Improvement of Phosphate Solubilization and Medicago Plant Yield by an Indole-3-Acetic Acid-Overproducing Strain of Sinorhizobium meliloti

ABSTRACT Nitrogen (N) and phosphorus (P) are the most limiting factors for plant growth. Some microorganisms improve the uptake and availability of N and P, minimizing chemical fertilizer dependence. It has been published that the RD64 strain, a Sinorhizobium meliloti 1021 strain engineered to overproduce indole-3-acetic acid (IAA), showed improved nitrogen fixation ability compared to the wild-type 1021 strain. Here, we present data showing that RD64 is also highly effective in mobilizing P from insoluble sources, such as phosphate rock (PR). Under P-limiting conditions, the higher level of P-mobilizing activity of RD64 than of the 1021 wild-type strain is connected with the upregulation of genes coding for the high-affinity P transport system, the induction of acid phosphatase activity, and the increased secretion into the growth medium of malic, succinic, and fumaric acids. Medicago truncatula plants nodulated by RD64 (Mt-RD64), when grown under P-deficient conditions, released larger amounts of another P-solubilizing organic acid, 2-hydroxyglutaric acid, than plants nodulated by the wild-type strain (Mt-1021). It has already been shown that Mt-RD64 plants exhibited higher levels of dry-weight production than Mt-1021 plants. Here, we also report that P-starved Mt-RD64 plants show significant increases in both shoot and root fresh weights when compared to P-starved Mt-1021 plants. We discuss how, in a Rhizobium-legume model system, a balanced interplay of different factors linked to bacterial IAA overproduction rather than IAA production per se stimulates plant growth under stressful environmental conditions and, in particular, under P starvation.

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Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

Applied and Environmental Microbiology ◽

10.1128/aem.69.6.3244-3250.2003 ◽

2003 ◽

Vol 69 (6) ◽

pp. 3244-3250 ◽

Cited By ~ 119

Author(s):

Daniel Kadouri ◽

Edouard Jurkevitch ◽

Yaacov Okon

Keyword(s):

Plant Growth ◽

Mutant Strain ◽

Azospirillum Brasilense ◽

Type Strain ◽

Wild Type Strain ◽

Root Colonization ◽

Cell Aggregation ◽

Wild Type ◽

Phb Production ◽

Promote Plant Growth

ABSTRACT When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.

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Environmental Regulation of Exopolysaccharide Production in Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.182.3.599-606.2000 ◽

2000 ◽

Vol 182 (3) ◽

pp. 599-606 ◽

Cited By ~ 92

Author(s):

Kiprian E. Mendrygal ◽

Juan E. González

Keyword(s):

Molecular Weight ◽

Type Strain ◽

Sinorhizobium Meliloti ◽

Wild Type Strain ◽

Root Nodules ◽

Weight Fraction ◽

Wild Type ◽

Exopolysaccharide Production ◽

Successful Establishment ◽

High Phosphate

ABSTRACT Exopolysaccharide production by Sinorhizobium melilotiis required for invasion of root nodules on alfalfa and successful establishment of a nitrogen-fixing symbiosis between the two partners.S. meliloti wild-type strain Rm1021 requires production of either succinoglycan, a polymer of repeating octasaccharide subunits, or EPS II, an exopolysaccharide of repeating dimer subunits. The reason for the production of two functional exopolysaccharides is not clear. Earlier reports suggested that low-phosphate conditions stimulate the production of EPS II in Rm1021. We found that phosphate concentrations determine which exopolysaccharide is produced by S. meliloti. The low-phosphate conditions normally found in the soil (1 to 10 μM) stimulate EPS II production, while the high-phosphate conditions inside the nodule (20 to 100 mM) block EPS II synthesis and induce the production of succinoglycan. Interestingly, the EPS II produced by S. meliloti in low-phosphate conditions does not allow the invasion of alfalfa nodules. We propose that this invasion phenotype is due to the lack of the active molecular weight fraction of EPS II required for nodule invasion. An analysis of the function of PhoB in this differential exopolysaccharide production is presented.

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Megaplasmid pRme2011a of Sinorhizobium meliloti Is Not Required for Viability

Journal of Bacteriology ◽

10.1128/jb.182.12.3582-3586.2000 ◽

2000 ◽

Vol 182 (12) ◽

pp. 3582-3586 ◽

Cited By ~ 71

Author(s):

Ivan J. Oresnik ◽

Shu-Lin Liu ◽

Christopher K. Yost ◽

Michael F. Hynes

Keyword(s):

Gel Electrophoresis ◽

Type Strain ◽

Sinorhizobium Meliloti ◽

Wild Type Strain ◽

Sole Source ◽

Pulsed Field Gel Electrophoresis ◽

Selection Strategy ◽

Wild Type ◽

Defined Media

ABSTRACT We report the curing of the 1,360-kb megaplasmid pRme2011a fromSinorhizobium meliloti strain Rm2011. With a positive selection strategy that utilized Tn5B12-S containing thesacB gene, we were able to cure this replicon by successive rounds of selecting for deletion formation in vivo. Subsequent Southern blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were consistent with the hypothesis that the resultant strain was indeed missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number of substrates as a sole source of carbon on defined media.

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Lack ofHXK2Induces Localization of Active Ras in Mitochondria and Triggers Apoptosis in the YeastSaccharomyces cerevisiae

Oxidative Medicine and Cellular Longevity ◽

10.1155/2013/678473 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 23

Author(s):

Loredana Amigoni ◽

Enzo Martegani ◽

Sonia Colombo

Keyword(s):

Cell Death ◽

Acetic Acid ◽

Plasma Membrane ◽

Programmed Cell Death ◽

Mitochondrial Dysfunction ◽

Type Strain ◽

Wild Type Strain ◽

Ros Production ◽

Ras Proteins ◽

Wild Type

We recently showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type cells growing exponentially on glucose, while in thehxk2Δ strain they accumulated mainly in mitochondria. An aberrant accumulation of activated Ras in these organelles was previously reported and correlated to mitochondrial dysfunction, accumulation of ROS, and cell death. Here we show that addition of acetic acid to wild-type cells results in a rapid recruitment of Ras-GTP from the nucleus and the plasma membrane to the mitochondria, providing a further proof that Ras proteins might be involved in programmed cell death. Moreover, we show that Hxk2 protects against apoptosis inS. cerevisiae. In particular, cells lackingHXK2and showing a constitutive accumulation of activated Ras at the mitochondria are more sensitive to acetic-acid-induced programmed cell death compared to the wild type strain. Indeed, deletion ofHXK2causes an increase of apoptotic cells with several morphological and biochemical changes that are typical of apoptosis, including DNA fragmentation, externalization of phosphatidylserine, and ROS production. Finally, our results suggest that apoptosis induced by lack of Hxk2 may not require the activation of Yca1, the metacaspase homologue identified in yeast.

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The Sinorhizobium meliloti Lon Protease Is Involved in Regulating Exopolysaccharide Synthesis and Is Required for Nodulation of Alfalfa

Journal of Bacteriology ◽

10.1128/jb.182.9.2551-2558.2000 ◽

2000 ◽

Vol 182 (9) ◽

pp. 2551-2558 ◽

Cited By ~ 15

Author(s):

Michael L. Summers ◽

Lina M. Botero ◽

Scott C. Busse ◽

Timothy R. McDermott

Keyword(s):

Mutant Strain ◽

Type Strain ◽

Sinorhizobium Meliloti ◽

Wild Type Strain ◽

Insertion Site ◽

Normal Growth ◽

Phosphate Limitation ◽

Nuclear Magnetic Resonance Analysis ◽

Lon Protease ◽

Wild Type

ABSTRACT While screening for Sinorhizobium meliloti Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the lonmutant total EPS was 5.0 ± 0.2 (mean ± standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 ± 0.5. These data suggested that in addition to EPSI, thelon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced byS. meliloti, but typically is expressed only under conditions of phosphate limitation. 13C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type lon gene was present intrans. The results of this study suggest that the S. meliloti Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.

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A Single-Nucleotide Insertion in a Drug Transporter Gene Induces a Thermotolerance Phenotype inGluconobacter frateuriiby Increasing the NADPH/NADP+Ratio via Metabolic Change

Applied and Environmental Microbiology ◽

10.1128/aem.00354-18 ◽

2018 ◽

Vol 84 (10) ◽

Cited By ~ 6

Author(s):

Nami Matsumoto ◽

Hiromi Hattori ◽

Minenosuke Matsutani ◽

Chihiro Matayoshi ◽

Hirohide Toyama ◽

...

Keyword(s):

Acetic Acid ◽

High Temperatures ◽

Type Strain ◽

Wild Type Strain ◽

Metabolic Change ◽

Drug Transporter ◽

Transporter Gene ◽

Wild Type ◽

Content Type ◽

In The Wild

ABSTRACTThermotolerant microorganisms are beneficial to the fermentation industry because they reduce the need for cooling and offer other operational advantages. Previously, we obtained a thermally adaptedGluconobacter frateuriistrain by experimental evolution. In the present study, we found only a single G insertion in the adapted strain, which causes a frameshift in a gene encoding a putative drug transporter. A mutant derivative strain with the single G insertion in the transporter gene (Wild-G) was constructed from the wild-type strain and showed increased thermotolerance. We found that the thermotolerant strains accumulated substantial intracellular trehalose and manifested a defect in sorbose assimilation, suggesting that the transporter is partly involved in trehalose efflux and sorbose uptake and that the defect in the transporter can improve thermotolerance. The ΔotsABstrain, constructed by elimination of the trehalose synthesis gene in the wild type, showed no trehalose production but, unexpectedly, much better growth than the adapted strain at high temperatures. The ΔotsABmutant produced more acetate as the final metabolite than the wild-type strain did. We hypothesized that trehalose does not contribute to thermotolerance directly; rather, a metabolic change including increased carbon flux to the pentose phosphate pathway may be the key factor. The NADPH/NADP+ratio was higher in strain Wild-G, and much higher in the ΔotsABstrain, than in the wild-type strain. Levels of reactive oxygen species (ROS) were lower in the thermotolerant strains. We propose that the defect of the transporter causes the metabolic flux to generate more NADPH, which may enhance thermotolerance inG. frateurii.IMPORTANCEThe biorefinery industry has to ensure that microorganisms are robust and retain their viability and function at high temperatures. Here we show thatGluconobacterfrateurii, an industrially important member of the acetic acid bacteria, exhibited enhanced thermotolerance through the reduction of trehalose excretion after thermal adaptation. Although intracellular trehalose may play a key role in thermotolerance, the molecular mechanisms of action of trehalose in thermotolerance are a matter of debate. Our mutated strain that was defective in trehalose synthase genes, producing no trehalose but a larger amount of acetic acid as the end metabolite instead, unexpectedly showed higher thermotolerance than the wild type. Our adapted and mutated thermotolerant strains showed increased NADPH/NADP+ratios and reductions in ROS levels. We concluded that inG. frateurii, trehalose does not contribute to thermotolerance directly; rather, the metabolic change increases the NADPH/NADP+ratio to enhance thermotolerance.

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Functional Characterization of the Sinorhizobium meliloti Acetate Metabolism Genes aceA, SMc00767, and glcB

Journal of Bacteriology ◽

10.1128/jb.00385-07 ◽

2007 ◽

Vol 189 (16) ◽

pp. 5875-5884 ◽

Cited By ~ 14

Author(s):

J. A. Ramírez-Trujillo ◽

S. Encarnación ◽

E. Salazar ◽

A. García de los Santos ◽

M. F. Dunn ◽

...

Keyword(s):

Type Strain ◽

Sinorhizobium Meliloti ◽

Wild Type Strain ◽

Isocitrate Lyase ◽

Functional Characterization ◽

Malate Synthase ◽

Acetate Metabolism ◽

Transcriptional Level ◽

Wild Type

ABSTRACT The genes encoding malate synthase (glcB) and isocitrate lyase (aceA) and a 240-bp open reading frame (SMc00767) located downstream of aceA were isolated and functionally characterized in Sinorhizobium meliloti. Independent and double interposon mutants of each gene were constructed, and the corresponding phenotypes were analyzed. aceA mutants failed to grow on acetate, and mutants deficient in SMc00767 were also affected in acetate utilization. In contrast, mutants deficient in glcB grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed that aceA and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants. aceA-glcB, aceA-SMc00767, and glcB-SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-type aceA-glcB or aceA-SMc00767 loci were introduced into the double mutants. These data confirm the functional role of aceA and SMc00767 and show that glcB, in the absence of SMc00767, is required for acetate metabolism. Isocitrate lyase and malate synthase activities were measured in strain Rm5000, the mutant derivatives, and complemented strains. aceA and glcB were able to complement the enzymatic activity lacking in the corresponding single mutants. The enzymatic activities also showed that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirmed the repressor role of SMc00767, which regulates aceA expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant and wild-type strain Rm5000 showed that SMc00767 represses the expression of a moderate number of open reading frames, including aceA; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism in S. meliloti. Genetic and functional analyses indicated that aceA and SMc00767 constitute a functional two-gene operon, which is conserved in other α-proteobacteria. Alfalfa plants infected with the aceA and glcB mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in the Rhizobium-legume symbiosis.

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Two New Sinorhizobium meliloti LysR-Type Transcriptional Regulators Required for Nodulation

Journal of Bacteriology ◽

10.1128/jb.187.13.4562-4572.2005 ◽

2005 ◽

Vol 187 (13) ◽

pp. 4562-4572 ◽

Cited By ~ 44

Author(s):

Li Luo ◽

Shi-Yi Yao ◽

Anke Becker ◽

Silvia Rüberg ◽

Guan-Qiao Yu ◽

...

Keyword(s):

Cell Growth ◽

Type Strain ◽

Sinorhizobium Meliloti ◽

Wild Type Strain ◽

Nitrogenase Activity ◽

Transcriptional Regulators ◽

Wild Type ◽

Free Living ◽

Multiple Points ◽

Regulator Genes

ABSTRACT The establishment of an effective nitrogen-fixing symbiosis between Sinorhizobium meliloti and its legume host alfalfa (Medicago sativa) depends on the timely expression of nodulation genes that are controlled by LysR-type regulators. Ninety putative genes coding for LysR-type transcriptional regulators were identified in the recently sequenced S. meliloti genome. All 90 putative lysR genes were mutagenized using plasmid insertions as a first step toward determining their roles in symbiosis. Two new LysR-type symbiosis regulator genes, lsrA and lsrB, were identified in the screening. Both the lsrA and lsrB genes are expressed in free-living S. meliloti cells, but they are not required for cell growth. An lsrA1 mutant was defective in symbiosis and elicited only white nodules that exhibited no nitrogenase activity. Cells of the lsrA1 mutant were recovered from the white nodules, suggesting that the lsrA1 mutant was blocked early in nodulation. An lsrB1 mutant was deficient in symbiosis and elicited a mixture of pink and white nodules on alfalfa plants. These plants exhibited lower overall nitrogenase activity than plants inoculated with the wild-type strain, which is consistent with the fact that most of the alfalfa plants inoculated with the lsrB1 mutant were short and yellow. Cells of the lsrB1 mutant were recovered from both pink and white nodules, suggesting that lsrB1 mutants could be blocked at multiple points during nodulation. The identification of two new LysR-type symbiosis transcriptional regulators provides two new avenues for understanding the complex S. meliloti-alfalfa interactions which occur during symbiosis.

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The Sinorhizobium meliloti EmrR Regulator Is Required for Efficient Colonization of Medicago sativa Root Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-13-0284-r ◽

2014 ◽

Vol 27 (4) ◽

pp. 388-399 ◽

Cited By ~ 9

Author(s):

Mário R. Santos ◽

Andreia T. Marques ◽

Jörg D. Becker ◽

Leonilde M. Moreira

Keyword(s):

Cell Wall ◽

Heat Shock ◽

Medicago Sativa ◽

Stress Responses ◽

Type Strain ◽

Sinorhizobium Meliloti ◽

Wild Type Strain ◽

Major Facilitator Superfamily ◽

Wild Type ◽

Expression Of Genes

The nitrogen-fixing bacterium Sinorhizobium meliloti must adapt to diverse conditions encountered during its symbiosis with leguminous plants. We characterized a new symbiotically relevant gene, emrR (SMc03169), whose product belongs to the TetR family of repressors and is divergently transcribed from emrAB genes encoding a putative major facilitator superfamily–type efflux pump. An emrR deletion mutant produced more succinoglycan, displayed increased cell-wall permeability, and exhibited higher tolerance to heat shock. It also showed lower tolerance to acidic conditions, a reduced production of siderophores, and lower motility and biofilm formation. The simultaneous deletion of emrA and emrR genes restored the mentioned traits to the wild-type phenotype, except for survival under heat shock, which was lower than that displayed by the wild-type strain. Furthermore, the ΔemrR mutant as well as the double ΔemrAR mutant was impaired in symbiosis with Medicago sativa; it formed fewer nodules and competed poorly with the wild-type strain for nodule colonization. Expression profiling of the ΔemrR mutant showed decreased expression of genes involved in Nod-factor and rhizobactin biosynthesis and in stress responses. Expression of genes directing the biosynthesis of succinoglycan and other polysaccharides were increased. EmrR may therefore be involved in a regulatory network targeting membrane and cell wall modifications in preparation for colonization of root hairs during symbiosis.

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