scholarly journals Characterization of Two Inducible Phosphate Transport Systems in Rhizobium tropici

2000 ◽  
Vol 66 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Lina M. Botero ◽  
Thamir S. Al-Niemi ◽  
Timothy R. McDermott
Keyword(s):  
Transport System ◽  
Response Regulator ◽  
Insertion Site ◽  
Dry Weight ◽  
Type Systems ◽  
Phosphate Transport ◽  
Transport Systems ◽  
Atpase Inhibitor ◽  
Rhizobium Tropici ◽  
High Affinity

ABSTRACT Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropiciassociation is sensitive to the availability of phosphate (Pi). To better understand phosphorus movement between the bacteroid and the host plant, Pi transport was characterized in R. tropici. We observed two Pitransport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. TheKm and V max values for the low-affinity system were estimated to be 34 ± 3 μM Pi and 118 ± 8 nmol of Pi · min−1 · mg (dry weight) of cells−1, respectively, and the Km andV max values for the high-affinity system were 0.45 ± 0.01 μM Pi and 86 ± 5 nmol of Pi · min−1 · mg (dry weight) of cells−1, respectively. Both systems were inducible by Pi starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but Pi transport through both systems was eliminated by the ATPase inhibitor N,N′-dicyclohexylcarbodiimide; the Pi transport rate was correlated with the intracellular ATP concentration. Also, Pi movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both Pi transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology withkdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.

scholarly journals Molecular Mechanisms of Phosphate Sensing, Transport and Signalling in Streptomyces and Related Actinobacteria

2021 ◽  
Vol 22 (3) ◽  
pp. 1129
Author(s):  
Juan Francisco Martín ◽  
Paloma Liras
Keyword(s):  
Transport System ◽  
Inorganic Phosphate ◽  
Molecular Mechanisms ◽  
Phosphate Transport ◽  
Transport Systems ◽  
Outer Side ◽  
Phosphate Binding ◽  
Streptomyces Species ◽  
High Affinity ◽  
Sugar Phosphates

Phosphorous, in the form of phosphate, is a key element in the nutrition of all living beings. In nature, it is present in the form of phosphate salts, organophosphates, and phosphonates. Bacteria transport inorganic phosphate by the high affinity phosphate transport system PstSCAB, and the low affinity PitH transporters. The PstSCAB system consists of four components. PstS is the phosphate binding protein and discriminates between arsenate and phosphate. In the Streptomyces species, the PstS protein, attached to the outer side of the cell membrane, is glycosylated and released as a soluble protein that lacks its phosphate binding ability. Transport of phosphate by the PstSCAB system is drastically regulated by the inorganic phosphate concentration and mediated by binding of phosphorylated PhoP to the promoter of the PstSCAB operon. In Mycobacterium smegmatis, an additional high affinity transport system, PhnCDE, is also under PhoP regulation. Additionally, Streptomyces have a duplicated low affinity phosphate transport system encoded by the pitH1–pitH2 genes. In this system phosphate is transported as a metal-phosphate complex in simport with protons. Expression of pitH2, but not that of pitH1 in Streptomyces coelicolor, is regulated by PhoP. Interestingly, in many Streptomyces species, three gene clusters pitH1–pstSCAB–ppk (for a polyphosphate kinase), are linked in a supercluster formed by nine genes related to phosphate metabolism. Glycerol-3-phosphate may be transported by the actinobacteria Corynebacterium glutamicum that contains a ugp gene cluster for glycerol-3-P uptake, but the ugp cluster is not present in Streptomyces genomes. Sugar phosphates and nucleotides are used as phosphate source by the Streptomyces species, but there is no evidence of the uhp gene involved in the transport of sugar phosphates. Sugar phosphates and nucleotides are dephosphorylated by extracellular phosphatases and nucleotidases. An isolated uhpT gene for a hexose phosphate antiporter is present in several pathogenic corynebacteria, such as Corynebacterium diphtheriae, but not in non-pathogenic ones. Phosphonates are molecules that contains phosphate linked covalently to a carbon atom through a very stable C–P bond. Their utilization requires the phnCDE genes for phosphonates/phosphate transport and genes for degradation, including those for the subunits of the C–P lyase. Strains of the Arthrobacter and Streptomyces genera were reported to degrade simple phosphonates, but bioinformatic analysis reveals that whole sets of genes for putative phosphonate degradation are present only in three Arthrobacter species and a few Streptomyces species. Genes encoding the C–P lyase subunits occur in several Streptomyces species associated with plant roots or with mangroves, but not in the laboratory model Streptomyces species; however, the phnCDE genes that encode phosphonates/phosphate transport systems are frequent in Streptomyces species, suggesting that these genes, in the absence of C–P lyase genes, might be used as surrogate phosphate transporters. In summary, Streptomyces and related actinobacteria seem to be less versatile in phosphate transport systems than Enterobacteria.

Proton gradient-dependent renal transport of glycine: evidence for vesicle studies

1990 ◽  
Vol 258 (2) ◽  
pp. F388-F396 ◽  
Author(s):  
H. Roigaard-Petersen ◽  
H. Jessen ◽  
S. Mollerup ◽  
K. E. Jorgensen ◽  
C. Jacobsen ◽  
...  
Keyword(s):  
Transport System ◽  
Membrane Vesicles ◽  
Proton Gradient ◽  
Transport Systems ◽  
Rabbit Kidney ◽  
Renal Transport ◽  
High Affinity ◽  
Luminal Membrane ◽  
Pars Recta ◽  
Pars Convoluta

The characteristics of renal transport of glycine by luminal membrane vesicles isolated from either proximal convoluted part (pars convoluta) or proximal straight part (pars recta) of rabbit proximal tubule were investigated. In vesicles from pars convoluta two transport systems have been characterized: a Na(+)-dependent system with intermediate affinity (half-saturation 3.64 mM) and a Na(+)-independent system that, in the presence of H+ gradient (extravesicular greater than intravesicular), can accelerate the transport of glycine into these vesicles. This is the first demonstration of H(+)-glycine cotransport across the luminal membrane of rabbit kidney proximal convoluted tubule. By contrast, in membrane vesicles from pars recta, transport of glycine was strictly dependent on Na+ and occurred via a dual transport system, namely a high-affinity (half-saturation 0.34 mM) and a low-affinity system (half-saturation 8.56 mM). The demonstration of competition between the H(+)-gradient dependent uptake of glycine, L-alanine, and L-proline, but insignificant inhibition with L-phenylalanine in vesicles from pars convoluta suggests that glycine, L-proline, and L-alanine probably share a common proton gradient-dependent transport system. In vesicles from pars recta, the Na(+)-dependent uptake of glycine was inhibited by low concentrations of L-alanine and L-phenylalanine, whereas addition of L-proline to the incubation medium did not significantly alter the uptake of glycine, suggesting that the Na(+)-dependent high-affinity system for glycine located in pars recta is shared with the high-affinity L-alanine and L-phenylalanine but not L-proline transport system.

Effect of pH on Na(+)-dependent phosphate transport in renal outer cortical and outer medullary BBMV

1990 ◽  
Vol 258 (2) ◽  
pp. F356-F363 ◽  
Author(s):  
G. A. Quamme
Keyword(s):  
Brush Border ◽  
Sodium Phosphate ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Transport Systems ◽  
Outer Cortex ◽  
Ph Change ◽  
High Affinity ◽  
Medullary Tissue ◽  
Maximum Uptake Rate

The influence of pH on sodium-phosphate cotransport was determined in brush-border membrane vesicles (BBMV) isolated from outer cortical and outer medullary tissue of porcine kidneys. Two transport systems are apparent in outer cortical brush-border vesicles, and one process is apparent in outer medullary vesicles at all pH values. The apparent maximum uptake rate (Vmax) of the low-affinity system in outer cortex vesicles decreased from 8.3 +/- 1.7 to 3.2 +/- 0.05 nmol.mg protein-1.min-1 with pH change of 8.0 to 6.0, and the high-affinity process changed from 1.3 +/- 0.2 to 0.1 +/- 0.01 nmol.mg protein-1.min-1. The respective affinity values (Km) also decreased 5.5 +/- 0.9 to 0.6 +/- 0.01 mM and 0.08 +/- 0.005 to 0.01 +/- 0.005 mM, respectively, with acidification. In outer medullary vesicles a decrease in pH diminished the apparent Km, 0.28 +/- 0.03 to 0.02 +/- 0.003 mM, and mean Vmax from 3.0 +/- 0.07 to 0.5 +/- 0.1 nmol.mg protein-1.min-1. The mean KNaD values were 22.1 +/- 4.2 mM in outer cortical vesicles (low-affinity system) and 58.7 +/- 7.2 mM in outer medullary vesicles (high-affinity system) and were not altered by pH, suggesting that H+ does not affect the sodium interactive site. The data suggest that the vesicles prepared from outer cortical and outer medullary tissue possess distinctive sodium-phosphate transporters that are sensitive to external H+ concentrations.

Identification of a system that allows a Rhizobium tropici dctA mutant to grow on succinate, but not on other C4-dicarboxylates

2001 ◽  
Vol 47 (6) ◽  
pp. 509-518 ◽  
Author(s):  
Silvia Batista ◽  
Ana I Catalán ◽  
Ismael Hernández-Lucas ◽  
Esperanza Martínez-Romero ◽  
O Mario Aguilar ◽  
...  
Keyword(s):  
Transport System ◽  
Biochemical Characterization ◽  
Dry Weight ◽  
Acetylene Reduction Activity ◽  
Insertion Mutant ◽  
Rhizobium Tropici ◽  
Gene Encoding ◽  
Shoot Dry Weight ◽  
Reduction Activity ◽  
Bean Plants

A defined insertion mutant of a gene encoding a homolog of the rhizobial C4-dicarboxylate permease (dctA) was constructed in Rhizobium tropici strain CIAT899. This mutant (GA1) was unable to grow on fumarate or malate; however, in contrast with other rhizobial dctA mutants, it retained a limited ability to grow on succinate with ammonia as a nitrogen source. Our results suggest the presence of a novel succinate-specific transport system in R. tropici. Biochemical characterization indicated that this alternative transport system in GA1 is active and dependent on an energized membrane. It was also induced by succinate and aspartate, and was repressed by glucose and glycerol. Bean plants inoculated with GA1 showed a reduced nitrogen-fixing ability, achieving only 29% of the acetylene reduction activity determined in CIAT899 strain nodules, 33 days after inoculation. Also, bean plants inoculated with GA1 had reduced shoot dry weight compared with plants inoculated with the wild-type strain.Key words: succinate transport, Rhizobium tropici, C4-dicarboxylate uptake.

Characterization of inorganic phosphate transport in osteoclast-like cells

2005 ◽  
Vol 288 (4) ◽  
pp. C921-C931 ◽  
Author(s):  
Mikiko Ito ◽  
Naoko Matsuka ◽  
Michiyo Izuka ◽  
Sakiko Haito ◽  
Yuko Sakai ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Transport System ◽  
Inorganic Phosphate ◽  
Phosphate Transport ◽  
Transport Systems ◽  
Raw264.7 Cells ◽  
Transport Activity ◽  
Phosphonoformic Acid ◽  
Carbonyl Cyanide ◽  
Bone Particles

Osteoclasts possess inorganic phosphate (Pi) transport systems to take up external Pi during bone resorption. In the present study, we characterized Pi transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-κB ligand (RANKL). In undifferentiated RAW264.7 cells, Pi transport into the cells was Na+ dependent, but after treatment with RANKL, Na+-independent Pi transport was significantly increased. In addition, compared with neutral pH, the activity of the Na+-independent Pi transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na+-independent system consisted of two components with Km of 0.35 mM and 7.5 mM. The inhibitors of Pi transport, phosphonoformic acid, and arsenate substantially decreased Pi transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K+ ionophore, valinomycin, significantly suppressed Pi transport activity. Analysis of BCECF fluorescence indicated that Pi transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K+ ion stimulated Pi transport, suggesting that membrane voltage is involved in the regulation of Pi transport activity. Finally, bone particles significantly increased Na+-independent Pi transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a Pi transport system with characteristics that are different from those of other Na+-dependent Pi transporters. We conclude that stimulation of Pi transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment.

scholarly journals Differential Regulation of High-Affinity Phosphate Transport Systems of Mycobacterium smegmatis: Identification of PhnF, a Repressor of the phnDCE Operon

2007 ◽  
Vol 190 (4) ◽  
pp. 1335-1343 ◽  
Author(s):  
Susanne Gebhard ◽  
Gregory M. Cook
Keyword(s):  
Mycobacterium Smegmatis ◽  
Constitutive Expression ◽  
Phosphate Transport ◽  
Phosphate Limitation ◽  
Transport Systems ◽  
New Members ◽  
High Affinity ◽  
Expression Of Genes ◽  
Two Component ◽  
Genes Encoding

ABSTRACT The uptake of phosphate into the cell via high-affinity, phosphate-specific transport systems has been studied with several species of mycobacteria. All of these species have been shown to contain several copies of such transport systems, which are synthesized in response to phosphate limitation. However, the mechanisms leading to the expression of the genes encoding these transporters have not been studied. This study reports on the investigation of the regulation of the pstSCAB and the phnDCE operons of Mycobacterium smegmatis. The phn locus contains an additional gene, phnF, encoding a GntR-like transcriptional regulator. Expression analyses of a phnF deletion mutant demonstrated that PhnF acts as a repressor of the phnDCE operon but does not affect the expression of pstSCAB. The deletion of pstS, which is thought to cause the constitutive expression of genes regulated by the two-component system SenX3-RegX3, led to the constitutive expression of the transcriptional fusions pstS-lacZ, phnD-lacZ, and phnF-lacZ, suggesting that phnDCE and phnF are conceivably new members of the SenX3-RegX3 regulon of M. smegmatis. Two presumptive binding sites for PhnF in the intergenic region between phnD and phnF were identified and shown to be required for the repression of phnD and phnF, respectively. We propose a model in which the transcription of pstSCAB is controlled by the two-component SenX3-RegX3 system, while phnDCE and phnF are subject to dual control by SenX3-RegX3 and PhnF.

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 High-Affinity Vanadate Transport System in the Cyanobacterium Anabaena variabilis ATCC 29413

2006 ◽  
Vol 188 (2) ◽  
pp. 464-468 ◽  
Author(s):  
Brenda S. Pratte ◽  
Teresa Thiel
Keyword(s):  
Transport System ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Rhodopseudomonas Palustris ◽  
Methanosarcina Barkeri ◽  
Anabaena Variabilis ◽  
Transport Systems ◽  
Bacterial Strains ◽  
High Affinity ◽  
Major Cluster

ABSTRACT High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 × 10−9 M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri.

Sign in / Sign up

Export Citation Format

Share Document