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.
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.
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]
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
Bacteria have branched aerobic respiratory chains that terminate at different terminal oxidases. These terminal oxidases have varying properties such as their affinity for oxygen, transcriptional regulation, and proton pumping ability. The focus of this study was on a quinol oxidase, encoded by cyoABCD. Although this oxidase (Cyo) is widespread among bacteria, not much is known about its role in the cell, particularly in bacteria that contain both cytochrome c oxidases and quinol oxidases. Using Rhizobium etli CFN42 as a model organism, a Cyo- mutant was analyzed for its ability to grow in batch cultures at high (21%) and low (1% and 0.1%) ambient oxygen concentrations. In comparison to other oxidase mutants, the Cyo- had a significantly longer lag phase at low oxygen conditions. Using a cyo:lacZ transcriptional fusion, it was shown that cyo expression in the wild type peaks between 1-2.5% oxygen. In addition, it was shown with q-RT-PCR that cyoB is up-regulated approximately 5-fold in 1% oxygen compared to fully aerobic (21%) conditions. Analysis of the Cyo- mutant during symbiosis with Phaseolous vulgaris indicated that Cyo is utilized during early development of the symbiosis. Although Cyo is commonly thought to be utilized only in higher oxygen concentrations, the results from this study indicate Cyo is important for adaptation to and sustained growth at low oxygen.
Lunak ZR, Noel KD (2014). Microbiology. Nov 4. [Epub ahead of print]
Sinorhizobium meliloti strains unable to utilize galactose as a sole carbon source, due to mutations in the De-Ley Doudoroff pathway (dgoK), were previously shown to be more competitive for nodule occupancy. In this work, we show that strains carrying this mutation have galactose-dependent exopolysaccharide (EPS) phenotypes that were manifested as aberrant Calcofluor staining as well as decreased mucoidy when in an expR+ genetic background. The aberrant Calcofluor staining was correlated with changes in the pH of the growth medium. Strains carrying dgoK mutations were subsequently demonstrated to show earlier acidification of their growth medium that was correlated with an increase expression of genes associated with succinoglycan biosynthesis as well as increased accumulation of high and low molecular weight EPS in the medium. In addition, it was shown that the acidification of the medium was dependent on the inability of S. meliloti strains to initiate the catabolism of galactose. To more fully understand why strains carrying the dgoK allele were more competitive for nodule occupancy, early nodulation phenotypes were investigated. It was found that strains carrying the dgoK allele had a faster rate of nodulation. In addition, nodule competition experiments using genetic backgrounds unable to synthesize either succinoglycan or EPSII were consistent with the hypothesis that the increased competition phenotype was dependent upon the synthesis of succinoglycan. Fluorescent microscopy experiments on infected root-hair cells, using the acidotropic dye Lysotracker Red DND-99, provide evidence that the colonized curled root hair is an acidic compartment.
Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as Vibrio cholerea and Brucella abortis as well as in many N2-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria Sinorhizobium meliloti. Understanding the evolution and role of this multipartite genome organization will provide significant insight into these important organisms; yet this knowledge remains incomplete, in part, because technical challenges of large-scale genome manipulations have limited experimental analyses. The distinct evolutionary histories and characteristics of the three replicons that constitute the S. meliloti genome (the chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good model to examine this topic. We transferred essential genes from pSymB into the chromosome, and constructed strains that lack pSymB as well as both pSymA and pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a prokaryotic genome to date and the first removal of an essential chromid. Strikingly, strains lacking pSymA and pSymB (ΔpSymAB) lost the ability to utilize 55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur, yet the ΔpSymAB strain grew well in minimal salts media and in sterile soil. This suggests that the core chromosome is sufficient for growth in a bulk soil environment and that the pSymA and pSymB replicons carry genes with more specialized functions such as growth in the rhizosphere and interaction with the plant. These experimental data support a generalized evolutionary model, in which non-chromosomal replicons primarily carry genes with more specialized functions. These large secondary replicons increase the organism's niche range, which offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution with the chromosome then leads to the formation of a chromid through the acquisition of functions core to all niches (e.g. pSymB).
George C. diCenzo, Allyson M. MacLean, Branislava Milunovic, G. Brian Golding, and Turlough M. Finan (2014). PLoS Genet 10(10): e1004742.
Legumes are a highly important source of food, feed, and biofuel crops. With a few exceptions, these plants can enter into a complex symbiotic relationship with specific soil bacteria collectively called rhizobia. This interaction leads to formation of a new, highly specialized root organ – the nodule, inside which bacteria, differentiated into bacteroids, reduce atmospheric dinitrogen into forms of nitrogen that are useable by the plant. The legume–rhizobium association is highly specific and tightly controlled mainly by the host plant, in such a way that a particular legume interacts with only a limited set of rhizobial species. The establishment of symbiosis is a complex process, which involves a coordinated exchange of multiple signals between the host plant and its microsymbiont; among them, flavonoids secreted by legume roots and rhizobial lipochitin oligosaccharides (Nod factors) play a crucial role. Also, other plant-derived and bacterial cell-surface components and low-molecular-weight metabolites are engaged in the signaling. Among these, there are several proteins derived from the host plants (Nod factor receptors, signal transduction cascade proteins, lectins, trifolins, remorins), non-flavonoid inducers of rhizobial nod genes, H2O2, NO, and phytohormones. Additionally, rhizobia are actively involved in extracellular signaling to their host legumes to initiate infection and nodule morphogenesis. These bacteria produce and secrete many different compounds including hopanoids, indole-3-acetic acid, quorum sensors, bradyoxetin, lumichrome, H2O2 and NO, several types of surface polysaccharides (extracellular polysaccharide, lipopolysaccharide, capsular polysaccharide, cyclic β-glucan, glucomannan, gel-forming-polysaccharide, cellulose), and proteins secreted via types I and III secretion systems (glycanases, rhicadhesins, NodO, and Nops – nodulation outer proteins). They all contribute to various stages of symbiotic interactions, e.g., attachment to roots, host recognition, infection thread formation, and invasion of nodules. This review summarizes many aspects of the roles of extracellular signals, proteins, and polysaccharides in the early stages of legume–rhizobium symbiosis, showing high complexity in this “molecular dialog” and its interconnection with other cellular processes.
Monika Janczarek, Kamila Rachwał, Anna Marzec, Jarosław Grządziel, Marta Palusińska-Szysz (2015). Appl Soil Ecol 85: 94-113
The objectives of this research were to select the appropriate plant growth promoting rhizobacteria (PGPR) and evaluate their influence in promoting nodulation and N2-fixing efficiency of soybean (Glycine max) by coinoculation with Bradyrhizobium diazoefficiens USDA110 and B. japonicum THA6 strains. Selected 12 PGPR performed a significant capability of promoting N2-fixation, nodule number, nodule and plant dry weight with both of the commercial bradyrhizobial strains, USDA110 and THA6 (P < 0.05). Furthermore, isolates S141 and S222, which are closely related to Bacillus subtilis and Staphylococcus sp., were selected for coinoculation with USDA110 and THA6. The effective coinoculation doses of PGPR:Bradyrhizobium on soybean were 106:106 colony forming unit (CFU) ml−1 seed−1. The expression levels of soybean and Bradyrhizobium related genes including Glyma17g07330, otsA, phbC, dctA and nifH in nodule discontinuously triggered both up- and down-regulation at different time frames (2–7 WAI). The transmission electron microscopy (TEM) micrograph of coinoculated soybean nodule showed the compact cluster of bacteroids which was densely packed with poly-ß-hydroxybutyrate (PHB) granules. The amounts of PHBs remained in mature nodule of coinoculation treatments whilst single inoculation nodules were senescence. The induction of soybean root subsequently increased the nodulation signaling and then activated the trehalose accumulation and the transport of carbon that represented an increase in PHB accumulation, resulting in the enhancement of the nodulation and N2-fixation in soybean. These results were accordingly related with phenotypic characters in Leonard’s jar experiment in terms of enhancing the nodulation and N2-fixation in soybean. The effect of coinoculation experiment under field condition could increase 9.7–43.6% of seed yield per hectare which was higher than those of uninoculation or single inoculation of PGPR or Bradyrhizobium. Therefore, the efficiency to enhance soybean N2-fixation by coinoculation of S141 and S222 with Bradyrhizobium strategy could be developed for supreme soybean inoculants.
BACKGROUND: Regulation of transcription is essential for any organism and Rhizobium etli (a multi-replicon, nitrogen-fixing symbiotic bacterium) is no exception. This bacterium is commonly found in the rhizosphere (free-living) or inside of root-nodules of the common bean (Phaseolus vulgaris) in a symbiotic relationship. Abiotic stresses, such as high soil temperatures and salinity, compromise the genetic stability of R. etli and therefore its symbiotic interaction with P. vulgaris. However, it is still unclear which genes are up- or down-regulated to cope with these stress conditions. The aim of this study was to identify the genes and non-coding RNAs (ncRNAs) that are differentially expressed under heat and saline shock, as well as the promoter regions of the up-regulated loci.
RESULTS:Analysing the heat and saline shock responses of R. etli CE3 through RNA-Seq, we identified 756 and 392 differentially expressed genes, respectively, and 106 were up-regulated under both conditions. Notably, the set of genes over-expressed under either condition was preferentially encoded on plasmids, although this observation was more significant for the heat shock response. In contrast, during either saline shock or heat shock, the down-regulated genes were principally chromosomally encoded. Our functional analysis shows that genes encoding chaperone proteins were up-regulated during the heat shock response, whereas genes involved in the metabolism of compatible solutes were up-regulated following saline shock. Furthermore, we identified thirteen and nine ncRNAs that were differentially expressed under heat and saline shock, respectively, as well as eleven ncRNAs that had not been previously identified. Finally, using an in silico analysis, we studied the promoter motifs in all of the non-coding regions associated with the genes and ncRNAs up-regulated under both conditions.
CONCLUSIONS: Our data suggest that the replicon contribution is different for different stress responses and that the heat shock response is more complex than the saline shock response. In general, this work exemplifies how strategies that not only consider differentially regulated genes but also regulatory elements of the stress response provide a more comprehensive view of bacterial gene regulation.
López-Leal G, Tabche ML, Castillo-Ramírez S, Mendoza-Vargas A, Ramírez-Romero MA, Dávila G. (2014). BMC Genomics. Sep 8;15:770.
The stabilization of host–symbiont mutualism against the emergence of parasitic individuals is pivotal to the evolution of cooperation. One of the most famous symbioses occurs between legumes and their colonizing rhizobia, in which rhizobia extract nutrients (or benefits) from legume plants while supplying them with nitrogen resources produced by nitrogen fixation (or costs). Natural environments, however, are widely populated by ineffective rhizobia that extract benefits without paying costs and thus proliferate more efficiently than nitrogen-fixing cooperators. How and why this mutualism becomes stabilized and evolutionarily persists has been extensively discussed. To better understand the evolutionary dynamics of this symbiosis system, we construct a simple model based on the continuous snowdrift game with multiple interacting players. We investigate the model using adaptive dynamics and numerical simulations. We find that symbiotic evolution depends on the cost–benefit balance, and that cheaters widely emerge when the cost and benefit are similar in strength. In this scenario, the persistence of the symbiotic system is compatible with the presence of cheaters. This result suggests that the symbiotic relationship is robust to the emergence of cheaters, and may explain the prevalence of cheating rhizobia in nature. In addition, various stabilizing mechanisms, such as partner fidelity feedback, partner choice, and host sanction, can reinforce the symbiotic relationship by affecting the fitness of symbionts in various ways. This result suggests that the symbiotic relationship is cooperatively stabilized by various mechanisms. In addition, mixed nodule populations are thought to encourage cheater emergence, but our model predicts that, in certain situations, cheaters can disappear from such populations. These findings provide a theoretical basis of the evolutionary dynamics of legume–rhizobia symbioses, which is extendable to other single-host, multiple-colonizer systems
Horizontal gene transfer (HGT) is an important mode of adaptation and diversification of prokaryotes and eukaryotes and a major event underlying the emergence of bacterial pathogens and mutualists. Yet it remains unclear how complex phenotypic traits such as the ability to fix nitrogen with legumes have successfully spread over large phylogenetic distances. Here we show, using experimental evolution coupled with whole genome sequencing, that co-transfer of imuABC error-prone DNA polymerase genes with key symbiotic genes accelerates the evolution of a soil bacterium into a legume symbiont. Following introduction of the symbiotic plasmid of Cupriavidus taiwanensis, the Mimosa symbiont, into pathogenic Ralstonia solanacearum we challenged transconjugants to become Mimosa symbionts through serial plant-bacteria co-cultures. We demonstrate that a mutagenesis imuABC cassette encoded on the C. taiwanensis symbiotic plasmid triggered a transient hypermutability stage in R. solanacearum transconjugants that occurred before the cells entered the plant. The generated burst in genetic diversity accelerated symbiotic adaptation of the recipient genome under plant selection pressure, presumably by improving the exploration of the fitness landscape. Finally, we show that plasmid imuABC cassettes are over-represented in rhizobial lineages harboring symbiotic plasmids. Our findings shed light on a mechanism that may have facilitated the dissemination of symbiotic competency among α- and β-proteobacteria in natura and provide evidence for the positive role of environment-induced mutagenesis in the acquisition of a complex lifestyle trait. We speculate that co-transfer of complex phenotypic traits with mutagenesis determinants might frequently enhance the ecological success of HGT.
Horizontal gene transfer has an extraordinary impact on microbe evolution and diversification, by allowing exploration of new niches such as higher organisms. This is the case for rhizobia, a group of phylogenetically diverse bacteria that form a nitrogen-fixing symbiotic relationship with most leguminous plants. While these arose through horizontal transfer of symbiotic plasmids, this in itself is usually unproductive, and full expression of the acquired traits needs subsequent remodeling of the genome to ensure the ecological success of the transfer. Here we uncover a mechanism that accelerates the evolution of a soil bacterium into a legume symbiont. We show that key symbiotic genes are co-transferred with genes encoding stress-responsive error-prone DNA polymerases that transiently elevate the mutation rate in the recipient genome. This burst in genetic diversity accelerates the symbiotic evolution process under selection pressure from the host plant. A more widespread involvement of plasmid mutagenesis cassettes in rhizobium evolution is supported by their overrepresentation in rhizobia-containing lineages. Our findings provide evidence for the role of environment-induced mutagenesis in the acquisition of a complex lifestyle trait and predict that co-transfer of complex phenotypic traits with mutagenesis determinants might help successful horizontal gene transfer.
Remigi P, Capela D, Clerissi C, Tasse L, Torchet R, et al. (2014) Transient Hypermutagenesis Accelerates the Evolution of Legume Endosymbionts following Horizontal Gene Transfer. PLoS Biol 12(9): e1001942
Bacterial surface components, especially exopolysaccharides, in combination with bacterial Quorum Sensing signals are crucial for the formation of biofilms in most species studied so far. Biofilm formation allows soil bacteria to colonize their surrounding habitat and survive common environmental stresses such as desiccation and nutrient limitation. This mode of life is often essential for survival in bacteria of the genera Mesorhizobium,Sinorhizobium, Bradyrhizobium, and Rhizobium. The role of biofilm formation in symbiosis has been investigated in detail for Sinorhizobium meliloti and Bradyrhizobium japonicum. However, for S. fredii this process has not been studied. In this work we have demonstrated that biofilm formation is crucial for an optimal root colonization and symbiosis between S. fredii SMH12 and Glycine max cv Osumi. In this bacterium, nod-gene inducing flavonoids and the NodD1 protein are required for the transition of the biofilm structure from monolayer to microcolony. Quorum Sensing systems are also required for the full development of both types of biofilms. In fact, both the nodD1 mutant and the lactonase strain (the lactonase enzyme prevents AHL accumulation) are defective in soybean root colonization. The impairment of the lactonase strain in its colonization ability leads to a decrease in the symbiotic parameters. Interestingly, NodD1 together with flavonoids activates certain quorum sensing systems implicit in the development of the symbiotic biofilm. Thus, S. fredii SMH12 by means of a unique key molecule, the flavonoid, efficiently forms biofilm, colonizes the legume roots and activates the synthesis of Nod factors, required for successfully symbiosis.
Pérez-Montaño F, Jiménez-Guerrero I, Del Cerro P, Baena-Ropero I, López-Baena FJ, Ollero FJ, Bellogín R, Lloret J, Espuny R. (2014). PLoS One. 2014 Aug 28;9(8):e105901.
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
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.
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].
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]
In Sinorhizobium meliloti, timing of Quorum Sensing-dependent gene expression is controlled at multiple levels. RNA binding protein Hfq contributes to the regulation of QS signal production, and this regulation is exerted both in the manner that involves AHL receptor ExpR, and via expR-independent mechanisms. In the expR+ strain of S. meliloti, deletion of hfq resulted in hyper-accumulation of QS signals at low population densities, increased diversity of the QS signals in mid-to-late exponential phase and then led to a sharp decrease in QS signal accumulation in stationary phase. qPCR revealed that the accumulation of expR, and sinI (but not sinR) mRNA was increased in the late exponential phase in an hfq-dependent manner. A translational, but not transcriptional, expR-uidA reporter was controlled by hfq, while both transcriptional and translational sinI-uidA reporters were regulated in the hfq-dependent manner. In co-immunoprecipation experiments, expR mRNA was bound to and then released from Hfq, similarly to the positive controls (small regulatory RNA SmrC9, SmrC15, SmrC16 and SmrC45). Neither sinI, nor sinR transcripts were detected in the pool of RNA heat-released from Hfq-RNA complexes. Therefore, post-transcriptional regulator Hfq controls production and perception of QS signals and at higher population densities, this control is mediated directly via interactions with expR.
Gao M\, Tang M, Guerich L, Salas-Gonzalez I, Teplitski M. (2014).
Environ Microbiol Rep. Nov 10. [Epub ahead of print]
We are honored to host the 19th International Congress on Nitrogen Fixation (19th ICNF). The meeting will be held at the beautiful Asilomar Conference Grounds in Pacific Grove, CA
AsilomarAsilomar State Beach and Conference Grounds is a breathtaking 107 acres of ecologically diverse beachfront land. It is conveniently located just eight (8) miles from The Monterey Peninsula Airport (MRY), 80 miles form San Jose Airport (SJC), and 110 miles south of the San Francisco Airport (SFO).
THE Genetics Society of America’s Thomas Hunt Morgan Medal is awarded to an individual GSA member for lifetime achievement in the field of genetics. The 2014 recipient is Frederick Ausubel, whose 40-year career has centered on host–microbe interactions and host innate immunity. He is widely recognized as a key scientist responsible for establishing the modern postrecombinant DNA field of host–microbe interactions using simple nonvertebrate hosts. He has used genetic approaches to conduct pioneering work that spawned six related areas of research: the evolution and regulation of Rhizobiumgenes involved in symbiotic nitrogen fixation; the regulation of Rhizobium genes by two-component regulatory systems involving histidine kinases; the establishment of Arabidopsis thaliana as a worldwide model system; the identification of a large family of plant disease resistance genes; the identification of so-called multi-host bacterial pathogens; and the demonstration that Caenorhabditis elegans has an evolutionarily conserved innate immune system that shares features of both plant and mammalian immunity.
Production of extracellular polysaccharides is a complex process engaging proteins localized in different subcellular compartments, yet communicating with each other or even directly interacting in multicomponent complexes. Proteins involved in polymerization and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum are encoded within the chromosomal Pss-I cluster. However, genes implicated in polysaccharide synthesis are common in rhizobia, with several homologues of pss genes identified in other regions of the R. leguminosarum genome. One such region is chromosomally located Pss-II encoding proteins homologous to known components of the Wzx/Wzy-dependent polysaccharide synthesis and transport systems. The pssP2 gene encodes a protein similar to polysaccharide co-polymerases involved in determination of the length of polysaccharide chains in capsule and O-antigen biosynthesis. In this work, a mutant with a disrupted pssP2 gene was constructed and its capabilities to produce EPS and enter into a symbiotic relationship with clover were studied. The pssP2 mutant, while not altered in lipopolysaccharide (LPS), displayed changes in molecular mass distribution profile of EPS. Lack of the full-length PssP2 protein resulted in a reduction of high molecular weight EPS, yet polymerized to a longer length than in the RtTA1 wild type. The mutant strain was also more efficient in symbiotic performance. The functional interrelation between PssP2 and proteins encoded within the Pss-I region was further supported by data from bacterial two-hybrid assays providing evidence for PssP2 interactions with PssT polymerase, as well as glycosyltransferase PssC. A possible role for PssP2 in a complex involved in EPS chain-length determination is discussed.
Marczak M, Matysiak P, Kutkowska J, Skorupska A (2014). PLoS One. Sep 30;9(9):e109106.
Autoregulatory negative-feedback loops play important roles in fine-balancing tissue and organ development. Such loops are composed of short-range intercellular signaling pathways via cell–cell communications. On the other hand, leguminous plants use a long-distance negative-feedback system involving root–shoot communication to control the number of root nodules, root lateral organs that harbor symbiotic nitrogen-fixing bacteria known as rhizobia. This feedback system, known as autoregulation of nodulation (AON), consists of two long-distance mobile signals: root-derived and shoot-derived signals. Two Lotus japonicus CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE)-related small peptides, CLE ROOT SIGNAL1 (CLE-RS1) and CLE-RS2, function as root-derived signals and are perceived by a shoot-acting AON factor, the HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) receptor protein, an ortholog of Arabidopsis CLAVATA1, which is responsible for shoot apical meristem homeostasis. This peptide–receptor interaction is necessary for systemic suppression of nodulation. How the onset of nodulation activates AON and how optimal nodule numbers are maintained remain unknown, however. Here we show that an RWP-RK–containing transcription factor, NODULE INCEPTION (NIN), which induces nodule-like structures without rhizobial infection when expressed ectopically, directly targets CLE-RS1 and CLE-RS2. Roots constitutively expressing NIN systemically repress activation of endogenous NIN expression in untransformed roots of the same plant in a HAR1-dependent manner, leading to systemic suppression of nodulation and down-regulation of CLE expression. Our findings provide, to our knowledge, the first molecular evidence of a long-distance autoregulatory negative-feedback loop that homeostatically regulates nodule organ formation.
In bacteria, membrane transporters of the cation diffusion facilitator (CDF) family participate in Zn2+, Fe2+, Mn2+, Co2+ and Ni2+ homeostasis. The functional role during infection processes for several members has been shown to be linked to the specificity of transport. Sinorhizobium meliloti has two homologous CDF genes with unknown transport specificity. Here we evaluate the role played by the CDF SMc02724 (SmYiiP). The deletion mutant strain of SmYiiP (ΔsmyiiP) showed reduced in vitro growth fitness only in the presence of Mn2+. Incubation of ΔsmyiiP and WT cells with sub-lethal Mn2+ concentrations resulted in a 2-fold increase of the metal only in the mutant strain. Normal levels of resistance to Mn2+ were attained by complementation with the gene SMc02724 under regulation of its endogenous promoter. In vitro, liposomes with incorporated heterologously expressed pure protein accumulated several transition metals. However, only the transport rate of Mn2+ was increased by imposing a transmembrane H+ gradient. Nodulation assays in alfalfa plants showed that the strain ΔsmyiiP induced a lower number of nodules than in plants infected with the WT strain. Our results indicate that Mn2+ homeostasis in S. meliloti is required for full infection capacity, or nodule function, and that the specificity of transport in vivo of SmYiiP is narrowed down to Mn2+ by a mechanism involving the proton motive force.
Raimunda D and Elso-Berberián G (2014). Biochim Biophys Acta. 2014 Sep 18.
Legume root nodules are induced by N-fixing rhizobium bacteria that are hosted in an intracellular manner. These nodules are formed by reprogramming differentiated root cells. The model legume Medicago truncatula forms indeterminate nodules with a meristem at their apex. This organ grows by the activity of the meristem that adds cells to the different nodule tissues. In Medicago sativa it has been shown that the nodule meristem is derived from the root middle cortex. During nodule initiation, inner cortical cells and pericycle cells are also mitotically activated. However, whether and how these cells contribute to the mature nodule has not been studied. Here, we produce a nodule fate map that precisely describes the origin of the different nodule tissues based on sequential longitudinal sections and on the use of marker genes that allow the distinction of cells originating from different root tissues. We show that nodule meristem originates from the third cortical layer, while several cell layers of the base of the nodule are directly formed from cells of the inner cortical layers, root endodermis and pericycle. The latter two differentiate into the uninfected tissues that are located at the base of the mature nodule, whereas the cells derived from the inner cortical cell layers form about eight cell layers of infected cells. This nodule fate map has then been used to re-analyse several mutant nodule phenotypes. This showed, among other things, that intracellular release of rhizobia in primordium cells and meristem daughter cells are regulated in a different manner.
Salicylic acid is an important signalling molecule in plant-microbe defence and symbiosis. We analysed the transcriptional responses of the nitrogen fixing plant symbiont, Rhizobium leguminosarum bv viciae 3841 to salicylic acid. Two MFS-type multicomponent efflux systems were induced in response to salicylic acid, rmrAB and the hitherto undescribed system salRAB. Based on sequence similarity salA and salB encode a membrane fusion and inner membrane protein respectively. salAB are positively regulated by the LysR regulator SalR. Disruption of salA significantly increased the sensitivity of the mutant to salicylic acid, while disruption of rmrA did not. A salA/rmrA double mutation did not have increased sensitivity relative to the salA mutant. Pea plants nodulated by salA or rmrA strains did not have altered nodule number or nitrogen fixation rates, consistent with weak expression of salA in the rhizosphere and in nodule bacteria. However, BLAST analysis revealed seventeen putative efflux systems in Rlv3841 and several of these were highly differentially expressed during rhizosphere colonisation, host infection and bacteroid differentiation. This suggests they have an integral role in symbiosis with host plants.
Tett AJ, Karunakaran R, Poole PS (2014). PLoS One. 2014 Aug 18;9(8):e103647
The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti carries a large number of toxin-antitoxin (TA) modules both on the chromosome and megaplasmids. One of them, the vapBC-5 module that belongs to the type II systems was characterized here. It encodes an active toxin vapC-5, and was shown to be controlled negatively by the complex of its own proteins. Different mutants of the vapBC-5genes exhibited diverse effects on symbiotic efficiency during interaction with the host plant Medicago sativa. The absence of the entirevapBC-5 region had no influence on nodule formation and nitrogen fixation properties. The strain carrying an insertion in the antitoxin gene showed a reduced nitrogen fixation capacity resulting in a lower plant yield. In contrast, when the toxin gene was mutated, the strain developed more efficient symbiosis with the host plant. The nitrogen fixing root nodules had a delayed senescent phenotype and contained elevated level of plant-derived molecules characteristic of later steps of nodule development. The longer bacteroid viability and abundance of active nitrogen fixing zone resulted in increased production of plant material. These data indicate that modification of the toxin/antitoxin production may influence bacteroid metabolism and may have an impact on the adaptation to changing environmental conditions.
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