Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones

Sinorhizobium meliloti is a soil bacterium which can establish a nitrogen-fixing symbiosis with the legume Medicago sativa. Recent work has identified a pair of genes, sinR and sinI, which represent a potential quorum-sensing system and are responsible for the production of N-acyl homoserine lactones (AHLs) in two S. meliloti strains, Rm1021 and Rm41. In this work, we characterize the sinRI locus and show that these genes are responsible for the synthesis of several long-chain AHLs ranging from 12 to 18 carbons in length. Four of these, 3-oxotetradecanoyl HL, 3-oxohexadecenoyl HL, hexadecenoyl HL, and octadecanoyl HL, have novel structures. This is the first report of AHLs having acyl chains longer than 14 carbons. We show that a disruption in sinI eliminates these AHLs and that a sinR disruption results in only basal levels of the AHLs. Moreover, the same sinI and sinR mutations also lead to a decrease in the number of pink nodules during nodulation assays, as well as a slight delay in the appearance of pink nodules, indicating a role for quorum sensing in symbiosis. We also show that sinI and sinR mutants are still capable of producing several short-chain AHLs, one of which was identified as octanoyl HL. We believe that these short-chain AHLs are evidence of a second quorum-sensing system in Rm1021, which we refer to here as the mel system, for "S. meliloti."     

Effector-stimulated single molecule protein-DNA interactions of a quorum-sensing system in Sinorhizobium meliloti

Intercellular communication by means of small signal molecules coordinates gene expression among bacteria. This population density-dependent regulation is known as quorum sensing. The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti Rm1021 possesses the Sin quorum sensing system based on N-acyl homoserine lactones (AHL) as signal molecules. Here, we demonstrate that the LuxR-type regulator ExpR binds specifically to a target sequence in the sinRI locus in the presence of different AHLs with acyl side chains from 8 to 20 carbons. Dynamic force spectroscopy based on the atomic force microscope provided detailed information about the molecular mechanism of binding upon activation by six different AHLs. These single molecule experiments revealed that the mean lifetime of the bound protein-DNA complex varies depending on the specific effector molecule. The small differences between individual AHLs also had a pronounced influence on the structure of protein-DNA interaction: The reaction length of dissociation varied from 2.6 to 5.8 A. In addition, dynamic force spectroscopy experiments indicate that N-heptanoyl-DL-homoserine lactone binds to ExpR but is not able to stimulate protein-DNA interaction.     

L-Canavanine made by Medicago sativa interferes with quorum sensing in Sinorhizobium meliloti

Sinorhizobium meliloti is a gram-negative soil bacterium, capable of establishing a nitrogen-fixing symbiosis with its legume host, alfalfa (Medicago sativa). Quorum sensing plays a crucial role in this symbiosis, where it influences the nodulation process and the synthesis of the symbiotically important exopolysaccharide II (EPS II). S. meliloti has three quorum-sensing systems (Sin, Tra, and Mel) that use N-acyl homoserine lactones as their quorum-sensing signal molecule. Increasing evidence indicates that certain eukaryotic hosts involved in symbiotic or pathogenic relationships with gram-negative bacteria produce quorum-sensing-interfering (QSI) compounds that can cross-communicate with the bacterial quorum-sensing system. Our studies of alfalfa seed exudates suggested the presence of multiple signal molecules capable of interfering with quorum-sensing-regulated gene expression in different bacterial strains. In this work, we choose one of these QSI molecules (SWI) for further characterization. SWI inhibited violacein production, a phenotype that is regulated by quorum sensing in Chromobacterium violaceum. In addition, this signal molecule also inhibits the expression of the S. meliloti exp genes, responsible for the production of EPS II, a quorum-sensing-regulated phenotype. We identified this molecule as l-canavanine, an arginine analog, produced in large quantities by alfalfa and other legumes.     

RIVET-a tool for in vivo analysis of symbiotically relevant gene expression in Sinorhizobium meliloti

Despite significant advances in the development of sensitive tools for studying genetics and signal exchange in legume-rhizobium symbioses, many uncertainties remain about the in vivo role of bacterial and plant signals in symbiotic gene regulation. In this study, we adapted TnpR recombinase-based in vivo expression technology (RIVET) to document gene regulation in Sinorhizobium meliloti. The substrate for TnpR, the res1-tet-res1 cassette, is stably inherited when cloned into a neutral site of the S. meliloti genome. Bicistronic promoterless tnpR-beta-glucuronidase (GUS) reporters were constructed to track expression ("resolution") of symbiotically relevant S. meliloti genes during different stages of the interaction. In proof of principle experiments, the resolution of the nodC::tnpR reporter was detected within 4 h of exposure to micromolar levels of the nod operon inducer luteolin and after overnight incubation in the rhizosphere. RIVET demonstrated that cell division gene ftsZ2 was not strongly expressed in the rhizosphere but was activated inside the nodules and on agar surfaces. Rhizosphere expression of the N-acyl homoserine lactone (AHL) synthase sinI::tnpR-GUS reporter was modest in prequorate microcolonies, and then increased with time. AHL synthase sinI and an AHL-regulated gene, expG, were activated inside the nodules.     

Identification of two quorum-sensing systems in Sinorhizobium meliloti

Sinorhizobium meliloti is a free-living soil bacterium which is capable of establishing a symbiotic relationship with the alfalfa plant (Medicago sativa). This symbiosis involves a network of bacterium-host signaling, as well as the potential for bacterium-bacterium communication, such as quorum sensing. In this study, we characterized the production of N-acyl homoserine lactones (AHLs) by two commonly used S. meliloti strains, AK631 and Rm1021. We found that AK631 produces at least nine different AHLs, while Rm1021 produces only a subset of these molecules. To address the difference in AHL patterns between the strains, we developed a novel screening method to identify the genes affecting AHL synthesis. With this screening method, chromosomal groEL (groELc) was shown to be required for synthesis of the AHLs that are unique to AK631 but not for synthesis of the AHLs that are made by both AK631 and Rm1021. We then used the screening procedure to identify a mutation in a gene homologous to traM of Agrobacterium tumefaciens, which was able to suppress the phenotype of the groELc mutation. A traR homolog was identified immediately upstream of traM, and we propose that its gene product requires a functional groELc for activity and is also responsible for inducing the synthesis of the AHLs that are unique to AK631. We show that the traR/traM locus is part of a quorum-sensing system unique to AK631 and propose that this locus is involved in regulating conjugal plasmid transfer. We also present evidence for the existence of a second quorum-sensing system, sinR/sinI, which is present in both AK631 and Rm1021.     

Competitive and cooperative effects in quorum-sensing-regulated galactoglucan biosynthesis in Sinorhizobium meliloti

The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti possesses the Sin quorum-sensing system based on N-acyl homoserine lactones (AHLs) as signal molecules. The Sin system consists of SinI, the AHL synthase, and SinR, the LuxR-type regulator. This system regulates the expression of a multitude of S. meliloti genes through ExpR, another LuxR-type regulator. Analysis of the activity of the sinI promoter showed that the expression of sinI is dependent on sinR and enhanced by a combination of expR and Sin AHLs. The characterization of the ExpR binding site upstream of sinI and the identification of binding sites upstream of the galactoglucan biosynthesis genes wgaA (expA1) and wgeA (expE1) allowed the definition of a consensus sequence for these binding sites. Based on this consensus, two additional ExpR binding sites in the promoter regions of exoI and exsH, two genes related to the production of succinoglycan, were found. The specific binding of ExpR to the wgaA and wgeA promoters was enhanced in the presence of oxo-C(14)-HL. Positive regulation of the galactoglucan biosynthesis genes by ExpR was shown to be dependent on WggR (ExpG) and influenced by MucR, both of which are previously characterized regulators of these genes. Based on these results, a reworked model of the Sin-ExpR quorum-sensing regulation scheme of galactoglucan production in S. meliloti is suggested.     

Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajan

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.     

EPS II-Dependent Autoaggregation of Sinorhizobium meliloti Planktonic Cells

Planktonic cells of Sinorhizobium meliloti, a Gram-negative symbiotic bacterium, display autoaggregation under static conditions. ExpR is a LuxR-type regulator that controls many functions in S. meliloti, including synthesis of two exopolysaccharides, EPS I (succinoglycan) and EPS II (galactoglucan). Since exopolysaccharides are important for bacterial attachment, we studied the involvement of EPS I and II in autoaggregation of S. meliloti. Presence of an intact copy of the expR locus was shown to be necessary for autoaggregation. A mutant incapable of producing EPS I displayed autoaggregation percentage similar to that of parental strain, whereas autoaggregation was significantly lower for a mutant defective in biosynthesis of EPS II. Our findings clearly indicate that EPS II is the essential component involved in autoaggregation of planktonic S. meliloti cells, and that EPS I plays no role in such aggregation.     

Regulation of succinoglycan and galactoglucan biosynthesis in Sinorhizobium meliloti

Sinorhizobium meliloti (Rhizobium meliloti) 2011 has the ability to produce the two acidic exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II). EPS I is a branched heteropolysaccharide composed of octasaccharide repeating units, whereas EPS II is a linear heteropolysaccharide consisting of disaccharide subunits. The exo-exs and exp gene clusters are involved in the biosynthesis of EPSI and EPSII, respectively. EPSI and EPSII biosynthesis genes are differentially expressed resulting in a complex regulation of EPS production in S. meliloti. The phosphate concentration was identified as an important factor affecting the expression of exp genes.     

Multiple N-Acyl Homoserine Lactone Signals of Rhizobium leguminosarum Are Synthesized in a Distinct Temporal Pattern

A common form of bacterial quorum sensing involves the production and release of acyl homoserine lactone (AHL) signal metabolites. The nitrogen-fixing symbiont Rhizobium leguminosarum reportedly produces at least six different AHLs, but little is known about the regulation of biosynthesis of these molecules. We used a radiolabeling protocol to quantify the relative amounts of AHLs synthesized over time by R. leguminosarum cells with and without the symbiosis plasmid pRL1JI. Cells containing pRL1JI were found to produce three predominant signals. In decreasing order of abundance, these were N-(3-oxo)octanoyl homoserine lactone [(3-O)C(8)HSL], N-octanoyl homoserine lactone, and N-hexanoyl homoserine lactone. Cells without pRL1JI produced only two major signals, N-(3-hydroxy-7-cis)tetradecanoyl homoserine lactone [(3-OH)C(14:1)HSL] and (3-O)C(8)HSL. Each AHL exhibited a distinct temporal pattern of synthesis, suggesting that each AHL is subject to unique regulatory mechanisms. While (3-O)C(8)HSL was produced in both cultures, the patterns of synthesis were different in cells with and without pRL1JI, possibly as a result of redundant gene functions that are present on both the chromosome and the symbiosis plasmid. None of the AHLs appeared to regulate its own biosynthesis, although exogenous (3-OH)C(14:1)HSL did activate synthesis of the three AHLs made by cells containing pRL1JI. These results indicate that the synthesis of multiple AHLs in R. leguminosarum is regulated by complex mechanisms that operate independently of quorum sensing itself but that (3-OH)C(14:1)HSL can supersede these controls in pRL1JI-containing cells. This work provides an important global perspective for AHL regulation that both complements and contrasts with the results of previous studies performed with isolated gene systems.     

Alfalfa root nodule invasion efficiency is dependent on Sinorhizobium meliloti polysaccharides

The soil bacterium Sinorhizobium meliloti is capable of entering into a nitrogen-fixing symbiosis with Medicago sativa (alfalfa). Particular low-molecular-weight forms of certain polysaccharides produced by S. meliloti are crucial for establishing this symbiosis. Alfalfa nodule invasion by S. meliloti can be mediated by any one of three symbiotically important polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). Using green fluorescent protein-labeled S. meliloti cells, we have shown that there are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both infection thread initiation and extension. However, EPS II is significantly less efficient than succinoglycan at mediating both invasion steps, and K antigen is significantly less efficient than succinoglycan at mediating infection thread extension. In the case of EPS II-mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen-producing strains. Additionally, EPS II- and K-antigen-mediated infection threads are 8 to 10 times more likely to have aberrant morphologies than those mediated by succinoglycan. These data have important implications for understanding how S. meliloti polysaccharides are functioning in the plant-bacterium interaction, and models are discussed.     

The LuxR homolog ExpR, in combination with the Sin quorum sensing system, plays a central role in Sinorhizobium meliloti gene expression

Quorum sensing, a population density-dependent mechanism for bacterial communication and gene regulation, plays a crucial role in the symbiosis between alfalfa and its symbiont Sinorhizobium meliloti. The Sin system, one of three quorum sensing systems present in S. meliloti, controls the production of the symbiotically active exopolysaccharide EPS II. Based on DNA microarray data, the Sin system also seems to regulate a multitude of S. meliloti genes, including genes that participate in low-molecular-weight succinoglycan production, motility, and chemotaxis, as well as other cellular processes. Most of the regulation by the Sin system is dependent on the presence of the ExpR regulator, a LuxR homolog. Gene expression profiling data indicate that ExpR participates in additional cellular processes that include nitrogen fixation, metabolism, and metal transport. Based on our microarray analysis we propose a model for the regulation of gene expression by the Sin/ExpR quorum sensing system and another possible quorum sensing system(s) in S. meliloti.     

Cloning and characterization of Rhizobium meliloti loci required for symbiotic root nodule invasion

An immunological assay of root nodule polypeptides was used to analyze the nodules induced by 25 symbiotically defective Rhizobium meliloti mutants. Differences in polypeptide accumulation in these nodules were used to divide the mutants into three subsets. One subset, containing two mutant strains, was further analyzed. Nodules induced by these mutant strains lack both infection threads and bacteria. The kinetics of nodule formation by these mutant strains, by an exoB mutant, and by mixed mutant inocula suggest that the gene products required for nodule invasion may also influence nodule meristem induction. One of the two mutants characterized in this study contains a transposon Tn5 insertion in the ndvB locus, which probably results in the loss of beta-glucan synthesis. The second mutant contains a transposon in a previously uncharacterized locus. RNA analysis suggests that the newly identified locus is transcribed in free-living cultures of ndvB and exoB strains, as well as in the parental R. meliloti strain. Southern blot analysis suggests that at least a portion of this locus is duplicated. This duplication may explain the apparently leaky phenotype of the mutant strain.