Medicae WSM419 Research Articles

Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-Rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system

The host-produced nodule specific cysteine-rich (NCR) peptides control the terminal differentiation of endosymbiotic rhizobia in the nodules of IRLC legumes. Although the Medicago truncatula genome encodes about 700 NCR peptides, only few of them have been proven to be crucial for nitrogen-fixing symbiosis. In this study, we applied the CRISPR/Cas9 gene editing technology to generate knockout mutants of NCR genes for which no genetic or functional data were previously available. We have developed a workflow to analyse the mutation and the symbiotic phenotype of individual nodules formed on Agrobacterium rhizogenes-mediated transgenic hairy roots. The selected NCR genes were successfully edited by the CRISPR/Cas9 system and nodules formed on knockout hairy roots showed wild type phenotype indicating that peptides NCR068, NCR089, NCR128 and NCR161 are not essential for symbiosis between M. truncatula Jemalong and Sinorhizobium medicae WSM419. We regenerated stable mutants edited for the NCR068 from hairy roots obtained by A. rhizogenes-mediated transformation. The analysis of the symbiotic phenotype of stable ncr068 mutants showed that peptide NCR068 is not required for symbiosis with S. meliloti strains 2011 and FSM-MA either. Our study reports that gene editing can help to elicit the role of certain NCRs in symbiotic nitrogen fixation.

Specialization in a Nitrogen-Fixing Symbiosis: Proteome Differences Between Sinorhizobium medicae Bacteria and Bacteroids.

Using tandem mass spectrometry (MS/MS), we analyzed the proteome of Sinorhizobium medicae WSM419 growing as free-living cells and in symbiosis with Medicago truncatula. In all, 3,215 proteins were identified, over half of the open reading frames predicted from the genomic sequence. The abundance of 1,361 proteins displayed strong lifestyle bias. In total, 1,131 proteins had similar levels in bacteroids and free-living cells, and the low levels of 723 proteins prevented statistically significant assignments. Nitrogenase subunits comprised approximately 12% of quantified bacteroid proteins. Other major bacteroid proteins included symbiosis-specific cytochromes and FixABCX, which transfer electrons to nitrogenase. Bacteroids had normal levels of proteins involved in amino acid biosynthesis, glycolysis or gluconeogenesis, and the pentose phosphate pathway; however, several amino acid degradation pathways were repressed. This suggests that bacteroids maintain a relatively independent anabolic metabolism. Tricarboxylic acid cycle proteins were highly expressed in bacteroids and no other catabolic pathway emerged as an obvious candidate to supply energy and reductant to nitrogen fixation. Bacterial stress response proteins were induced in bacteroids. Many WSM419 proteins that are not encoded in S. meliloti Rm1021 were detected, and understanding the functions of these proteins might clarify why S. medicae WSM419 forms a more effective symbiosis with M. truncatula than S. meliloti Rm1021.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative Genomics across Three Ensifer Species Using a New Complete Genome Sequence of the Medicago Symbiont Sinorhizobium (Ensifer) meliloti WSM1022.

Here, we report an improved and complete genome sequence of Sinorhizobium (Ensifer) meliloti strain WSM1022, a microsymbiont of Medicago species, revealing its tripartite structure. This improved genome sequence was generated combining Illumina and Oxford nanopore sequencing technologies to better understand the symbiotic properties of the bacterium. The 6.75 Mb WSM1022 genome consists of three scaffolds, corresponding to a chromosome (3.70 Mb) and the pSymA (1.38 Mb) and pSymB (1.66 Mb) megaplasmids. The assembly has an average GC content of 62.2% and a mean coverage of 77X. Genome annotation of WSM1022 predicted 6058 protein coding sequences (CDSs), 202 pseudogenes, 9 rRNAs (3 each of 5S, 16S, and 23S), 55 tRNAs, and 4 ncRNAs. We compared the genome of WSM1022 to two other rhizobial strains, closely related Sinorhizobium (Ensifer) meliloti Sm1021 and Sinorhizobium (Ensifer) medicae WSM419. Both WSM1022 and WSM419 species are high-efficiency rhizobial strains when in symbiosis with Medicago truncatula, whereas Sm1021 is ineffective. Our findings report significant genomic differences across the three strains with some similarities between the meliloti strains and some others between the high efficiency strains WSM1022 and WSM419. The addition of this high-quality rhizobial genome sequence in conjunction with comparative analyses will help to unravel the features that make a rhizobial symbiont highly efficient for nitrogen fixation.

Sinorhizobium medicae WSM419 Genes That Improve Symbiosis between Sinorhizobium meliloti Rm1021 and Medicago truncatula Jemalong A17 and in Other Symbiosis Systems.

Some soil bacteria, called rhizobia, can interact symbiotically with legumes, in which they form nodules on the plant roots, where they can reduce atmospheric dinitrogen to ammonia, a form of nitrogen that can be used by growing plants. Rhizobium-plant combinations can differ in how successful this symbiosis is: for example, Sinorhizobium meliloti Rm1021 forms a relatively ineffective symbiosis with Medicago truncatula Jemalong A17, but Sinorhizobium medicae WSM419 is able to support more vigorous plant growth. Using proteomic data from free-living and symbiotic S. medicae WSM419, we previously identified a subset of proteins that were not closely related to any S. meliloti Rm1021 proteins and speculated that adding one or more of these proteins to S. meliloti Rm1021 would increase its effectiveness on M. truncatula A17. Three genes, Smed_3503, Smed_5985, and Smed_6456, were cloned into S. meliloti Rm1021 downstream of the E. coli lacZ promoter. Strains with these genes increased nodulation and improved plant growth, individually and in combination with one another. Smed_3503, renamed iseA (increased symbiotic effectiveness), had the largest impact, increasing M. truncatula biomass by 61%. iseA homologs were present in all currently sequenced S. medicae strains but were infrequent in other Sinorhizobium isolates. Rhizobium leguminosarum bv. viciae 3841 containing iseA led to more nodules on pea and lentil. Split-root experiments with M. truncatula A17 indicated that S. meliloti Rm1021 carrying the S. medicae iseA is less sensitive to plant-induced resistance to rhizobial infection, suggesting an interaction with the plant's regulation of nodule formation. IMPORTANCE Legume symbiosis with rhizobia is highly specific. Rhizobia that can nodulate and fix nitrogen on one legume species are often unable to associate with a different species. The interaction can be more subtle. Symbiotically enhanced growth of the host plant can differ substantially when nodules are formed by different rhizobial isolates of a species, much like disease severity can differ when conspecific isolates of pathogenic bacteria infect different cultivars. Much is known about bacterial genes essential for a productive symbiosis, but less is understood about genes that marginally improve performance. We used a proteomic strategy to identify Sinorhizobium genes that contribute to plant growth differences that are seen when two different strains nodulate M. truncatula A17. These genes could also alter the symbiosis between R. leguminosarum bv. viciae 3841 and pea or lentil, suggesting that this approach identifies new genes that may more generally contribute to symbiotic productivity.

Evolution of a multi-step phosphorelay signal transduction system in Ensifer: recruitment of the sigma factor RpoN and a novel enhancer-binding protein triggers acid-activated gene expression.

Most Ensifer strains are comparatively acid sensitive, compromising their persistence in low pH soils. In the acid-tolerant strain Ensifer medicae WSM419, the acid-activated expression of lpiA is essential for enhancing survival in lethal acidic conditions. Here we characterise a multi-step phosphorelay signal transduction pathway consisting of TcsA, TcrA, FsrR, RpoN and its cognate enhancer-binding protein EbpA, which is required for the induction of lpiA and the downstream acvB gene. The fsrR, tcrA, tcsA and rpoN genes were constitutively expressed, whereas lpiA and acvB were strongly acid-induced. RACE mapping revealed that lpiA/acvB were co-transcribed as an operon from an RpoN promoter. In most Ensifer species, lpiA/acvB is located on the chromosome and the sequence upstream of lpiA lacks an RpoN-binding site. Nearly all Ensifer meliloti strains completely lack ebpA, tcrA, tcsA and fsrR regulatory loci. In contrast, E. medicae strains have lpiA/acvB and ebpA/tcrA/tcsA/fsrR co-located on the pSymA megaplasmid, with lpiA/acvB expression coupled to an RpoN promoter. Here we provide a model for the expression of lpiA/acvB in E. medicae. This unique acid-activated regulatory system provides insights into an evolutionary process which may assist the adaptation of E. medicae to acidic environmental niches.

Colonization of Microbial Biofilms in Pipeline of Water Reuse

Aim of this study was to investigate biofilms attached in pipeline of water reuse from the MBR system treating sewage without chlorination in correlation to the outflow water quality. Two general pipe materials: polyvinyl chloride (PVC) and polyethylene (PE) were employed in the experiment. The peak growths were found at week 4 in both pipes. The maximum biofilms in PE pipe was 33 mgVSS/cm 2 with the growth rate of 4.75 mgVSS/cm 2 -d which was significant higher than that of PVC pipe. Biofilms examined by PCR-DGGE technique revealed five bacterial species in PE biofilms namely Sinorhizobium medicae WSM419, Sinorhizobium fredii NGR234, Geobacter sp. M18, Parachlamydia acanthamoebae UV-7, and Mycobacterium chubuense NBB4. The VSS concentrations in outflow had directly correlated to the biofilm attachment and detachment. High COD concentrations of outflow appeared during biofilm detaching phase. In summary, water quality of reuse water corresponded to the biofilms attachment and detachment in the pipeline.

Nodule carbohydrate catabolism is enhanced in the Medicago truncatula A17-Sinorhizobium medicae WSM419 symbiosis.

The symbiotic association between Medicago truncatula and Sinorhizobium meliloti is a well-established model system in the legume–Rhizobium community. Despite its wide use, the symbiotic efficiency of this model has been recently questioned and an alternative microsymbiont, S. medicae, has been proposed. However, little is known about the physiological mechanisms behind the higher symbiotic efficiency of S. medicae WSM419. In the present study, we inoculated M. truncatula Jemalong A17 with either S. medicae WSM419 or S. meliloti 2011 and compared plant growth, photosynthesis, N2-fixation rates, and plant nodule carbon and nitrogen metabolic activities in the two systems. M. truncatula plants in symbiosis with S. medicae showed increased biomass and photosynthesis rates per plant. Plants grown in symbiosis with S. medicae WSM419 also showed higher N2-fixation rates, which were correlated with a larger nodule biomass, while nodule number was similar in both systems. In terms of plant nodule metabolism, M. truncatula–S. medicae WSM419 nodules showed increased sucrose-catabolic activity, mostly associated with sucrose synthase, accompanied by a reduced starch content, whereas nitrogen-assimilation activities were comparable to those measured in nodules infected with S. meliloti 2011. Taken together, these results suggest that S. medicae WSM419 is able to enhance plant carbon catabolism in M. truncatula nodules, which allows for the maintaining of high symbiotic N2-fixation rates, better growth and improved general plant performance.

Enhanced Nodulation and Nodule Development by nolR Mutants of Sinorhizobium medicae on Specific Medicago Host Genotypes

The nolR gene encodes a negatively acting, transcriptional regulatory protein of core Nod-factor biosynthetic genes in the sinorhizobia. Although previous reports showed that nolR modulates Nod-factor production and enhances nodulation speed of Sinorhizobium meliloti on alfalfa, there have been no reports for the symbiotic function of this gene in the S. medicae-Medicago truncatula symbiosis. Here, we constructed an nolR mutant of S. medicae WSM419 and evaluated mutant and wild-type strains for their nodulation ability, competitiveness, host specificity, and density-dependent nodulation phenotypes. When the mutant was inoculated at low and medium population densities, it showed enhanced nodule formation during the initial stages of nodulation. Results of quantitative competitive nodulation assays indicated that an nolR mutant had 2.3-fold greater competitiveness for nodulation on M. truncatula 'A17' than did the wild-type strain. Moreover, the nodulation phenotype of the nolR mutant differed among Medicago genotypes and showed significantly enhanced nodule development on M. tricycla. Taken together, these results indicated that mutation of nolR in S. medicae positively influenced nodule initiation, competitive nodulation, and nodule development at later nodulation stages. This may allow nolR mutants of S. medicae to have a selective advantage under field conditions.

RptA, a novel gene fromEnsifer(Sinorhizobium)melilotiinvolved in conjugal transfer

We approached the identification of Ensifer (Sinorhizobium) meliloti conjugal functions by random Tn5-B13 mutagenesis of the pSmeLPU88a plasmid of E.meliloti strain LPU88 and the subsequent selection of those mutants that had lost the ability to mobilize the small plasmid pSmeLPU88b. The Tn5-B13-insertion site of one of the mutants was cloned as an EcoRI-restricted DNA fragment that after subsequent isolation and sequencing demonstrated that a small open reading frame of 522bp (designated rptA, for rhizobium plasmid transfer A) had been disrupted. The predicted gene product encoded by the rptA sequence shows a significant similarity to two hypothetical proteins of the plasmid pSmed03 of Ensifer medicae WSM419 and other rhizobia plasmids. No significant similarity was found to any protein sequence of known function registered in the databases. Although the rptA gene was required for pSmeLPU88b-plasmid mobilization in the strain 2011 background, it was not required in the original strain LPU88 background.

Enhanced nodulation and symbiotic effectiveness of Medicago truncatula when co-inoculated with Pseudomonas fluorescens WSM3457 and Ensifer (Sinorhizobium) medicae WSM419

Low numbers of rhizobia in soil or inoculants delay nodulation and decrease symbiotic legume productivity. This study investigated the effect of co-inoculation with a helper bacterium, Pseudomonas fluorescens WSM3457 on the Medicago truncatula - Ensifer (Sinorhizobium) medicae WSM419 symbiosis challenged by a low inoculum dose. In a glasshouse experiment the effect of co-inoculation with WSM3457 on the kinetics of nodule initiation and development was assessed 5, 7, 10, 14, 17, 21, and 42 days after inoculation of M. truncatula cv. Caliph with 103 cells/plant of E. medicae WSM419. Co-inoculated plants had enhanced rate of nodule initiation and development, greater numbers of larger crown nodules, and by day 42 accumulated more N than plants inoculated with E. medicae WSM419 alone. Nodule development was altered by co-inoculation. Approximately 25% of nodule initials on co-inoculated plants formed in closely associated pairs, young nodules were larger with multiple meristems and developed into cluster-like multi-lobed nodules compared to those on WSM419 inoculated plants. Molecular typing showed WSM3457 occupied a significant proportion of root nodules on co-inoculated plants. Co-inoculation with P. fluorescens WSM3457 enhanced symbiotic effectiveness of M. truncatula when inoculated with a low inoculum dose of E. medicae WSM419.

The complete genome sequence of the dominant Sinorhizobium meliloti field isolate SM11 extends the S. meliloti pan-genome

Isolates of the symbiotic nitrogen-fixing species Sinorhizobium meliloti usually contain a chromosome and two large megaplasmids encoding functions that are absolutely required for the specific interaction of the microsymbiont with corresponding host plants leading to an effective symbiosis. The complete genome sequence, including the megaplasmids pSmeSM11c (related to pSymA) and pSmeSM11d (related to pSymB), was established for the dominant, indigenous S. meliloti strain SM11 that had been isolated during a long-term field release experiment with genetically modified S. meliloti strains. The chromosome, the largest replicon of S. meliloti SM11, is 3,908,022bp in size and codes for 3785 predicted protein coding sequences. The size of megaplasmid pSmeSM11c is 1,633,319bp and it contains 1760 predicted protein coding sequences whereas megaplasmid pSmeSM11d is 1,632,395bp in size and comprises 1548 predicted coding sequences. The gene content of the SM11 chromosome is quite similar to that of the reference strain S. meliloti Rm1021. Comparison of pSmeSM11c to pSymA of the reference strain revealed that many gene regions of these replicons are variable, supporting the assessment that pSymA is a major hot-spot for intra-specific differentiation. Plasmids pSymA and pSmeSM11c both encode unique genes. Large gene regions of pSmeSM11c are closely related to corresponding parts of Sinorhizobium medicae WSM419 plasmids. Moreover, pSmeSM11c encodes further novel gene regions, e.g. additional plasmid survival genes (partition, mobilisation and conjugative transfer genes), acdS encoding 1-aminocyclopropane-1-carboxylate deaminase involved in modulation of the phytohormone ethylene level and genes having predicted functions in degradative capabilities, stress response, amino acid metabolism and associated pathways. In contrast to Rm1021 pSymA and pSmeSM11c, megaplasmid pSymB of strain Rm1021 and pSmeSM11d are highly conserved showing extensive synteny with only few rearrangements. Most remarkably, pSmeSM11b contains a new gene cluster predicted to be involved in polysaccharide biosynthesis. Compilation of the S. meliloti SM11 genome sequence contributes to an extension of the S. meliloti pan-genome.

Identification of Sinorhizobium (Ensifer) medicae based on a specific genomic sequence unveiled by M13-PCR fingerprinting.

A collection of nodule isolates from Medicago polymorpha obtained from southern and central Portugal was evaluated by M13-PCR fingerprinting and hierarchical cluster analysis. Several genomic clusters were obtained which, by 16S rRNA gene sequencing of selected representatives, were shown to be associated with particular taxonomic groups of rhizobia and other soil bacteria. The method provided a clear separation between rhizobia and co-isolated non-symbiotic soil contaminants. Ten M13-PCR groups were assigned to Sinorhizobium (Ensifer) medicae and included all isolates responsible for the formation of nitrogen-fixing nodules upon re-inoculation of M. polymorpha test-plants. In addition, enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting indicated a high genomic heterogeneity within the major M13- PCR clusters of S. medicae isolates. Based on nucleotide sequence data of an M13-PCR amplicon of ca. 1500 bp, observed only in S. medicae isolates and spanning locus Smed_3707 to Smed_3709 from the pSMED01 plasmid sequence of S. medicae WSM419 genome's sequence, a pair of PCR primers was designed and used for direct PCR amplification of a 1399-bp sequence within this fragment. Additional in silico and in vitro experiments, as well as phylogenetic analysis, confirmed the specificity of this primer combination and therefore the reliability of this approach in the prompt identification of S. medicae isolates and their distinction from other soil bacteria.

The model legume Medicago truncatula A17 is poorly matched for N2 fixation with the sequenced microsymbiont Sinorhizobium meliloti 1021

Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021-M. truncatula symbiosis at fixing N(2) was evaluated. N(2) fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed. Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells. The Sm1021-M. truncatula symbiosis is poorly matched for N(2) fixation and the strain could possess broader N(2) fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N(2) fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.

Sequence analysis of the 181-kb accessory plasmid pSmeSM11b, isolated from a dominantSinorhizobium melilotistrain identified during a long-term field release experiment

The 181 251 bp accessory plasmid pSmeSM11b of Sinorhizobium meliloti strain SM11, belonging to a dominant indigenous S. meliloti subpopulation identified during a long-term field release experiment, was sequenced. This plasmid has 166 coding sequences (CDSs), 42% of which encode proteins with homology to proteins of known function. Plasmid pSmeSM11b is a member of the repABC replicon family and contains a large gene region coding for a conjugation system similar to that of other self-transmissible plasmids in Rhizobium and Agrobacterium. Another pSmeSM11b gene region, possibly involved in sugar metabolism and polysaccharide catabolism, resembled a region of S. meliloti 1021 megaplasmid pSymB and in the genome of Sinorhizobium medicae WSM419. Another module of plasmid pSmeSM11b encodes proteins similar to those of the nitrogen-fixing actinomycete Frankia CcI3, and which are likely to be involved in the synthesis of a secondary metabolite. Several ORFs of pSmeSM11b were predicted to play a role in nonribosomal peptide synthesis. Plasmid pSmeSM11b has many mobile genetic elements, which contribute to the mosaic composition of the plasmid.

The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor-regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.