Rhizobium leguminosarum bv. trifolii NodD2 Enhances Competitive Nodule Colonization in the Clover-Rhizobium Symbiosis

ABSTRACT Establishment of the symbiotic relationship that develops between rhizobia and their legume hosts is contingent upon an interkingdom signal exchange. In response to host legume flavonoids, NodD proteins from compatible rhizobia activate expression of nodulation genes that produce lipochitin oligosaccharide signaling molecules known as Nod factors. Root nodule formation commences upon legume recognition of compatible Nod factor. Rhizobium leguminosarum was previously considered to contain one copy of nodD; here, we show that some strains of the Trifolium (clover) microsymbiont R. leguminosarum bv. trifolii contain a second copy designated nodD2. nodD2 genes were present in 8 out of 13 strains of R. leguminosarum bv. trifolii, but were absent from the genomes of 16 R. leguminosarum bv. viciae strains. Analysis of single and double nodD1 and nodD2 mutants in R. leguminosarum bv. trifolii strain TA1 revealed that NodD2 was functional and enhanced nodule colonization competitiveness. However, NodD1 showed significantly greater capacity to induce nod gene expression and infection thread formation. Clover species are either annual or perennial and this phenological distinction is rarely crossed by individual R. leguminosarum bv. trifolii microsymbionts for effective symbiosis. Of 13 strains with genome sequences available, 7 of the 8 effective microsymbionts of perennial hosts contained nodD2, whereas the 3 microsymbionts of annual hosts did not. We hypothesize that NodD2 inducer recognition differs from NodD1, and NodD2 functions to enhance competition and effective symbiosis, which may discriminate in favor of perennial hosts. IMPORTANCE Establishment of the rhizobium-legume symbiosis requires a highly specific and complex signal exchange between both participants. Rhizobia perceive legume flavonoid compounds through LysR-type NodD regulators. Often, rhizobia encode multiple copies of nodD, which is one determinant of host specificity. In some species of rhizobia, the presence of multiple copies of NodD extends their symbiotic host-range. Here, we identified and characterized a second copy of nodD present in some strains of the clover microsymbiont Rhizobium leguminosarum bv. trifolii. The second nodD gene contributed to the competitive ability of the strain on white clover, an important forage legume. A screen for strains containing nodD2 could be utilized as one criterion to select strains with enhanced competitive ability for use as inoculants for pasture production.

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Lifestyle adaptations of Rhizobium from rhizosphere to symbiosis

10.1101/2020.05.07.082560 ◽

2020 ◽

Cited By ~ 1

Author(s):

Rachel M. Wheatley ◽

Brandon L. Ford ◽

Li Li ◽

Samuel T. N. Aroney ◽

Hayley E. Knights ◽

...

Keyword(s):

Rhizobium Leguminosarum ◽

Root Colonization ◽

Glycogen Synthesis ◽

Rapid Expansion ◽

Metabolic Adaptation ◽

Nodule Formation ◽

Transposon Insertion ◽

Legume Symbiosis ◽

Transposon Insertion Sequencing ◽

Multiple Stages

AbstractBy analyzing successive lifestyle stages of a model Rhizobium-legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N2-fixing bacteroids and release from legume (pea) nodules. While only 27 genes are annotated as nif and fix in Rhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 tRNAs and 5 RNA features) are required for the competitive ability to nodulate pea and fix N2. Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signalling, N2 fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism and glutamine synthesis (GlnII). There are separate lifestyle adaptations specific to rhizosphere growth (17) and root colonization (23), distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of a Rhizobium-legume symbiosis.SignificanceRhizobia are soil-dwelling bacteria that form symbioses with legumes and provide biologically useable nitrogen as ammonium for the host plant. High-throughput DNA sequencing has led to a rapid expansion in publication of complete genomes for numerous rhizobia, but analysis of gene function increasingly lags gene discovery. Mariner-based transposon insertion sequencing (INSeq) has allowed us to characterize the fitness contribution of bacterial genes and determine those functionally important in a Rhizobium-legume symbiosis at multiple stages of development.

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Lifestyle adaptations ofRhizobiumfrom rhizosphere to symbiosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2009094117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23823-23834

Author(s):

Rachel M. Wheatley ◽

Brandon L. Ford ◽

Li Li ◽

Samuel T. N. Aroney ◽

Hayley E. Knights ◽

...

Keyword(s):

Rhizobium Leguminosarum ◽

Root Colonization ◽

Cell Envelope ◽

Glycogen Synthesis ◽

Metabolic Adaptation ◽

Nodule Formation ◽

Transposon Insertion ◽

Legume Symbiosis ◽

Glutamine Synthesis ◽

Transposon Insertion Sequencing

By analyzing successive lifestyle stages of a modelRhizobium–legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N2-fixing bacteroids, and release from legume (pea) nodules. While only 27 genes are annotated asnifandfixinRhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 transfer RNAs, and 5 RNA features) are required for the competitive ability to nodulate pea and fix N2. Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signaling, N2fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism, and glutamine synthesis (GlnII). There are 17 separate lifestyle adaptations specific to rhizosphere growth and 23 to root colonization, distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of aRhizobium–legume symbiosis.

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The Rhizobium-Legume Symbiosis: Co-opting Successful Stress Management

Frontiers in Plant Science ◽

10.3389/fpls.2021.796045 ◽

2022 ◽

Vol 12 ◽

Author(s):

Justin P. Hawkins ◽

Ivan J. Oresnik

Keyword(s):

Model System ◽

Low Ph ◽

Complex Signal ◽

Tolerance Mechanisms ◽

Positive Interaction ◽

Bacterial Response ◽

Plant Peptides ◽

Legume Symbiosis ◽

Growth Inhibiting ◽

Signal Exchange

The interaction of bacteria with plants can result in either a positive, negative, or neutral association. The rhizobium-legume interaction is a well-studied model system of a process that is considered a positive interaction. This process has evolved to require a complex signal exchange between the host and the symbiont. During this process, rhizobia are subject to several stresses, including low pH, oxidative stress, osmotic stress, as well as growth inhibiting plant peptides. A great deal of work has been carried out to characterize the bacterial response to these stresses. Many of the responses to stress are also observed to have key roles in symbiotic signaling. We propose that stress tolerance responses have been co-opted by the plant and bacterial partners to play a role in the complex signal exchange that occurs between rhizobia and legumes to establish functional symbiosis. This review will cover how rhizobia tolerate stresses, and how aspects of these tolerance mechanisms play a role in signal exchange between rhizobia and legumes.

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From Ossau and Iraty to PDO Ossau-Iraty

British Food Journal ◽

10.1108/bfj-10-2018-0719 ◽

2019 ◽

Vol 121 (12) ◽

pp. 3062-3075 ◽

Cited By ~ 1

Author(s):

Morgane Millet

Keyword(s):

Local Development ◽

Qualitative Interviews ◽

Pasture Production ◽

Content Type ◽

Farm Production ◽

Protected Designation Of Origin ◽

Summer Pasture ◽

On Farm ◽

Term Analysis

Purpose The purpose of this paper is to understand how a geographical indication (GI) is built through time and how its (non)appropriation by local producers shapes it. The reciprocity of such process is also considered: how the creation of a GI changes local relationships between producers, within the GI and out of it? The case of Ossau-Iraty is relevant: in south-west of France, this protected designation of origin (PDO) has been based on two distinct regions: Bearn (Ossau) and Pays Basque (Iraty). Since then, most producers of Bearn have rejected this PDO. Design/methodology/approach The author adopts a diachronic perspective: the trajectory of the local dairy ewe sector is described, focusing on the trajectory of on-farm cheese makers from Bearn and Pays Basque and the trajectory of Ossau-Iraty. Based on different methods (qualitative interviews and archive research), this paper aims at analyzing the interactions within such heterogeneous networks. Findings When the PDO was created (1980), the opposition between producers of Bearn and Pays Basque was based on strong senses of place, which would be translated in a different perception of tradition: to Bearn producers, PDO Ossau-Iraty would be an industrial cheese, in which they did not recognize their product and themselves. With time, the producers who have been involved in the PDO worked on its specifications. The recognition of symbolic practices such as on-farm production or Summer pasture production, the recognition of differences between Basque cheese and Bearn cheese are changes that contribute to the evolution of perceptions within the local producers’ community. The author observes a recent convergence between Basque producers and Bearn producers, as their distinct products share common and strong qualifications within PDO Ossau-Iraty that contribute to their respective valorization. However, it seems to occur at an institutional level and the adhesion of the local producers might still be at stakes. Research limitations/implications A statistical study could reinforce the author’s exploratory and historical research. Furthermore, it would have been relevant to take local inhabitants and local consumers into account, as they have participated in the products’ qualifications as well. Originality/value A long-term analysis (40 years) contributes to better understand how cheeses are valorized and how such process is based on controversial processes. It contributes to root GIs into local histories, which are nor as consensual neither as uniform as we would primarily think, and to identity levers for sustainable local development.

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Important Late-Stage Symbiotic Role of theSinorhizobium melilotiExopolysaccharide Succinoglycan

Journal of Bacteriology ◽

10.1128/jb.00665-17 ◽

2018 ◽

Vol 200 (13) ◽

pp. e00665-17 ◽

Cited By ~ 10

Author(s):

Markus F. F. Arnold ◽

Jon Penterman ◽

Mohammed Shabab ◽

Esther J. Chen ◽

Graham C. Walker

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Late Stage ◽

Sinorhizobium Meliloti ◽

Antimicrobial Action ◽

Protective Function ◽

Content Type ◽

Early Stages ◽

Symbiotic Interactions ◽

Legume Symbiosis

ABSTRACTSinorhizobium melilotienters into beneficial symbiotic interactions withMedicagospecies of legumes. Bacterial exopolysaccharides play critical signaling roles in infection thread initiation and growth during the early stages of root nodule formation. After endocytosis ofS. melilotiby plant cells in the developing nodule, plant-derived nodule-specific cysteine-rich (NCR) peptides mediate terminal differentiation of the bacteria into nitrogen-fixing bacteroids. Previous transcriptional studies showed that the intensively studied cationic peptide NCR247 induces expression of theexogenes that encode the proteins required for succinoglycan biosynthesis. In addition, genetic studies have shown that someexomutants exhibit increased sensitivity to the antimicrobial action of NCR247. Therefore, we investigated whether the symbiotically activeS. melilotiexopolysaccharide succinoglycan can protectS. melilotiagainst the antimicrobial activity of NCR247. We discovered that high-molecular-weight forms of succinoglycan have the ability to protectS. melilotifrom the antimicrobial action of the NCR247 peptide but low-molecular-weight forms of wild-type succinoglycan do not. The protective function of high-molecular-weight succinoglycan occurs via direct molecular interactions between anionic succinoglycan and the cationic NCR247 peptide, but this interaction is not chiral. Taken together, our observations suggest thatS. melilotiexopolysaccharides not only may be critical during early stages of nodule invasion but also are upregulated at a late stage of symbiosis to protect bacteria against the bactericidal action of cationic NCR peptides. Our findings represent an important step forward in fully understanding the complete set of exopolysaccharide functions during legume symbiosis.IMPORTANCESymbiotic interactions between rhizobia and legumes are economically important for global food production. The legume symbiosis also is a major part of the global nitrogen cycle and is an ideal model system to study host-microbe interactions. Signaling between legumes and rhizobia is essential to establish symbiosis, and understanding these signals is a major goal in the field. Exopolysaccharides are important in the symbiotic context because they are essential signaling molecules during early-stage symbiosis. In this study, we provide evidence suggesting that theSinorhizobium melilotiexopolysaccharide succinoglycan also protects the bacteria against the antimicrobial action of essential late-stage symbiosis plant peptides.

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A Sulfoglycolytic Entner-Doudoroff Pathway in Rhizobium leguminosarum bv. trifolii SRDI565

Applied and Environmental Microbiology ◽

10.1128/aem.00750-20 ◽

2020 ◽

Vol 86 (15) ◽

Cited By ~ 1

Author(s):

Jinling Li ◽

Ruwan Epa ◽

Nichollas E. Scott ◽

Dominik Skoneczny ◽

Mahima Sharma ◽

...

Keyword(s):

Rhizobium Leguminosarum ◽

Glycolytic Enzyme ◽

Proteomics Data ◽

Free Living ◽

Content Type ◽

Biochemical Processes ◽

Symbiotic Relationships ◽

Rhizobium Isolate ◽

Rhizobial Isolate ◽

Metabolomics Analysis

ABSTRACT Rhizobia are nitrogen-fixing bacteria that engage in symbiotic relationships with plant hosts but can also persist as free-living bacteria in the soil and rhizosphere. Here, we show that free-living Rhizobium leguminosarum SRDI565 can grow on the sulfosugar sulfoquinovose (SQ) or the related glycoside SQ-glycerol using a sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway, resulting in production of sulfolactate (SL) as the major metabolic end product. Comparative proteomics supports the involvement of a sulfo-ED operon encoding an ABC transporter, sulfo-ED enzymes, and an SL exporter. Consistent with an oligotrophic lifestyle, proteomics data revealed little change in expression of the sulfo-ED proteins during growth on SQ versus mannitol, a result confirmed through biochemical assay of sulfoquinovosidase activity in cell lysates. Metabolomics analysis showed that growth on SQ involves gluconeogenesis to satisfy metabolic requirements for glucose-6-phosphate and fructose-6-phosphate. Metabolomics analysis also revealed the unexpected production of small amounts of sulfofructose and 2,3-dihydroxypropanesulfonate, which are proposed to arise from promiscuous activities of the glycolytic enzyme phosphoglucose isomerase and a nonspecific aldehyde reductase, respectively. The discovery of a rhizobium isolate with the ability to degrade SQ builds our knowledge of how these important symbiotic bacteria persist within soil. IMPORTANCE Sulfonate sulfur is a major form of organic sulfur in soils but requires biomineralization before it can be utilized by plants. Very little is known about the biochemical processes used to mobilize sulfonate sulfur. We show that a rhizobial isolate from soil, Rhizobium leguminosarum SRDI565, possesses the ability to degrade the abundant phototroph-derived carbohydrate sulfonate SQ through a sulfoglycolytic Entner-Doudoroff pathway. Proteomics and metabolomics demonstrated the utilization of this pathway during growth on SQ and provided evidence for gluconeogenesis. Unexpectedly, off-cycle sulfoglycolytic species were also detected, pointing to the complexity of metabolic processes within cells under conditions of sulfoglycolysis. Thus, rhizobial metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the soil and to mobilization of sulfur in the pedosphere.

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Ceftazidime-Avibactam Resistance Associated with Increased blaKPC-3 Gene Copy Number Mediated by pKpQIL Plasmid Derivatives in Sequence Type 258 Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01816-19 ◽

2020 ◽

Vol 64 (4) ◽

Cited By ~ 5

Author(s):

Marco Coppi ◽

Vincenzo Di Pilato ◽

Francesco Monaco ◽

Tommaso Giani ◽

Pier Giulio Conaldi ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Copy Number ◽

Acquired Resistance ◽

Gene Copy Number ◽

Gene Copy ◽

Content Type ◽

Outer Membrane Porins ◽

Long Read ◽

Multiple Copies

ABSTRACT This study reports on the characterization of two ceftazidime-avibactam (CZA)-resistant KPC-producing Klebsiella pneumoniae strains (KP-14159 and KP-8788) sequentially isolated from infections occurred in a patient never treated with CZA. Whole-genome sequencing characterization using a combined short- and long-read sequencing approach showed that both isolates belonged to the same ST258 strain, had altered outer membrane porins (a truncated OmpK35 and an Asp137Thr138 duplication in the L3 loop of OmpK36), and carried novel pKpQIL plasmid derivatives (pIT-14159 and pIT-8788, respectively) harboring two copies of the Tn4401a KPC-3-encoding transposon. Plasmid pIT-8788 was a cointegrate of pIT-14159 with a ColE replicon (that was also present in KP-14159) apparently evolved in vivo during infection. pIT-8788 was maintained at a higher copy number than pIT-14159 and, upon transfer to Escherichia coli DH10B, was able to increase the CZA MIC by 32-fold. The present findings provide novel insights about the mechanisms of acquired resistance to CZA, underscoring the role that the evolution of broadly disseminated pKpQIL plasmid derivatives may have in increasing the blaKPC gene copy number and KPC-3 expression in bacterial hosts. Although not self-transferable, similar elements, with multiple copies of Tn4401 and maintained at a high copy number, could mediate transferable CZA resistance upon mobilization.

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Genome Sequences of vB_RleM_RL38JI and vB_RleM_RL2RES, Two Virulent Rhizobium leguminosarum Transducing Phages

Microbiology Resource Announcements ◽

10.1128/mra.01589-19 ◽

2020 ◽

Vol 9 (11) ◽

Cited By ~ 1

Author(s):

K. M. Damitha Gunathilake ◽

Supriya V. Bhat ◽

Christopher K. Yost ◽

Michael F. Hynes

Keyword(s):

Rhizobium Leguminosarum ◽

Nucleotide Identity ◽

Genome Sequences ◽

Content Type ◽

Generalized Transduction

Phages vB_RleM_RL38JI and vB_RleM_RL2RES are known to mediate generalized transduction in Rhizobium leguminosarum. The RL38JI genome consists of 158,577 nucleotides and 270 predicted genes, whereas RL2RES has a 156,878-bp genome with 262 predicted genes. The two genomes are similar, with 82.88% nucleotide identity to each other.

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Nod Factor-Induced Expression of Leghemoglobin to Study the Mechanism of NH4NO3 Inhibition on Root Hair Deformation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1997.10.2.215 ◽

1997 ◽

Vol 10 (2) ◽

pp. 215-220 ◽

Cited By ~ 52

Author(s):

Renze Heidstra ◽

Gerd Nilsen ◽

Francisco Martinez-Abarca ◽

Ab van Kammen ◽

Ton Bisseling

Keyword(s):

Gene Expression ◽

Root Hair ◽

Rhizobium Leguminosarum ◽

Cortical Cell ◽

Brefeldin A ◽

Marker Genes ◽

Nodule Formation ◽

Cdna Clones ◽

Nod Factor ◽

Root Hair Deformation

Nod factors secreted by Rhizobium leguminosarum bv. viciae induce root hair deformation, the formation of nodule primordia, and the expression of early nodulin genes in Vicia sativa (vetch). Root hair deformation is induced within 3 h in a small, susceptible zone (±2 mm) of the root. NH4NO3, known to be a potent blocker of nodule formation, inhibits root hair deformation, initial cortical cell divisions, and infection thread formation. To test whether NH4NO3 affects the formation of a component of the Nod factor perception-transduction system, we studied Nod factor-induced gene expression. The differential display technique was used to search for marker genes, which are induced within 1 to 3 h after Nod factor application. Surprisingly, one of the isolated cDNA clones was identified as a leghemoglobin gene (VsLb1), which is induced in vetch roots within 1 h after Nod factor application. By using the drug brefeldin A, it was then shown that VsLb1 activation does not require root hair deformation. The pVsLb1 clone was used as a marker to show that in vetch plants grown in the presence of NH4NO3 Nod factor perception and transduction leading to gene expression are unaffected.

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Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by Sinorhizobium meliloti or Rhizobium leguminosarum

Applied and Environmental Microbiology ◽

10.1128/aem.01561-17 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 10

Author(s):

Michael J. Mitsch ◽

George C. diCenzo ◽

Alison Cowie ◽

Turlough M. Finan

Keyword(s):

Nitrogen Fixation ◽

Carbon Source ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Symbiotic Nitrogen Fixation ◽

Sequencing Analysis ◽

Wild Type ◽

Content Type ◽

Transcriptional Changes ◽

Symbiotic Properties

ABSTRACTSymbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C4-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C4-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) inSinorhizobium melilotiRm1021dctmutants unable to transport C4-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF withMedicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-typeS. meliloti.Rhizobium leguminosarumbv. viciae 3841dctmutants unable to transport C4-dicarboxylates but expressing themaePtransporter had strong symbiotic properties, withPisum sativumplants inoculated with these strains appearing similar to plants inoculated with wild-typeR. leguminosarum. This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combinedP. sativum-R. leguminosarumnodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses,l-malate is likely the primary C4-dicarboxylate provided to the bacterium.IMPORTANCESymbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C4-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.

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