scholarly journals Environmental Regulation of Exopolysaccharide Production in Sinorhizobium meliloti

2000 ◽  
Vol 182 (3) ◽  
pp. 599-606 ◽  
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.

scholarly journals Effect of phosphate levels on the synthesis of acid phosphatases (EC 3.1.3.2) inNeurospora crassa

1985 ◽  
Vol 45 (3) ◽  
pp. 239-249 ◽  
Author(s):  
Sergio A. Rodrigues ◽  
Antonio Rossi
Keyword(s):  
Molecular Weight ◽  
Type Strain ◽  
Wild Type Strain ◽  
Deae Cellulose ◽  
Phosphate Starvation ◽  
Acid Phosphatases ◽  
Wild Type ◽  
Specific Activities ◽  
Phosphate Medium ◽  
High Phosphate

SummaryWhen grown on high-phosphate medium, the wild-type strain 74A ofN. crassasynthesized two acid phosphatases, as shown by DEAE -cellulose chromatography. These purified enzymes showed heterogeneity on PAGE, low specific activities towards PNP-P, molecular weight values of at least 300000, no deviation from Michaelian behaviour, and great stability in 50 mM sodium acetate buffer, at pH 5·4, when kept at 54 °C. These acid phosphatases were synthesized in reduced amounts or not at all when the mould was grown under conditions of phosphate starvation, indicating that the level of phosphate also regulates the synthesis of the high molecular weight enzyme forms. When grown on high phosphate medium, thepho-3mutant strain also synthesized two acid phosphatases, whose purified enzymes showed no pronounced differences when compared to those synthesized by the wild-type strain in terms of electrophoretic analysis, specific activities towards PNP-P, molecular weight values, and Michaelian behaviour. However, one enzyme form had a higherKmvalue and a lower heat stability than the corresponding enzyme of the wild-type strain. Even though thepho-3locus might not be responsible for an alteration in the primary structure of the repressible acid phosphatase, it seems clear that the enzymes synthesized by the mould grown on low-or high-phosphate medium must share some structural features. Thus, the drastic differences observed in the molecular properties of the enzymes synthesized by the mould grown under conditions of phosphate starvation as opposed to phosphate repression might be due to an effect exerted by the level of inorganic phosphate in regulating the translation, post-translational modifications and/or excretion, but not necessarily the gene-directed synthesis of distinct mRNAs.

A Stable 44S Intermediate in the Autodigestion of 50S Ribosomal Subunits

1971 ◽  
Vol 29 ◽  
pp. 430-431
Author(s):  
John H. Nisbet ◽  
Henry S. Slayter
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Ribosomal Rna ◽  
Gel Electrophoresis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Ribosomal Subunits ◽  
Type Strains

Wild - type strains of Escherichia coli are known to contain as many as four endogenous nucleases (Ref. 1). These are commonly found associated with the ribosomes after extraction from the cell, but may be removed, with the exception of RNase IV, by washing the ribosomes in NH4Cl (at 0.2 M and higher concentrations). We have examined the effect of these nucleases on the 50S ribosomal subunit of one wild-type strain, K12 (Hfr 3000), by incubating the unwashed particles at 37° in the presence of varying magnesium concentrations.At 10-4 molar magnesium (slower at 10-3 molar), the 50S particle is converted to a species sedimenting at about 44S. About 20% of the total O.D260 is liberated at the same time. Continued incubation leads to the release of more O.D260 material while the RNA remaining in the 44S (Fig. 1) particle is progressively cleaved, eventually to the point where it consists of one principal fragment of molecular weight 0.42 x 106 daltons and several lesser fragments. The ribosomal RNA and proteins have been characterized by acrylamide gel electrophoresis.

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

2010 ◽  
Vol 76 (14) ◽  
pp. 4626-4632 ◽  
Author(s):  
Carmen Bianco ◽  
Roberto Defez
Keyword(s):  
Acetic Acid ◽  
Plant Growth ◽  
Type Strain ◽  
Sinorhizobium Meliloti ◽  
Wild Type Strain ◽  
Dry Weight ◽  
Limiting Factors ◽  
Wild Type ◽  
System A ◽  
Indole 3 Acetic Acid

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.

scholarly journals Megaplasmid pRme2011a of Sinorhizobium meliloti Is Not Required for Viability

2000 ◽  
Vol 182 (12) ◽  
pp. 3582-3586 ◽  
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.

scholarly journals A ClpB Chaperone Knockout Mutant of Mesorhizobium ciceri Shows a Delay in the Root Nodulation of Chickpea Plants

2012 ◽  
Vol 25 (12) ◽  
pp. 1594-1604 ◽  
Author(s):  
Clarisse Brígido ◽  
Marta Robledo ◽  
Esther Menéndez ◽  
Pedro F. Mateos ◽  
Solange Oliveira
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Root Nodules ◽  
Strain Analysis ◽  
Wild Type ◽  
Knockout Mutant ◽  
Mesorhizobium Ciceri ◽  
Acid Shock ◽  
Green Fluorescent

Several molecular chaperones are known to be involved in bacteria stress response. To investigate the role of chaperone ClpB in rhizobia stress tolerance as well as in the rhizobia-plant symbiosis process, the clpB gene from a chickpea microsymbiont, strain Mesorhizobium ciceri LMS-1, was identified and a knockout mutant was obtained. The ClpB knockout mutant was tested to several abiotic stresses, showing that it was unable to grow after a heat shock and it was more sensitive to acid shock than the wild-type strain. A plant-growth assay performed to evaluate the symbiotic performance of the clpB mutant showed a higher proportion of ineffective root nodules obtained with the mutant than with the wild-type strain. Nodulation kinetics analysis showed a 6- to 8-day delay in nodule appearance in plants inoculated with the ΔclpB mutant. Analysis of nodC gene expression showed lower levels of transcript in the ΔclpB mutant strain. Analysis of histological sections of nodules formed by the clpB mutant showed that most of the nodules presented a low number of bacteroids. No differences in the root infection abilities of green fluorescent protein–tagged clpB mutant and wild-type strains were detected. To our knowledge, this is the first study that presents evidence of the involvement of the chaperone ClpB from rhizobia in the symbiotic nodulation process.

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

2000 ◽  
Vol 182 (9) ◽  
pp. 2551-2558 ◽  
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.

MenNC1: A PUTATIVE REGULATORY MUTANT OF NADP-MALIC ENZYME IN DROSOPHILA MELANOGASTER

1982 ◽  
Vol 24 (4) ◽  
pp. 409-416 ◽  
Author(s):  
John H. Williamson
Keyword(s):  
Molecular Weight ◽  
Malic Enzyme ◽  
Type Strain ◽  
Wild Type Strain ◽  
Specific Protein ◽  
Activity Levels ◽  
Wild Type ◽  
Type Population ◽  
Temperature Optima ◽  
Substrate Kinetics

MenNC1 was isolated from a wild-type population as a putative NADP-Malic enzyme null-activity mutant. Detailed analysis of this mutant revealed that MenNC1 homozygotes exhibit 3 to 5% of normal levels of NADP-ME activity throughout development. NADP-ME purified from MenNC1 homozygotes is not different from NADP-ME purified from a standard wild-type strain when electrophoretic mobility, molecular weight, substrate kinetics and temperature optima are compared. Quantitative rocket immunoelectrophoresis has demonstrated reduction of NADP-ME-specific protein in MenNC1 homozygotes proportionate to activity levels. It is suggested that MenNC1 may represent a regulatory locus controlling levels of NADP-ME in Drosophila.

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

2007 ◽  
Vol 189 (16) ◽  
pp. 5875-5884 ◽  
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.

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

2005 ◽  
Vol 187 (13) ◽  
pp. 4562-4572 ◽  
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.

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

2014 ◽  
Vol 27 (4) ◽  
pp. 388-399 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document