A New Genetic Locus in Sinorhizobium meliloti Is Involved in Stachydrine Utilization

1998 ◽  
Vol 64 (10) ◽  
pp. 3954-3960 ◽  
Author(s):  
Donald A. Phillips ◽  
Eve S. Sande ◽  
J. A. C. Vriezen ◽  
Frans J. de Bruijn ◽  
Daniel Le Rudulier ◽  
...  
Keyword(s):  
Salt Stress ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Genetic Locus ◽  
Wild Type ◽  
Single Strain ◽  
Reading Frame ◽  
Carbon And Nitrogen ◽  
Promoter Probe ◽  
Inducible Genes

ABSTRACT Stachydrine, a betaine released by germinating alfalfa seeds, functions as an inducer of nodulation genes, a catabolite, and an osmoprotectant in Sinorhizobium meliloti. Two stachydrine-inducible genes were found in S. meliloti1021 by mutation with a Tn5-luxAB promoter probe. Both mutant strains (S10 and S11) formed effective alfalfa root nodules, but neither grew on stachydrine as the sole carbon and nitrogen source. When grown in the absence or presence of salt stress, S10 and S11 took up [14C]stachydrine as well as wild-type cells did, but neither used stachydrine effectively as an osmoprotectant. In the absence of salt stress, both S10 and S11 took up less [14C]proline than wild-type cells did. S10 and S11 appeared to colonize alfalfa roots normally in single-strain tests, but when mixed with the wild-type strain, their rhizosphere counts were reduced more than 50% (P ≤ 0.01) relative to the wild type. These results suggest that stachydrine catabolism contributes to root colonization. DNA sequence analysis identified the mutated locus in S11 as putA, and the luxABfusion in that gene was induced by proline as well as stachydrine. DNA that restored the capacity of mutant S10 to catabolize stachydrine contained a new open reading frame, stcD. All data are consistent with the concept that stcD codes for an enzyme that produces proline by demethylation of N-methylproline, a degradation product of stachydrine.

scholarly journals The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

2007 ◽  
Vol 20 (11) ◽  
pp. 1421-1430 ◽  
Author(s):  
Christian Sohlenkamp ◽  
Kanaan A. Galindo-Lagunas ◽  
Ziqiang Guan ◽  
Pablo Vinuesa ◽  
Sally Robinson ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Minimal Medium ◽  
Polymyxin B ◽  
Growth Conditions ◽  
Membrane Lipid ◽  
Low Ph ◽  
Wild Type ◽  
Rhizobium Tropici ◽  
Gram Negative ◽  
Inducible Genes

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed lpiA, have been identified in the gram-negative α-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.

scholarly journals Malic Enzyme Cofactor and Domain Requirements for Symbiotic N2 Fixation by Sinorhizobium meliloti

2006 ◽  
Vol 189 (1) ◽  
pp. 160-168 ◽  
Author(s):  
Michael J. Mitsch ◽  
Alison Cowie ◽  
Turlough M. Finan
Keyword(s):  
Amino Acid ◽  
Malic Enzyme ◽  
Sinorhizobium Meliloti ◽  
Symbiotic Nitrogen Fixation ◽  
Root Nodules ◽  
High Ratio ◽  
Terminal Region ◽  
Wild Type ◽  
Mutant Strains ◽  
Alfalfa Root

ABSTRACT The NAD+-dependent malic enzyme (DME) and the NADP+-dependent malic enzyme (TME) of Sinorhizobium meliloti are representatives of a distinct class of malic enzymes that contain a 440-amino-acid N-terminal region homologous to other malic enzymes and a 330-amino-acid C-terminal region with similarity to phosphotransacetylase enzymes (PTA). We have shown previously that dme mutants of S. meliloti fail to fix N2 (Fix−) in alfalfa root nodules, whereas tme mutants are unimpaired in their N2-fixing ability (Fix+). Here we report that the amount of DME protein in bacteroids is 10 times greater than that of TME. We therefore investigated whether increased TME activity in nodules would allow TME to function in place of DME. The tme gene was placed under the control of the dme promoter, and despite elevated levels of TME within bacteroids, no symbiotic nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme from the tme promoter resulted in a large reduction in DME activity and symbiotic N2 fixation. Hence, TME cannot replace the symbiotic requirement for DME. In further experiments we investigated the DME PTA-like domain and showed that it is not required for N2 fixation. Thus, expression of a DME C-terminal deletion derivative or the Escherichia coli NAD+-dependent malic enzyme (sfcA), both of which lack the PTA-like region, restored wild-type N2 fixation to a dme mutant. Our results have defined the symbiotic requirements for malic enzyme and raise the possibility that a constant high ratio of NADPH + H+ to NADP in nitrogen-fixing bacteroids prevents TME from functioning in N2-fixing bacteroids.

scholarly journals Rhizobium etli Mutant Modulates Carbon and Nitrogen Metabolism in Phaseolus vulgaris Nodules

2002 ◽  
Vol 15 (7) ◽  
pp. 728-733 ◽  
Author(s):  
Sonia Silvente ◽  
Lourdes Blanco ◽  
Alberto Camas ◽  
José-Luis Ortega ◽  
Mario Ramírez ◽  
...  
Keyword(s):  
Root Nodule ◽  
Root Nodules ◽  
Glutamate Synthase ◽  
Rhizobium Etli ◽  
Wild Type ◽  
Respiratory Capacity ◽  
Carbon And Nitrogen ◽  
N Assimilation ◽  
Mutant Strains ◽  
Carbon And Nitrogen Metabolism

The aim of this study was to evaluate the biochemical events in root nodules which lead to increased yield when bean is inoculated with a Rhizobium etli mutant (CFN037) having increased respiratory capacity. CFN037-inoculated plants had 22% more nitrogen (N) than did wild-type (CE3)-inoculated plants. Root nodule enzymes involved in nodule carbon and nitrogen assimilation as well as in ureides and amides synthesis were assessed in plants inoculated with CFN037 and the CE3. Our results show that the xylem ureides content was lower while that of amino acids was higher in CFN037- compared with CE3-inoculated plants. Supporting these results, enzymes involved in ureide synthesis were reduced while activity of aspartate aminotransferase, glutamate synthase, sucrose synthase, and glucose-6-P dehydrogenase were increased in CFN037- induced nodules. Glutamate synthase and phosphoenolpyruvate carboxylase transcripts were detected early in the development of nodules induced by CFN037 compared with CE3. However, plants inoculated with strain CE3-vhb, which express the Vitreoscilla sp. hemoglobin and also displays increased respiratory capacity, did not have altered ureide transport in N2-fixing plants. The data suggest that inoculation with special selected mutant strains of R. etli can modulate nodule N assimilation and N transport compounds.

scholarly journals ROS-Scavengers, Osmoprotectants and Violaxanthin De-Epoxidation in Salt-Stressed Arabidopsis thaliana with Different Tocopherol Composition

2021 ◽  
Vol 22 (21) ◽  
pp. 11370
Author(s):  
Ewa Surówka ◽  
Dariusz Latowski ◽  
Michał Dziurka ◽  
Magdalena Rys ◽  
Anna Maksymowicz ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Salt Stress ◽  
Transgenic Line ◽  
Wild Type ◽  
Carbon And Nitrogen ◽  
Nacl Concentration ◽  
Tocopherol Composition ◽  
Ros Scavengers ◽  
Excessive Accumulation

To determine the role of α- and γ-tocopherol (TC), this study compared the response to salt stress (200 mM NaCl) in wild type (WT) Arabidopsis thaliana (L.) Heynh. And its two mutants: (1) totally TC-deficient vte1; (2) vte4 accumulating γ-TC instead of α-TC; and (3) tmt transgenic line overaccumulating α-TC. Raman spectra revealed that salt-exposed α-TC accumulating plants were more flexible in regulating chlorophyll, carotenoid and polysaccharide levels than TC deficient mutants, while the plants overaccumulating γ-TC had the lowest levels of these biocompounds. Tocopherol composition and NaCl concentration affected xanthophyll cycle by changing the rate of violaxanthin de-epoxidation and zeaxanthin formation. NaCl treated plants with altered TC composition accumulated less oligosaccharides than WT plants. α-TC deficient plants increased their oligosaccharide levels and reduced maltose amount, while excessive accumulation of α-TC corresponded with enhanced amounts of maltose. Salt-stressed TC-deficient mutants and tmt transgenic line exhibited greater proline levels than WT plants, lower chlorogenic acid levels, and lower activity of catalase and peroxidases. α-TC accumulating plants produced more methylated proline- and glycine- betaines, and showed greater activity of superoxide dismutase than γ-TC deficient plants. Under salt stress, α-TC demonstrated a stronger regulatory effect on carbon- and nitrogen-related metabolites reorganization and modulation of antioxidant patterns than γ-TC. This suggested different links of α- and γ-TCs with various metabolic pathways via various functions and metabolic loops.

scholarly journals Phosphate Assimilation in Rhizobium(Sinorhizobium) meliloti: Identification of apit-Like Gene

1998 ◽  
Vol 180 (16) ◽  
pp. 4219-4226 ◽  
Author(s):  
Sylvie D. Bardin ◽  
Ralf T. Voegele ◽  
Turlough M. Finan
Keyword(s):  
Transport System ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Meliloti ◽  
Root Nodules ◽  
Phosphate Transport ◽  
Wild Type ◽  
Transcription Start ◽  
Present Evidence ◽  
Suppressor Mutations ◽  
Mutant Cells

ABSTRACT Rhizobium meliloti mutants defective in thephoCDET-encoded phosphate transport system form root nodules on alfalfa plants that fail to fix nitrogen (Fix−). We have previously reported that two classes of second-site mutations can suppress the Fix− phenotype ofphoCDET mutants to Fix+. Here we show that one of these suppressor loci (sfx1) contains two genes, orfA and pit, which appear to form an operon transcribed in the order orfA-pit. The Pit protein is homologous to various phosphate transporters, and we present evidence that three suppressor mutations arose from a single thymidine deletion in a hepta-thymidine sequence centered 54 nucleotides upstream of the orfA transcription start site. This mutation increased the level of orfA-pit transcription. These data, together with previous biochemical evidence, show that theorfA-pit genes encode a Pi transport system that is expressed in wild-type cells grown with excess Pibut repressed in cells under conditions of Pi limitation. In phoCDET mutant cells, orfA-pitexpression is repressed, but this repression is alleviated by the second-site suppressor mutations. Suppression increasesorfA-pit expression compensating for the deficiencies in phosphate assimilation and symbiosis of the phoCDETmutants.

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 A luxR Homolog, aviR, in Agrobacterium vitis Is Associated with Induction of Necrosis on Grape and a Hypersensitive Response on Tobacco

2003 ◽  
Vol 16 (7) ◽  
pp. 650-658 ◽  
Author(s):  
Desen Zheng ◽  
Hongsheng Zhang ◽  
Sigrid Carle ◽  
Guixia Hao ◽  
Michele R. Holden ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Polymerase Chain Reaction Analysis ◽  
Homoserine Lactone ◽  
Wild Type ◽  
Agrobacterium Vitis ◽  
Reading Frame ◽  
Homologous Genes ◽  
Luxr Homolog ◽  
Genes Encoding ◽  
Tn5 Mutant

A Tn5 mutant of Agrobacterium vitis F2/5 (M1154) differs from the wild-type strain in that it has lost its abilities to cause necrosis on grape and a hypersensitive-like response (HR) on tobacco. The Tn5 insertion occurred in an open reading frame (ORF) aviR that is homologous to genes encoding the LuxR family of transcriptional regulators, thereby suggesting that the HR and necrosis are regulated by a quorum-sensing system. Fewer N-acyl-homoserine lactone autoinducers were detected in extracts from M1154 compared with extracts from F2/5 and from aviR-complemented M1154. The complemented mutant regained full ability to cause grape necrosis and HR. Eighteen ORFs located on a 36.6-kb insert in cosmid clone CPB221, which includes aviR, were sequenced and aligned with homologous genes from A. tumefaciens C58 and Sinorhizobium meliloti Rm1021. The order of several clustered genes is conserved among the bacteria; however, rearrangements are also apparent. Reverse transcriptase-polymerase chain reaction analysis indicated that ORF2 and ORF14 may be regulated by an aviR-encoded transcriptional regulator. Single site-directed mutations in each of the ORFs, however, had no effect on expression of HR or necrosis as compared with the wild-type parent.

scholarly journals Identification of a Hydroxyproline Transport System in the Legume Endosymbiont Sinorhizobium meliloti

2009 ◽  
Vol 22 (9) ◽  
pp. 1116-1127 ◽  
Author(s):  
Allyson M. MacLean ◽  
Catharine E. White ◽  
Jane E. Fowler ◽  
Turlough M. Finan
Keyword(s):  
Transport System ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Sole Source ◽  
In Planta ◽  
Carbon And Nitrogen ◽  
Meliloti Strain ◽  
Ecological Success ◽  
Hydroxyproline Metabolism ◽  
Alfalfa Root

Hydroxyproline-rich proteins in plants offer a source of carbon and nitrogen to soil-dwelling microorganisms in the form of root exudates and decaying organic matter. This report describes an ABC-type transport system dedicated to the uptake of hydroxyproline in the legume endosymbiont Sinorhizobium meliloti. We have designated genes involved in hydroxyproline metabolism as hyp genes and show that an S. meliloti strain lacking putative transport genes (ΔhypMNPQ) is unable to grow with or transport trans-4-hydroxy-l-proline when this compound is available as a sole source of carbon. Expression of hypM is upregulated in the presence of trans-4-hydroxy-l-proline and cis-4-hydroxy-d-proline, as modulated by a repressor (HypR) of the GntR/FadR subfamily. Although alfalfa root nodules contain hydroxyproline-rich proteins, we demonstrate that the transport system is not highly expressed in nodules, suggesting that bacteroids are not exposed to high levels of free hydroxyproline in planta. In addition to hypMNPQ, we report that S. meliloti encodes a second independent mechanism that enables transport of trans-4-hydroxy-l-proline. This secondary transport mechanism is induced in proline-grown cells and likely entails a system involved in l-proline uptake. This study represents the first genetic description of a prokaryotic hydroxyproline transport system, and the ability to metabolize hydroxyproline may contribute significantly toward the ecological success of plant-associated bacteria such as the rhizobia.

scholarly journals Regulation and Properties of PstSCAB, a High-Affinity, High-Velocity Phosphate Transport System of Sinorhizobium meliloti

2006 ◽  
Vol 188 (3) ◽  
pp. 1089-1102 ◽  
Author(s):  
Ze-Chun Yuan ◽  
Rahat Zaheer ◽  
Turlough M. Finan
Keyword(s):  
Transport System ◽  
High Velocity ◽  
Sinorhizobium Meliloti ◽  
Protein Transport ◽  
Root Nodules ◽  
Phosphate Transport ◽  
Site Directed Mutagenesis ◽  
Uptake System ◽  
Wild Type ◽  
High Affinity

ABSTRACT The properties and regulation of the pstSCAB-encoded Pi uptake system from the alfalfa symbiont Sinorhizobium meliloti are reported. We present evidence that the pstSCAB genes and the regulatory phoUB genes are transcribed from a single promoter that contains two PhoB binding sites and that transcription requires PhoB. S. meliloti strain 1021 (Rm1021) and its derivatives were found to carry a C deletion frameshift mutation in the pstC gene (designated pstC1021) that severely impairs activity of the PstSCAB Pi transport system. This mutation is absent in RCR2011, the parent of Rm1021. Correction of the pstC1021 mutation in Rm1021 by site-directed mutagenesis revealed that PstSCAB is a Pi-specific, high-affinity (Km , 0.2 μM), high-velocity (V max, 70 nmol/min/mg protein) transport system. The pstC1021 allele was shown to generate a partial pho regulon constitutive phenotype, in which transcription is activated by PhoB even under Pi-excess conditions that render PhoB inactive in a wild-type background. The previously reported symbiotic Fix− phenotype of phoCDET mutants was found to be dependent on the pstC1021 mutation, as Rm1021 phoCDET mutants formed small white nodules on alfalfa that failed to reduce N2, whereas phoCDET mutant strains with a corrected pstC allele (RmP110) formed pink nodules on alfalfa that fixed N2 like the wild type. Alfalfa root nodules formed by the wild-type RCR2011 strain expressed the low-affinity orfA-pit-encoded Pi uptake system and neither the pstSCAB genes nor the phoCDET genes. Thus, metabolism of alfalfa nodule bacteroids is not Pi limited.

Regulation of Phosphate Assimilation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1689-1700 ◽  
Author(s):  
Sylvie D Bardin ◽  
Turlough M Finan
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Phosphate Transport ◽  
Wild Type ◽  
Pi Transport ◽  
Regulatory Systems ◽  
Mutant Strains ◽  
Insertion Mutations

Abstract We report the isolation of phoB and phoU mutants of the bacterium Rhizobium (Sinorhizobium) meliloti. These mutants form N2-fixing nodules on the roots of alfalfa plants. R. meliloti mutants defective in the phoCDET (ndvF) encoded phosphate transport system grow slowly in media containing 2 mm Pi, and form nodules which fail to fix nitrogen (Fix−). We show that the transfer of phoB or phoU insertion mutations into phoC mutant strains restores the ability of these mutants to: (i) form normal N2-fixing root-nodules, and (ii) grow like the wild type in media containing 2 mm Pi. We also show that expression of the alternate orfA pit encoded Pi transport system is negatively regulated by the phoB gene product, whereas phoB is required for phoCDET expression. We suggest that in R. meliloti cells growing under Pi limiting conditions, PhoB protein activates phoCDET transcription and represses orfA pit transcription. Our results suggest that there are major differences between the Escherichia coli and R. meliloti phosphate regulatory systems.

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