Rhizobium Research
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Choice of hydrogen uptake (Hup) status in legume-rhizobia symbioses

Choice of hydrogen uptake (Hup) status in legume-rhizobia symbioses | Rhizobium Research | Scoop.it

The H2 is an obligate by-product of N-fixation. Recycling of H2 through uptake hydrogenase (Hup) inside the root nodules of leguminous plants is often considered an advantage for plants. However, many of the rhizobium-legume symbioses found in nature, especially those used in agriculture are shown to be Hup−, with the plants releasing H2 produced by nitrogenase activity from root nodules into the surrounding rhizosphere. Recent studies have suggested that, H2 induces plant-growth-promoting rhizobacteria, which may explain the widespread of Hup− symbioses in spite of the low energy efficiency of such associations. Wild legumes grown in Nova Scotia, Canada, were surveyed to determine if any plant-growth characteristics could give an indication of Hup choice in leguminous plants. Out of the plants sampled, two legumes, Securigera varia and Vicia cracca, showed Hup+ associations. Securigera varia exhibited robust root structure as compared with the other plants surveyed. Data from the literature and the results from this study suggested that plants with established root systems are more likely to form the energy-efficient Hup+ symbiotic relationships with rhizobia. Conversely, Hup− associations could be beneficial to leguminous plants due to H2-oxidizing plant-growth-promoting rhizobacteria that allow plants to compete successfully, early in the growing season. However, some nodules from V. cracca tested Hup+, while others were Hup−. This was similar to that observed in Glycine max and Pisum sativum, giving reason to believe that Hup choice might be affected by various internal and environmental factors.

 

Henry Annan, Amber-Leigh Golding, Yinping Zhao, and Zhongmin Dong (2012). Ecol Evol 2(9): 2285–2290.

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Rhizobium Research
Plant-Microbe interactions focusing on Rhizobium
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Welcome to Rhizobium Research!

I really didn't know what to expect when I started playing with this web site. I tried a few different names before settling on Rhizobium Research (note the typo in the URL, late night, tired eyes). The site represents literature that I find interesting, papers that I think I should have read, and papers that I plan to read in the near future. It turns out that other people have similar, or overlapping interests (usually doesn't happen locally). My searches are relatively simple. If I have missed something you think should be posted, please feel free to suggest it so that it can be posted. If you have a comments, please keep them constructive and/or positive. If you are an author of a paper that has been posted and want to leave a comment, I can see how this can lead to some interesting discussions. The long and short, I hope you enjoy the site. I hope that it works to build an on-line community of people with similar interests, so please let other people know of this site. I have been encouraged by early comments that I have received so I will try to keep this site current.

Ivan
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Muhammad Afridi's comment, September 20, 2013 10:41 AM
well done
Chang Fu Tian's comment, March 24, 2014 10:20 PM
Very helpful.
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Sinorhizobium meliloti YbeY is an endoribonuclease with unprecedented catalytic features, acting as silencing enzyme in riboregulation

Sinorhizobium meliloti YbeY is an endoribonuclease with unprecedented catalytic features, acting as silencing enzyme in riboregulation | Rhizobium Research | Scoop.it
Structural and biochemical features suggest that the almost ubiquitous bacterial YbeY protein may serve catalytic and/or Hfq-like protective functions central to small RNA (sRNA)-mediated regulation and RNA metabolism. We have biochemically and genetically characterized the YbeY ortholog of the legume symbiont Sinorhizobium meliloti (SmYbeY). Co-immunoprecipitation (CoIP) with a FLAG-tagged SmYbeY yielded a poor enrichment in RNA species, compared to Hfq CoIP-RNA uncovered previously by a similar experimental setup. Purified SmYbeY behaved as a monomer that indistinctly cleaved single- and double-stranded RNA substrates, a unique ability among bacterial endoribonucleases. SmYbeY-mediated catalysis was supported by the divalent metal ions Mg2+, Mn2+ and Ca2+, which influenced in a different manner cleavage efficiency and reactivity patterns, with Ca2+ specifically blocking activity on double-stranded and some structured RNA molecules. SmYbeY loss-of-function compromised expression of core energy and RNA metabolism genes, whilst promoting accumulation of motility, late symbiotic and transport mRNAs. Some of the latter transcripts are known Hfq-binding sRNA targets and might be SmYbeY substrates. Genetic reporter and in vitro assays confirmed that SmYbeY is required for sRNA-mediated down-regulation of the amino acid ABC transporter prbA mRNA. We have thus discovered a bacterial endoribonuclease with unprecedented catalytic features, acting also as gene silencing enzyme.

Margarida Saramago, Alexandra Peregrina, Marta Robledo, Rute G. Matos, Rolf Hilker, Javier Serrania, Anke Becker, Cecilia M. Arraiano and José I. Jiménez-Zurdo (2016) Nucleic Acids Res. Dec 6. pii: gkw1234. [Epub ahead of print]
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The Use of Transposon Insertion Sequencing to Interrogate the Core Functional Genome of the Legume Symbiont Rhizobium leguminosarum.

The Use of Transposon Insertion Sequencing to Interrogate the Core Functional Genome of the Legume Symbiont Rhizobium leguminosarum. | Rhizobium Research | Scoop.it

The free-living legume symbiont Rhizobium leguminosarum is of significant economic value because of its ability to provide fixed nitrogen to globally important leguminous food crops, such as peas and lentils. Discovery based research into the genetics and physiology of R. leguminosarum provides the foundational knowledge necessary for understanding the bacterium's complex lifestyle, necessary for augmenting its use in an agricultural setting. Transposon insertion sequencing (INSeq) facilitates high-throughput forward genetic screening at a genomic scale to identify individual genes required for growth in a specific environment. In this study we applied INSeq to screen the genome of R. leguminosarum bv. viciae strain 3841 (RLV3841) for genes required for growth on minimal mannitol containing medium. Results from this study were contrasted with a prior INSeq experiment screened on peptide rich media to identify a common set of functional genes necessary for basic physiology. Contrasting the two growth conditions indicated that approximately 10% of the chromosome was required for growth, under both growth conditions. Specific genes that were essential to singular growth conditions were also identified. Data from INSeq screening on mannitol as a sole carbon source were used to reconstruct a metabolic map summarizing growth impaired phenotypes observed in the Embden-Meyerhof-Parnas pathway, Entner-Doudoroff pathway, pentose phosphate pathway, and tricarboxylic acid cycle. This revealed the presence of mannitol dependent and independent metabolic pathways required for growth, along with identifying metabolic steps with isozymes or possible carbon flux by-passes. Additionally, genes were identified on plasmids pRL11 and pRL12 that are likely to encode functional activities important to the central physiology of RLV3841.


Perry BJ, Akter MS, Yost CK (2016). .Front Microbiol. 2016 Nov 22;7:1873. eCollection 2016.

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Type IV Effector Proteins Involved in the Sinorhizobium-Medicago Symbiosis | Molecular Plant-Microbe Interactions

Type IV Effector Proteins Involved in the Sinorhizobium-Medicago Symbiosis | Molecular Plant-Microbe Interactions | Rhizobium Research | Scoop.it
In this study, we investigated genetic elements of the type IV secretion system (T4SS) found in Sinorhizobium and the role they play in symbiosis. Sinorhizobium meliloti and S. medicae each contain a putative T4SS, similar to that used by Agrobacterium tumefaciens during pathogenesis. The Cre reporter assay for translocation (CRAfT) system was used to validate potential effector proteins. Both S. meliloti and S. medicae contained the effector protein TfeA, which was translocated into the host plant. Sequence analysis revealed the presence of a nod box, involved in transcriptional activation of symbiosis-related genes, upstream of the transcriptional regulator (virG) in the Sinorhizobium T4SS. Replicate qRT-PCR analyses indicated that luteolin, released by roots and seeds of Medicago truncatula, upregulated transcription of tfeA and virG. Mutations in the T4SS apparatus, or tfeA alone, resulted in reduced numbers of nodules formed on M. truncatula genotypes. In addition, S. meliloti strain KH46c, which contains a deletion in the T4SS, was less competitive for nodule formation when co-inoculated with an equal number of cells of the wild-type strain. To our knowledge, TfeA is the first T4SS effector protein identified in Sinorhizobium. Our results indicate that Sinorhizobium 1) uses a T4SS during initiation of symbiosis with Medicago, and 2) alters Medicago cells in planta during symbiosis. This study also offers additional bioinformatic evidence that several different rhizobial species may use the T4SS in symbiosis with other legumes.

Nelson MS, Chun CL, Sadowsky MJ (2016).Mol Plant Microbe Interact. 2016 Dec 5. [Epub ahead of print]
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DNA double-strand break repair is involved in desiccation resistance of Sinorhizobium meliloti, but is not essential for its symbiotic interaction with Medicago truncately

DNA double-strand break repair is involved in desiccation resistance of Sinorhizobium meliloti, but is not essential for its symbiotic interaction with Medicago truncately | Rhizobium Research | Scoop.it

The soil bacterium Sinorhizobium meliloti, a nitrogen-fixing symbiont of legume plants, is exposed to numerous stress conditions in nature, some of which cause the formation of harmful DNA double strand breaks (DSB). In particular, the reactive oxygen (ROS) and nitrogen (RNS) species produced during symbiosis, and the desiccation occurring in dry soils, are conditions which induce DSB. Two major systems of DSB repair are known in S. meliloti: homologous recombination (HR) and non-homologous end-joining (NHEJ). However, their role in the resistance to ROS, RNS and desiccation has never been examined in this bacterial species, and the importance of DSB repair in the symbiotic interaction has not been properly evaluated. Here, we constructed S. meliloti strains deficient in HR (by deleting the recA gene) or in NHEJ (by deleting the four ku genes) or both. Interestingly, we observed that ku and/or recA genes are involved in S. meliloti resistance to ROS and RNS. Nevertheless, a S. meliloti strain deficient in both HR and NHEJ was not altered in its ability to establish and maintain an efficient nitrogen-fixing symbiosis with Medicago truncatula, showing that rhizobial DSB repair is not essential for this process. This result suggests either that DSB formation in S. meliloti is efficiently prevented during symbiosis, or that DSB are not detrimental for symbiosis efficiency. In contrast, we found for the first time that both recA and ku genes are involved in S. meliloti resistance to desiccation, suggesting that DSB repair could be important for rhizobium persistence in the soil.



Dupuy P, Gourion B, Sauviac L, Bruant C (2016).  Nov 23. doi: 10.1099/mic.0.000400. [Epub ahead of print]

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A NodD-like protein activates transcription of genes involved with naringenin degradation in a flavonoid-dependent manner in Herbaspirillum seropedicae

A NodD-like protein activates transcription of genes involved with naringenin degradation in a flavonoid-dependent manner in Herbaspirillum seropedicae | Rhizobium Research | Scoop.it

Herbaspirillum seropedicae is an associative, endophytic non-nodulating diazotrophic bacterium that colonises several grasses. An ORF encoding a LysR-type transcriptional regulator, very similar to NodD proteins of rhizobia, was identified in its genome. This nodD-like gene, named fdeR, is divergently transcribed from an operon encoding enzymes involved in flavonoid degradation (fde operon). Apigenin, chrysin, luteolin and naringenin strongly induce transcription of the fde operon, but not that of the fdeR, in an FdeR-dependent manner. The intergenic region between fdeR and fdeA contains several generic LysR consensus sequences (T-N11 -A) and we propose a binding site for FdeR, which is conserved in other bacteria. DNase I foot-printing revealed that the interaction with the FdeR binding site is modified by the four flavonoids that stimulate transcription of the fde operon. Moreover, FdeR binds naringenin and chrysin as shown by isothermal titration calorimetry. Interestingly, FdeR also binds in vitro to the nod-box from the nodABC operon of Rhizobium sp. NGR234 and is able to activate its transcription in vivo. These results show that FdeR exhibits two features of rhizobial NodD proteins: nod-box recognition and flavonoid-dependent transcription activation, but its role in H. seropedicae and related organisms seems to have evolved to control flavonoid metabolism. 


Wassem R, Marin AM, Daddaoua A, Monteiro RA, Chubatsu LS, Ramos JL, Deakin WJ, Broughton WJ, Pedrosa FO, Souza EM. (2016) Environ Microbiol. Nov 23. doi: 10.1111/1462-2920.13604. [Epub ahead of print]

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Elucidation of gibberellin biosynthesis in bacteria reveals convergent evolution.

Elucidation of gibberellin biosynthesis in bacteria reveals convergent evolution. | Rhizobium Research | Scoop.it

Gibberellins (GAs) are crucial phytohormones involved in many aspects of plant growth and development, including plant-microbe interactions, which has led to GA production by plant-associated fungi and bacteria as well. While the GA biosynthetic pathways in plants and fungi have been elucidated and found to have arisen independently through convergent evolution, little has been uncovered about GA biosynthesis in bacteria. Some nitrogen-fixing, symbiotic, legume-associated rhizobia, including Bradyrhizobium japonicum-the symbiont of soybean-and Sinorhizobium fredii-a broad-host-nodulating species-contain a putative GA biosynthetic operon, or gene cluster. Through functional characterization of five unknown genes, we demonstrate that this operon encodes the enzymes necessary to produce GA9, thereby elucidating bacterial GA biosynthesis. The distinct nature of these enzymes indicates that bacteria have independently evolved a third biosynthetic pathway for GA production. Furthermore, our results also reveal a central biochemical logic that is followed in all three convergently evolved GA biosynthetic pathways. 


Nett RS, Montanares M, Marcassa A, Lu X, Nagel R, Charles TC, Hedden P, Rojas MC, Peters RJ.(2016). Nat Chem Biol. 2016 Nov 14. doi: 10.1038/nchembio.2232. [Epub ahead of print]

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A putative 3-hydroxyisobutyryl-CoA hydrolase is required for efficient symbiotic nitrogen fixation in Sinorhizobium meliloti and Sinorhizobium fredii NGR234

A putative 3-hydroxyisobutyryl-CoA hydrolase is required for efficient symbiotic nitrogen fixation in Sinorhizobium meliloti and Sinorhizobium fredii NGR234 | Rhizobium Research | Scoop.it

We report that the smb20752 gene of the alfalfa symbiont Sinorhizobium meliloti is a novel symbiotic gene required for full N2 -fixation. Deletion of smb20752 resulted in lower nitrogenase activity and smaller nodules without impacting overall nodule morphology. Orthologs of smb20752 were present in all alpha and beta rhizobia, including the ngr_b20860 gene of Sinorhizobium fredii NGR234. A ngr_b20860 mutant formed Fix- determinate nodules that developed normally to a late stage of the symbiosis on the host plants Macroptillium atropurpureum and Vigna unguiculata. However an early symbiotic defect was evident during symbiosis with Leucaena leucocephala, producing Fix- indeterminate nodules. The smb20752 and ngr_b20860 genes encode putative 3-hydroxyisobutyryl-CoA (HIB-CoA) hydrolases. HIB-CoA hydrolase are required for L-valine catabolism and appear to prevent the accumulation of toxic metabolic intermediates, particularly methacrylyl-CoA. Evidence presented here and elsewhere (Curson et al., 2014. PLoS ONE 9:e97660) demonstrated that Smb20752 and NGR_b20860 can also prevent metabolic toxicity, are required for L-valine metabolism, and play an undefined role in 3-hydroxybutyrate catabolism. We present evidence that the symbiotic defect of the HIB-CoA hydrolase mutants is independent of the inability to catabolize L-valine and suggest it relates to the toxicity resulting from metabolism of other compounds possibly related to 3-hydroxybutyric acid. 


Zamani M, diCenzo GC, Milunovic B, Finan TM (2016) Environ Microbiol. 2016 Oct 11. doi: 10.1111/1462-2920.13570. [Epub ahead of print]

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Sinorhizobium fredii HH103 invades Lotus burttii by crack entry in a Nod-factor and surface polysaccharides dependent manner

Sinorhizobium fredii HH103 invades Lotus burttii by crack entry in a Nod-factor and surface polysaccharides dependent manner | Rhizobium Research | Scoop.it

Sinorhizobium fredii HH103-Rifr, a broad host-range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod-factors are strictly necessary for this crack-entry mode and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod-factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic capacity while mutants affected in production of exopolysaccharides and/or capsular polysaccharides were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rifr fla mutants nodulated L. burttii. None of the S. fredii HH103-Rifr surface-polysaccharide mutants gained effective nodulation with L. japonicas. 


Acosta-Jurado S, Rodríguez-Navarro DN, Kawaharada Y, Fernández-Perea J, Gil-Serrano A, Jin H, An Q, Rodriguez-Carvajal MA, Andersen SU, Sandal N, Stougaard J, Vinardell JM, Ruiz-Sainz JE (2016). Mol Plant Microbe Interact. Nov 9. [Epub ahead of print]

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Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities

Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities | Rhizobium Research | Scoop.it

Significance

Legumes are known as pioneer plants colonizing marginal soils, and as enhancers of the nutritional status in cultivated soils. This beneficial activity has been explained by their capacity to engage in symbiotic relationship with nitrogen-fixing rhizobia. We performed a community profiling analysis of Lotus japonicus wild type and mutants to investigate the role of the nodulation pathway on the structure of the root-associated bacterial microbiota. We found that several bacterial orders were almost entirely depleted from the mutant roots, and that an intact symbiosis is needed for the establishment of taxonomically diverse and distinctive bacterial communities in the root and rhizosphere. Our findings imply that a symbiosis-linked bacterial community, rather than dinitrogen-fixing rhizobia alone, contributes to legume growth and ecological performance.


Abstract

Lotus japonicus has been used for decades as a model legume to study the establishment of binary symbiotic relationships with nitrogen-fixing rhizobia that trigger root nodule organogenesis for bacterial accommodation. Using community profiling of 16S rRNA gene amplicons, we reveal that in Lotus, distinctive nodule- and root-inhabiting communities are established by parallel, rather than consecutive, selection of bacteria from the rhizosphere and root compartments. Comparative analyses of wild-type (WT) and symbiotic mutants in Nod factor receptor5 (nfr5), Nodule inception (nin) and Lotus histidine kinase1 (lhk1) genes identified a previously unsuspected role of the nodulation pathway in the establishment of different bacterial assemblages in the root and rhizosphere. We found that the loss of nitrogen-fixing symbiosis dramatically alters community structure in the latter two compartments, affecting at least 14 bacterial orders. The differential plant growth phenotypes seen between WT and the symbiotic mutants in nonsupplemented soil were retained under nitrogen-supplemented conditions that blocked the formation of functional nodules in WT, whereas the symbiosis-impaired mutants maintain an altered community structure in the nitrogen-supplemented soil. This finding provides strong evidence that the root-associated community shift in the symbiotic mutants is a direct consequence of the disabled symbiosis pathway rather than an indirect effect resulting from abolished symbiotic nitrogen fixation. Our findings imply a role of the legume host in selecting a broad taxonomic range of root-associated bacteria that, in addition to rhizobia, likely contribute to plant growth and ecological performance.


Via Christophe Jacquet
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Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island

Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island | Rhizobium Research | Scoop.it
Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICEAc) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICEAc-located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity.

Ling J, Wang H, Wu P, Li T, Tang Y, Naseer N, Zheng H, Masson-Boivin C, Zhong Z, Zhu J (2016). Proc Natl Acad Sci U S A. Nov 14.[Epub ahead of print]
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An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis.

An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis. | Rhizobium Research | Scoop.it

In the nitrogen-fixing symbiosis between legume hosts and rhizobia, the bacteria are engulfed by a plant cell membrane to become intracellular organelles. In the model legume Medicago truncatula, internalization and differentiation of Sinorhizobium (also known as Ensifer) meliloti is a prerequisite for nitrogen fixation. The host mechanisms that ensure the long-term survival of differentiating intracellular bacteria (bacteroids) in this unusual association are unclear. The M. truncatula defective nitrogen fixation4 (dnf4) mutant is unable to form a productive symbiosis, even though late symbiotic marker genes are expressed in mutant nodules. We discovered that in the dnf4 mutant, bacteroids can apparently differentiate, but they fail to persist within host cells in the process. We found that the DNF4 gene encodes NCR211, a member of the family of nodule-specific cysteine-rich (NCR) peptides. The phenotype of dnf4 suggests that NCR211 acts to promote the intracellular survival of differentiating bacteroids. The greatest expression of DNF4 was observed in the nodule interzone II-III, where bacteroids undergo differentiation. A translational fusion of DNF4 with GFP localizes to the peribacteroid space, and synthetic NCR211 prevents free-living S. meliloti from forming colonies, in contrast to mock controls, suggesting that DNF4 may interact with bacteroids directly or indirectly for its function. Our findings indicate that a successful symbiosis requires host effectors that not only induce bacterial differentiation, but also that maintain intracellular bacteroids during the host-symbiont interaction. The discovery of NCR211 peptides that maintain bacterial survival inside host cells has important implications for improving legume crops.

 

 

Kim M, Chen Y, Xi J, Waters C, Chen R, Wang D (2015) Proc Natl Acad Sci U S A. . [Epub ahead of print]

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NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103.

NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103. | Rhizobium Research | Scoop.it

Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103.

 

Jiménez-Guerrero I, Pérez-Montaño F, Medina C, Ollero FJ, López-Baena FJ (2015). .PLoS One. Nov 16;10(11):e0142866. 

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Interaction between Nitrogen and Phosphate Stress Responses in Sinorhizobium meliloti

Interaction between Nitrogen and Phosphate Stress Responses in Sinorhizobium meliloti | Rhizobium Research | Scoop.it

Bacteria have developed various stress response pathways to improve their assimilation and allocation of limited nutrients, such as nitrogen and phosphate. While both the Nitrogen Stress Response (NSR) and Phosphate Stress Response (PSR) have been studied individually, there are few experiments reported that characterize effects of multiple stresses on one or more pathways in Sinorhizobium meliloti, a facultatively symbiotic, nitrogen-fixing bacteria. The PII proteins, GlnB and GlnK, regulate the NSR activity, but analysis of global transcription changes in a PII deficient mutant suggest that the S. meliloti PII proteins may also regulate the PSR. PII double deletion mutants grow very slowly and pseudoreversion of the slow growth phenotype is common. To understand this phenomenon better, transposon mutants were isolated that had a faster growing phenotype. One mutation was in phoB, the response regulator for a two component regulatory system that is important in the PSR. phoB::Tn5 mutants had different phenotypes in the wild type compared to a PII deficient background. This led to the hypothesis that phosphate stress affects the NSR and conversely, that nitrogen stress affects the PSR. Our results show that phosphate availability affects glutamine synthetase activity and expression, which are often used as indicators of NSR activity, but that nitrogen availability did not affect alkaline phosphatase activity and expression, which are indicators of PSR activity. We conclude that the NSR is co-regulated by nitrogen and phosphate, whereas the PSR does not appear to be co-regulated by nitrogen in addition to its known phosphate regulation.


Hagberg KL, Yurgel SN, Mulder M, Kahn ML (2016). Front Microbiol. Nov 30;7:1928. eCollection 2016.

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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 expression.

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 expression. | Rhizobium Research | Scoop.it

Most Ensifer strains are comparatively acid sensitive, compromising their persistence in low pH soils. In the acid-tolerant strain E. 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 E. 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 Ensifer medicae to acidic environmental niches. 


Tian R, Heiden S, Osman WA1, Ardley JK, James EK, Gollagher MM, Tiwari R, Seshadri R, Kyrpides NC, Reeve WG. Mol Microbiol. 2016 Dec 9. doi: 10.1111/mmi.13592. [Epub ahead of print]

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The Absence of the N-acyl-homoserine-lactone Autoinducer Synthase Genes traI and ngrI Increases the Copy Number of the Symbiotic Plasmid in Sinorhizonium fredii NGR234

The Absence of the N-acyl-homoserine-lactone Autoinducer Synthase Genes traI and ngrI Increases the Copy Number of the Symbiotic Plasmid in Sinorhizonium fredii NGR234 | Rhizobium Research | Scoop.it

Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria. Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signaling cascades. 


Grote J, Krysciak D, Petersen K, Güllert S, Schmeisser C, Förstner KU, Krishnan HB, Schwalbe H, Kubatova N, Streit WR (2016).Front Microbiol. 2016 Nov 18;7:1858. eCollection 2016.

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Working with rhizobia

Working with rhizobia | Rhizobium Research | Scoop.it

This manual brings together state-of-the-art methods for the study of root-nodule bacteria, both in the free-living state and in symbiosis with legumes. In each chapter, the manual introduces a topic and provides guidance on how study of the symbiosis might best be tackled.


Detailed descriptions of the protocols that need to be followed, potential problems and pitfalls are provided. Topics covered include acquiring, recognising, growing and storing rhizobia, experimenting with strains in the laboratory, glasshouse and field, and applying contemporary molecular and genetic methodologies to assist in the study of rhizobia.


Ricardo Silva Araujo, Julie Ardley, Robert Abaidoo, Abdullahi Bala, Roz Deaker, Michael Dilworth, Greg Gemell, Ken Giller, Elizabeth Hartley, David Herridge, John Howleson, Marlangela Hungrla, Nancy Karanja, Karunakaran Ramakrishnan, Vanessa Melino, Sofle De Meyer, Graham O'Hara, Philip Poole, Wayne Reeve, Janet Sprent, Jason Terpolilli, Ravl Tlwarl, Paul Woomer, Ron Yates and Jerry Zilli Howieson J.G. and Dilworth M.J. (Eds.)

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Defining Substrate Specificities for Lipase and Phospholipase Candidates.

Defining Substrate Specificities for Lipase and Phospholipase Candidates. | Rhizobium Research | Scoop.it

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase. 


Sahonero-Canavesi DX, Zavaleta-Pastor M, Martínez-Aguilar L, López-Lara IM, Geiger O (2016).  J Vis Exp. 2016 Nov 23;(117). doi: 10.3791/54613.

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Chasing unicorns: Nodulation origins and the paradox of novelty

Chasing unicorns: Nodulation origins and the paradox of novelty | Rhizobium Research | Scoop.it
A radio series on the history of music called “Composers Datebook” ends each vignette by reminding listeners that “All music was once new.” Well, in evolutionary terms, every tissue and every organ was once an innovation, assembled de novo or from bits and pieces of pre-existing parts. How novelty arises is a fundamental question in the field of developmental evolution. In plants, the legume nodule is a fascinating system for studying the process by which a novel structure evolves and is modified in diverse lineages.


Via Jean-Michel Ané
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Jean-Michel Ané's curator insight, November 26, 2016 12:21 PM

Great review. I love it.

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nifDK Clusters Located on the Chromosome and Megaplasmid of Bradyrhizobium sp. Strain DOA9 Contribute Differently to Nitrogenase Activity During Symbiosis and Free-Living Growth

nifDK Clusters Located on the Chromosome and Megaplasmid of Bradyrhizobium sp. Strain DOA9 Contribute Differently to Nitrogenase Activity During Symbiosis and Free-Living Growth | Rhizobium Research | Scoop.it

Bradyrhizobium sp. strain DOA9 contains two copies of the nifDK genes, nifDKc, located on the chromosome, and nifDKp, located on a symbiotic megaplasmid. Unlike most rhizobia, this bacterium displays nitrogenase activity under both free-living and symbiotic conditions. Transcriptional analysis using gusA reporter strains showed that both nifDK operons were highly expressed under symbiosis, whereas nifDKc was the most abundantly expressed under free-living conditions. During free-living growth, the nifDKp mutation did not affect nitrogenase activity, whereas nitrogenase activity was drastically reduced with the nifDKc mutant. This led us to suppose that nifDKc is the main contributor of nitrogenase activity in the free-living state. In contrast, during symbiosis, no effect of the nifDKc mutation was observed and the nitrogen-fixation efficiency of plants inoculated with the nifDKp mutant was reduced. This suggests that nifDKp plays the main role in nitrogenase enzyme activity during symbiosis. Together, these data suggest that Bradyrhizobium sp. strain DOA9 contains two functional copies of nifDK genes that are regulated differently and that, depending on their lifestyle, contribute differently to nitrogenase activity. 


Wongdee J, Songwattana P, Nouwen N, Noisangiam R, Fardoux J, Chaintreuil C, Teaumroong N, Tittabutr P, Giraud E.(2016) Mol Plant Microbe Interact. Oct;29(10):767-773. 

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Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti. 

Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti.  | Rhizobium Research | Scoop.it

The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes. 



diCenzo GC, Checcucci A, Bazzicalupo M, Mengoni A, Viti C, Dziewit L, Finan TM, Galardini M, Fondi M (2016).  Nat Commun. Jul 22;7:12219. doi: 10.1038/ncomms12219.

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Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids. - PubMed - NCBI

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids. - PubMed - NCBI | Rhizobium Research | Scoop.it

Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the tricarboxylic acid (TCA) cycle to generate NAD(P)H for reduction of N2 Metabolic flux analysis of laboratory-grown Rhizobium leguminosarum showed that the flux from [(13)C]succinate was consistent with respiration of an obligate aerobe growing on a TCA cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady-state labeling under N2-fixing conditions. Therefore, comparative metabolomic profiling was used to compare free-living R. leguminosarum with pea bacteroids. While the TCA cycle was shown to be essential for maximal rates of N2 fixation, levels of pyruvate (5.5-fold reduced), acetyl coenzyme A (acetyl-CoA; 50-fold reduced), free coenzyme A (33-fold reduced), and citrate (4.5-fold reduced) were much lower in bacteroids. Instead of completely oxidizing acetyl-CoA, pea bacteroids channel it into both lipid and the lipid-like polymer poly-β-hydroxybutyrate (PHB), the latter via a type III PHB synthase that is active only in bacteroids. Lipogenesis may be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules. Direct reduction by NAD(P)H of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance the production of NAD(P)H from oxidation of acetyl-CoA in the TCA cycle with its storage in PHB and lipids. IMPORTANCE: Biological nitrogen fixation by symbiotic bacteria (rhizobia) in legume root nodules is an energy-expensive process. Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the TCA cycle to generate NAD(P)H for reduction of N2 However, direct reduction of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance oxidation of plant-derived dicarboxylates in the TCA cycle with lipid synthesis. Pea bacteroids channel acetyl-CoA into both lipid and the lipid-like polymer poly-β-hydroxybutyrate, the latter via a type II PHB synthase. Lipogenesis is likely to be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules.


Terpolilli JJ, Masakapalli SK, Karunakaran R, Webb IU, Green R, Watmough NJ, Kruger NJ, Ratcliffe RG, Poole PS (2016).  J Bacteriol. Sep 22;198(20):2864-75. 

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The role of O2 in the growth of Rhizobium leguminosarum bv. viciae 3841 on glucose and succinate. 

The role of O2 in the growth of Rhizobium leguminosarum bv. viciae 3841 on glucose and succinate.  | Rhizobium Research | Scoop.it
Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O2 was used to understand how O2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 (eryD) and RL0547 (phoB)), five on succinate (pRL100388, RL1641, RL1642, RL3427 and RL4524 (ecfL)) and three on 1% O2 (pRL110072, RL0545 (phoU) and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmid-less rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the ED pathway and TCA-cycle for optimal growth on glucose. Surprisingly the ED pathway was required for growth on succinate, suggesting sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (gatB) and pRL120419 (opaA, omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis including ribose-phosphate pyrophosphokinase (RL3468 (prs)) and a cytosine deaminase (pRL90208 (codA)). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (glnB, nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O2, conditions similar to those experienced by N2-fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting consumption of 2-ketoglutarate may increase flux through the TCA-cycle, leading to excess reductant that cannot be reoxidized at 1% O2 and cell death.

Wheatley RM, Ramachandran VK, Geddes BA, Perry BJ, Yost CK2, Poole PS. (2016). J Bacteriol. 2016 Oct 17. pii: JB.00572-16. [Epub ahead of print]

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Discovery of Novel Plant Interaction Determinants from the Genomes of 163 Root Nodule Bacteria

Discovery of Novel Plant Interaction Determinants from the Genomes of 163 Root Nodule Bacteria | Rhizobium Research | Scoop.it
Root nodule bacteria (RNB) or “rhizobia” are a type of plant growth promoting bacteria, typified by their ability to fix nitrogen for their plant host, fixing nearly 65% of the nitrogen currently utilized in sustainable agricultural production of legume crops and pastures. In this study, we sequenced the genomes of 110 RNB from diverse hosts and biogeographical regions, and undertook a global exploration of all available RNB genera with the aim of identifying novel genetic determinants of symbiotic association and plant growth promotion. Specifically, we performed a subtractive comparative analysis with non-RNB genomes, employed relevant transcriptomic data, and leveraged phylogenetic distribution patterns and sequence signatures based on known precepts of symbiotic- and host-microbe interactions. A total of 184 protein families were delineated, including known factors for nodulation and nitrogen fixation, and candidates with previously unexplored functions, for which a role in host-interaction, -regulation, biocontrol, and more, could be posited. These analyses expand our knowledge of the RNB purview and provide novel targets for strain improvement in the ultimate quest to enhance plant productivity and agricultural sustainability.

Via Christophe Jacquet
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Symbiosis within Symbiosis: Evolving Nitrogen-Fixing Legume Symbionts

Symbiosis within Symbiosis: Evolving Nitrogen-Fixing Legume Symbionts | Rhizobium Research | Scoop.it
Bacterial accessory genes are genomic symbionts with an evolutionary history and future that is different from that of their hosts. Packages of accessory genes move from strain to strain and confer important adaptations, such as interaction with eukaryotes. The ability to fix nitrogen with legumes is a remarkable example of a complex trait spread by horizontal transfer of a few key symbiotic genes, converting soil bacteria into legume symbionts. Rhizobia belong to hundreds of species restricted to a dozen genera of the Alphaproteobacteria and Betaproteobacteria, suggesting infrequent successful transfer between genera but frequent successful transfer within genera. Here we review the genetic and environmental conditions and selective forces that have shaped evolution of this complex symbiotic trait.

Via Jean-Michel Ané
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Quinol oxidase encoded by cyoABCD in Rhizobium etli CFN42 is regulated by ActSR and is crucial for growth at low pH or low iron conditions.

Quinol oxidase encoded by cyoABCD in Rhizobium etli CFN42 is regulated by ActSR and is crucial for growth at low pH or low iron conditions. | Rhizobium Research | Scoop.it

Rhizobium etli aerobically respires with several terminal oxidases. The quinol oxidase (Cyo) encoded by cyoABCD is needed for efficient adaptation to low oxygen conditions and cyo transcription is upregulated at low oxygen. This study sought to determine how transcription of the cyo operon is regulated. The 5' sequence upstream of cyo was analysed in silico and revealed putative binding sites for ActR of the ActSR two-component regulatory system. The expression of cyo was decreased in an actSR mutant regardless of the oxygen condition. As ActSR is known to be important for growth under low pH in another rhizobial species, the effect of growth medium pH on cyo expression was tested. As the pH of the media was incrementally decreased, cyo expression gradually increased in the WT, eventually reaching ∼10-fold higher levels at low pH (4.8) compared with neutral pH (7.0) conditions. This upregulation of cyo under decreasing pH conditions was eliminated in the actSR mutant. Both the actSR and cyo mutants had severe growth defects at low pH (4.8). Lastly, the actSR and cyo mutants had severe growth defects when grown in media treated with an iron chelator. Under these conditions, cyo was upregulated in the WT, whereas cyo was not induced in the actSR mutant. Altogether, the results indicated cyo expression is largely dependent on the ActSR two-component system. This study also demonstrated additional physiological roles for Cyo in R. etli CFN42, in which it is the preferred oxidase for growth under acidic and low iron conditions. 

 Lunak ZR, Dale Noel K. (2015). Microbiology. Sep;161(9):1806-15. Epub 2015 Jul 9.
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