Rhizobium Research
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Rhizobia with 16S rRNA and nifH Similar to M. huakuii but Novel recA, glnII, nodA and nodC Genes Are Symbionts of New Zealand Carmichaelinae

Rhizobia with 16S rRNA and nifH Similar to M. huakuii but Novel recA, glnII, nodA and nodC Genes Are Symbionts of New Zealand Carmichaelinae | Rhizobium Research | Scoop.it

New Zealand became geographically isolated about 80 million years ago and this separation gave rise to a unique native flora including four genera of legume, Carmichaelia, Clianthus and Montigena in the Carmichaelinae clade, tribe Galegeae, and Sophora, tribe Sophoreae, sub-family Papilionoideae. Ten bacterial strains isolated from NZ Carmichaelinae growing in natural ecosystems grouped close to the Mesorhizobium huakuii type strain in relation to their 16S rRNA and nifH gene sequences. However, the ten strains separated into four groups on the basis of their recA and glnII sequences: all groups were clearly distinct from all Mesorhizobium type strains. The ten strains separated into two groups on the basis of their nodA sequences but grouped closely together in relation to nodC sequences; all nodA and nodC sequences were novel. Seven strains selected and the M. huakuii type strain (isolated from Astragalus sinicus) produced functional nodules on Carmichaelia spp., Clianthus puniceus and A. sinicus but did not nodulate two Sophora species. We conclude that rhizobia closely related to M. huakuii on the basis of 16S rRNA and nifH gene sequences, but with variable recA and glnII genes and novel nodA and nodC genes, are common symbionts of NZ Carmichaelinae.

 

Heng Wee Tan, Bevan S. Weir, Noel Carter, Peter B. Heenan, Hayley J. Ridgway, Euan K. James, Janet I. Sprent, J. Peter W. Young, Mitchell Andrews (2012).  PLoS ONE 7(10): e47677. 

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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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Andres Zurita's comment, August 21, 2013 8:58 AM
Nice site indeed! I keep following your suggestions from a rootology perspective ;)
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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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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Proline auxotrophy in Sinorhizobium meliloti results in a plant-specific symbiotic phenotype.

In order to effectively manipulate the rhizobium - legume symbiosis for our benefit, it is crucial to first gain a complete understanding of the underlying genetics and metabolism. Studies with rhizobium auxotrophs have provided insight into the requirement for amino acid biosynthesis during the symbiosis; however, a paucity of available L-proline auxotrophs has limited our understanding of the role of L-proline biosynthesis. Here, we examine the symbiotic phenotypes of a recently described Sinorhizobium meliloti L-proline auxotroph. Proline auxotrophy was observed to result in a host plant specific phenotype. The S. meliloti auxotroph displayed reduced symbiotic capability with alfalfa (Medicago sativa) due to a decrease in nodule mass formed and therefore a reduction in nitrogen fixed per plant. However, the proline auxotroph formed nodules on white sweet clover (Melilotus alba) that failed to fix N2. The rate of M. alba nodulation by the auxotroph was slightly delayed but the final number of nodules per plant was not impacted. Examination of M. alba nodules by confocal microscopy and transmission electron microscopy revealed the presence of the S. meliloti proline auxotroph cells within the host-legume cells, but few differentiated bacteroids were identified compared to the bacteroid-filled plant cells of wildtype nodules. Overall, these results indicated that L-proline biosynthesis is a general requirement for a fully effective nitrogen fixing symbiosis, likely due to a transient requirement during bacteroid differentiation.

 

diCenzo GC, Zamani M, Cowie A, Finan TM (2015). Microbiology. Sep 21. doi: 10.1099/mic.0.000182. [Epub ahead of print]

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The Sugar Kinase that is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

The Sugar Kinase that is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport | Rhizobium Research | Scoop.it

Rhamanose catabolism in Rhizobium leguminosarum was found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a CUT 2 ABC transporter encoded by rhaSTPQ, and that the transport of rhamnose was also dependent on the presence of RhaK, a protein known to have a sugar kinase activity. A linker scanning mutagenesis of rhaK showed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the alleles rhaK72 and rhaK73 were able to uncouple RhaK transport and kinase activities such that kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C-terminus and the N-terminus of RhaK respectively. Taken together the data led to the hypothesis that RhaK might interact either directly, or indirectly, with the ABC transporter defined by rhaSTPQ.In this work we show that both an N- and C- terminal fragment of RhaK are capable of interacting with the N-terminal fragment of the ABC-protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either the rhaK72, or rhaK73 alleles were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N-terminus of RhaK may represent a putative binding domain. Alanine scanning mutagenesis of this region followed by 2-hybrid analysis revealed a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments suggesting that the sugar kinase RhaK and, the ABC protein RhaT interact directly.

IMPORTANCE: ABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters are the carbohydrate uptake transporter 1 and 2 class (CUT1 and CUT2 respectively). This work provides the first evidence that a kinase that is necessary for the catabolism of a sugar can directly interact with a domain from the ABC protein that is necessary for the its transport.


Rivers DM, Oresnik IJ. (2015). J Bacteriol. Sep 28. pii: JB.00510-15. [Epub ahead of print]

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The plant microbiome explored: implications for experimental botany

The plant microbiome explored: implications for experimental botany | Rhizobium Research | Scoop.it
The importance of microbial root inhabitants for plant growth and health was recognized as early as 100 years ago. Recent insights reveal a close symbiotic relationship between plants and their associated microorganisms, and high structural and functional diversity within plant microbiomes. Plants provide microbial communities with specific habitats, which can be broadly categorized as the rhizosphere, phyllosphere, and endosphere. Plant-associated microbes interact with their host in essential functional contexts. They can stimulate germination and growth, help plants fend off disease, promote stress resistance, and influence plant fitness. Therefore, plants have to be considered as metaorganisms within which the associated microbes usually outnumber the cells belonging to the plant host. The structure of the plant microbiome is determined by biotic and abiotic factors but follows ecological rules. Metaorganisms are co-evolved species assemblages. The metabolism and morphology of plants and their microbiota are intensively connected with each other, and the interplay of both maintains the functioning and fitness of the holobiont. Our study of the current literature shows that analysis of plant microbiome data has brought about a paradigm shift in our understanding of the diverse structure and functioning of the plant microbiome with respect to the following: (i) the high interplay of bacteria, archaea, fungi, and protists; (ii) the high specificity even at cultivar level; (iii) the vertical transmission of core microbiomes; (iv) the extraordinary function of endophytes; and (v) several unexpected functions and metabolic interactions. The plant microbiome should be recognized as an additional factor in experimental botany and breeding strategies.

Via Jean-Michel Ané
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Microbiomes in light of traits: A phylogenetic perspective

Microbiomes in light of traits: A phylogenetic perspective | Rhizobium Research | Scoop.it
Microbial communities—microbiomes—are intricately linked to human health and critical ecosystem services. New technologies allow the rapid characterization of hundreds of samples at a time and provide a sweeping perspective on microbiome patterns. However, a systematic understanding of what determines microbiome diversity and composition and its implications for system functioning is still lacking. A focus on the phenotypic characteristics of microorganisms—their traits—offers a path for interpreting the growing amount of microbiome data. Indeed, a variety of trait-based approaches have been proposed for plants and animal communities, and this approach has helped to clarify the mechanisms underlying community assembly, diversity-process relationships, and ecosystem responses to environmental change.

Although there is a growing emphasis on microbial traits, the concept has not been fully appreciated in microbiology. However, a trait focus for microorganisms may present an even larger research opportunity than for macro-organisms. Not only do microorganisms play a central role in nutrient and energy cycling in most systems, but the techniques used to characterize microbiomes usually provide extensive molecular and phylogenetic information.

ADVANCES
One major difference between macro- and microorganisms is the potential for horizontal gene transfer (HGT) in microbes. Higher rates of HGT mean that many microbial traits might be unrelated to the history of the vertically descended parts of the genome. If true, then the taxonomic composition of a microbiome might reveal little about the health or functioning of a system. We first review key aspects of microbial traits and then recent studies that document the distribution of microbial traits onto the tree of life. A synthesis of these studies reveals that, despite the promiscuity of HGT, microbial traits appear to be phylogenetically conserved, or not distributed randomly across the tree of life. Further, microbial traits appear to be conserved in a hierarchical fashion, possibly linked to their biochemical and genetic complexity. For instance, traits such as pH and salinity preference are relatively deeply conserved, such that taxa within deep clades tend to share the trait. In contrast, other traits like the ability to use simple carbon substrates or to take up organic phosphorus are shallowly conserved, and taxa share these traits only within small, shallow clades.

OUTLOOK
The phylogenetic, trait-based framework that emerges offers a path to interpret microbiome variation and its connection to the health and functioning of environmental, engineered, and human systems. In particular, the taxonomic resolution of biogeographic patterns provides information about the traits under selection, even across entirely different systems. Parallels observed among human and free-living communities support this idea. For instance, microbial traits related to growth on different substrates (e.g., proteins, fats, and carbohydrates) in the human gut appear to be conserved at approximately the genus level, a resolution associated with the level of conservation of glycoside hydrolase genes in bacteria generally. A focus on two particular types of traits—response and effect traits—may also aid in microbiome management, whether that means maintaining human health or mitigating climate change impacts. Future work on microbial traits must consider three challenges: the influence of different trait measurements on cross-study comparisons; correlations between traits within and among microorganisms; and interactions among microbial traits, the environment, and other organisms. Our conclusions also have implications for the growing field of community phylogenetics beyond applications to microorganisms.

Via Jean-Michel Ané
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Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant.

Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant. | Rhizobium Research | Scoop.it

Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula.

 
Horváth B, Domonkos Á, Kereszt A, Szűcs A, Ábrahám E, Ayaydin F, Bóka K, Chen Y, Chen R, Murray JD, Udvardi MK, Kondorosi É, Kaló P. (2015) Proc Natl Acad Sci U S A. 2015 Sep 23. [Epub ahead of print]
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The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis

The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis | Rhizobium Research | Scoop.it

BACKGROUND: Strigolactones are a class of plant hormones whose biosynthesis is activated in response to phosphate starvation. This involves several enzymes, including the carotenoid cleavage dioxygenases 7 (CCD7) and CCD8 and the carotenoid isomerase DWARF27 (D27). D27 expression is known to be responsive to phosphate starvation. In Medicago truncatula and rice (Oryza sativa) this transcriptional response requires the GRAS-type proteins NSP1 and NSP2; both proteins are essential for rhizobium induced root nodule formation in legumes. In line with this, we questioned whether MtNSP1-MtNSP2 dependent MtD27 regulation is co-opted in rhizobium symbiosis.RESULTS: We provide evidence that MtD27 is involved in strigolactone biosynthesis in M. truncatula roots upon phosphate stress. Spatiotemporal expression studies revealed that this gene is also highly expressed in nodule primordia and subsequently becomes restricted to the meristem and distal infection zone of a mature nodules. A similar expression pattern was found for MtCCD7 and MtCCD8. Rhizobium lipo-chitooligosaccharide (LCO) application experiments revealed that of these genes MtD27 is most responsive in an MtNSP1 and MtNSP2 dependent manner. Symbiotic expression of MtD27 requires components of the symbiosis signaling pathway; including MtDMI1, MtDMI2, MtDMI3/MtCCaMK and in part MtERN1. This in contrast to MtD27 expression upon phosphate starvation, which only requires MtNSP1 and MtNSP2.CONCLUSION: Our data show that the phosphate-starvation responsive strigolactone biosynthesis gene MtD27 is also rapidly induced by rhizobiumLCO signals in an MtNSP1 and MtNSP2-dependent manner. Additionally, we show that MtD27 is co-expressed with MtCCD7 and MtCCD8 in nodule primordia and in the infection zone of mature nodules.

 

van Zeijl A, Liu W2, Xiao TT, Kohlen W, Yang WC, Bisseling T, Geurts R (2015).BMC Plant Biol. 2015 Oct 26;15(1):260. 

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Rhizobial peptidase HrrP cleaves host-encoded signaling peptides and mediates symbiotic compatibility.

Rhizobial peptidase HrrP cleaves host-encoded signaling peptides and mediates symbiotic compatibility. | Rhizobium Research | Scoop.it

Legume-rhizobium pairs are often observed that produce symbiotic root nodules but fail to fix nitrogen. Using the Sinorhizobium meliloti and Medicago truncatula symbiotic system, we previously described several naturally occurring accessory plasmids capable of disrupting the late stages of nodule development while enhancing bacterial proliferation within the nodule. We report here that host range restriction peptidase (hrrP), a gene found on one of these plasmids, is capable of conferring both these properties. hrrP encodes an M16A family metallopeptidase whose catalytic activity is required for these symbiotic effects. The ability of hrrP to suppress nitrogen fixation is conditioned upon the genotypes of both the host plant and the hrrP-expressing rhizobial strain, suggesting its involvement in symbiotic communication. Purified HrrP protein is capable of degrading a range of nodule-specific cysteine-rich (NCR) peptides encoded by M. truncatula. NCR peptides are crucial signals used by M. truncatula for inducing and maintaining rhizobial differentiation within nodules, as demonstrated in the accompanying article [Horváth B, et al. (2015) Proc Natl Acad Sci USA, 10.1073/pnas.1500777112]. The expression pattern of hrrP and its effects on rhizobial morphology are consistent with the NCR peptide cleavage model. This work points to a symbiotic dialogue involving a complex ensemble of host-derived signaling peptides and bacterial modifier enzymes capable of adjusting signal strength, sometimes with exploitative outcomes.


Price PA1, Tanner HR1, Dillon BA1, Shabab M2, Walker GC2, Griffitts JS, ( 2015)  Proc Natl Acad Sci U S A. Sep 23. pii: 201417797. [Epub ahead of print]

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Engineering the plant rhizosphere

Engineering the plant rhizosphere | Rhizobium Research | Scoop.it
Plant natural products are low molecular weight compounds playing important roles in plant survival under biotic and abiotic stresses. In the rhizosphere, several groups of plant natural products function as semiochemicals that mediate the interactions of plants with other plants, animals and microorganisms. The knowledge on the biosynthesis and transport of these signaling molecules is increasing fast. This enables us to consider to optimize plant performance by changing the production of these signaling molecules or their exudation into the rhizosphere. Here we discuss recent advances in the understanding and metabolic engineering of these rhizosphere semiochemicals.

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Molecular modeling and computational analyses suggests that the Sinorhizobium meliloti periplasmic regulator protein ExoR adopts a superhelical fold and is controlled by a unique mechanism of prote...

Molecular modeling and computational analyses suggests that the Sinorhizobium meliloti periplasmic regulator protein ExoR adopts a superhelical fold and is controlled by a unique mechanism of prote... | Rhizobium Research | Scoop.it

The Sinorhizobium meliloti periplasmic ExoR protein and the ExoS/ChvI two-component system form a regulatory mechanism that directly controls the transformation of free-living to host-invading cells. In the absence of crystal structures, understanding the molecular mechanism of interaction between ExoR and the ExoS sensor, which is thought to drive the key regulatory step in the invasion process, remains a major challenge. In this study, we present a theoretical structural model of the active form of ExoR protein, ExoRm , generated using computational methods. Our model suggests that ExoR possesses a super-helical fold comprising twelve α-helices forming six Sel1-like repeats, including two that were unidentified in previous studies. This fold is highly conducive to mediating protein-protein interactions and this is corroborated by the identification of putative protein binding sites on the surface of the ExoRm protein. Our studies reveal two novel insights: (a) an extended conformation of the third Sel1-like repeat that might be important for ExoR regulatory function and (b) a buried proteolytic site that implies a unique proteolytic mechanism. This study provides new and interesting insights into the structure of S. meliloti ExoR, lays the groundwork for elaborating the molecular mechanism of ExoRm cleavage, ExoRm -ExoS interactions, and studies of ExoR homologs in other bacterial host interactions.


Wiech EM1, Cheng HP, Singh SM. (2014). Protein Sci. Dec 9.  [Epub ahead of print]

Wiech EM1, Cheng HP, Singh SM.


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Construction of a mariner-based transposon vector for use in insertion sequence mutagenesis in selected members of the Rhizobiaceae

Construction of a mariner-based transposon vector for use in insertion sequence mutagenesis in selected members of the Rhizobiaceae | Rhizobium Research | Scoop.it

The Rhizobiaceae family of Gram-negative bacteria often engage in symbiosis with plants of economic importance. Historically, genetic studies to identify the function of individual genes, and characterize the biology of these bacteria have relied on the use of classical transposon mutagenesis. To increase the rate of scientific discovery in the Rhizobiaceae there is a need to adapt high-throughput genetic screens like insertion sequencing for use in this family of bacteria. Here we describe a Rhizobiaceae compatible MmeI-adapted mariner transposon that can be used with insertion sequencing for high-throughput genetic screening.

 

Benjamin J Perry and Christopher K Yost (2014), BMC Microbiology, 14:298 

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State of the art insertion mutagenesis and sequence analysis of relevant genes.

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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.

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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.

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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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MgtE from Rhizobium leguminosarum is a Mg2+ channel essential for growth at low pH and N2-fixation on specific plants.

MgtE from Rhizobium leguminosarum is a Mg2+ channel essential for growth at low pH and N2-fixation on specific plants. | Rhizobium Research | Scoop.it

MgtE is predicted to be a Rhizobium leguminosarum channel that is essential for growth when both Mg2+ is limiting and the pH is low. N2 was only fixed at 8% of the rate of wild type when the crop legume Pisum sativum was inoculated with an mgtE mutant of R. leguminosarum and although bacteroids were present, they were few in number and not fully developed. R. leguminosarum MgtE was also essential for N2-fixation on the native legume Vicia hirsuta but not when in symbiosis with Vicia faba. The importance of MgtE and the relevance of the contrasting phenotypes is discussed.

 
Hood G, Karunakaran R, Downie A, Poole P (2015) Mol Plant Microbe Interact. 2015 Sep 30. [Epub ahead of print]
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Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase.

Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase. | Rhizobium Research | Scoop.it

Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion). These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology. 

 
Liu T, Tian CF, Chen WX (2015) PLoS One. 2015 Sep 24;10(9):e0139143. 
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The Plasmid Mobilome of the Model Plant-Symbiont Sinorhizobium meliloti: Coming up with New Questions and Answers.

The Plasmid Mobilome of the Model Plant-Symbiont Sinorhizobium meliloti: Coming up with New Questions and Answers. | Rhizobium Research | Scoop.it

Rhizobia are Gram-negative Alpha- and Betaproteobacteria living in the underground which have the ability to associate with legumes for the establishment of nitrogen-fixing symbioses. Sinorhizobium meliloti in particular-the symbiont of Medicago, Melilotus, and Trigonella spp.-has for the past decades served as a model organism for investigating, at the molecular level, the biology, biochemistry, and genetics of a free-living and symbiotic soil bacterium of agricultural relevance. To date, the genomes of seven different S. meliloti strains have been fully sequenced and annotated, and several other draft genomic sequences are also available. The vast amount of plasmid DNA that S. meliloti frequently bears (up to 45% of its total genome), the conjugative ability of some of those plasmids, and the extent of the plasmid diversity has provided researchers with an extraordinary system to investigate functional and structural plasmid molecular biology within the evolutionary context surrounding a plant-associated model bacterium. Current evidence indicates that the plasmid mobilome in S. meliloti is composed of replicons varying greatly in size and having diverse conjugative systems and properties along with different evolutionary stabilities and biological roles. While plasmids carrying symbiotic functions (pSyms) are known to have high structural stability (approaching that of chromosomes), the remaining plasmid mobilome (referred to as the non-pSym, functionally cryptic, or accessory compartment) has been shown to possess remarkable diversity and to be highly active in conjugation. In light of the modern genomic and current biochemical data on the plasmids of S. meliloti, the current article revises their main structural components, their transfer and regulatory mechanisms, and their potential as vehicles in shaping the evolution of the rhizobial genome. 

 
 

Lagares A, Sanjuán J, Pistorio M. Microbiol Spectr. 2014 Oct;2(5). doi: 10.1128/microbiolspec.PLAS-0005-2013. Research Support, Non-U.S. Gov't

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Jean-Michel Ané's curator insight, November 8, 2015 8:51 AM

The mobilome... Excellent!

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Sinorhizobium fredii HH103 bacteroids are not terminally differentiated and show altered O-antigen in nodules of the IRLC legume Glycyrrhiza uralensis

Sinorhizobium fredii HH103 bacteroids are not terminally differentiated and show altered O-antigen in nodules of the IRLC legume Glycyrrhiza uralensis | Rhizobium Research | Scoop.it

In rhizobial species that nodulate IRLC (inverted repeat-lacking clade) legumes, such as the interaction between Sinorhizobium meliloti and Medicago, bacteroid differentiation is driven by an endoreduplication event that is induced by host nodule-specific cysteine rich (NCR) antimicrobial peptides and requires the participation of the bacterial protein BacA. We have studied bacteroid differentiation of Sinorhizobium fredii HH103 in three host plants: Glycine max, Cajanus cajan and the IRLC legume Glycyrrhiza uralensis. Flow cytometry, microscopy analyses and viability studies of bacteroids as well as confocal microscopy studies carried out in nodules showed that S. fredii HH103 bacteroids, regardless of the host plant, had DNA contents, cellular sizes and survival rates similar to those of free living bacteria. Contrary to S. meliloti, S. fredii HH103 showed little or no sensitivity to Medicago NCR247 and NCR335 peptides. Inactivation of S. fredii HH103 bacA neither affected symbiosis with Glycyrrhiza nor increased bacterial sensitivity to Medicago NCRs. Finally, HH103 bacteroids isolated from Glycyrrhiza, but not those isolated from Cajanus or Glycine, showed an altered lipopolysaccharide. Our studies indicate that, in contrast to the S. meliloti-Medicago model symbiosis, bacteroids in the S. fredii HH103-Glycyrrhiza symbiosis do not undergo NCR-induced and bacA-dependent terminal differentiation.

 

Crespo-Rivas JC, Guefrachi I, Mok KC, Villaécija-Aguilar JA,, Acosta-Jurado S, Pierre O, Ruiz-Sainz JE, Taga ME, Mergaert P, Vinardell JM. (2015) Environ Microbiol. Nov 2. [Epub ahead of print]
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Algal ancestor of land plants was preadapted for symbiosis

Algal ancestor of land plants was preadapted for symbiosis | Rhizobium Research | Scoop.it
Significance

Colonization of land by plants was a critical event for the emergence of extant ecosystems. The innovations that allowed the algal ancestor of land plants to succeed in such a transition remain unknown. Beneficial interaction with symbiotic fungi has been proposed as one of these innovations. Here we show that the genes required for this interaction appeared in a stepwise manner: Some evolved before the colonization of land by plants and others first appeared in land plants. We thus propose that the algal ancestor of land plants was preadapted for interaction with beneficial fungi and employed these gene networks to colonize land successfully.
Abstract

Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

Via Christophe Jacquet
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Construction and pilot screening of a signature-tagged mutant library of Sinorhizobium fredii

Construction and pilot screening of a signature-tagged mutant library of Sinorhizobium fredii | Rhizobium Research | Scoop.it

Sinorhizobium fredii is well known for its ability to establish symbiosis with diverse legumes such as Glycine max (soybean, determinate nodules) and Cajanus cajan (pigeon pea, indeterminate nodules). In order to make screening of S. fredii genes related to symbiosis cost-effective, we constructed a large Tn5 insertion mutant library of S. fredii CCBAU45436 using the signature-tagged mutagenesis (STM) technique. This STM library contains a total of 25,500 independent mutants distributed in 17 sublibraries tagged by corresponding distinct DNA bar-code sequences. After the pilot screening of 255 mutants in 15 batches, Tag85-4, Tag4-17, Tag4-11 and Tag10-13 were found to have attenuated competitiveness (0–30 % in nodule occupation) compared to the wild-type strain when inoculated on soybean. Further characterization of these mutants suggests that Tag4-11 (a pyrC mutant) and Tag10-13 (a nrdJ mutant) are defective in establishing symbiosis with soybean. The pyrC mutant induced uninfected pseudonodules while the nrdJ mutant formed significantly more nodules containing bacteroids with poor persistence ability. When these two mutants were tested on pigeon pea, host-specific symbiotic defects were found. These results demonstrated the STM library as a valuable resource for identifying S. fredii genes relevant to symbiosis.

 

Dan Wang , Yuan Chun Wang , Li Juan Wu, Jian Xin Liu, Pan Zhang, Jian Jiao, Hui Yan, Tao Liu, Chang Fu Tian, Wen Xin Chen (2015). Arch Microbiol. Oct 15. [Epub ahead of print]

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Genetic redundancy is prevalent within the 6.7 Mb Sinorhizobium meliloti genome

Genetic redundancy is prevalent within the 6.7 Mb Sinorhizobium meliloti genome | Rhizobium Research | Scoop.it

Biological pathways are frequently identified via a genetic loss-of-function approach. While this approach has proven to be powerful, it is imperfect as illustrated by well-studied pathways continuing to have missing steps. One potential limiting factor is the masking of phenotypes through genetic redundancy. The prevalence of genetic redundancy in bacterial species has received little attention, although isolated examples of functionally redundant gene pairs exist. Here, we made use of a strain of Sinorhizobium meliloti whose genome was reduced by 45 % through the complete removal of a megaplasmid and a chromid (3 Mb of the 6.7 Mb genome was removed) to begin quantifying the level of genetic redundancy within a large bacterial genome. A mutagenesis of the strain with the reduced genome identified a set of transposon insertions precluding growth of this strain on minimal medium. Transfer of these mutations to the wild-type background revealed that 10–15 % of these chromosomal mutations were located within duplicated genes, as they did not prevent growth of cells with the full genome. The functionally redundant genes were involved in a variety of metabolic pathways, including central carbon metabolism, transport, and amino acid biosynthesis. These results indicate that genetic redundancy may be prevalent within large bacterial genomes. Failing to account for redundantly encoded functions in loss-of-function studies will impair our understanding of a broad range of biological processes and limit our ability to use synthetic biology in the construction of designer cell factories.

 

George C. diCenzo, Turlough M. Finan (2015). Mol Genet Genomics Published on line DOI 10.1007/s00438-015-0998-6



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Structure and Biological Roles of Sinorhizobium fredii HH103 Exopolysaccharide

Structure and Biological Roles of Sinorhizobium fredii HH103 Exopolysaccharide | Rhizobium Research | Scoop.it
Abstract Here we report that the structure of the Sinorhizobium fredii HH103 exopolysaccharide (EPS) is composed of glucose, galactose, glucuronic acid, pyruvic acid, in the ratios 5∶2∶2∶1 and is partially acetylated. A S. fredii HH103 exoA mutant (SVQ530), unable to produce EPS, not only forms nitrogen fixing nodules with soybean but also shows increased competitive capacity for nodule occupancy. Mutant SVQ530 is, however, less competitive to nodulate Vigna unguiculata. Biofilm formation was reduced in mutant SVQ530 but increased in an EPS overproducing mutant. Mutant SVQ530 was impaired in surface motility and showed higher osmosensitivity compared to its wild type strain in media containing 50 mM NaCl or 5% (w/v) sucrose. Neither S. fredii HH103 nor 41 other S. fredii strains were recognized by soybean lectin (SBL). S. fredii HH103 mutants affected in exopolysaccharides (EPS), lipopolysaccharides (LPS), cyclic glucans (CG) or capsular polysaccharides (KPS) were not significantly impaired in their soybean-root attachment capacity, suggesting that these surface polysaccharides might not be relevant in early attachment to soybean roots. These results also indicate that the molecular mechanisms involved in S. fredii attachment to soybean roots might be different to those operating in Bradyrhizobium japonicum.


Rodríguez-Navarro DN1, Rodríguez-Carvajal MA2, Acosta-Jurado S3, Soto MJ4, Margaret I3, Crespo-Rivas JC3, Sanjuan J4, Temprano F1, Gil-Serrano A2, Ruiz-Sainz JE3, Vinardell JM3 (2014).  PLoS One. 2014 Dec 18;9(12):e11539.

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Genetic analysis of signal integration by the Sinorhizobium meliloti sensor kinase FeuQ

Genetic analysis of signal integration by the Sinorhizobium meliloti sensor kinase FeuQ | Rhizobium Research | Scoop.it

Two-component signaling systems allow bacteria to recognize and respond to diverse environmental stimuli. Auxiliary proteins can provide an additional layer of control to these systems. The Sinorhizobium meliloti FeuPQ two-component system is required for symbiotic development and is negatively regulated by the auxiliary small periplasmic protein FeuN. This study explores the mechanistic basis of this regulation. We provide evidence that FeuN directly interacts with the sensor kinase FeuQ. The isolation and characterization of an extensive set of FeuN-insensitive and FeuN-mimicking variants of FeuQ reveal specific FeuQ residues (periplasmic and intracellular) that control the transmission of FeuN-specific signaling information. Similar analysis of the FeuN protein highlights short patches of compatibly charged residues on each protein that likely engage one another, giving rise to the downstream effects on target gene expression. The accumulated evidence suggests that the periplasmic interaction between FeuN and FeuQ introduces an intracellular conformational change in FeuQ, resulting in an increase in its ability to remove phosphate from its cognate response regulator FeuP. These observations underscore the complex manner in which membrane-spanning sensor kinases interface with the extracytoplasmic environment and convert that information to changes in intracellular processes.


VanYperen RD, Orton TS, Griffitts JS. (2014).  Microbiology Dec 5  [Epub ahead of print].

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A previously uncharacterized tetratricopeptide repeat containing protein is involved in cell envelope function in Rhizobium leguminosarum

A previously uncharacterized tetratricopeptide repeat containing protein is involved in cell envelope function in Rhizobium leguminosarum | Rhizobium Research | Scoop.it

Rhizobium leguminosarum is a soil bacterium that is an intracellular symbiont of leguminous plants through the formation of nitrogen-fixing root nodules. Due to the changing environments that rhizobia encounter, the cell is often faced with a variety of cell altering stressors that can compromise the cell envelope integrity. A previously uncharacterized operon (RL3499-RL3502) has been linked to proper cell envelope function, and mutants display pleiotropic phenotypes including an inability to grow on peptide rich media. In order to identify functional partners to the operon, gain of function suppressor mutants capable of growth on complex, peptide-rich media were isolated. A suppressor mutant (38KN52) restoring gain of function in mutant with a non-polar mutation to RL3500 was chosen for further characterization. Transposon mutagenesis, screening for loss of the suppressor phenotype, led to the identification of a Tn5 insertion in an uncharacterized tetratricopeptide repeat containing protein RL0936. Furthermore, RL0936 had a 3.5-fold increase in gene expression in the suppressor strain when compared to the wild-type and a 1.5-fold increase in the original RL3500 mutant. Mutation of RL0936 decreased desiccation tolerance and lowered the ability to form biofilms when compared to the wild-type strain. This work has identified a potential interaction between RL0936 and the RL3499-RL3502 operon that is involved in cell envelope development in R. leguminosarum, and has described phenotypic activities to a previously annotated uncharacterized conserved gene.

 

Neudorf KD, Vanderlinde EM, Tambalo DD, Yost CK (2014). Microbiology. Nov 4.  [Epub ahead of print]

   
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