scholarly journals Symbiotic Performance of Sinorhizobium meliloti Lacking ppGpp Depends on the Medicago Host Species

2019 ◽  
Vol 32 (6) ◽  
pp. 717-728 ◽  
Author(s):  
Kathrin Wippel ◽  
Sharon R. Long
Keyword(s):  
Nitrogen Fixation ◽  
Stress Responses ◽  
Sinorhizobium Meliloti ◽  
Root Nodule ◽  
Stringent Response ◽  
Wild Type ◽  
Exopolysaccharide Biosynthesis ◽  
Fixation Capacity ◽  
Late Stages ◽  
Nodule Symbiosis

Host specificity in the root-nodule symbiosis between legumes and rhizobia is crucial for the establishment of a successful interaction and ammonia provision to the plant. The specificity is mediated by plant-bacterial signal exchange during early stages of interaction. We observed that a Sinorhizobium meliloti mutant ∆relA, which is deficient in initiating the bacterial stringent response, fails to nodulate Medicago sativa (alfalfa) but successfully infects Medicago truncatula. We used biochemical, histological, transcriptomic, and imaging approaches to compare the behavior of the S. meliloti ∆relA mutant and wild type (WT) on the two plant hosts. ∆relA performed almost WT-like on M. truncatula, except for reduced nitrogen-fixation capacity and a disorganized positioning of bacteroids within nodule cells. In contrast, ∆relA showed impaired root colonization on alfalfa and failed to infect nodule primordia. Global transcriptome analyses of ∆relA cells treated with the alfalfa flavonoid luteolin and of mature nodules induced by the mutant on M. truncatula revealed normal nod gene expression but overexpression of exopolysaccharide biosynthesis genes and a slight suppression of plant defense-like reactions. Many RelA-dependent transcripts overlap with the hypo-osmolarity–related FeuP regulon or are characteristic of stress responses. Based on our findings, we suggest that RelA is not essential until the late stages of symbiosis with M. truncatula, in which it may be involved in processes that optimize nitrogen fixation.

scholarly journals Sinorhizobium meliloti Mutants Deficient in Phosphatidylserine Decarboxylase Accumulate Phosphatidylserine and Are Strongly Affected during Symbiosis with Alfalfa

2008 ◽  
Vol 190 (20) ◽  
pp. 6846-6856 ◽  
Author(s):  
Miguel Angel Vences-Guzmán ◽  
Otto Geiger ◽  
Christian Sohlenkamp
Keyword(s):  
Sinorhizobium Meliloti ◽  
Minimal Medium ◽  
Root Nodule ◽  
Membrane Lipids ◽  
Nodule Formation ◽  
Deficient Mutant ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Root Nodule Symbiosis ◽  
Nodule Symbiosis

ABSTRACT Sinorhizobium meliloti contains phosphatidylglycerol, cardiolipin, phosphatidylcholine, and phosphatidylethanolamine (PE) as major membrane lipids. PE is formed in two steps. In the first step, phosphatidylserine synthase (Pss) condenses serine with CDP-diglyceride to form phosphatidylserine (PS), and in the second step, PS is decarboxylated by phosphatidylserine decarboxylase (Psd) to form PE. In this study we identified the sinorhizobial psd gene coding for Psd. A sinorhizobial mutant deficient in psd is unable to form PE but accumulates the anionic phospholipid PS. Properties of PE-deficient mutants lacking either Pss or Psd were compared with those of the S. meliloti wild type. Whereas both PE-deficient mutants grew in a wild-type-like manner on many complex media, they were unable to grow on minimal medium containing high phosphate concentrations. Surprisingly, the psd-deficient mutant could grow on minimal medium containing low concentrations of inorganic phosphate, while the pss-deficient mutant could not. Addition of choline to the minimal medium rescued growth of the pss-deficient mutant, CS111, to some extent but inhibited growth of the psd-deficient mutant, MAV01. When the two distinct PE-deficient mutants were analyzed for their ability to form a nitrogen-fixing root nodule symbiosis with their alfalfa host plant, they behaved strikingly differently. The Pss-deficient mutant, CS111, initiated nodule formation at about the same time point as the wild type but did form about 30% fewer nodules than the wild type. In contrast, the PS-accumulating mutant, MAV01, initiated nodule formation much later than the wild type and formed 90% fewer nodules than the wild type. The few nodules formed by MAV01 seemed to be almost devoid of bacteria and were unable to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 were yellowish, indicating that the plants were starved for nitrogen. Therefore, changes in lipid composition, including the accumulation of bacterial PS, prevent the establishment of a nitrogen-fixing root nodule symbiosis.

Differential responses of the sunn4 and rdn1-1 super-nodulation mutants of Medicago truncatula to elevated atmospheric CO2

2021 ◽  
Author(s):  
Yunfa Qiao ◽  
Shujie Miao ◽  
Jian Jin ◽  
Ulrike Mathesius ◽  
Caixian Tang
Keyword(s):  
Nitrogen Fixation ◽  
Elevated Co2 ◽  
Medicago Truncatula ◽  
N2 Fixation ◽  
Sink Strength ◽  
Wild Type ◽  
Total N ◽  
Autoregulation Of Nodulation ◽  
Nodulation Mutants ◽  
Fixation Capacity

Abstract Background and Aims Nitrogen fixation in legumes requires tight control of carbon and nitrogen balance. Thus, legumes control nodule numbers via an autoregulation mechanism. ‘Autoregulation of nodulation’ mutants super-nodulate and are thought to be carbon-limited due to the high carbon-sink strength of excessive nodules. This study aimed to examine the effect of increasing carbon supply on the performance of super-nodulation mutants. Methods We compared the responses of Medicago truncatula super-nodulation mutants (sunn-4 and rdn1-1) and wild type to five CO2 levels (300-850 μmol mol -1). Nodule formation and N2 fixation were assessed in soil-grown plants at 18 and 42 days after sowing. Key results Shoot and root biomass, nodule number and biomass, nitrogenase activity and fixed-N per plant of all genotypes increased with increasing CO2 concentration and reached the maximum around 700 μmol mol -1. While the sunn-4 mutant showed strong growth-retardation compared to wild-type plants, elevated CO2 increased shoot biomass and total N content of rdn1-1 mutant up to two-fold. This was accompanied by a four-fold increase in nitrogen fixation capacity in the rdn1-1 mutant. Conclusions These results suggest that the super-nodulation phenotype per se did not limit growth. The additional nitrogen fixation capacity of the rdn1-1 mutant may enhance the benefit of elevated CO2 on plant growth and N2 fixation.

scholarly journals Antioxidant ability of glutaredoxins and their role in symbiotic nitrogen fixation in Rhizobium leguminosarum bv. viciae 3841

2020 ◽  
Author(s):  
Qian Zou ◽  
Yanlin Zhou ◽  
Guojun Cheng ◽  
Yang Peng ◽  
Sha Luo ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Quantitative Proteomics ◽  
Rhizobium Leguminosarum ◽  
Symbiotic Nitrogen Fixation ◽  
Proteome Analysis ◽  
Wild Type ◽  
Triple Mutant ◽  
Antioxidant Proteins ◽  
Mixed Disulfides ◽  
Nodule Symbiosis

Glutaredoxins (Grx) are redoxin family proteins that reduce disulfides and mixed disulfides between glutathione and proteins. Rhizobium leguminosarum bv. Viciae 3841 contains three genes coding for glutaredoxins: RL4289 (grxA) codes for a dithiolic glutaredoxin, RL2615 (grxB) codes for a monothiol glutaredoxin, while RL4261 (grxC) codes for a glutaredoxin-like NrdH protein. We generated mutants interrupted in one, two, or three glutaredoxin genes. These mutants had no obvious differences in growth phenotypes from the wild type RL3841. However, while a mutant of grxC did not affect the antioxidant or symbiotic capacities of R. leguminosarum, grxA-derived or grxB mutants decreased antioxidant and nitrogen fixation capacities. Furthermore, grxA mutants were severely impaired in rhizosphere colonization, and formed smaller nodules with defects of bacteroid differentiation, whereas nodules induced by grxB mutants contained abnormally thick cortices and prematurely senescent bacteroids. The grx triple mutant had the greatest defect in antioxidant and symbiotic capacities of R. leguminosarum and quantitative proteomics revealed it had 56 up-regulated and 81 down-regulated proteins relative to wildtype. Of these proteins, twenty-eight are involved in transporter activity, twenty are related to stress response and virulence, and sixteen are involved in amino acid metabolism. Overall, R. leguminosarum glutaredoxins behave as antioxidant proteins mediating root nodule symbiosis. IMPORTANCE Glutaredoxin catalyzes glutathionylation/deglutathionylation reactions, protects SH-groups from oxidation and restores functionally active thiols. Three glutaredoxins exist in R. leguminosarum and their properties were investigated in free-living bacteria and during nitrogen-fixing symbiosis. All the glutaredoxins were necessary for oxidative stress defense. Dithiol GrxA affects nodulation and nitrogen fixation of bacteroids by altering deglutathionylation reactions, monothiol GrxB is involved in symbiotic nitrogen fixation by regulating Fe-S cluster biogenesis, and GrxC may participate in symbiosis by an unknown mechanism. Proteome analysis provides clues to explain the differences between the grx triple mutant and wild-type nodules.

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

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Michael J. Mitsch ◽  
George C. diCenzo ◽  
Alison Cowie ◽  
Turlough M. Finan
Keyword(s):  
Nitrogen Fixation ◽  
Carbon Source ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Symbiotic Nitrogen Fixation ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Content Type ◽  
Transcriptional Changes ◽  
Symbiotic Properties

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

Characterisation of dry and mucoid colonies isolated from Australian rhizobial inoculant strains for Medicago species

2005 ◽  
Vol 45 (3) ◽  
pp. 151 ◽  
Author(s):  
A. McInnes ◽  
P. Holford ◽  
J. E. Thies
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Dry Weight ◽  
Sequence Element ◽  
Shoot Dry Weight ◽  
Single Colony ◽  
Agar Media ◽  
Fixation Capacity ◽  
Medicago Species ◽  
K Antigen

The presence of dry and mucoid colonies in cultures of rhizobial strains used in the production of commercial Australian inoculants is of concern for quality assurance because of the possibility of altered capacity for nodulation and nitrogen fixation by the different colony types. In this study, single colony isolates obtained from dry and mucoid colonies present in commercial cultures of Sinorhizobium meliloti were investigated to identify stability in culture, genetic identity and changes in exopolysaccharide (EPS) production, nodulation and nitrogen fixation. The 2 strains studied were WSM688 and WSM826 (Australian inoculant strains for annual and perennial medics, respectively), both of which produced only mucoid colonies on agar media when originally isolated from nodules. Dry and mucoid single colony isolates from the ‘mother cultures’ of the 2 strains exhibited stable colony phenotypes during successive subculturing in our laboratory and were shown to be most closely related to S. meliloti using 16S rRNA partial sequencing. All isolates produced at least 1 of 3 exopolysaccharides (succinoglycan, EPS II and K antigen) that are required for successful nodulation of Medicago species by S. meliloti strains, as indicated by nodulation of host legumes. Strain WSM826 isolates probably produce succinoglycan, as shown by similarity to the succinoglycan-producing strain Rm1021 in a calcofluor binding assay. In contrast to published work, there was no evidence that loss of mucoidy in dry colony isolates of either strain was associated with the presence of an insertion sequence element in the expR gene that inhibits EPS II production. For strain WSM688, dry and mucoid isolates were identical by PCR fingerprinting and showed a similar capacity to nodulate and fix nitrogen with the target host legume M. truncatula in glasshouse tests. In contrast, strain WSM826 mucoid isolates produced PCR fingerprints that were different from each other and from the WSM826 dry colony isolates. Dry and mucoid colonies may have arisen from substantial genetic change or through contamination of cultures by other S. meliloti strains. One WSM826 mucoid isolate (826-3) produced significantly lower shoot dry weight when inoculated onto both the target host M. sativa and non-target host M. truncatula, even though the capacity to nodulate both hosts was retained. This suggests that this isolate was affected in its nitrogen fixation capacity. Further research is required to identify the origin and extent of colony variation in commercial S. meliloti cultures.

scholarly journals Disruption of a Gene Essential for Sulfoquinovosyldiacylglycerol Biosynthesis in Sinorhizobium meliloti Has No Detectable Effect on Root Nodule Symbiosis

2000 ◽  
Vol 13 (6) ◽  
pp. 666-672 ◽  
Author(s):  
Barbara Weissenmayer ◽  
Otto Geiger ◽  
Christoph Benning
Keyword(s):  
Sinorhizobium Meliloti ◽  
Root Nodule ◽  
Membrane Lipids ◽  
Growth Conditions ◽  
Laboratory Culture ◽  
Culture Conditions ◽  
Detectable Effect ◽  
Purple Bacterium ◽  
Root Nodule Symbiosis ◽  
Nodule Symbiosis

The sulfolipid sulfoquinovosyldiacylglycerol is commonly found in the thylakoid membranes of photosynthetic bacteria and plants. While there is a good correlation between the occurrence of sulfolipid and photosynthesis, a number of exceptions are known. Most recently, sulfoquinovosyldiacylglycerol was discovered in the non-photosynthetic, root nodule-forming bacterium Sinorhizobium meliloti. This discovery raised the questions of the phylogenetic origin of genes essential for the biosynthesis of this lipid in S. meliloti and of a function of sulfolipid in root nodule symbiosis. To begin to answer these questions, we isolated and inactivated the sqdB gene of S. meliloti. This gene and two other genes located directly 3′ of sqdB are highly similar to the sqdB, sqdC, and sqdD genes known to be essential for sulfolipid biosynthesis in the photosynthetic, purple bacterium Rhodobacter sphaeroides. This observation confirms the close phylogenetic kinship between these two species. Furthermore, the reduced similarity of sqdB to the plant ortholog SQD1 of Arabidopsis thaliana does not support a previous sqd gene transfer from the plant as a consequence of close symbiosis. A sul-folipid-deficient mutant of S. meliloti disrupted in sqdB is capable of inducing functional nodules and does not show an obvious disadvantage under different laboratory culture conditions. Thus far, no specific function can be assigned to bacterial sulfolipid, in either nodule-associated or free-living cells. S. meliloti contains a rich set of polar membrane lipids some of which, including sulfolipid, may become critical only under growth conditions that still need to be discovered.

scholarly journals Asymmetric Redundancy of Soybean Nodule Inception (NIN) Genes in Root Nodule Symbiosis

2021 ◽  
Author(s):  
Mengdi Fu ◽  
Jiafeng Sun ◽  
Xiaolin Li ◽  
Yuefeng Guan ◽  
Fang Xie
Keyword(s):  
Root Nodule ◽  
Minor Role ◽  
Nodule Formation ◽  
Wild Type ◽  
Promoter Regions ◽  
Triple Mutant ◽  
Symbiotic Interaction ◽  
Root Nodule Symbiosis ◽  
A Minor ◽  
Nodule Symbiosis

NIN is one of the most important root nodule symbiotic genes as it is required for both infection and nodule organogenesis in legume. Unlike most legumes with a sole NIN gene, there are four putative NIN genes in soybean (Glycine max). Whether and how these orthologs NIN genes contribute to soybean-rhizobia symbiotic interaction remain unknown. In this study, we found that all four GmNIN genes are induced by rhizobia, and that conserved CE and CYC binding motifs in their promoter regions are required for their expression in the nodule formation process. By generation of multiplex Gmnin mutants, we found that Gmnin1a nin2a nin2b triple mutant and Gmnin1a nin1b nin2a nin2b quadruple mutant displayed similar defects in rhizobia infection and root nodule formation, Gmnin2a nin2b produced less nodules but displayed hyper infection phenotype than wild type, while a Gmnin1a nin1b double mutant nodulated as wild type. Overexpression of GmNIN1a, GmNIN1b, GmNIN2a, and GmNIN2b reduced nodule numbers after rhizobia inoculation, with GmNIN1b overexpression having the weakest effect. In addition, overexpression of GmNIN1a, GmNIN2a, or GmNIN2b, but not GmNIN1b, produced malformed pseudo-nodule like structures without rhizobia inoculation. In conclusion, GmNIN1a, GmNIN2a and GmNIN2b play functionally redundant yet complicated roles for soybean nodulation. GmNIN1b, although is expressed at comparable level with other homologs, plays a minor role in root nodule symbiosis. Our work provides insight into the understanding of asymmetrically redundant function of GmNIN genes in soybean.

scholarly journals Differential Regulation of Two Divergent Sinorhizobium meliloti Genes for HPII-Like Catalases during Free-Living Growth and Protective Role of Both Catalases during Symbiosis

1999 ◽  
Vol 181 (8) ◽  
pp. 2634-2639 ◽  
Author(s):  
Samuel Sigaud ◽  
Vanessa Becquet ◽  
Pierre Frendo ◽  
Alain Puppo ◽  
Didier Hérouart
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Protective Role ◽  
Transcription Level ◽  
Dramatic Decrease ◽  
Free Living ◽  
Gene Encoding ◽  
Fixation Capacity ◽  
Minimum Medium

ABSTRACT Two catalases, KatA and KatB, have been detected inSinorhizobium meliloti growing on rich medium. Here we characterize a new catalase gene encoding a third catalase (KatC). KatC activity was detectable only at the end of the stationary phase inS. meliloti growing in minimum medium, whereas KatA activity was found during the exponential phase. Analysis with akatC-lacZ fusion demonstrated that katCexpression is mainly regulated at the transcription level. An increase of catalase activity correlating with KatA induction was detected in bacteroids. A dramatic decrease of nitrogen fixation capacity in a katA katC double mutant was observed, suggesting that these catalases are very important for the protection of the nitrogen fixation process.

scholarly journals A Thioredoxin of Sinorhizobium meliloti CE52G Is Required for Melanin Production and Symbiotic Nitrogen Fixation

2007 ◽  
Vol 20 (8) ◽  
pp. 986-993 ◽  
Author(s):  
Susana Castro-Sowinski ◽  
Ofra Matan ◽  
Paula Bonafede ◽  
Yaacov Okon
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Redox State ◽  
Wild Type Strain ◽  
Symbiotic Nitrogen Fixation ◽  
Mrna Level ◽  
Wild Type ◽  
Melanin Production ◽  
In The Wild ◽  
Reducing Activity

A miniTn5-induced mutant of a melanin-producing strain of Sinorhizobium meliloti (CE52G) that does not produce melanin was mapped to a gene identified as a probable thioredoxin gene. It was proved that the thiol-reducing activity of the mutant was affected. Addition to the growth medium of substrates that induce the production of melanin (l-tyrosine, guaiacol, orcinol) increased the thioredoxin-like (trxL) mRNA level in the wild-type strain. The mutant strain was affected in the response to paraquat-induced oxidative stress, symbiotic nitrogen fixation, and both laccase and tyrosinase activities. The importance of thioredoxin in melanin production in bacteria, through the regulation of laccase or tyrosinase activities, or both, by the redox state of structural or catalytic SH groups, is discussed.

scholarly journals Thiamine Is Synthesized by a Salvage Pathway in Rhizobium leguminosarum bv. viciae Strain 3841

2006 ◽  
Vol 188 (18) ◽  
pp. 6661-6668 ◽  
Author(s):  
R. Karunakaran ◽  
K. Ebert ◽  
S. Harvey ◽  
M. E. Leonard ◽  
V. Ramachandran ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
De Novo ◽  
Fluorescent Microscopy ◽  
Good Growth ◽  
Wild Type ◽  
Salvage Pathway ◽  
Mesorhizobium Loti ◽  
Reverse Transcription Pcr

ABSTRACT In the absence of added thiamine, Rhizobium leguminosarum bv. viciae strain 3841 does not grow in liquid medium and forms only “pin” colonies on agar plates, which contrasts with the good growth of Sinorhizobium meliloti 1021, Mesorhizobium loti 303099, and Rhizobium etli CFN42. These last three organisms have thiCOGE genes, which are essential for de novo thiamine synthesis. While R. leguminosarum bv. viciae 3841 lacks thiCOGE, it does have thiMED. Mutation of thiM prevented formation of pin colonies on agar plates lacking added thiamine, suggesting thiamine intermediates are normally present. The putative functions of ThiM, ThiE, and ThiD are 4-methyl-5-(β-hydroxyethyl) thiazole (THZ) kinase, thiamine phosphate pyrophosphorylase, and 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) kinase, respectively. This suggests that a salvage pathway operates in R. leguminosarum, and addition of HMP and THZ enabled growth at the same rate as that enabled by thiamine in strain 3841 but elicited no growth in the thiM mutant (RU2459). There is a putative thi box sequence immediately upstream of the thiM, and a gfp-mut3.1 fusion to it revealed the presence of a promoter that is strongly repressed by thiamine. Using fluorescent microscopy and quantitative reverse transcription-PCR, it was shown that thiM is expressed in the rhizosphere of vetch and pea plants, indicating limitation for thiamine. Pea plants infected by RU2459 were not impaired in nodulation or nitrogen fixation. However, colonization of the pea rhizosphere by the thiM mutant was impaired relative to that of the wild type. Overall, the results show that a thiamine salvage pathway operates to enable growth of Rhizobium leguminosarum in the rhizosphere, allowing its survival when thiamine is limiting.

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