Phosphate Uptake Systems Research Articles

A Phosphate Uptake System Is Required for Xanthomonas citri pv. glycines Virulence in Soybean.

The genes encoding the phosphate uptake system in Xanthomonas citri pv. glycines 12-2 were previously found to be upregulated when in soybean leaves. This study thus explored the role of the phosphate uptake system on its virulence to soybean. While phoB and pstSCAB mutants were greatly impaired in both inciting disease symptoms and growth in soybean, the virulence and growth in soybean of a phoU mutant was not reduced when compared with the wild-type strain. The expression of phoB and pstSCAB was highly induced in phosphate-deficient media. In addition, the expression of phoB, assessed with a fusion to a promoterless ice nucleation reporter gene, was greatly increased in soybean leaves, confirming that the soybean apoplast is a phosphorus-limited habitat for X. citri pv. glycines. Global gene expression profiles of phoB and phoU mutants of X. citri pv. glycines conducted under phosphate-limitation conditions invitro, using RNA-seq, revealed that PhoB positively regulated genes involved in signal transduction, the xcs cluster type II secretion system, cell motility, and chemotaxis, while negatively regulating cell wall and membrane biogenesis, DNA replication and recombination and repair, and several genes with unknown function. PhoU also positively regulated the same genes involved in cell motility and chemotaxis. The severity of bacterial pustule disease was decreased in soybean plants grown under high phosphate fertilization conditions, demonstrating that high phosphate availability in soybean plants can affect infection by X. citri pv. glycines by modulation of the expression of phosphate uptake systems. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Genome-guided prediction of acid resistance mechanisms in acidophilic methanotrophs of phylogenetically deep-rooted Verrucomicrobia isolated from geothermal environments.

Verrucomicrobia are a group of microorganisms that have been proposed to be deeply rooted in the Tree of Life. Some are methanotrophs that oxidize the potent greenhouse gas methane and are thus important in decreasing atmospheric concentrations of the gas, potentially ameliorating climate change. They are widespread in various environments including soil and fresh or marine waters. Recently, a clade of extremely acidophilic Verrucomicrobia, flourishing at pH < 3, were described from high-temperature geothermal ecosystems. This novel group could be of interest for studies about the emergence of life on Earth and to astrobiologists as homologs for possible extraterrestrial life. In this paper, we describe predicted mechanisms for survival of this clade at low pH and suggest its possible evolutionary trajectory from an inferred neutrophilic ancestor. Extreme acidophiles are defined as organisms that thrive in extremely low pH environments (≤ pH 3). Many are polyextremophiles facing high temperatures and high salt as well as low pH. They are important to study for both providing fundamental insights into biological mechanisms of survival and evolution in such extreme environments and for understanding their roles in biotechnological applications such as industrial mineral recovery (bioleaching) and mitigation of acid mine drainage. They are also, potentially, a rich source of novel genes and pathways for the genetic engineering of microbial strains. Acidophiles of the Verrucomicrobia phylum are unique as they are the only known aerobic methanotrophs that can grow optimally under acidic (pH 2–3) and moderately thermophilic conditions (50–60°C). Three moderately thermophilic genera, namely Methylacidiphilum, Methylacidimicrobium, and Ca. Methylacidithermus, have been described in geothermal environments. Most of the investigations of these organisms have focused on their methane oxidizing capabilities (methanotrophy) and use of lanthanides as a protein cofactor, with no extensive study that sheds light on the mechanisms that they use to flourish at extremely low pH. In this paper, we extend the phylogenetic description of this group of acidophiles using whole genome information and we identify several mechanisms, potentially involved in acid resistance, including “first line of defense” mechanisms that impede the entry of protons into the cell. These include the presence of membrane-associated hopanoids, multiple copies of the outer membrane protein (Slp), and inner membrane potassium channels (kup, kdp) that generate a reversed membrane potential repelling the intrusion of protons. Acidophilic Verrucomicrobia also display a wide array of proteins potentially involved in the “second line of defense” where protons that evaded the first line of defense and entered the cell are expelled or neutralized, such as the glutamate decarboxylation (gadAB) and phosphate-uptake systems. An exclusive N-type ATPase F0-F1 was identified only in acidophiles of Verrucomicrobia and is predicted to be a specific adaptation in these organisms. Phylogenetic analyses suggest that many predicted mechanisms are evolutionarily conserved and most likely entered the acidophilic lineage of Verrucomicrobia by vertical descent from a common ancestor. However, it is likely that some defense mechanisms such as gadA and kup entered the acidophilic Verrucomicrobia lineage by horizontal gene transfer.

High External K+ Concentrations Impair Pi Nutrition, Induce the Phosphate Starvation Response, and Reduce Arsenic Toxicity in Arabidopsis Plants.

Potassium (K+) and phosphorous (Pi) are two of the most important nutrients required by plants and there is an interest in studying how they are acquired. Most studies have focused on the characterization of the mechanisms involved in K+ and Pi uptake and their distribution within the plants, as well as the regulatory mechanisms involved. Evidence is emerging which points to interactions in the nutrition of different nutrients and to the existence of crosstalk in the signaling cascades regulating their acquisition. However, the interaction between K+ and Pi has been scarcely studied. Here we show that high concentrations of K+ in the external solution inhibit Pi uptake and impair Pi nutrition in Arabidopsis plants, resulting in the induction of phosphate starvation response (PSR) and the upregulation of genes encoding root phosphate uptake systems. The high K+-induced PSR depends on the PHR1 and PHL1 transcription factors that are key pieces of Pi signaling in Arabidopsis. Importantly, high K+ reduces arsenic accumulation in plants and its toxic effects. The results presented may help to design strategies to reduce Pi deficiency as well as the accumulation of arsenic in crops.

Phosphate uptake by the phosphonate transport system PhnCDE

BackgroundPhosphate is a fundamental nutrient for all creatures. It is thus not surprising that a single bacterium carries different transport systems for this molecule, each usually operating under different environmental conditions. The phosphonate transport system of E. coli K-12 is cryptic due to an 8 bp insertion in the phnE ORF.ResultsHere we report that an E. coli K-12 strain carrying the triple knockout ΔpitAΔpstΔugp reverted the phnE mutation when plated on complex medium containing phosphate as the main phosphorus source. It is also shown that PhnCDE takes up orthophosphate with transport kinetics compatible with that of the canonical transport system PitA and that Pi-uptake via PhnCDE is sufficient to enable bacterial growth. Ugp, a glycerol phosphate transporter, is unable to take up phosphate.ConclusionsThe phosphonate transport system, which is normally cryptic in E. coli laboratory strains is activated upon selection in rich medium and takes up orthophosphate in the absence of the two canonical phosphate-uptake systems. Based on these findings, the PhnCDE system can be considered a genuine phosphate transport system.

Dynamic Phosphate Uptake in Arbuscular Mycorrhizal Roots Under Field Conditions

Many crops are colonized with arbuscular mycorrhizal fungi (AMF), which can efficiently absorb nutrients such as phosphate from the soil. The utilization of mycorrhizal roots is one of the most promising options for developing resource-saving and sustainable agricultural systems. Most laboratory studies have illustrated the roles of AM symbiosis by inoculating plants with limited AMF isolates. In the field, however, the roots of crops are co-colonized with multiple AMF species, which are difficult to separate and identify and may have different abilities regarding phosphate uptake. Given advances in high-throughput sequencing technologies, such multi-component phosphate uptake systems under field conditions are the new frontier in mycorrhizal research and are crucial for managing the phosphate nutrition of crops. However, it is still difficult to understand which AMF are functional due to the dynamics of AMF colonization processes and the largely unknown genomic structure. This review summarizes key discoveries supporting the importance of the dynamics of AM colonization and genomic structure, which potentially influence the characteristics of AM phosphate uptake. Moreover, this review aims to identify the research direction necessary to obtain a better understanding of the phosphate uptake systems of crops in the field.

Nitrogen availability facilitates phosphorus acquisition by bloom-forming cyanobacteria.

Cyanobacterial blooms are threatening freshwater ecosystems. The physiological basis involved in the onset of cyanobacterial bloom is fundamental to advance in bloom predictions. Generally, cyanobacteria grow until the availability of nitrogen (N), phosphorus (P) or both nutrients becomes limited. Population survival may depend on physiological adjustments to nutrient deficiency as well as on the efficient use of episodic N and P inputs. This study investigated the effect of N inputs on phosphate uptake affinity and activity of N-deficient bloom-forming cyanobacteria. Lake samples dominated by filamentous cyanobacteria were preincubated with and without nitrate addition, and the uptake of [32P] phosphate pulses was measured in the following days. Phosphate uptake kinetics were analyzed with a flow-force model that provides the threshold concentration, reflecting phosphate uptake affinity, and the membrane conductivity coefficient that corresponds to the activity of uptake systems. After 24 h of nitrate preincubation, phosphate uptake kinetics showed a progressive increase in affinity (nanomolar [Pe]A) and activity (25-fold) concomitant with cyanobacterial growth. It was demonstrated that the alleviation of N-deficiency by N inputs boosts the activation of phosphate uptake systems of non-N2-fixing cyanobacteria to sustain growth. Therefore, reduction of dissolved inorganic N levels in lakes is also mandatory to limit cyanobacterial phosphate uptake affinity and activity capabilities.

Regulation of phosphate uptake reveals cyanobacterial bloom resilience to shifting N:P ratios

Abstract The resilience of cyanobacterial blooms challenges lake restoration programmes based on nutrient load reduction. The survival of organisms may depend on episodic short‐term fluctuations of nitrogen (N) and phosphorus (P). Insight into physiological responses to shifts in nutrient limitation will improve our understanding of cyanobacterial bloom resilience. This study investigated the resilience and collapse of a long‐term cyanobacterial bloom dominated by Planktothrix agardhii and Raphidiopsis mediterranea, subjected to different periods of dissolved inorganic nitrogen (DIN) and phosphorus (SRP) inputs in Lago Rodó, a polymictic, hypereutrophic (6.0 ± 1.6 μmol total P/L) and turbid lake (attenuation coefficient: 4.7 ± 1.7 m−1). Phytoplankton composition, nutrient dynamics and physiological monitoring of [32P] phosphate uptake kinetics were studied over four summers. The influence of environmental nutrient supply on phytoplankton net phosphate uptake kinetics was analysed with a flow–force model that gives the threshold value of phosphate uptake ([Pe]A), a limiting concentration below which incorporation is not energetically possible by organisms, and the membrane conductivity coefficient (LP). The [Pe]A reflects the affinity and LP the activity of the cellular uptake systems. Nutrient supply into the lake sustained high DIN:SRP ratios and high cyanobacterial biomass that showed P‐deficiency features characterised by low [Pe]A (5.4 ± 0.5 nm [32P] phosphate) and high LP. High cyanobacterial biomass persisted after external nutrient inflows were interrupted for 14 months, which resulted in low dissolved DIN:SRP ratios; however, N2‐fixers did not develop. The collapse of cyanobacterial populations occurred under the lowest N:P ratios, low affinity and activity of phosphate uptake systems and strong water outflows. Our study suggests that the alleviation of N‐deficiency under high N:P ratios enhances the activation of phosphate uptake systems by cyanobacteria. The flexible physiological response results in more efficient exploitation of limiting nutrients favouring cyanobacterial resilience under suboptimal conditions.

Role of Phosphate Transport System Component PstB1 in Phosphate Internalization by Nostoc punctiforme.

In bacteria, limited phosphate availability promotes the synthesis of active uptake systems, such as the Pst phosphate transport system. To understand the mechanisms that facilitate phosphate accumulation in the cyanobacterium Nostoc punctiforme, phosphate transport systems were identified, revealing a redundancy of Pst phosphate uptake systems that exists across three distinct operons. Four separate PstB system components were identified. pstB1 was determined to be a suitable target for creating phenotypic mutations that could result in the accumulation of excessive levels of phosphate through its overexpression or in a reduction of the capacity to accumulate phosphate through its deletion. Using quantitative real-time PCR (qPCR), it was determined that pstB1 mRNA levels increased significantly over 64 h in cells cultured in 0 mM added phosphate and decreased significantly in cells exposed to high (12.8 mM) phosphate concentrations compared to the level in cells cultured under normal (0.8 mM) conditions. Possible compensation for the loss of PstB1 was observed when pstB2, pstB3, and pstB4 mRNA levels increased, particularly in cells starved of phosphate. The overexpression of pstB1 increased phosphate uptake by N. punctiforme and was shown to functionally complement the loss of PstB in E. coli PstB knockout (PstB-) mutants. The knockout of pstB1 in N. punctiforme did not have a significant effect on cellular phosphate accumulation or growth for the most part, which is attributed to the compensation for the loss of PstB1 by alterations in the pstB2, pstB3, and pstB4 mRNA levels. This study provides novel in vivo evidence that PstB1 plays a functional role in phosphate uptake in N. punctiforme IMPORTANCE: Cyanobacteria have been evolving over 3.5 billion years and have become highly adept at growing under limiting nutrient levels. Phosphate is crucial for the survival and prosperity of all organisms. In bacteria, limited phosphate availability promotes the synthesis of active uptake systems. The Pst phosphate transport system is one such system, responsible for the internalization of phosphate when cells are in phosphate-limited environments. Our investigations reveal the presence of multiple Pst phosphate uptake systems that exist across three distinct operons in Nostoc punctiforme and functionally characterize the role of the gene product PstB1 as being crucial for the maintenance of phosphate accumulation. We demonstrate that the genes pstB2, pstB3, and pstB4 show alterations in expression to compensate for the deletion of pstB1 The overall outcomes of this work provide insights as to the complex transport mechanisms that exist in cyanobacteria like N. punctiforme, allowing them to thrive in low-phosphate environments.

Phosphorus depletion in forest soils shapes bacterial communities towards phosphorus recycling systems.

Phosphorus (P) is an important macronutrient for all biota on earth but similarly a finite resource. Microorganisms play on both sides of the fence as they effectively mineralize organic and solubilize precipitated forms of soil phosphorus but conversely also take up and immobilize P. Therefore, we analysed the role of microbes in two beech forest soils with high and low P content by direct sequencing of metagenomic deoxyribonucleic acid. For inorganic P solubilization, a significantly higher microbial potential was detected in the P-rich soil. This trait especially referred to Candidatus Solibacter usiatus, likewise one of the dominating species in the data sets. A higher microbial potential for efficient phosphate uptake systems (pstSCAB) was detected in the P-depleted soil. Genes involved in P starvation response regulation (phoB, phoR) were prevalent in both soils. This underlines the importance of effective phosphate (Pho) regulon control for microorganisms to use alternative P sources during phosphate limitation. Predicted genes were primarily harboured by Rhizobiales, Actinomycetales and Acidobacteriales.

Phosphorus depletion in forest soils shapes bacterial communities towards phosphorus recycling systems.

Phosphorus (P) is an important macronutrient for all biota on earth but similarly a finite resource. Microorganisms play on both sides of the fence as they effectively mineralize organic and solubilize precipitated forms of soil phosphorus but conversely also take up and immobilize P. Therefore, we analysed the role of microbes in two beech forest soils with high and low P content by direct sequencing of metagenomic deoxyribonucleic acid. For inorganic P solubilization, a significantly higher microbial potential was detected in the P-rich soil. This trait especially referred to Candidatus Solibacter usiatus, likewise one of the dominating species in the data sets. A higher microbial potential for efficient phosphate uptake systems (pstSCAB) was detected in the P-depleted soil. Genes involved in P starvation response regulation (phoB, phoR) were prevalent in both soils. This underlines the importance of effective phosphate (Pho) regulon control for microorganisms to use alternative P sources during phosphate limitation. Predicted genes were primarily harboured by Rhizobiales, Actinomycetales and Acidobacteriales.

The molecular basis of phosphate discrimination in arsenate-rich environments

Arsenate and phosphate are abundant on Earth and have striking similarities: nearly identical pK(a) values, similarly charged oxygen atoms, and thermochemical radii that differ by only 4% (ref. 3). Phosphate is indispensable and arsenate is toxic, but this extensive similarity raises the question whether arsenate may substitute for phosphate in certain niches. However, whether it is used or excluded, discriminating phosphate from arsenate is a paramount challenge. Enzymes that utilize phosphate, for example, have the same binding mode and kinetic parameters as arsenate, and the latter's presence therefore decouples metabolism. Can proteins discriminate between these two anions, and how would they do so? In particular, cellular phosphate uptake systems face a challenge in arsenate-rich environments. Here we describe a molecular mechanism for this process. We examined the periplasmic phosphate-binding proteins (PBPs) of the ABC-type transport system that mediates phosphate uptake into bacterial cells, including two PBPs from the arsenate-rich Mono Lake Halomonas strain GFAJ-1. All PBPs tested are capable of discriminating phosphate over arsenate at least 500-fold. The exception is one of the PBPs of GFAJ-1 that shows roughly 4,500-fold discrimination and its gene is highly expressed under phosphate-limiting conditions. Sub-angstrom-resolution structures of Pseudomonas fluorescens PBP with both arsenate and phosphate show a unique mode of binding that mediates discrimination. An extensive network of dipole-anion interactions, and of repulsive interactions, results in the 4% larger arsenate distorting a unique low-barrier hydrogen bond. These features enable the phosphate transport system to bind phosphate selectively over arsenate (at least 10(3) excess) even in highly arsenate-rich environments.

The molecular basis of phosphate discrimination in arsenate-rich environments

Arsenate and phosphate are abundant on Earth and have striking similarities: nearly identical pK(a) values, similarly charged oxygen atoms, and thermochemical radii that differ by only 4% (ref. 3). Phosphate is indispensable and arsenate is toxic, but this extensive similarity raises the question whether arsenate may substitute for phosphate in certain niches. However, whether it is used or excluded, discriminating phosphate from arsenate is a paramount challenge. Enzymes that utilize phosphate, for example, have the same binding mode and kinetic parameters as arsenate, and the latter's presence therefore decouples metabolism. Can proteins discriminate between these two anions, and how would they do so? In particular, cellular phosphate uptake systems face a challenge in arsenate-rich environments. Here we describe a molecular mechanism for this process. We examined the periplasmic phosphate-binding proteins (PBPs) of the ABC-type transport system that mediates phosphate uptake into bacterial cells, including two PBPs from the arsenate-rich Mono Lake Halomonas strain GFAJ-1. All PBPs tested are capable of discriminating phosphate over arsenate at least 500-fold. The exception is one of the PBPs of GFAJ-1 that shows roughly 4,500-fold discrimination and its gene is highly expressed under phosphate-limiting conditions. Sub-ångström-resolution structures of Pseudomonas fluorescens PBP with both arsenate and phosphate show a unique mode of binding that mediates discrimination. An extensive network of dipole-anion interactions, and of repulsive interactions, results in the 4% larger arsenate distorting a unique low-barrier hydrogen bond. These features enable the phosphate transport system to bind phosphate selectively over arsenate (at least 10(3) excess) even in highly arsenate-rich environments.

Thalassospira sp. Isolated from the Oligotrophic Eastern Mediterranean Sea Exhibits Chemotaxis toward Inorganic Phosphate during Starvation

The eastern Mediterranean Sea represents an ultraoligotrophic environment where soluble phosphate limits the growth of bacterioplankton. Correspondingly, genes coding for high-affinity phosphate uptake systems and for organophosphonate utilization are highly prevalent in the plankton metagenome. Chemotaxis toward inorganic phosphate constitutes an alternative strategy to cope with phosphate limitation, but so far has only been demonstrated for two bacterial pathogens and an archaeon, and not in any free-living planktonic bacterium. In the present study, bacteria affiliated with the genus Thalassospira were found to constitute a regular, low-abundance member of the bacterioplankton that can be detected throughout the water column of the eastern Mediterranean Sea. A representative (strain EM) was isolated in pure culture and exhibited a strong positive chemotaxis toward inorganic phosphate that was induced exclusively in phosphate-starved cultures. Phosphate-depleted cells were 2-fold larger than in exponentially growing cultures, and 43% of the cells retained their motility even during prolonged starvation over 10 days. In addition, Thalassospira sp. strain EM was chemotactically attracted by complex substrates (yeast extract and peptone), amino acids, and 2-aminoethylphosphonate but not by sugar monomers. Similarly to the isolate from the eastern Mediterranean, chemotaxis toward phosphate was observed in starved cultures of the other two available isolates of the genus, T. lucentensis DSM 14000T and T. profundimaris WP0211T. Although Thalassospira sp. represents only up to 1.2% of the total bacterioplankton community in the water column of the eastern Mediterranean Sea, its chemotactic behavior potentially leads to an acceleration of nutrient cycling and may also explain the persistence of marine copiotrophs in this extremely nutrient-limited environment.

Evidence for a sodium efflux mechanism in the leaf cells of the seagrass Zostera marina L.

The efficient exclusion of excess Na + from the cytoplasm is of vital importance in cells from the seagrass Zostera marina L., a vascular plant that lives in seawater and exhibits Na +-dependent high affinity nitrate and phosphate uptake systems. Intracellular measurements with Na +-selective microelectrodes show a low cytosolic Na + concentration in leaf cells (13.2 ± 2.8 mM Na +) of this species. This value, similar to the concentrations detected in terrestrial glycophyte plants, increases about 10 mM in the presence of respiration inhibitors. A rise in sodium concentration is also detected when external pH is increased, whereas a decrease is recorded at lower external pH levels. These results suggest that H +-dependent systems are involved in the maintenance of a low cytosolic Na + concentration in Z. marina cells. To investigate this further, plasma membrane vesicles were isolated from leaves and cation/H + antiport activities were analyzed using a fluorescent probe. Na +/H + exchange activity was detected and showed saturation around 35 mM Na +. This activity was highly selective for Na + as no exchange activity was detected with Rb + or TMA +, whereas Li + showed only 25% of the Na + activity. Furthermore, K + did not affect Na +/H + exchange activity, which showed similar K m values in the presence of 5 mM K + (4.1 ± 2.1 mM Na +) or 50 mM K + (3.9 ± 2.6 mM Na +). These K m values are lower than the mean sodium concentration measured in the cytoplasm of Z. marina cells, indicating that the presence of Na +/H + exchangers in the plasma membrane, whose activity has been detected for the first time in a seagrass, can be a very effective Na + efflux mechanism in this plant. This activity can be crucial both for salinity tolerance and for the energetization of nutrient uptake in this species.

Adaptive phosphate uptake behaviour of phytoplankton to environmental phosphate fluctuations

When phytoplankton growth in lakes is limited by the available phosphate, the external phosphate concentration fluctuates around a threshold value at which available energy is insufficient to drive phosphate incorporation into a polyphosphate pool. As a result, occasional increases in the external concentration are experienced by phytoplankton as a series of phosphate pulses. Based on [(32) P] phosphate uptake experiments with lake phytoplankton, we show that a community is able to process information about the experienced pattern of phosphate pulses via a complex regulation of the kinetic and energetic properties of cellular phosphate uptake systems. As a result, physiological adaptation to alterations of ambient phosphate concentration depends on the pattern of phosphate fluctuations to which the community had been exposed during its previous growth. In this process, the entire community exhibits coherent uptake behaviour with respect to a common threshold value. Thereby, different threshold values result from different antecedent pulse patterns, apparently unrestrained by the amount of previously stored phosphate. The coherent behaviour observed contradicts the basic assumptions of the competitive exclusion principle and provides an alternative perspective for explaining the paradoxical coexistence of many phytoplankton species.

The SPX domain of the yeast low‐affinity phosphate transporter Pho90 regulates transport activity

Yeast has two phosphate-uptake systems that complement each other: the high-affinity transporters (Pho84 and Pho89) are active under phosphate starvation, whereas Pho87 and Pho90 are low-affinity transporters that function when phosphate is abundant. Here, we report new regulatory functions of the amino-terminal SPX domain of Pho87 and Pho90. By studying truncated versions of Pho87 and Pho90, we show that the SPX domain limits the phosphate-uptake velocity, suppresses phosphate efflux and affects the regulation of the phosphate signal transduction pathway. Furthermore, split-ubiquitin assays and co-immunoprecipitation suggest that the SPX domain of both Pho90 and Pho87 interacts physically with the regulatory protein Spl2. This work suggests that the SPX domain inhibits low-affinity phosphate transport through a physical interaction with Spl2.

The low-affinity phosphate transporter PitA is dispensable for in vitro growth of Mycobacterium smegmatis

BackgroundMycobacteria have been shown to contain an apparent redundancy of high-affinity phosphate uptake systems, with two to four copies of such systems encoded in all mycobacterial genomes sequenced to date. In addition, all mycobacteria also contain at least one gene encoding the low-affinity phosphate transporter, Pit. No information is available on a Pit system from a Gram-positive microorganism, and the importance of this system in a background of multiple other phosphate transporters is unclear.ResultsThe aim of this study was to determine the physiological role of the PitA phosphate transporter in Mycobacterium smegmatis. Expression of pitA was found to be constitutive under a variety of growth conditions. An unmarked deletion mutant in pitA of M. smegmatis was created. The deletion did not affect in vitro growth or phosphate uptake of M. smegmatis. Expression of the high-affinity transporters, PstSCAB and PhnDCE, was increased in the pitA deletion strain.ConclusionPitA is the only low-affinity phosphate transport system annotated in the genome of M. smegmatis. The lack of phenotype of the pitA deletion strain shows that this system is dispensable for in vitro growth of this organism. However, increased expression of the remaining phosphate transporters in the mutant indicates a compensatory mechanism and implies that PitA is indeed used for the uptake of phosphate in M. smegmatis.

The extended N-terminal region of SphS is required for detection of external phosphate levels in Synechocystis sp. PCC 6803

A novel 47 amino acid extension at the N-terminus of the SphS histidine kinase has been identified in the cyanobacterium Synechocystis sp. PCC 6803. Here, we demonstrate this region is required for activation of the SphS–SphR phosphate-sensing two-component system under phosphate-limiting conditions and mutants lacking this extension do not show constitutive alkaline phosphatase activity when the negative regulator SphU is inactivated. We have also identified a putative membrane-associated domain within this region involved in control of the Pho regulon. In addition, there are two high-affinity ABC-type phosphate uptake systems in this organism. Our results demonstrate that the Pst1 system, but not the Pst2 system, is required for suppression of the Pho regulon under phosphate-sufficient conditions. Deletion of the pst1 operon and disruption of the membrane-spanning domain may both target the same control mechanism since constitutive alkaline phosphatase activity is similar in the double and single mutants.

The Effect of Polymeric Substances on Apatite Reactivity in the Presence of a Freshwater Cyanobacterium

Marine cyanobacteria have evolved phosphate uptake systems to live where P is limiting. Ultimately, P derives from apatite in rocks but little is known about apatite solubilization by cyanobacteria. Fluorapatite (FAP) was added to a P-free culture medium with and without Anabaena, as well as in cell-free supernatant to test reports that the cyanobacterium Anabaena PCC 7120 enhances the dissolution of fluorapatite (FAP). Anabaena, a fresh-water organism, is an analogue to harder-to-grow marine cyanobacteria. The initial abiotic release rate ratios (Ca/P) are significantly higher than the similar stoichiometric ratio of apatite. The majority of the “missing” P in abiotic experiments is sorbed to precipitates formed from Fe added as a nutrient to the culture medium. In contrast to literature reports, Ca release from FAP in the presence of Anabaena or culture supernatant was lower than abiotic release. However, Anabaena cultures aggregate and cells attach to the FAP when P-limited. Adhesion is explained by surface charge rather than P content because significant adhesion on boehmite occurred in similar experiments. X-ray photoelectron spectroscopic (XPS) analysis of the FAP surface after incubation with Anabaena is consistent with sorbed proteins that promote adhesion. These released polymers are also inferred to complex with Ca during dissolution, contributing to nucleation of Ca-P precipitates in experiments with cells or supernatant. These experiments emphasize that for Anabaena growing under P-limited conditions, polymeric material is important for cellular adhesion to mineral particles and for the concentration of phosphate as Ca-or Fe-complexes or nanoparticles.

Ssh4, Rcr2 and Rcr1 affect plasma membrane transporter activity in Saccharomyces cerevisiae.

Nutrient uptake in the yeast Saccharomyces cerevisiae is a highly regulated process. Cells adjust levels of nutrient transporters within the plasma membrane at multiple stages of the secretory and endosomal pathways. In the absence of the ER-membrane-localized chaperone Shr3, amino acid permeases (AAP) inefficiently fold and are largely retained in the ER. Consequently, shr3 null mutants exhibit greatly reduced rates of amino acid uptake due to lower levels of AAPs in their plasma membranes. To further our understanding of mechanisms affecting AAP localization, we identified SSH4 and RCR2 as high-copy suppressors of shr3 null mutations. The overexpression of SSH4, RCR2, or the RCR2 homolog RCR1 increases steady-state AAP levels, whereas the genetic inactivation of these genes reduces steady-state AAP levels. Additionally, the overexpression of any of these suppressor genes exerts a positive effect on phosphate and uracil uptake systems. Ssh4 and Rcr2 primarily localize to structures associated with the vacuole; however, Rcr2 also localizes to endosome-like vesicles. Our findings are consistent with a model in which Ssh4, Rcr2, and presumably Rcr1, function within the endosome-vacuole trafficking pathway, where they affect events that determine whether plasma membrane proteins are degraded or routed to the plasma membrane.