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.

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.

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.

scholarly journals The Sinorhizobium meliloti ABC Transporter Cho Is Highly Specific for Choline and Expressed in Bacteroids from Medicago sativa Nodules

2004 ◽  
Vol 186 (18) ◽  
pp. 5988-5996 ◽  
Author(s):  
Laurence Dupont ◽  
Isabelle Garcia ◽  
Marie-Christine Poggi ◽  
Geneviève Alloing ◽  
Karine Mandon ◽  
...  
Keyword(s):  
Medicago Sativa ◽  
Abc Transporter ◽  
Sinorhizobium Meliloti ◽  
Lipid Membrane ◽  
Site Directed Mutagenesis ◽  
Choline Uptake ◽  
Uptake System ◽  
Choline Transport ◽  
High Affinity ◽  
Uptake Activity

ABSTRACT In Sinorhizobium meliloti, choline is the direct precursor of phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family, and glycine betaine, an important osmoprotectant. Moreover, choline is an efficient energy source which supports growth. Using a PCR strategy, we identified three chromosomal genes (choXWV) which encode components of an ABC transporter: ChoX (binding protein), ChoW (permease), and ChoV (ATPase). Whereas the best homology scores were obtained with components of betaine ProU-like systems, Cho is not involved in betaine transport. Site-directed mutagenesis of choX strongly reduced (60 to 75%) the choline uptake activity, and purification of ChoX, together with analysis of the ligand-binding specificity, showed that ChoX binds choline with a high affinity (K D , 2.7 μM) and acetylcholine with a low affinity (K D , 145 μM) but binds none of the betaines. Uptake competition experiments also revealed that ectoine, various betaines, and choline derivatives were not effective competitors for Cho-mediated choline transport. Thus, Cho is a highly specific high-affinity choline transporter. Choline transport activity and ChoX expression were induced by choline but not by salt stress. Western blotting experiments with antibodies raised against ChoX demonstrated the presence of ChoX in bacteroids isolated from nitrogen-fixing nodules obtained from Medicago sativa roots. The choX mutation did not have an effect on growth under standard conditions, and neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that the remaining choline uptake system(s) still present in the mutant strain can compensate for the lack of Cho transporter.

scholarly journals The Phn system of Mycobacterium smegmatis: a second high-affinity ABC-transporter for phosphate

Microbiology ◽  
2006 ◽  
Vol 152 (11) ◽  
pp. 3453-3465 ◽  
Author(s):  
Susanne Gebhard ◽  
Sieu L. Tran ◽  
Gregory M. Cook
Keyword(s):  
Transport System ◽  
Mycobacterium Smegmatis ◽  
Single Copy ◽  
Phosphate Transport ◽  
Phosphate Limitation ◽  
Wild Type ◽  
High Affinity ◽  
Genes Encoding ◽  
Limiting Nutrient ◽  
In The Wild

Uptake of inorganic phosphate, an essential but often limiting nutrient, in bacteria is usually accomplished by the high-affinity ABC-transport system Pst. Pathogenic species of mycobacteria contain several copies of the genes encoding the Pst system (pstSCAB), and two of the encoded proteins, PstS1 and PstS2, have been shown to be virulence factors in Mycobacterium tuberculosis. The fast-growing Mycobacterium smegmatis contains only a single copy of the pst operon. This study reports the biochemical and molecular characterization of a second high-affinity phosphate transport system, designated Phn. The Phn system is encoded by a three-gene operon that constitutes the components of a putative ABC-type phosphonate/phosphate transport system. Expression studies using phnD– and pstS–lacZ transcriptional fusions showed that both operons were induced when the culture entered phosphate limitation, indicating a role for both systems in phosphate uptake at low extracellular concentrations. Deletion mutants in either phnD or pstS failed to grow in minimal medium with a 10 mM phosphate concentration, while the isogenic wild-type strain mc2155 grew at micromolar phosphate concentrations. Analysis of the kinetics of phosphate transport in the wild-type and mutant strains led to the proposal that the Phn and Pst systems are both high-affinity phosphate transporters with similar affinities for phosphate (i.e. apparent K m values between 40 and 90 μM Pi). The Phn system of M. smegmatis appears to be unique in that, unlike previously identified Phn systems, it does not recognize phosphonates or phosphite as substrates.

scholarly journals Overexpression of BetS, a Sinorhizobium meliloti High-Affinity Betaine Transporter, in Bacteroids from Medicago sativa Nodules Sustains Nitrogen Fixation During Early Salt Stress Adaptation

2006 ◽  
Vol 19 (8) ◽  
pp. 896-903 ◽  
Author(s):  
Alexandre Boscari ◽  
Ghislaine Van de Sype ◽  
Daniel Le Rudulier ◽  
Karine Mandon
Keyword(s):  
Nitrogen Fixation ◽  
Salt Stress ◽  
Glycine Betaine ◽  
Sinorhizobium Meliloti ◽  
Uptake System ◽  
Wild Type ◽  
In Planta ◽  
Rapid Acquisition ◽  
High Affinity ◽  
Nitrogen Fixation Activity

Sinorhizobium meliloti possesses several betaine transporters to cope with salt stress, and BetS represents a crucial high-affinity glycine and proline betaine uptake system involved in the rapid acquisition of betaines by cells subjected to osmotic upshock. Using a transcriptional lacZ (β-galactosidase) fusion, we showed that betS is expressed during the establishment of the symbiosis and in mature nitrogen-fixing nodules. However, neither Nod nor Fix phenotypes were impaired in a betS mutant. BetS is functional in isolated bacteroids, and its activity is strongly activated by high osmolarity. In bacteroids from a betS mutant, glycine betaine and proline betaine uptake was reduced by 85 to 65%, indicating that BetS is a major component of the overall betaine uptake activity in bacteroids in response to osmotic stress. Upon betS overexpression (strain UNA349) in free-living cells, glycine betaine transport was 2.3-fold higher than in the wild-type strain. Interestingly, the accumulation of proline betaine, the endogenous betaine synthesized by alfalfa plants, was 41% higher in UNA349 bacteroids from alfalfa plants subjected to 1 week of salinization (0.3 M NaCl) than in wild-type bacteroids. In parallel, a much better maintenance of nitrogen fixation activity was observed in 7-day-salinized plants nodulated with the over-expressing strain than in wild-type nodulated plants. Taken altogether, these results are consistent with the major role of BetS as an emergency system involved in the rapid uptake of betaines in isolated and in planta osmotically stressed bacteroids of S. meliloti.

Phosphate uptake by renal membrane vesicles of rabbits adapted to high and low phosphorus diets

1983 ◽  
Vol 245 (2) ◽  
pp. F175-F180 ◽  
Author(s):  
L. Cheng ◽  
C. T. Liang ◽  
B. Sacktor
Keyword(s):  
Transport System ◽  
Phosphate Uptake ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Uptake System ◽  
Low Phosphorus ◽  
Dietary Phosphate ◽  
Preferred Species ◽  
Uptake Medium ◽  
Phosphate Carrier

Renal adaptation to changes in phosphate intake was studied by comparing phosphate uptake by proximal tubule brush border membrane vesicles from rabbits on a relatively high or low phosphorus diet. The low phosphorus diet increased Na+ gradient-dependent phosphate uptake. Uptake in the absence of Na+ and in the presence of Na+, but no gradient, was not significantly affected. The phosphorus diet did not alter Na+ gradient-dependent D-glucose and L-proline uptake. The low phosphorus diet increased Vmax; affinity for phosphate was not appreciably changed. At all concentrations of extravesicular Na+, phosphate uptake was higher in membrane vesicles from animals fed the low phosphorus diet; the kinetics of the phosphate uptake system, with respect to Na+, was also altered by the change in dietary phosphate. These findings suggest that adaptation involves an alteration in the rate of translocation of the Na+-phosphate carrier when energized by a Na+ gradient driving force rather than a change in the number of Na+-phosphate carrier sites. With membrane vesicles from rabbits fed a low phosphorus diet, phosphate uptake increased several-fold when the pH of the uptake medium was raised, whereas with membrane vesicles from animals fed a high phosphorus diet the enhancement of uptake with alkalinization was relatively small. Irrespective of the diet, divalent phosphate was the probable preferred species for transport. Dietary adaptation was associated, however, with an alteration in the pH dependency of the transport system per se. These findings provide evidence that the adaptation of the kidney phosphate transport system to dietary phosphate load involves an intrinsic change in the Na+-phosphate carrier.

scholarly journals Regulation of Copper Metabolism by Nitrogen Utilization in Saccharomyces cerevisiae

2021 ◽  
Vol 7 (9) ◽  
pp. 756
Author(s):  
Suzie Kang ◽  
Hyewon Seo ◽  
Min-Gyu Lee ◽  
Cheol-Won Yun
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Iron Uptake ◽  
Nitrogen Starvation ◽  
Copper Metabolism ◽  
Nitrogen Utilization ◽  
Deletion Mutants ◽  
Uptake System ◽  
Wild Type ◽  
High Affinity ◽  
Uptake Activity

To understand the relationship between carbon or nitrogen utilization and iron homeostasis, we performed an iron uptake assay with several deletion mutants with partial defects in carbon or nitrogen metabolism. Among them, some deletion mutants defective in carbon metabolism partially and the MEP2 deletion mutant showed lower iron uptake activity than the wild type. Mep2 is known as a high-affinity ammonia transporter in Saccharomyces cerevisiae. Interestingly, we found that nitrogen starvation resulted in lower iron uptake activity than that of wild-type cells without downregulation of the genes involved in the high-affinity iron uptake system FET3/FTR1. However, the gene expression of FRE1 and CTR1 was downregulated by nitrogen starvation. The protein level of Ctr1 was also decreased by nitrogen starvation, and addition of copper to the nitrogen starvation medium partially restored iron uptake activity. However, the expression of MAC1, which is a copper-responsive transcriptional activator, was not downregulated by nitrogen starvation at the transcriptional level but was highly downregulated at the translational level. Mac1 was downregulated dramatically under nitrogen starvation, and treatment with MG132, which is an inhibitor of proteasome-dependent protein degradation, partially attenuated the downregulation of Mac1. Taken together, these results suggest that nitrogen starvation downregulates the high-affinity iron uptake system by degrading Mac1 in a proteasome-dependent manner and eventually downregulates copper metabolism.

scholarly journals Characterization of a Sinorhizobium melilotiATP-Binding Cassette Histidine Transporter Also Involved in Betaine and Proline Uptake

2000 ◽  
Vol 182 (13) ◽  
pp. 3717-3725 ◽  
Author(s):  
Eric Boncompagni ◽  
Laurence Dupont ◽  
Tam Mignot ◽  
Magne Østeräs ◽  
Annie Lambert ◽  
...  
Keyword(s):  
Glycine Betaine ◽  
Sinorhizobium Meliloti ◽  
Binding Protein ◽  
Compatible Solutes ◽  
Site Directed Mutagenesis ◽  
Uptake System ◽  
Periplasmic Binding Protein ◽  
Gene Encoding ◽  
Inhibition Of Growth

ABSTRACT The symbiotic soil bacterium Sinorhizobium melilotiuses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli(ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed inhut mutants (hutX and hutH2). Expression analysis of the hut operon determined using ahutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

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.

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