Gene cloning and expression of the Pseudomonas aeruginosa periplasmic phosphate-binding protein

Abstract The pstS gene, encoding the Pseudomonas aeruginosa phosphate-binding protein, was cloned onto a cosmid vector into Escherichia coli , and localized by subcloning, mapping the insertion site of Tn 501 in a P. aeruginosa pstS ::Tn 501 mutant, and hybridization to an oligonucleotide pool synthesized according to the aminoterminal amino acid sequence of the purified protein. The cloned pstS gene was transferred to P. aeruginosa pstS mutants. The P. aeruoginosa phosphate-binding protein was also expressed and secreted into the periplasm of E. coli pstS mutants.     

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2 (PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.Abbreviations Pi inorganic phosphate - PiBP2 phosphate binding protein 2 obtained from Candida tropicalis - Tris Tris(hydroxymethyl)-aminoethane - MES [2-(N-Morpholino)] ethanesulfonic acid - EDTA ethylene diamine tetraacetic acid, disoldium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Phosphate uptake by the yeast, Rhodotorula rubra, and the green alga, Selenastrum capricornutum Printz, after phosphate additions to steady-state continuous cultures

Abstract We examined phosphate (P_i) uptake by two well-characterized microorganisms: a green alga ( Selenastrum capricornutum ) and a heterotrophic yeast ( Rhodotorula rubra ). Phosphate uptake was measured in dual- and single-species continuous cultures after perturbation of a phosphorus (P)-limited steady-state culture by additions of varying concentrations of P_i. We found that, under these conditions, both organisms had very high transport rates for P_i. The yeast was able to attain higher internal P concentrations than predicted from either steady-state or from P-starved batch culture data. Because the yeast was able to sequester and store P_i more efficiently than the alga under dilute P_i continuous culture conditions, co-existence of the two organisms was ultimately controlled by the concentration of carbon available for growth of the yeast.     

Recombination after protoplast fusion in the yeast Candida tropicalis

Candida tropicalis protoplasts obtained by snail enzyme treatment were induced to fuse by the use of polyethylene-glycol. Heterokaryons formed by two auxotrophic strains were selected by complementation on minimal medium. These heterokaryons were unstable and readily dissociated into their nuclear components. Under appropriate conditions, the parental nuclei of an heterokaryon fused. The homokaryon so obtained was unstable and segregated into various types of auxotrophic and prototrophic recombinants.List of Abbreviations Used MM minimal medium - YEA yeast extract agar (complete medium) - YPGT yeast-peptone-glucosethiol (medium for protoplast preparation) - PTP medium for cell pretreatment (used before the action of snail enzyme) - PEG polyethylene glycol - p-FPA para-fluorophenylalanine - 5-FC 5-fluorocytosine     

Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb⁺ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the low-affinity system has decreased for about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.     

Evidence for multiple nitrate uptake systems in the yeast Hansenula polymorpha

Hansenula polymorpha mutants disrupted in the high-affinity nitrate transporter gene (YNT1) are still able to grow in nitrate. To detect the nitrate transporter(s) responsible for this growth a strain containing disruption of the nitrate assimilation gene cluster and expressing nitrate reductase gene (YNR1) under the control of H. polymorpha MOX1 (methanol oxidase) promoter was used (FM31 strain). In this strain nitrate taken up is transformed into nitrite by nitrate reductase and excreted to the medium where it is easily detected. Nitrate uptake which is neither induced by nitrate nor repressed by reduced nitrogen sources was detected in the FM31 strain. Likewise, nitrate uptake detected in the strain FM31 is independent of both Ynt1p and Yna1p and is not affected by ammonium, glutamine or chlorate. The inhibition of nitrite extrusion by extracellular nitrite suggests that the nitrate uptake system shown in the FM31 strain could also be involved in nitrite uptake.     

Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae

Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5′-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast λEMBL3 library. The nucleotide sequence of the gene encoding C. tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPR-ICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae. Received: 20 November 1996 / Accepted: 25 February 1997     

Vanadate-resistant mutants of Candida albicans show alterations in phosphate uptake

Phosphate uptake studies in different strains of the dimorphic pathogenic yeast Candida albicans were undertaken to show that this yeast actively transported phosphate with an apparent Km in the range of 90-170 microM. The uptake was pH dependent and derepressible under phosphate starvation. Vanadate-resistant (van) mutants of C. albicans showed a 20-70% reduction in the rate of phosphate uptake in high phosphate medium and was associated with an increased Km and reduced Vmax. The magnitude of derepression under phosphate starvation was different between van mutants. These results demonstrate that van mutants may have developed resistance by modifying the rate of entry of vanadate.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH,cations and phosphate

The uptake of Ca²⁺ and Sr²⁺ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics.The rate of Ca²⁺ and Sr²⁺ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentrations of Ca²⁺ and Sr²⁺ at low pH may be explained by a decrease of the surface potential.The inhibition of Ca²⁺ and Sr²⁺ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane.Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

Effect of phosphorus supply on phosphate uptake and alkaline phosphatase activity in Rhizobia

The effect of P nutrition on phosphate uptake and alkaline phosphatase activity was studied in chemostat culture for four rhizobial and three bradyrhizobial species. Phosphate-limited cells took up phosphate 10- to 180-fold faster than phosphate-rich cells. The four fast-growing rhizobial strains contained high levels of alkaline phosphatase activity under P-limited conditions compared to the repressed levels found in P-rich cells; alkaline phosphatase activity could not be detected in three slow-growing rhizobial strains, regardless of their P-status.Glycerol 1-phosphate-uptake in the cowpea Rhizobium NGR234 was derepressed over 50-fold under P-limited conditions, and appeared to be co-regulated with phosphate uptake.The phosphate-uptake system appeared similar in all strains with apparent K _m values ranging from 1.6 M to 6.0 M phosphate and maximum activities from 17.2 to 126 nmol · min^-1 · (mg dry weight of cells)^-1. Carbonyl cyanide m-chlorophenyl hydrazone strongly inhibited phosphate uptake in all strains and a number of other metabolic inhibitors also decreased phosphate uptake in the cowpea Rhizobium NGR234. The phosphate uptake system in all strains failed to catalyse exchange of ³²P label in preloaded cells or efflux of phosphate. The results suggest a single, repressible, unidirectional and energy-dependent system for the transport of phosphate into rhizobia.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulphonic acid     

Information about previous phosphate fluctuations is stored via an adaptive response of the high-affinity phosphate uptake system of the cyanobacterium Anacystis nidulans

The high-affinity uptake system of phosphatelimited cyanobacterium Anacystis nidulans [Synechococcus leopoliensis (Raciborski) Komarek] is characterized by a threshold value below which uptake cannot occur. Here it is shown that, if phosphate-limited cyanobacteria are challenged with a short pulse of high phosphate concentration that appreciably exceeds this threshold value, the uptake system undergoes an adaptive response, leading to the attainment of new kinetic properties and a new threshold value. These new properties are maintained for several hours after the pulse. A notable characteristic of this new state is a wide linear dependence of the uptake rate on the external phosphate potential that is a function of the driving force of the uptake process. According to theoretical arguments it is shown that this “linear operation mode” can be explained by the simultaneous operation of several uptake systems with different, staggered threshold values and kinetic properties. Moreover, the new linear uptake properties, in turn, reflect the prehistory of phosphate supply experienced by the population. The consequences of this result with regard to environmental fluctuations of the phosphate concentration in lakes are discussed.     

The role of microorganisms in mediating and facilitating the uptake of plant nutrients from soil

Summary No root systems in nature are without a microbial population. These may be freeliving or symbiotic.The incidence and nutrition of the freeliving microorganisms is discussed. Shortage of substrate makes it unlikely that the N-fixers in the population can fix useful amounts of N. There is a possibility that P supply is improved, but an analysis of possible processes shows them to be rather unlikely, and evidence for them to be poor. Manganese and iron uptake can be altered by microbial activity. Growth of plants can be affected by non-nutritional bacterial effects.The ecology of Rhizobium in the soil is briefly discussed, and the varying needs of different identified strains is stressed.Mycorrhizal infection of plants leads to large growth increases in appropriate conditions. This is almost always linked to increased P uptake, but zinc and copper nutrition can also be improved. The processes involved are briefly discussed. Rapid and extensive infection is important; it is very sensitive to temperature. New modelling methods are now becoming available to measure the behaviour of the fungal infections. The microorganisms require C compounds from the plant, and new measurements of this cost are discussed. The possibility of practical use of mycorrhizal fungi seem to be improving.Keynote address     

Biochemical and structural characterization of the cyanophage-encoded phosphate-binding protein: implications for enhanced phosphate uptake of infected cyanobacteria

To acquire phosphorus, cyanobacteria use the typical bacterial ABC-type phosphate transporter, which is composed of a periplasmic high-affinity phosphate-binding protein PstS and a channel formed by two transmembrane proteins PstC and PstA. A putative pstS gene was identified in the genomes of cyanophages that infect the unicellular marine cyanobacteria Prochlorococcus and Synechococcus. However, it has not been determined whether the cyanophage PstS protein is functional during infection to enhance the phosphate uptake rate of host cells. Here we showed that the cyanophage P-SSM2 PstS protein was abundant in the infected Prochlorococcus NATL2A cells and the host phosphate uptake rate was enhanced after infection. This is consistent with our biochemical and structural analyses showing that the phage PstS protein is indeed a high-affinity phosphate-binding protein. We further modelled the complex structure of phage PstS with host PstCA and revealed three putative interfaces that may facilitate the formation of a chimeric ABC transporter. Our results provide insights into the molecular mechanism by which cyanophages enhance the phosphate uptake rate of cyanobacteria. Phosphate acquisition by infected bacteria can increase the phosphorus contents of released cellular debris and virus particles, which together constitute a significant proportion of the marine dissolved organic phosphorus pool.     

Molecular cloning and functional analysis of a H+-dependent phosphate transporter gene from the ectomycorrhizal fungus Boletus edulis in southwest China

Phosphate transporters (PTs), as entry points for phosphorus (P) in organisms, are involved in a number of P nutrition processes such as phosphate uptake, transport, and transfer. In the study, a PT gene 1632 bp long (named BePT) was cloned, identified, and functionally characterized from Boletus edulis. BePT was expected to encode a polypeptide with 543 amino acid residues. The BePT polypeptide belonged to the major facilitator superfamily and showed a high degree of sequence identity to the Pht1 family. A topology model revealed that BePT exhibited 12 transmembrane helices, divided into two halves, and connected by a large hydrophilic loop in the middle. A yeast mutant complementation analysis suggested that BePT was a functional PT which mediated orthophosphate uptake of yeast at micromolar concentrations. Green fluorescent protein–BePT fusion proteins expressed were extensively restricted to the plasma membrane in BePT transformed yeast, and its activity was dependent on electrochemical membrane potential. In vitro, quantitative PCR confirmed that the expression of BePT was significantly upregulated at lower phosphorus availability, which may enhance phosphate uptake and transport under phosphate starvation. Our results suggest that BePT plays a key role in phosphate acquisition in the ectomycorrhizal fungus B. edulis.     

A novel peroxisomal nonspecific lipid-transfer protein from Candida tropicalis. Gene structure, purification and possible role in beta-oxidation

We have sequenced the nucleotides of the gene POX18 that encodes PXP-18, a major peroxisomal polypeptide inducible by oleic acid in the yeast Candida tropicalis. POX18 had a single open reading frame of 127 amino acids. Some 33% of the amino acid sequence of the predicted basic polypeptide (13,805 Da), was identical to that of the nonspecific lipid-transfer protein (sterol carrier protein 2) from rat liver. PXP-18, purified to near homogeneity from isolated peroxisomes, had an amino-terminal sequence identical to that of the predicted polypeptide except for the initiator methionine, and had nonspecific lipid-transfer activity comparable to that of its mammalian equivalents. Unexpectedly, PXP-18 lacked the cysteine residue thought to be essential for the activity of this protein in mammals. RNA blot analysis showed that the POX18 gene was expressed exclusively in cells grown on oleic acid, suggesting that PXP-18 has a role in the beta-oxidation of long-chain fatty acids. PXP-18 modulated acyl-coenzyme A oxidase activity at low pH.