scholarly journals A conserved rhizobial peptidase that interacts with host-derived symbiotic peptides

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alex B. Benedict ◽  
Prithwi Ghosh ◽  
Samuel M. Scott ◽  
Joel S. Griffitts
Keyword(s):  
Sinorhizobium Meliloti ◽  
Core Genome ◽  
Root Nodule ◽  
Root Nodules ◽  
Intracellular Bacteria ◽  
Chemical Signaling ◽  
Free Living ◽  
Weak Expression ◽  
Free Living Conditions

AbstractIn the Medicago truncatula-Sinorhizobium meliloti symbiosis, chemical signaling initiates rhizobial infection of root nodule tissue, where a large portion of the bacteria are endocytosed into root nodule cells to function in nitrogen-fixing organelles. These intracellular bacteria are subjected to an arsenal of plant-derived nodule-specific cysteine-rich (NCR) peptides, which induce the physiological changes that accompany nitrogen fixation. NCR peptides drive these intracellular bacteria toward terminal differentiation. The bacterial peptidase HrrP was previously shown to degrade host-derived NCR peptides and give the bacterial symbionts greater fitness at the expense of host fitness. The hrrP gene is found in roughly 10% of Sinorhizobium isolates, as it is carried on an accessory plasmid. The objective of the present study is to identify peptidase genes in the core genome of S. meliloti that modulate symbiotic outcome in a manner similar to the accessory hrrP gene. In an overexpression screen of annotated peptidase genes, we identified one such symbiosis-associated peptidase (sap) gene, sapA (SMc00451). When overexpressed, sapA leads to a significant decrease in plant fitness. Its promoter is active in root nodules, with only weak expression evident under free-living conditions. The SapA enzyme can degrade a broad range of NCR peptides in vitro.

scholarly journals Paraburkholderia phymatum STM815 σ54 Controls Utilization of Dicarboxylates, Motility, and T6SS-b Expression

Nitrogen ◽  
2020 ◽  
Vol 1 (2) ◽  
pp. 81-98
Author(s):  
Martina Lardi ◽  
Yilei Liu ◽  
Sebastian Hug ◽  
Samanta Bolzan de Campos ◽  
Leo Eberl ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Nitrogenase Activity ◽  
Secretion Systems ◽  
Free Living ◽  
Major Life ◽  
Life Styles ◽  
Type Vi Secretion ◽  
Flagellar Biosynthesis ◽  
Free Living Conditions

Rhizobia have two major life styles, one as free-living bacteria in the soil, and the other as bacteroids within the root/stem nodules of host legumes where they convert atmospheric nitrogen into ammonia. In the soil, rhizobia have to cope with changing and sometimes stressful environmental conditions, such as nitrogen limitation. In the beta-rhizobial strain Paraburkholderia phymatum STM815, the alternative sigma factor σ54 (or RpoN) has recently been shown to control nitrogenase activity during symbiosis with Phaseolus vulgaris. In this study, we determined P. phymatum’s σ54 regulon under nitrogen-limited free-living conditions. Among the genes significantly downregulated in the absence of σ54, we found a C4-dicarboxylate carrier protein (Bphy_0225), a flagellar biosynthesis cluster (Bphy_2926-64), and one of the two type VI secretion systems (T6SS-b) present in the P. phymatum STM815 genome (Bphy_5978-97). A defined σ54 mutant was unable to grow on C4 dicarboxylates as sole carbon source and was less motile compared to the wild-type strain. Both defects could be complemented by introducing rpoNin trans. Using promoter reporter gene fusions, we also confirmed that the expression of the T6SS-b cluster is regulated by σ54. Accordingly, we show that σ54 affects in vitro competitiveness of P. phymatum STM815 against Paraburkholderia diazotrophica.

scholarly journals GlnB/GlnK PII Proteins and Regulation of the Sinorhizobium meliloti Rm1021 Nitrogen Stress Response and Symbiotic Function

2010 ◽  
Vol 192 (10) ◽  
pp. 2473-2481 ◽  
Author(s):  
Svetlana N. Yurgel ◽  
Jennifer Rice ◽  
Monika Mulder ◽  
Michael L. Kahn
Keyword(s):  
Stress Response ◽  
Cellular Response ◽  
Protein Modification ◽  
Sinorhizobium Meliloti ◽  
Nitrogen Stress ◽  
Free Living ◽  
Sensor Protein ◽  
Pii Proteins ◽  
Nitrogen Exchange

ABSTRACT The Sinorhizobium meliloti Rm1021ΔglnD-sm2 mutant, which is predicted to make a GlnD nitrogen sensor protein truncated at its amino terminus, fixes nitrogen in symbiosis with alfalfa, but the plants cannot use this nitrogen for growth (S. N. Yurgel and M. L. Kahn, Proc. Natl. Acad. Sci. U. S. A. 105:18958-18963, 2008). The mutant also has a generalized nitrogen stress response (NSR) defect. These results suggest a connection between GlnD, symbiotic metabolism, and the NSR, but the nature of this connection is unknown. In many bacteria, GlnD modifies the PII proteins, GlnB and GlnK, as it transduces a measurement of bacterial nitrogen status to a cellular response. We have now constructed and analyzed Rm1021 mutants missing GlnB, GlnK, or both proteins. Rm1021ΔglnKΔglnB was much more defective in its NSR than either single mutant, suggesting that GlnB and GlnK overlap in regulating the NSR in free-living Rm1021. The single mutants and the double mutant all formed an effective symbiosis, indicating that symbiotic nitrogen exchange could occur without the need for either GlnB or GlnK. N-terminal truncation of the GlnD protein interfered with PII protein modification in vitro, suggesting either that unmodified PII proteins were responsible for the glnD mutant's ineffective phenotype or that connecting GlnD and appropriate symbiotic behavior does not require the PII proteins.

scholarly journals HPrK Regulates Succinate-Mediated Catabolite Repression in the Gram-Negative Symbiont Sinorhizobium meliloti

2008 ◽  
Vol 191 (1) ◽  
pp. 298-309 ◽  
Author(s):  
Catalina Arango Pinedo ◽  
Daniel J. Gage
Keyword(s):  
Catabolite Repression ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Phosphotransferase System ◽  
Free Living ◽  
Gene Encoding ◽  
Common Component ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Living Organisms

ABSTRACT The HPrK kinase/phosphatase is a common component of the phosphotransferase system (PTS) of gram-positive bacteria and regulates catabolite repression through phosphorylation/dephosphorylation of its substrate, the PTS protein HPr, at a conserved serine residue. Phosphorylation of HPr by HPrK also affects additional phosphorylation of HPr by the PTS enzyme EI at a conserved histidine residue. Sinorhizobium meliloti can live as symbionts inside legume root nodules or as free-living organisms and is one of the relatively rare gram-negative bacteria known to have a gene encoding HPrK. We have constructed S. meliloti mutants that lack HPrK or that lack key amino acids in HPr that are likely phosphorylated by HPrK and EI. Deletion of hprK in S. meliloti enhanced catabolite repression caused by succinate, as did an S53A substitution in HPr. Introduction of an H22A substitution into HPr alleviated the strong catabolite repression phenotypes of strains carrying ΔhprK or hpr(S53A) mutations, demonstrating that HPr-His22-P is needed for strong catabolite repression. Furthermore, strains with a hpr(H22A) allele exhibited relaxed catabolite repression. These results suggest that HPrK phosphorylates HPr at the serine-53 residue, that HPr-Ser53-P inhibits phosphorylation at the histidine-22 residue, and that HPr-His22-P enhances catabolite repression in the presence of succinate. Additional experiments show that ΔhprK mutants overproduce exopolysaccharides and form nodules that do not fix nitrogen.

scholarly journals Multiple groESL Operons Are Not Key Targets of RpoH1 and RpoH2 in Sinorhizobium meliloti

2006 ◽  
Vol 188 (10) ◽  
pp. 3507-3515 ◽  
Author(s):  
Alycia N. Bittner ◽  
Valerie Oke
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Fixation ◽  
High Temperature ◽  
Sigma Factor ◽  
Host Plants ◽  
Sinorhizobium Meliloti ◽  
Free Living ◽  
Genes Encoding ◽  
Multiple Copies ◽  
Free Living Conditions

ABSTRACT Among the rhizobia that establish nitrogen-fixing nodules on the roots of host plants, many contain multiple copies of genes encoding the sigma factor RpoH and the chaperone GroEL/GroES. In Sinorhizobium meliloti there are two rpoH genes, four groESL operons, and one groEL gene. rpoH1 mutants are defective for growth at high temperature and form ineffective nodules, rpoH1 rpoH2 double mutants are unable to form nodules, and groESL1 mutants form ineffective nodules. To explore the roles of RpoH1 and RpoH2, we identified mutants that suppress both the growth and nodulation defects. These mutants do not suppress the nitrogen fixation defect. This implies that the functions of RpoH1 during growth and RpoH1/RpoH2 during the initiation of symbiosis are similar but that there is a different function of RpoH1 needed later during symbiosis. We showed that, unlike in Escherichia coli, overexpression of groESL is not sufficient to bypass any of the RpoH defects. Under free-living conditions, we determined that RpoH2 does not control expression of the groE genes, and RpoH1 only controls expression of groESL5. Finally, we completed the series of groE mutants by constructing groESL3 and groEL4 mutants and demonstrated that they do not display symbiotic defects. Therefore, the only groESL operon required by itself for symbiosis is groESL1. Taken together, these results suggest that GroEL/GroES production alone cannot explain the requirements for RpoH1 and RpoH2 in S. meliloti and that there must be other crucial targets.

scholarly journals Effects of Engineered Sinorhizobium meliloti on Cytokinin Synthesis and Tolerance of Alfalfa to Extreme Drought Stress

2012 ◽  
Vol 78 (22) ◽  
pp. 8056-8061 ◽  
Author(s):  
Ji Xu ◽  
Xiao-Lin Li ◽  
Li Luo
Keyword(s):  
Nitrogen Fixation ◽  
Drought Stress ◽  
Sinorhizobium Meliloti ◽  
Nitrogenase Activity ◽  
Root Nodules ◽  
Severe Drought ◽  
Control Strain ◽  
Free Living ◽  
Content Type ◽  
Host Tolerance

ABSTRACTCytokinin is required for the initiation of leguminous nitrogen fixation nodules elicited by rhizobia and the delay of the leaf senescence induced by drought stress. A few free-living rhizobia have been found to produce cytokinin. However, the effects of engineered rhizobia capable of synthesizing cytokinin on host tolerance to abiotic stresses have not yet been described. In this study, two engineeredSinorhizobiumstrains overproducing cytokinin were constructed. The tolerance of inoculated alfalfa plants to severe drought stress was assessed. The engineered strains, which expressed theAgrobacterium iptgene under the control of different promoters, synthesized more zeatins than the control strain under free-living conditions, but their own growth was not affected. After a 4-week inoculation period, the effects of engineered strains on alfalfa growth and nitrogen fixation were similar to those of the control strain under nondrought conditions. After being subjected to severe drought stress, most of the alfalfa plants inoculated with engineered strains survived, and the nitrogenase activity in their root nodules showed no apparent change. A small elevation in zeatin concentration was observed in the leaves of these plants. The expression of antioxidant enzymes increased, and the level of reactive oxygen species decreased correspondingly. Although theiptgene was transcribed in the bacteroids of engineered strains, the level of cytokinin in alfalfa nodules was identical to that of the control. These findings suggest that engineeredSinorhizobiumstrains synthesizing more cytokinin could improve the tolerance of alfalfa to severe drought stress without affecting alfalfa nodulation or nitrogen fixation.

scholarly journals Cooperation, Competition, and Specialized Metabolism in a Simplified Root Nodule Microbiome

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Bridget L. Hansen ◽  
Rita de Cassia Pessotti ◽  
Monika S. Fischer ◽  
Alyssa Collins ◽  
Laila El-Hifnawi ◽  
...  
Keyword(s):  
Potential Role ◽  
Root Nodule ◽  
Root Nodules ◽  
Imaging Mass Spectrometry ◽  
In Planta ◽  
Content Type ◽  
Specialized Metabolites ◽  
Brevibacillus Brevis

ABSTRACT Microbiomes associated with various plant structures often contain members with the potential to make specialized metabolites, e.g., molecules with antibacterial, antifungal, or siderophore activities. However, when and where microbes associated with plants produce specialized metabolites, and the potential role of these molecules in mediating intramicrobiome interactions, is not well understood. Root nodules of legume plants are organs devoted to hosting symbiotic bacteria that fix atmospheric nitrogen and have recently been shown to harbor a relatively simple accessory microbiome containing members with the ability to produce specialized metabolites in vitro. On the basis of these observations, we sought to develop a model nodule microbiome system for evaluating specialized microbial metabolism in planta. Starting with an inoculum derived from field-grown Medicago sativa nodules, serial passaging through gnotobiotic nodules yielded a simplified accessory community composed of four members: Brevibacillus brevis, Paenibacillus sp., Pantoea agglomerans, and Pseudomonas sp. Some members of this community exhibited clear cooperation in planta, while others were antagonistic and capable of disrupting cooperation between other partners. Using matrix-assisted laser desorption ionization–imaging mass spectrometry, we found that metabolites associated with individual taxa had unique distributions, indicating that some members of the nodule community were spatially segregated. Finally, we identified two families of molecules produced by B. brevis in planta as the antibacterial tyrocidines and a novel set of gramicidin-type molecules, which we term the britacidins. Collectively, these results indicate that in addition to nitrogen fixation, legume root nodules are likely also sites of active antimicrobial production.

scholarly journals Metapopulation dominance and genomic-island acquisition of Bradyrhizobium with superior catabolic capabilities

2016 ◽  
Vol 283 (1829) ◽  
pp. 20160496 ◽  
Author(s):  
Amanda C. Hollowell ◽  
John U. Regus ◽  
David Turissini ◽  
Kelsey A. Gano-Cohen ◽  
Roxanne Bantay ◽  
...  
Keyword(s):  
Root Nodule ◽  
Carbon Sources ◽  
Natural Populations ◽  
Soil Environment ◽  
In Planta ◽  
Selective Force ◽  
Free Living ◽  
Chromosomal Genes ◽  
Key Driver ◽  
Free Living Conditions

Root nodule-forming rhizobia exhibit a bipartite lifestyle, replicating in soil and also within plant cells where they fix nitrogen for legume hosts. Host control models posit that legume hosts act as a predominant selective force on rhizobia, but few studies have examined rhizobial fitness in natural populations. Here, we genotyped and phenotyped Bradyrhizobium isolates across more than 800 km of the native Acmispon strigosus host range. We sequenced chromosomal genes expressed under free-living conditions and accessory symbiosis loci expressed in planta and encoded on an integrated ‘symbiosis island’ (SI). We uncovered a massive clonal expansion restricted to the Bradyrhizobium chromosome, with a single chromosomal haplotype dominating populations, ranging more than 700 km, and acquiring 42 divergent SI haplotypes, none of which were spatially widespread. For focal genotypes, we quantified utilization of 190 sole-carbon sources relevant to soil fitness. Chromosomal haplotypes that were both widespread and dominant exhibited superior growth on diverse carbon sources, whereas these patterns were not mirrored among SI haplotypes. Abundance, spatial range and catabolic superiority of chromosomal, but not symbiosis genotypes suggests that fitness in the soil environment, rather than symbiosis with hosts, might be the key driver of Bradyrhizobium dominance.

Confirmation of the infectivity of a free-living actinomycete isolated from Comptonia peregrina root nodules by immunological and ultrastructural studies

1978 ◽  
Vol 56 (21) ◽  
pp. 2621-2635 ◽  
Author(s):  
Maurice Lalonde
Keyword(s):  
Nitrogen Fixation ◽  
Pure Culture ◽  
Root Nodules ◽  
Free Living ◽  
Ultrastructural Studies ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity ◽  
Membrane Envelope ◽  
Freeze Etching

The inoculation of the host plant Comptonia peregrina (L.) Coult. (Myrica asplenifolia L.) by a pure culture of a free-living actinomycete, isolated from Comptonia root nodules by Callaham et al. (1978, Science, 199: 899–902), was successful. Short-term and long-term nodulation tests confirmed the infectivity of the Comptonia isolate. Acetylene reduction assays of the nodules induced by this prokaryote isolate demonstrated nitrogen fixation activity. This nitrogen fixation activity was able to sustain a prolific growth of the nodulated host plants growing in a N-free substrate. Indirect immufluorescence reactions, using specific gamma globulin against the actinomycetal isolate of the Comptonia root nodule, demonstrated the identity of this actinomycete in these in vitro produced Comptonia root nodules. Ultrastructure of the Comptonia isolate, developing as a free-living or endophytic actinomycete, was observed by light microscopy, freeze etching, and transmission electron microscopy. The free-living filamentous and sporulating isolate produced typical hyphae and vesicles when growing as an endophyte in the Comptonia nodule. These endophytic hyphae and vesicles were always encapsulated by a polysaccharide material which was surrounded by a host membrane envelope. A polysaccharide capsule was not demonstrated on the free-living Comptonia isolate. The endophytic vesicles were club shaped and highly septate. Such vesicles were never produced by the free-living isolate growing in an artificial medium. The Comptonia isolate is a spore former in pure culture and was able to sporulate in basal tissues of 5-month-old Comptonia nodules.

Behavior in tissue culture of nitrogen-fixing root nodules of Ceanothus integerrimus

1980 ◽  
Vol 58 (10) ◽  
pp. 1121-1128 ◽  
Author(s):  
George S. Ellmore ◽  
Ruth Strand ◽  
W. M. Laetsch
Keyword(s):  
Tissue Culture ◽  
Root Nodule ◽  
Root Nodules ◽  
Tissue Growth ◽  
Cultivated Plants ◽  
In Vitro Growth ◽  
Cell Cytoplasm ◽  
Host Cell Cytoplasm ◽  
Agar Media

This study documents the in vitro growth of N2-fixing root nodules from the dicotyledonous shrub Ceanothus integerrimus (Rhamnaceae). Root nodule lobes were removed from cultivated plants, surface sterilized, and cultured on agar media containing varied amounts of nitrogen (N). Uninfected cells inside the cultured nodules divide and form a callus which is visible after 7 days of growth. Cells infected by the N2-fixing actinomycete do not divide, but rather degenerate. Host cell cytoplasm disappears and organelles and the plasma membrane are no longer seen under the electron microscope. Endophyte structure also deteriorates. Nucleoid areas within the terminal vesicles become diffuse. Cross walls separating vesicles from the hyphae disappear and cytoplasm of the hyphae withdraws from the actinomycete wall. Ceanothus nodules readily form callus in tissue culture. Callus proliferates from uninfected cortical parenchyma outside the infection zone. The callus is free of living endophyte and is incapable of fixing N2 as measured by acetylene reduction. Nodule tissue growth in vitro is more vigorous on media with high N than on N-deficient media.

scholarly journals Differential Expression of Paraburkholderia phymatum Type VI Secretion Systems (T6SS) Suggests a Role of T6SS-b in Early Symbiotic Interaction

2021 ◽  
Vol 12 ◽  
Author(s):  
Sebastian Hug ◽  
Yilei Liu ◽  
Benjamin Heiniger ◽  
Aurélien Bailly ◽  
Christian H. Ahrens ◽  
...  
Keyword(s):  
Root Nodules ◽  
Rhizobial Strain ◽  
Secretion Systems ◽  
Phenotypic Analysis ◽  
Free Living ◽  
Symbiotic Interaction ◽  
Type Vi Secretion ◽  
Mutant Strains

Paraburkholderia phymatum STM815, a rhizobial strain of the Burkholderiaceae family, is able to nodulate a broad range of legumes including the agriculturally important Phaseolus vulgaris (common bean). P. phymatum harbors two type VI Secretion Systems (T6SS-b and T6SS-3) in its genome that contribute to its high interbacterial competitiveness in vitro and in infecting the roots of several legumes. In this study, we show that P. phymatum T6SS-b is found in the genomes of several soil-dwelling plant symbionts and that its expression is induced by the presence of citrate and is higher at 20/28°C compared to 37°C. Conversely, T6SS-3 shows homologies to T6SS clusters found in several pathogenic Burkholderia strains, is more prominently expressed with succinate during stationary phase and at 37°C. In addition, T6SS-b expression was activated in the presence of germinated seeds as well as in P. vulgaris and Mimosa pudica root nodules. Phenotypic analysis of selected deletion mutant strains suggested a role of T6SS-b in motility but not at later stages of the interaction with legumes. In contrast, the T6SS-3 mutant was not affected in any of the free-living and symbiotic phenotypes examined. Thus, P. phymatum T6SS-b is potentially important for the early infection step in the symbiosis with legumes.

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