Identification of Sinorhizobium meliloti Early Symbiotic Genes by Use of a Positive Functional Screen

The soil bacterium Sinorhizobium meliloti establishes nitrogen-fixing symbiosis with its leguminous host plant, alfalfa, following a series of continuous signal exchanges. The complexity of the changes of alfalfa root structures during symbiosis and the amount of S. meliloti genes with unknown functions raised the possibility that more S. meliloti genes may be required for early stages of the symbiosis. A positive functional screen of the entire S. meliloti genome for symbiotic genes was carried out using a modified in vivo expression technology. A group of genes and putative genes were found to be expressed in early stages of the symbiosis, and 23 of them were alfalfa root exudate inducible. These 23 genes were further separated into two groups based on their responses to apigenin, a known nodulation (nod) gene inducer. The group of six genes not inducible by apigenin included the lsrA gene, which is essential for the symbiosis, and the dgkA gene, which is involved in the synthesis of cyclic β-1,2-glucan required for the S. meliloti-alfalfa symbiosis. In the group of 17 apigenin-inducible genes, most have not been previously characterized in S. meliloti, and none of them belongs to the nod gene family. The identification of this large group of alfalfa root exudate-inducible S. meliloti genes suggests that the interactions in the early stages of the S. meliloti and alfalfa symbiosis could be complex and that further characterization of these genes will lead to a better understanding of the symbiosis.     

Growth-Related Substituent Changes in Exopolysaccharides of Fast-Growing Rhizobia

Pyruvic acid and O-acetyl groups are the major noncarbohydrate substituents in exopolysaccharides (EPS) produced by fast-growing species of Rhizobium. EPS substituent variations were observed among strains of the same species. The amounts of these substituents also varied with culture age; pyruvic acid increased in the EPS of all four species, whereas O-acetyl increased in Rhizobium trifolii and R. leguminosarum EPS, decreased in R. meliloti EPS, and remained constant in R. phaseoli EPS. The use of glycerol as a substrate for R. meliloti significantly increased EPS yields, whereas mannitol increased those of the other three Rhizobium species.     

Responses of the model legume Medicago truncatula to the rhizobial exopolysaccharide succinoglycan

Many species of rhizobial bacteria can invade their plant hosts and induce development of symbiotic nitrogen-fixing nodules only if they are able to produce an acidic exopolysaccharide (EPS) with certain structural and molecular weight characteristics.^1–3 Sinorhizobium meliloti that produces the functional form of the exopolysaccharide succinoglycan induces formation of invasion structures called infection threads in the root hair cells of its plant hosts alfalfa and Medicago truncatula. However, S. meliloti mutants that cannot produce succinoglycan are not able to induce infection thread formation, resulting in an early arrest of nodule development and in nitrogen starvation of the plant. Mounting evidence has suggested that succinoglycan acts as a signal to these host plants to permit the entry of S. meliloti. Now, our microarray screen and functional category analysis of differentially-expressed genes show that M. truncatula plants inoculated with wild type S. meliloti receive a signal to increase their translation capacity, alter their metabolic activity and prepare for invasion, while those inoculated with a succinoglycan-deficient mutant do not receive this signal, and also more strongly express plant defense genes.Key words: nitrogen fixation, nodule, succinoglycan, microarray, legume, rhizobial bacteria, Sinorhizobium meliloti, Medicago truncatula, infection thread, root hair     

Plant defence and delayed infection of alfalfa pseudonodules induced by an exopolysaccharide (EPS I)-deficient Rhizobium meliloti mutant

Mutants of the symbiotic soil bacterium Rhizobium meliloti that fail to synthesize the acidic exopolysaccharide EPS I were unable to induce infected root nodules on Medicago sativa L. (alfalfa). These strains, however, elicited pseudonodules that contained no infection threads or bacteroids. The cortical cell walls of the pseudonodules were abnormally thick and incrusted with an autofluorescent material. Parts of these cell walls and wall appositions contained callose. Biochemical analysis of nodules induced by the EPS I-deficient R. meliloti mutant revealed an increase of phenolic compounds bound to the nodule cell walls when compared with the wild-type strain. These microscopic and biochemical data indicated that a general plant defence response against the EPS I-deficient mutant of R. meliloti was induced in alfalfa pseudonodules. Following prolonged incubation with the EPS I-deficient R. meliloti mutant, the defence system of the alfalfa plant could be overcome by the rhizobium mutant. In the case of the delayed infections, the mutants colonized lobes of the pseudonodules, but the infection threads in these nodules had an abnormal morphology. They were greatly enlarged and did not contain the typical gum-like matrix inside. The bacteria were tightly packed. Based on the mechanism of phytopathogenic interactions, we propose that EPS I or a related compound may act as a suppressor of the alfalfa plant defence system, enabling R. meliloti to infect the plant.     

Phosphate concentration alters the effective bacterial quorum in the symbiosis of Medicago truncatula-Sinorhizobium meliloti

The symbiosis of Medicago truncatula-Sinorhizobium meliloti is affected by phosphate (P) deficiency in the environment. Quorum sensing (QS) is a regulatory pathway in S. meliloti that controls various functions of free-living and symbiotic bacteria in response to phosphate availability and regulation is mediated by a periplasmic protein PstS, and also bacterial density. The quorum sensing pathway of S. meliloti, involves three genes named sinI, sinR and expR and also some bacterial auto-inducers such as N-acyl homoserine lactones (AHLs). In the current study, the expression of the different genes of quorum sensing and pstS were evaluated under 0.1, 0.5 and 2 mM P. The qRT-PCR results showed an increased expression of pstS and also the quorum sensing genes sinI and sinR but not expR, following phosphate starvation. Indeed, the enhanced level of sinR induces the expression of sinI that is responsible for the N-acyl homoserine lactones (AHL) production in S. meliloti. The different response of expR may be due to its negative control on sinR expression. In the symbiosis of M. truncatula-S. meliloti, it was shown that the concentration of phosphate in the medium alters the effective inoculating bacterial quorum (density). By increasing the phosphate concentration in the medium from 0.1 to 0.5 and 2 mM, considering the optimal plant growth and pink nodule (nitrogen-fixing) formation, the effective inoculating bacterial densities were 10⁵, 10⁷ and 10⁹ CFU ml^?1, respectively. Therefore, low phosphate concentrations can compensate for a low bacterial density by inducing the quorum sensing pathway and establishing a symbiosis. Conversely, bacterial density plays the main role in the formation of symbiosis at high phosphate concentrations.     

Regulatory and DNA Repair Genes Contribute to the Desiccation Resistance of Sinorhizobium meliloti Rm1021

Sinorhizobium meliloti can form a nitrogen-fixing symbiotic relationship with alfalfa after bacteria in the soil infect emerging root hairs of the growing plant. To be successful at this, the bacteria must be able to survive in the soil between periods of active plant growth, including when conditions are dry. The ability of S. meliloti to withstand desiccation has been known for years, but genes that contribute to this phenotype have not been identified. Transposon mutagenesis was used in combination with novel screening techniques to identify four desiccation-sensitive mutants of S. meliloti Rm1021. DNA sequencing of the transposon insertion sites identified three genes with regulatory functions (relA, rpoE2, and hpr) and a DNA repair gene (uvrC). Various phenotypes of the mutants were determined, including their behavior on several indicator media and in symbiosis. All of the mutants formed an effective symbiosis with alfalfa. To test the hypothesis that UvrC-related excision repair was important in desiccation resistance, uvrA, uvrB, and uvrC deletion mutants were also constructed. These strains were sensitive to DNA damage induced by UV light and 4-NQO and were also desiccation sensitive. These data indicate that uvr gene-mediated DNA repair and the regulation of stress-induced pathways are important for desiccation resistance.     

Succinoglycan Production by Rhizobium meliloti Is Regulated through the ExoS-ChvI Two-Component Regulatory System

The Rhizobium meliloti exoS gene is involved in regulating the production of succinoglycan, which plays a crucial role in the establishment of the symbiosis between R. meliloti Rm1021 and its host plant, alfalfa. The exoS96::Tn5 mutation causes the upregulation of the succinoglycan biosynthetic genes, thereby resulting in the overproduction of succinoglycan. Through cloning and sequencing, we found that the exoS gene is a close homolog of the Agrobacterium tumefaciens chvG gene, which has been proposed to encode the sensor protein of the ChvG-ChvI two-component regulatory system, a member of the EnvZ-OmpR family. Further analyses revealed the existence of a newly discovered A. tumefaciens chvI homolog located just upstream of the R. meliloti exoS gene. R. meliloti ChvI may serve as the response regulator of ExoS in a two-component regulatory system. By using ExoS-specific antibodies, it was found that the ExoS protein cofractionated with membrane proteins, suggesting that it is located in the cytoplasmic membrane. By using the same antibodies, it was shown that the exoS96::Tn5 allele encodes an N-terminal truncated derivative of ExoS. The cytoplasmic histidine kinase domain of ExoS was expressed in Escherichia coli and purified, as was the R. meliloti ChvI protein. The ChvI protein autophosphorylated in the presence of acetylphosphate, and the ExoS cytoplasmic domain fragment autophosphorylated at a histidine residue in the presence of ATP. The ChvI protein was phosphorylated in the presence of ATP only when the histidine kinase domain of ExoS was also present. We propose a model for regulation of succinoglycan production by R. meliloti through the ExoS-ChvI two-component regulatory system.     

Nodules Initiated by Rhizobium meliloti Exopolysaccharide Mutants Lack a Discrete, Persistent Nodule Meristem

Infection of alfalfa with Rhizobium meliloti exo mutants deficient in exopolysaccharide results in abnormal root nodules that are devoid of bacteria and fail to fix nitrogen. Here we report further characterization of these abnormal nodules. Tightly curled root hairs or shepherd's crooks were found after inoculation with Rm 1021-derived exo mutants, but curling was delayed compared with wild-type Rm 1021. Infection threads were initiated in curled root hairs by mutants as well as by wild-type R. meliloti, but the exo mutant-induced threads aborted within the peripheral cells of the developing nodule. Also, nodules elicited by Rm 1021-derived exo mutants were more likely to develop on secondary roots than on the primary root. In contrast with wild-type R. meliloti-induced nodules, the exo mutant-induced nodules lacked a well defined apical meristem, presumably due to the abortion of the infection threads. The relationship of these findings to the physiology of nodule development is discussed.     

Null mutations in Sinorhizobium meliloti exoS and chvI demonstrate the importance of this two‐component regulatory system for symbiosis

Exopolysaccharides, either succinoglycan or galactoglucan, are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two‐component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid‐facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two‐component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. Other studied alpha‐proteobacteria ExoS/ChvI orthologues are required for the bacteria to invade or persist in host cells and thus we present more evidence that this two‐component regulatory system is essential for alpha‐proteobacterial host interaction.     

Analysis of the Rhizobium meliloti exoH/exoK/exoL fragment: ExoK shows homology to excreted endo-β-1,3-1,4-glucanases and ExoH resembles membrane proteins

Nucleotide sequencing of a 4.15 kb DNA fragment from megaplasmid 2 of Rhizobium meliloti 2011 revealed the location of the genes exoH, exoK and exoL. The putative proteins encoded by these genes have molecular weights of 41, 30, and 44 kDa, respectively. The hydrophobicity profile of the ExoH amino acid sequence resembles that of transmembrane proteins. The predicted exoL gene product does not contain hydrophobic regions, indicating a cytoplasmic localization. The exoK gene product is characterized by a putative signal peptide and exhibits significant homology to endo-β-1,3 1,4-glucanases of bacilli and Clostridium thermocellum. R. meliloti exoK mutants induced pink nodules and synthesized a reduced amount of exopolysaccharide (EPS). Colonies of this mutant showed a delay in the appearance of the Calcofluor white fluorescence. In addition, the formation of the characteristic halo was strongly delayed. R. meliloti exoL and exoH mutants induced pseudonodules. The exoH, but not the exoL mutant, synthesized an EPS that could be precipitated by cetyl pyridinium chloride (CPC) and also by ethanol. Plasmid integration mutagenesis revealed promoter regions preceding exoH, exoK and exoL.