔqsrR mutant treated with either 500 mg L^-1 WSHM or 2% alfalfa seed exudates (E), respectively. Alfalfa seed exudates were used to assess whether WSHM could repress expR expression in a manner similar with plant signal. Data are expressed as average ± SEM for three replicates. Different letters indicate statistical significance as assessed by Duncan tests (P < 0.05).

FIGURE 5

Electrophoretic mobility shift assays confirmed direct binding of QsrR to the expR promoter (pexpR). pexpR is a 286 bp DNA fragment from the translational start codon of gene expR. Each lane contained 0.005 nM of labeled pexpR. The labeled pexpR and a ∼100-fold excess of the unlabeled pexpR were used in competitive assays. Labeled non-specific DNA from Streptomyces avermitilis was used as negative control. The amount of His₆-QsrR added in each lane were 0 μM, 0.92 Mm, 1.65 μM, 2.38 μM, 3.3 μM, 4.77 μM, 4.77 μM, and 4.77 μM, respectively.

FIGURE 6

Bacterial-one-hybrid assays showing interaction between expR promoter (R1, R4) and QsrR which was potentiated by WSHM. (A) Schematic diagram of bacterial one-hybrid system. The HIS3/URA3 cistron is in the pH3U3 prey vector. The target DNA sequences were ligated to pH3U3. The binding of DNA-binding domain (DBD, ligated to pB1H1) with the target DNA sequence enables the transcription of HIS3 and URA3. The expression of HIS3 enabled E. coli USO grow on NM selective medium containing the desired concentration of 3-amino-triazole (3-AT), a competitive inhibitor of HIS3. The expression of URA3 prevented E. coli USO growth on medium containing 5- fluoroorotic acid (5-FOA). This figure was adopted from Meng et al. (2005). (B) Schematic diagram of the promoter region of gene expR which was cloned into pH3U3. “+1” in red was marked as the transcription start site of expR which was located in 87 bp upstream of ATG of expR (Charoenpanich et al., 2013). Regions R1, R2, and R4 were ligated to pH3U3 prey vector to detect the binding region of QsrR. Region R1 is the entire intergenic region between expR and SMc03900. Region R2 is the middle region of R1 which lost the 87bp right flank and the 76bp left flank of R1. Region R4 is the left flank of R1 (142 bp). The blue arrows indicate transcriptional direction of expR and SMc03900. Region R2 was excluded from further analyses because of the high level self-activation of (E. coli USO: pB1H1/pH3U3-R2) (see Materials and Methods for details). (C) The interaction of QsrR and pexpR was assayed by the growth of bacteria on selective medium (NM lacking histidine) supplemented with 2, 3, or 5 mM of 3-AT. The genetic information of test strains is labeled on the left. “+/–” indicated that qsrR gene was cloned into pB1H1 (+) or the plasmid was left empty (–), and similar information for pH3U3. Images were assembled and the marked lines were eliminated without modifying the observed data in Adobe Photoshop CS5.