Sole Phosphate Source Research Articles

Critical importance of phytase for yeast growth and alcohol fermentation in Japanese sake brewing

The high phytase producing mutant of Aspergillus oryzae (KL-38) previously isolated was employed for koji making, and the produced koji rice then supplied for sake brewing. The alcohol fermentation was improved compared to that with the parent strain (A. oryzae BP-1). The effects of two phytase isozymes (Phy I and Phy II) produced by A. oryzae on yeast growth and inorganic phosphate liberation were investigated using a synthetic medium containing phytic acid as a sole phosphate source. Yeast growth and the liberation of inorganic phosphate were both enhanced by the combination of Phy I and Phy II at a ratio of 1 to 3, which was compatible with the production ratio in KL-38. Based on these results, phytase plays important role in sake brewing, and that the maximum inorganic phosphate liberation from phytic acid can be obtained by a suitable combination of Phy I and Phy II.

Identification and characterisation of two distinct acid phosphatases in cell walls of roots of white clover

White clover ( Trifolium repens L.) plants were grown in liquid media and subjected to phosphate starvation by removal of the sole phosphate source. After 21 d, roots were harvested and ionically-bound (1 M salt-extractable) cell wall proteins isolated. Two distinct acid phosphatases (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2), designated APase I and II, were identified using hydrophobic column chromatography with each protein consisting of two isoforms resolved by ion-exchange column chromatography. For APase I, both isoforms (IA and IB) are glycosylated (as determined by binding to a monoclonal antibody, mAb 2.23, which is specific to xylose/fucose-containing complex-type N-linked glycans, and a Galanthus nivalis (GNA) lectin which recognises terminal mannose sugars), and exist as active monomers of 52 kDa as determined by SDS-PAGE and by gel filtration. APase IA and IB have pH optima for p-nitrophenyl phosphate of 5.8 and 6.2, and pIs of 7.3 and 6.5, respectively. For APase II, both isoforms exist as active monomers of 113 kDa by SDS-PAGE and 92 kDa by gel filtration, with pH optima of 5.8 and 6.8, and pIs of 4.4 and 5.2 to 5.3 for IIA and IIB, respectively. Isoform IIA was not recognised by the two glycan probes, while IIB was recognised by mAb 2.23. The activity of all four isoforms was severely inhibited by Cu 2+, Zn 2+ and Mo 2+, while each showed activity against a range of phosphate monoester substrates with highest substrate specificity ( V max/ K m) for ATP and PPi. Activity associated with APase I and II is detectable in roots isolated from plants maintained in P-containing media, but the activity of both is increased with the onset of P-deficiency. However, a temporal difference in response between APase I and II is observed over a 28-d time course of P-deprivation.

Phytase of the yeast Arxula adeninivorans

Of a number of yeasts screened for growth on phytic acid (inositol phosphates) as a sole source of carbon and phosphate, Arxula adeninivorans showed a particularly vigorous growth. This capacity was correlated with the presence of a high activity of secreted phytase. The crude enzyme showed an optimal temperature of at least 75 °C and an optimal pH of 4.5. The level of secreted enzyme far exceeded that of previously reported yeast phytases.

Cell wall proteins in white clover: influence of plant phosphate status

White clover ( Trifolium repens L.) plants were grown in either P-containing liquid media, or in media with the sole source of phosphate removed (P-deprived). At 29 d, plants were harvested and a water-soluble whole tissue extract and an ionically-bound (1 M salt-extractable) cell wall protein extract made from root and leaf tissue. Acid phosphatase activity was highest in all extracts from root and leaf tissue excised from P-deprived plants, with the biggest difference (4.5-fold) in root cell walls. The smallest fold-increase was observed in the leaf water-soluble extract. The relative intensity of several, although not all, acid phosphatase isoenzymes was also highest in extracts from P-deprived plants. Up to the limits of detection used, no new acid phosphatase isoenzymes could be detected in extracts from plants maintained either in P-deprived or P-containing media. The complement of ionically-bound (1 M salt-extractable) cell wall glycoproteins in leaf and root tissue extracts maintained in P-deprived and P-containing media was also compared. Using SDS-PAGE and immuno-recognition with mAb 2.23, a monoclonal antibody that specifically recognises xylose/fucose mixed-type N-linked glycans, glycoproteins of 30 and 31 kDa were identified which were more prevalent in the P-deprived root cell wall. Further, a protein of 60 kDa was identified, which was prevalent in root cell wall extracts from plants maintained in P-containing media. The GNA lectin, which detects oligomannose N-linked structures, identified a glycoprotein of 37 kDa which was more prevalent in the P-deprived root cell wall.

Identification of cell wall proteins in roots of phosphate-deprived white clover plants

White clover ( Trifolium repens L.) plants were grown in liquid media with the sole source of phosphate (KH 2PO 4) removed. After 21 days, the roots were harvested, extracted first with 25 mM 2-mercaptoethanol, the cell wall fraction washed thoroughly with water before incubation with 1.0 M NaCl to extract ionically-bound cell wall proteins. The cell wall proteins were separated using hydrophobic column chromatography, then SDS-PAGE, and selected proteins subjected to N-terminal sequencing. Two of these proteins have been identified using data-base searching. The first is a c. 22 kDa protein with identity to an α-fucosidase purified from Pisum sativum, while the second is a c. 35 kDa protein with identity to a class III endochitinase purified from Nicotiana tobaccum. Specific hydrophobic column fractions were purified further using ion-exchange column chromatography and a further protein identified as a c. 41 kDa protein with highest identity to polygalacturonase inhibitors purified from several plant species.

The pho regulon-dependent Ugp uptake system for glycerol-3-phosphate in Escherichia coli is trans inhibited by Pi.

sn-Glycerol-3-phosphate (G3P) or glyceryl phosphoryl phosphodiesters, the substrates of the phoB-dependent Ugp transport system, when transported exclusively through this system, can serve as a sole source of phosphate but not as a sole source of carbon (H. Schweizer, M. Argast, and W. Boos, J. Bacteriol. 150:1154-1163, 1982). In order to explain this phenomenon, we tested two possibilities: repression of the pho regulon by Ugp-mediated transport and feedback inhibition by internal G3P or its degradation product Pi. Using an ugp-lacZ fusion, we found that the expression of ugp does not decline upon exposure to G3P, in contrast to the repressing effect of transport of Pi via the Pst system. This indicated that the Ugp system becomes inhibited after the uptake and metabolism of G3P. Using 32P-labeled G3P, we observed that little Pi is released by cells taking up G3P via the Ugp system but large amounts of Pi are released when the cells are taking up G3P via the GlpT system. Using a glpD mutant that could not oxidize G3P but which could still phosphorylate exogenous glycerol to G3P after GlpF-mediated transport of glycerol, we could not find trans inhibition of Ugp-mediated uptake of exogenous 14C-G3P. However, when allowing uptake of Pi via Pst, we observed a time-dependent inhibition of 14C-G3P taken up by the Ugp transport system. Inhibition was half maximal after 2 min and could be elicited by Pi concentrations below 0.5 mM. Cells had to be starved for Pi in order to observe this inhibition. We conclude that the activity of the Ugp transport system is controlled by the level of internal phosphate.

Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli

In vivo 31P nuclear magnetic resonance analysis of Escherichia coli cells showed that the intracellular concentration of P(i) remained constant in wild-type and in a glpT mutant strain whether the cells were grown on excess (2 mM) P(i) or sn-glycerol-3-phosphate as a phosphate source. The function of the phoA promoter (measured by beta-galactosidase activity in a phoA-lacZ fusion strain) was repressed when glpT+ cells were utilizing sn-glycerol-3-phosphate as the sole source of phosphate. These cells were devoid of alkaline phosphatase activity. However, the phoA promoter was fully active in a glpT mutant. These results indicated that the repression of the enzyme synthesis was not due to a variation in the level of cytoplasmic P(i) but was due to the P(i) excreted into the periplasm and/or to the medium.

Role of energy metabolism in conversion of nonmucoid Pseudomonas aeruginosa to the mucoid phenotype.

Phosphatidylcholine, the major component of lung surfactant, when supplied as the sole source of phosphate for Pseudomonas aeruginosa PAO1, resulted in conversion of as much as 2% of the population to the mucoid phenotype under continuous culture conditions over a 24-day culture period. In addition, growth in phosphatidylcholine resulted in the highest yields of extracellular alginate compared with other environmental conditions. Iron limitation, another environmental condition relevant to the lungs of patients with cystic fibrosis, also resulted in conversion to mucoid. Since both conditions suggested the likelihood of an energy-deprived growth environment as a common variable, the effect of direct inhibition of energy generation by N,N'-dicyclohexylcarbodiimide or gramicidin on the conversion of nonmucoid P. aeruginosa to the mucoid phenotype was examined. Both inhibitors resulted in mucoid subpopulations (0.5 and 0.8%, respectively). Severe energy stress imposed by the combination of phosphate limitation and N,N'-dicyclohexylcarbodiimide treatment resulted in conversion of 55% of the population to mucoidy during a 7-day growth period. A growth advantage of the mucoid over the nonmucoid phenotype was observed under severe nutrient deprivation by growth on unsupplemented Noble agar or in a 1/2,500 dilution of a chemically defined medium. These results clearly demonstrate a significant role for the energy state of the cell in conversion to mucoid and in selection for the mucoid phenotype.

Utilization of phytate by some yeasts

Of 21 yeast strains screened for ability to hydrolyse phytic acid salts, nine strains grew on sodium phytate as sole source of inorganic phosphate. Of the five most interesting strains for their growth parameters tested and for their phytase activity in batch-culture,Schwanniomyces castellii CBS 2863 had the highest phytase activity in presence of 5 g phytate I−1.

Utilization of exogenous glucose-1-phosphate as a source of carbon or phosphate byEscherichia coli K12: respective roles of acid glucose-1-phosphatase, hexose-phosphate permease, phosphoglucomutase and alkaline phosphatase

The periplasmic acid glucose-1-phosphatase (G-1-Pase) encoded by gene agp is necessary for the growth of Escherichia coli in a minimal medium containing glucose-1-phosphate (G-1-P) as the sole source of carbon. From a mutant in which the agp gene was inactivated, suppressors were isolated which recovered the ability to utilize G-1-P as carbon source. The mutants constitutively expressed hexose phosphate permease activity (encoded by uhpT). The mutation involved mapped in the uhp region and, unlike those of wild-type strains, bacteria of the suppressed strains required phosphoglucomutase (pgm), to grow on G-1-P. Surprisingly, in a minimal medium deprived of inorganic phosphate, uhpT+ bacteria lacking the two enzymes, alkaline-phosphatase (phoA) and glucose-1-phosphatase (agp), could utilize G-1-P as the sole source of phosphate, and also as both the sole phosphate and carbon source provided the integrity of pgm and of uhpT was conserved. Although glucose-6-phosphate, the inducer of UhpT permease, was not present in the medium, the activity of uhpT was greatly stimulated by inorganic phosphate depletion. This phosphate-starvation-induced bypass of G-1-Pase by UhpT + Pgm systems shows that agp is essential for G-1-P assimilation as a carbon source only in a high-phosphate medium, a result in agreement with the lack of agp regulation by inorganic phosphate.

A topological analysis of subunit alpha from Escherichia coli F1F0-ATP synthase predicts eight transmembrane segments.

The membrane topology of subunit alpha from the Escherichia coli F1F0-ATP synthase was studied using a gene fusion technique. Fusion proteins linking different amino-terminal fragments of the alpha subunit with an enzymatically active fragment of alkaline phosphatase were constructed by both random transposition of TnphoA and site-directed mutagenesis. Those proteins with high levels of alkaline phosphatase activity are predicted to define periplasmic domains of alpha, and this was confirmed by testing for cell growth in minimal medium supplemented with polyphosphate (P greater than 75) as the sole source of phosphate. The enzymatic activity of some fusion proteins was shown to be sensitive to glucose present in the growth medium. Results from subcellular fractionation experiments suggest that these fusion proteins may be inactive even though they have a periplasmic alkaline phosphatase. The enzymatic activity appears dependent upon proteolytic release of the alkaline phosphatase moiety from its alpha subunit membrane anchor and suggests the target of glucose repression may be a protease present in the periplasm. For the topological analysis of the alpha subunit, a total of 28 unique fusion proteins were studied and the results were consistent with a model of alpha containing eight transmembrane segments, including periplasmic amino and carboxyl termini. Surprisingly, separate periplasmic domains were identified near amino acids 200, 233, and 270. These results suggest the flanking membrane spans are only 10-15 amino acids in length and not able to span a standard 30 A bilayer in an alpha-helical conformation. These short spans may have interesting mechanistic implications for the function of F0, because they contain several amino acids which appear critical for proton translocation. Finally, a fusion of alkaline phosphatase at amino acid 271, the carboxyl-terminal residue, but not at amino acid 260, was able to complement the strain RH305 (uncB-) for growth on succinate and suggests the last 11 amino acids of the alpha subunit are critical to the function of F1F0-ATP synthase.

Characterization of two tobacco cell lines selected to grow in the presence of either ionic Al or insoluble Al-phosphate

A tobacco cell line tolerant to ionic A1 (AIT) was selected at a frequency of 10-6 to 10-7 by plating the wild-type cells on agar medium containing 2.5 mm AICI3 and 0.1 mM phosphate at pH 4.0. Another cell line which is able to grow with insoluble Al-phosphate as a sole source of phosphate (IPU) was also selected by subculturing the wild-type cells for more than 10 passages in suspension culture in the presence of 2 mM phosphate and 4 mM A1C13 at pH 5.0. The IPU cell line also tolerated ionic A1 but the AIT line showed poor growth with insoluble Al-phosphate as a sole source of phosphate. Both cell lines excreted citrate into the media. The rate of citrate excretion by the IPU cell line, on a fresh weight basis, was higher than that by the AIT and wild-type cell lines.

Re‐evaluation of characteristics of a carrot cell line previously selected as aluminum‐tolerant cells

A carrot (Daucus carota L. cv. MS Yonsun) cell line previously selected to be tolerant to Al supply was re‐evaluated by culturing with hardly soluble or ionic Al. When insoluble Al‐phosphate (2.0 mM) was supplied as a sole source of phosphate at an initial pH of 5.6, the selected cell line, but not wild‐type cells, grew normally corresponding to the growth of the cells supplied with Na‐phosphate in the absence of Al Under these conditions, the selected cells excreted large amounts of citrate into the medium. Insoluble Fe‐phosphate could also be utilized as a phosphate source by the selected cells. Excretion of citrate was, however, not detected when the cells were cultured in the absence of insoluble phosphate. The selected cells were less tolerant against soluble. Al ions than the wild‐type cells.

Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins.

We observed that wild-type Escherichia coli utilized a linear polyphosphate with a chain length of 100 phosphate residues (poly-P100) as the sole source of phosphate in growth medium. A mutation in the gene phoA of alkaline phosphatase or phoB, the positive regulatory gene, prevented growth in this medium. Since no alkaline phosphatase activity was detected outside the wild-type cells, the periplasmic presence of the enzyme was necessary for the degradation of polyphosphate. A 90% reduction in the activity of periplasmic acid phosphatase with a pH optimum of 2.5 (delta appA mutants) did not affect polyphosphate utilization. Of the porins analyzed (OmpC, OmpF, and PhoE), the phoB-inducible porin PhoE was not essential since its absence did not prevent growth. To study how poly-P100 diffused into the cells, we used high-resolution 31P nuclear magnetic resonance (31P NMR) spectroscopy. The results suggest that poly-P100 entered the periplasm and remained in equilibrium between the periplasm and the medium. When present individually, porins PhoE and OmpF facilitated a higher permeability for poly-P100 than porin OmpC did. The degradation of polyphosphate by intact cells of E. coli observed by 31P NMR showed a time-dependent increase in cellular phosphate and a decrease in polyphosphate concentration.

Utilization of hydroxyapatite by Cyanobacteria as their sole source of phosphate and calcium

Two species of Cyanobacteria, (one Nostoc, one Anabaena) are described which are able to grow utilizing hydroxyapatite as their sole source of phosphate and calcium at pH=7.6.

Characterization of a binding protein involved in the transport of O-phosphorylethanolamine inPseudomonas aeruginosa

Pseudomonas aeruginosa, grown in a medium containing O-phosphorylethanolamine as the sole source of phosphate and carbon, is able to take up this ester phosphate via two kinetically distinct systems with apparent Km values of 2.5 and 55 μM, respectively. An inducible O-phosphorylethanolamine-binding protein was purified to homogeneity from the shock fluid of the bacteria. It has an average molecular weight of 38 kda and an isoelectric point of 5.35. One mole of substrate was bound per mole of protein with a dissociation constant of 1.2 μM. This binding protein appears very specific since neither phosphate, phosphoserine, phosphocholine,sn-glycerol-3-phosphate, 2-aminoethylphosphonic acid, nor ethanolamine could inhibit the binding of O-phosphoryl-[2-14C]ethanolamine when measured by equilibrium dialysis.

Mutagenicity of metal ions in bacteria

The mutagenicity of 24 metal salts was investigated in plate incorporation and fluctuation assays with Salmonella TA strains or Escherichia coli WP2 uvrA p Km 101. Chromate(VI) and selenate(VI) ions were found to be mutagenic in plate incorporation assays employing conventional media. On the other hand, cadmium(II), beryllium(II), chromate(VI), and metavanadate(V) ions were detected in conventional fluctuation assays, indicating the importance of this technique in detection of metal mutagens. Modified culture media, with trimetaphosphate ions in place of orthophosphate as the sole phosphate source for bacterial growth, were also used in this study. The media modifications prevented precipitation of metals such as nickel and cadmium as their insoluble phosphates, and allowed detection of the mutagenicity of metavanadate ions in plate incorporation assays. However, the fluctuation technique using standard media was shown to detect a wider range of mutagenic metal ions than tests with modified media. It is notable that metaarsenite(III), arsenate(V), and nickel(II) ions were not found to be mutagenic in any of the assays although they are known to be carcinogenic and are mutagenic in other test systems. Their lack of mutagenicity in the modified media indicates that precipitation of these ions as orthophosphates is not the reason for their lack of activity in standard bacterial assays.

Phosphate-Solubilizing Potentiality of the Microorganisms Capable of Utilizing Aluminium Phosphate as a Sole Phosphate Source

Eight bacteria, each of the genus Bacillus, two actinomycetes, each of the genus Streptomyces, and six fungi, one each of the genus Penicillium and Chaetomium and four of the genus Aspergillus, were isolated on AlPO4-sucrose agar from a typical Indian lateritic soil (Typic Ochragualf). All of them were capable of solubilizing Ca3(PO4)2 to a higher degree than AlPO4. Bacillus subtilis (B-7655), LAB4, Bacillus sp., LAB5, Penicillium sp., LAF2, and Aspergillus spp., LAF3 and LAF4, were solubilizing Ca3(PO4)2 very efficiently, but AlPO4 to a lesser degree. Bacillus spp., LAB1, LAB2, LAB5, LAB6 and LAB7, Chaetomium nigricolor, LAF1, and Aspergillus spp., LAF5 and LAF6, were unable to bring detectable amounts of soluble phosphorus to solution from AlPO4. Except Chaetomium, all the other organisms produced free aliphatic organic acid in detectable amounts. The organic acids produced were oxalic, succinic, citric, and 2-keto gluconic acid. 2-Keto gluconic acid, singly and in combination with succinic or citric acid, accounted for higher solubilization. Amount of free organic acids in the growth medium was not directly correlated with phosphate solubilization.

Participation of alkaline phosphatase in the active transport of phosphates in brush border membrane vesicles

Hamster and rat kidney brush border membrane vesicles were compared for their ability to transport phosphate in vitro . In presence of 0.1 mM inorganic phosphate, the Na + dependent uptake was greater in rat than in hamster but the Km values for uptake were respectively 0.32 mM and 0.12 mM for hamster and rat. In presence of glycerophosphate, as sole source of phosphate, only hydrolysed phosphate was transported but in a much greater amount (× 10) than the one expected from the actual concentration of inorganic phosphate released by the action of alkaline phosphatase during the course of the experiments. Although the Na + dependent uptake of phosphate from glycerophosphate relied on alkaline phosphatase action, the extent of transport did not seem to be correlated to the amount of catalytic activity present, pointing to a concerted action between alkaline phosphatase and the phosphate carrier.

Characterization of MMS-sensitive mutants of Neurospora crassa

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer resistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways (as indicated by their ability to utilize DNA as the sole phosphate source in the growth medium). On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. The first group includes three mutants, mus-(SC3), mus-(SC13), and mus-(SC28). These are slow growers, insensitive to histidine with no apparent meiotic defects and may have reduced frequency of spontaneous mutation. In addition, their mycelial growth is sensitive to MMS but the conidial viability following MMS, UV or X-ray treatment appears normal or only slightly more sensitive than the wild-type. The second group includes only one mutant, mus-(SC15); its mycelial growth is very sensitive to MMS but the conidial survival following treatment with MMS or UV appears normal; however, the conidial survival following exposure to X-ray is significantly reduced. This mutant shows an increase (more than 10-fold) frequency of spontaneous mutation, but behaves normal like the wild-type with respect to fertility, growth rate and insensitivity to histidine. The third group includes mutants mus-(SC10), mus-(SC25), and mus-(SC29). These mutants are very sensitive to UV, X-rays and MMS and to histadine but have normal growth rates on minimal medium. Mutant mus-(SC10), but not mus-(SC25) and mus-(SC29), has an increased (11 ×) frequency of spontaneous mutation. On the basis of data presented, the MMS sensitivity of the first group of mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to a number of DNA-damaging agents such as MMS, UV and/or X-ray, high frequencies of spontaneous mutation (mutator effects) and defects in meiotic behavior.