Growth and physiology of Candida utilis NCYC 321 in potassium-limited chemostat culture

When grown in a defined simple salts medium, plus vitamins, Candida utilis displayed an absolute requirement for potassium. But the potassium content of this yeast was exceedingly variable and, with aerobic chemostat cultures (grown at a dilution rate of 0.1 h^-1; 30° C; pH 5.5), was low (< 0.2%, w/w) when they were potassium-limited and high (> 2%, w/2) when glucose-limited. With potassium-limited cultures, the cell-bound potassium content also varied markedly with growth rate, though hardly at all with glucose-limited cultures; magnesium- and phosphate-limited cultures gave intermediate responses.Changes in cell-bound potassium content correlated only weakly with changes in the cellular contents of magnesium, phosphate and RNA, but strongly with changes in both the Y _glucose and Y _O values, indicating an involvement of potassium in the generation of energy by oxidative phosphorylation reactions and/or the utilization of this energy for growth processes.Studies with isolated mitochondria revealed that potassium-limited organisms had a changed content of cytochrome b relative to cytochrome a, and lacked coupling at either site 2 or site 3 of the respiratory chain.These results are discussed in relation to the reported functions of potassium in the growth of micro-organisms, and the organizational differences between prokaryotic and eukaryotic cells.     

The influence of different carbon sources and medium osmolarity on the potassium requirements of Candida utilis NCYC 321, growing in continuous culture

In order to study the influence of different carbon sources on the K⁺-requirements of Candida utilis NCYC 321, this yeast was grown at several different dilution rates in potassium-limited continuous cultures with either glucose, glycerol, ethanol, citrate or lactate serving as the carbon and energy source.It was found that the nature of the carbon source profoundly influenced the cellular potassium content, especially at low dilution rates, but that these differences could not be correlated with any differences in relative growth rate (i.e., / _max. And although small amounts of potassium seemingly were needed to serve in osmoregulation and in the cotransport of some acidic carbon sources (lactate and citrate), these requirements were negligible.Independent of carbon source, a strong correlation existed between the intracellular potassium concentration and the yield value on oxygen (Y _O), and between cellular potassium concentration and growth rate. From these two correlations it was concluded that potassium probably was involved mainly in processes associated with ATP synthesis in this yeast.Finally the effect of the addition of NaCl to the medium was tested with glucose-containing cultures that were either carbon- or potassium-limited. Up to a concentration of 20 g/l, NaCl was without influence on Y _O, Y _glucose and q _{O 2}, but effected a slight increase in the cellular potassium content of the potassium-limited cells and a decrease in that of the glucose-limited cells.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Rubidium as a probe for function and transport of potassium in the yeast Candida utilis NCYC 321, grown in chemostat culture

Attempts to grow the yeast Candida utilis in continuous culture, using media in which all the potassium had been replaced by other monovalent cations, revealed that neither lithium, sodium, caesium nor ammonium ions could functionally substitute for potassium. However, potassium could be effectively replaced by rubidium which gave (on a molar basis, and under conditions where cation availability limited growth) the same yield of cells as did potassium.Comparison of potassium- and rubidium-limited cultures showed them to be virtually identical in all the measured parameters, with the single exception of the maximum growth rate value which was considerably decreased in the rubidium-containing culture (0.35 h^-1 as compared with 0.55 h^-1).When, with variously-limited chemostat cultures, both potassium and rubidium were supplied in equimolar amounts, these ions were taken up by the cells in a ratio that varied with both the growth rate and the nature of the growth limitation. With glucose-, phosphate- or magnesium-limited cultures, the molar ratio K⁺:Rb⁺ was 1:0.6 (at D=0.1 h^-1) and 1:0.17 (at D=0.5 h^-1). In contrast, ammonia-limited cultures took up increased amounts of rubidium when growing at a low rate such that the ratio was 1:1.2, at D=0.1 h^-1, though still 1:0.17 at the higher growth rate value (D=0.5 h^-1).From a comparison of glucose- and ammonialimited cultures growing first with an equimolar mixture of potassium and rubidium, and then with rubidium alone, it was noted that the yield on oxygen was significantly decreased when potassium was absent.These results are discussed in relation to the transport and possible functions of monovalent cations in micro-organisms. It was concluded that, on the basis of these experiments, some objections could be raised against estimation of potassium transport rates by means of the tracer ⁸⁶Rb.     

Toxicity and accumulation of thallium in bacteria and yeast

Thallium sulphate inhibited microbial growth, withBacillus megaterium KM, more sensitive to the metal thanSaccharomyces cerevisiae andEscherichia coli. Inhibition ofB. megaterium KM andS. cerevisiae, but not ofE. coli, was alleviated by increasing the potassium concentration of the medium; inhibition of respiration ofS. cerevisiae, but not ofE. coli, was similarly alleviated. Thallium was rapidly bound, presumably to cell surfaces, byS. cerevisiae andE. coli, and was progressively accumulated by energy-dependent transport systems (probably concerned primarily with potassium uptake) with both organisms. Thallium uptake kinetics suggested more than one transport system operated in yeast, possibly reflecting a multiplicity of potassium transport systems. ApparentK _m andK _i values for competitive inhibition of thallium uptake by potassium indicatedS. cerevisiae to have a higher affinity for thallium uptake than for potassium, whileE. coli had a transport system with a higher affinity for potassium than for thallium. The likely systems for thallium transport are discussed. A mutant ofE. coli with tenfold decreased sensitivity to thallium was isolated and apparently effected surface binding of thallium in amounts equivalent to the wild type organism, but showed no subsequent uptake and accumulation of the metal from buffer, even though it was able to accumulate potassium to normal intracellular concentrations during growth. Abbreviations: Metal are referred to by their recognised atomic symbols (e.g. TI = Thallium; K = potassium; Co = cobalt)     

Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. Ferric iron reductase activity of 9 nmol Fe²⁺ mg^-1 protein min^-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe²⁺-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification).     

Effect of palmitoylcarnitine on mitochondrial activities

Mitochondria from pea (Pisum sativum L.) cotyledons and potato (Solanum tuberosum L.) tubers exhibited a palmitoyl carnitine-dependent, KCN-sensitive stimulation of the oxygen uptake measured in the presence of 0.2mmol·^–1 malate (sparker malate), provided a certain concentration range of palmitoylcarnitine was observed. Above this concentration range, which was dependent on the bovine serum albumin (BSA) concentration of the reaction mixture, the mitochondrial oxygen uptake was inhibited by palmitoylcarnitine. Palmitoylcarnitine (racemate) and palmitoyl-l-carnitine were equally effective in stimulating/inhibiting mitochondrial oxygen uptake in the presence of sparker malate. The mitochondrial membrane potential generated in the presence of sparker malate was partially dissipated by palmitoyl-lcarnitine concentrations stimulating the mitochondrial oxygen uptake. The formation of acid-soluble radioactivity in reaction mixtures provided with [1-¹⁴C]palmitoyll-carnitine was considerably lower than that expected minimally if the palmitoyl-l-carnitine-stimulated oxygen uptake resulted from palmitoyl-l-carnitine oxidation sparked by malate. Palmitoylcarnitine concentrations resulting in stimulation of the mitochondrial oxygen uptake in the presence of sparker malate also led to a stimulation of succinate-cytochrome c reductase activity, as well as to an increase in the measurable activities of mitochondrial matrix enzymes, indicating loss of both mitochondrial integrity and mitochondrial enzyme latency in the presence of palmitoylcarnitine. Correspondingly, malate-dependent NADH formation was stimulated by palmitoylcarnitine. Neither NAD reduction nor oxygen uptake were observed when the mitochondria were provided with palmitoylcarnitine only. The oxygen uptake due to glycine oxidation by mitochondria from green sunflower (Helianthus annuus L.) cotyledons was affected by palmitoylcarnitine in a similar manner to the oxygen uptake of pea cotyledon and potato tuber mitochondria in the presence of sparker malate. The results lead to the conclusion that the palmitoylcarnitine-dependent stimulation of mitochondrial oxygen uptake observed in the presence of sparker malate results substantially from an enhanced malate oxidation due to the detergent effect of palmitoylcarnitine on the mitochondrial membranes, rather than from palmitoylcarnitine -oxidation.Abbreviations BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophyenylhydrazone The work was supported by the Deutsche Forschungsgemeinschaft.     

Quantitative aspects of glucose metabolism by Escherichia coli B/r,grown in the presence of pyrroloquinoline quinone

Escherichia coli B/r was grown in chemostat cultures under various limitations with glucose as carbon source. Since E. coli only synthesized the glucose dehydrogenase (GDH) apo-enzyme and not the appropriate cofactor, pyrroloquinoline quinone (PQQ), no gluconate production could be observed. However, when cell-saturating amounts of PQQ (nmol to mol range) were pulsed into steady state glucose-excess cultures of E. coli, the organisms responded with an instantaneous formation of gluconate and an increased oxygen consumption rate. This showed that reconstitution of GDH in situ was possible.Hence, in order to examine the influence on glucose metabolism of an active GDH, E. coli was grown aerobically in chemostat cultures under various limitations in the presence of PQQ. It was found that the presence of PQQ indeed had a sizable effect: at pH 5.5 under phosphate- or sulphate- limited conditions more than 60% of the glucose consumed was converted to gluconate, which resulted in steady state gluconate concentrations up to 80 mmol/l. The specific rate of gluconate production (0.3–7.6 mmol·h^-1·(g dry wt cells)^-1) was dependent on the growth rate and the nature of the limitation. The production rate of other overflow metabolites such as acetate, pyruvate, and 2-oxoglutarate, was only slightly altered in the presence of PQQ. The fact that the cells were now able to use an active GDH apparently did not affect apo-enzyme synthesis.Abbreviations HEPES N-2-hydroxy-ethylpiperazine-N-2-ethane sulphonic acid - MES 2-morpholinoethane sulphonic acid - PQQ pyrroloquinoline quinone (systematic name: 2,7,9-tricarboxy-1H-pyrrolo-(2,3-f)-quinoline-4,5-dione) - WB Wurster's Blue (systematic name: 1,4-bis-(dimethylamino)-benzene perchlorate     

A recombinant vaccine against hydatidosis: production of the antigen in Escherichia coli

A commercial process was developed for producing a recombinant vaccine against hydatidosis in farm animals. The vaccine antigen consisting of a surface protein of the oncospheres of the hydatid worm (Echinococcus granulosus), was produced as inclusion bodies in Escherichia coli. Fed-batch cultures of E. coli using Terrific broth in stirred bioreactors at 37°C, pH 7.0, and a dissolved oxygen level of 30% of air saturation produced the highest volumetric concentrations of the final solubilized antigen. An exponential feeding strategy proved distinctly superior to feeding based on pH-stat and DO-stat methods. The plasmid coding for the antigen was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at 4 h after initiation of the culture. The minimum IPTG concentration for full induction was 0.1 mM.     

Ammonium regulation of urate uptake in Chlamydomonas reinhardtii

Urate was taken up at a negligible rate by Chlamydomonas reinhardtii cells grown on ammonium and transferred to media containing urate plus ammonium or urate plus chloral hydrate or cycloheximide. Addition of ammonium to cells actively consuming urate produced a rapid inhibition of urate uptake whereas the intracellular oxidation of urate was unaffected. Methylammonium but not glutamine or glutamate inhibited urate uptake. Addition of l-methionine-dl-sulfoximine to cells actively consuming urate provoked ammonium excretion, which was accompanied by a rapid inhibition of urate uptake. In cells growing on urate and exhibiting noticeable levels of nitrite-reductase activity, nitrite caused a sudden inhibition of urate uptake whereas nitrate required a time to induce nitrate reductase and to exert its inhibitory effect on uptake. The urate-uptake system did not require urate for induction since the urate-uptake capacity appeared in nitrogen-starved cells. From these results it is concluded that, in Chlamydomonas reinhardtii, ammonium inhibits urate uptake and also acts as co-repressor of the uptake system.     

Effect of polydimethylsiloxane oxygen carriers on the biological bleaching of hardwood kraft pulp by Trametes versicolor

Summary Improving the availability of oxygen by adding polydimethylsiloxanes (PDMS) oxygen carriers to Trametes versicolor cultures increased pulp brightening. The presence of the oxygen carriers in cultures of T. versicolor with hardwood kraft pulp increased the growth rate of the fungus, but not the ultimate biomass yield. The PDMS also stimulated brightening of hardwood kraft pulp by it T. versicolor immobilized in polyurethane foam. A threefold increase in the oxygen uptake rate in T. versicolor cultures with PDMS was observed. This increase can be explained by elevated oxygen transfer rate and attributed to the surfactant properties of PDMS. Offprint requests to: E. ZiomekIssued as NRCC 32760     

Differential effects of turgor on sucrose and potassium transport at the tonoplast and plasma membrane of sugar beet storage root tissue

When turgor was increased, by decreasing the concentration of mannitol bathing discs of sugar beet storage root tissue, the rates of sucrose and potassium uptake into the vacuole were decreased. At all external mannitol concentrations the rate of sucrose and potassium uptake across the plasma membrane was an order of magnitude greater than the rate of quasi-steady uptake into the vacuole, implying a very large efflux. Efflux of both sucrose and potassium was increased at high turgor. However, while increasing turgor decreased the rate of K⁺ uptake, the rate of sucrose uptake at the plasma membrane increased with time. Compartmental analysis of tracer exchange kinetics was used to determine unidirectional K⁺ fluxes. From these results, it was estimated that the increase in K⁺ efflux accompanying a 1.5 MPa increase in turgor could lead to a net increase of 140mol^?3h^?1 in the external potassium concentration. It is suggested that the turgor-imposed increase in solute efflux is a means of regulating intracellular osmotic pressure and/or turgor in sugar beet storage roots, but that sucrose is preferentially retrieved from the apoplast, even under conditions of excessively high turgor. However, much of this sucrose is probably lost from the cell, implying a ‘futile’ sucrose transport cycle at the plasma membrane. The turgor-stimulated leak of potassium could play a major role in the regulation of turgor pressure in sugar beet storage root tissue.     

Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria

Ammonium at low concentrations caused a rapid and effective inhibition of nitrate utilization in the light by the cyanobacterium Anacystis nidulans without affecting the cellular level of nitrate reductase activity. The inhibition was reversible, and the ability of the cells to utilize nitrate was restored immediately after ammonium had been exhausted. The inhibitory effect was dependent on consumption by the cells of the added ammonium which was rapidly incorporated into amino acids. In the presence of L-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase pathway, ammonium did not exhibit any inhibitory effect on nitrate utilization. Ammonium assimilation, rather than ammonium itself, seems to regulate nitrate utilization in A. nidulans. Short-term inhibition by ammonium of nitrate utilization and its prevention by MSX were also demonstrated in the filamentous cyanobacteria Anabaena and Nostoc.Abbreviations MSX L-Methionine-d-l-sulfoximine     

Parasitism of Iron-siderophore Receptors of Escherichia Coli by the Siderophore-peptide Microcin E492m and its Unmodified Counterpart

Microcin E492 (MccE492) is an antibacterial peptide naturally secreted by Klebsiella pneumoniae RYC492. Initially described as an 84-residue unmodified peptide, it was also recently isolated in a posttranslationally modified form, MccE492m. The production of MccE492m is dependent on the synthesis of enterobactin and the mceABCDEFGHIJ gene cluster. The posttranslational modification was characterized as a trimer of N-(2,3-dihydroxybenzoyl)-l-serine (DHBS) linked to the Ser84-carboxylate via a β-d-glucose moiety. MccE492m was shown to bind ferric ions through the trimer of DHBS. This is the first example of a novel type of antibacterial peptide termed siderophore-peptide. Recognition of MccE492m, but also of the unmodified MccE492, was shown to be mediated by the catecholate siderophore receptors FepA, Cir and Fiu at the outer membrane of E. coli. The siderophore-type modification was shown to be responsible for a significant enhancement of the microcin antibacterial activity. Therefore, we propose that MccE492 and MccE492m use iron-siderophore receptors for uptake into the target bacteria and that improvement of MccE492 antimicrobial activity upon modification results from an increase in the microcin/receptor affinity.     

Urea and thiourea transport in Aspergillus nidulans

Wild-type Aspergillus nidulans has an active transport system specific for urea which concentrates urea at least 50-fold relative to the extracellular concentration. It is substrate concentration dependent, with an apparent K _m of 3×10^–5 m for urea. Competition studies and the properties of mutants indicate that thiourea is taken up by the same system as urea. Thiourea is toxic at 5mm to wild-type cells of Aspergillus nidulans. Mutants, designated ureA1 to ureA16, resistant to thiourea have been isolated, and transport assays and growth tests show that they are specifically impaired in urea transport. The mutant ureA1 has a higher K _m value than the wild type for thiourea uptake. The ureA locus has been assigned to linkage group VIII. ureA1 is recessive for thiourea resistance while semidominant for the low uptake characteristic. The urea uptake system is under nitrogen regulation, with l-glutamine as the probable effector. The mutants, meaA8 and gdhA1, which are insensitive to ammonium control of many nitrogen-regulated metabolic systems, are also insensitive to ammonium control of urea uptake, but both are sensitive to l-glutamine regulation.Formerly at the Department of Genetics, University of Glasgow, Glasgow, Scotland.     

Dependence of fluorescence behaviour and other photosystem-II characteristics on the nature of the nitrogen source for the filamentous cyanobacterium Oscillatoria chalybea

The filamentous cyanobacterium Oscillatoria chalybea grows phototrophically on a mineral medium in the presence of either nitrate or ammonium ions as nitrogen source at similar growth rates. In the absence of any combined nitrogen source in the medium the cyanobacterium also grows, although at a reduced growth rate. The steady state rate of oxygen evolution by filaments from these three culture conditions is approximately constant if compared on an equal chlorophyll basis. Qualitative differences, however, emerge, if transient phenomena, e.g. the oxygen gush, are investigated. Only nitrate-and nitrogen-free-grown cultures show an oxygen gush, whereas ammonium sulfate-grown cultures do not show this phenomenon. Fluorescence induction in O. chalybea shows a fast monophasic rise, comparable to the fluorescence rise curves of higher plant chloroplasts in the presence of dithionite. The steady state level of fluorescence in ammonium sulfate-grown cells is up to seven times higher than in nitrate-grown cells when compared on an equal chlorophyll basis. In ammonium sulfate-grown cells, DCMU (N,N-3,4-Dichlorophenyl dimethylurea) causes a further increase in fluorescence level. In nitrate-grown cyanobacteria, however, the effect of DCMU consists of a decrease of the steady state level of fluorescence. In context with earlier research on Anabaena cylindrica, another filamentous cyanobacterium, it appears that the type of the nitrogen source used for growth determines the main location of the DCMU-block in this organism. It thus appears that in O. chalybea the site of DCMU inhibition lies on the oxygen-evolving side of photosystem II, if the organism is grown on nitrate. If grown on ammonium sulfate, no substantial difference of the location of the inhibition site when compared to algae or higher plant chloroplasts is found.Thylakoid preparations of O. chalybea perform the usual Hill reactions with ferricyanide, p-benzoquinone or silicomolybdate as electron acceptors. In each case it is seen that with thylakoids of nitrate-grown cells the steady-state level of fluorescence is lowered by DCMU in the presence of these acceptors, which should be the case, if DCMU inhibits electron transfer on the donor side of photosystem II. According to the literature silicomolybdate accepts electrons mainly before the DCMU-block in higher plant chloroplasts. Hence, in higher plants this reaction is mainly DCMU-insensitive. In thylakoids of O. chalybea, however, the Hill reaction with silicomolybdate is DCMU-sensitive which provides further evidence that the DCMU-block is on the oxygen-evolving side of photosystem II in O. chalybea provided the cells have been grown on nitrate.Abbreviations DCMU N-N-3,4-Dichlorophenyl dimethylurea     

Relationship between substrate concentration,growth rate,and respiration rate of Escherichia coli in continuous culture

Summary Using a continuous flow technique the relationship between growth rate and substrate concentration was investigated with glucose as the limiting factor of a culture of Escherichia coli. Graphical and numerical analysis of the experimental data demonstrated that the application of the Michaelis-Menten equation produced erroneous results, whereas, the constants obtained from the Teissier equation were in agreement with the experimental data. On this basis, new equations defining the steady state cell and substrate concentration in continuous flow cultures were developed and tested against experimental data.Comparison of the specific growth rates, substrate uptake rates and oxygen consumption rates demonstrated that all were directly proportional to each other and could be related to each other by mathematical equations. Specifically it was shown that as the growth rate increased from 0.06 to k _m =0.76 the substrate uptake rate increased from 134 to 1420 mg glucose per gram cell weight per hour and the oxygen consumption rate increased from 48.6 to 505 mg O₂ per gram cell weight per hour. Independent of the growth rate 37% of the carbohydrate consumed were oxidized. The yield factor varied from 0.44 at low growth rates to 0.54 at high growth rates. Analysis of the growth rate-substrate uptake rate relationship indicated that a minimum substrate uptake rate of 55 mg glucose per gram cell weight per hour existed below which cell reproduction would cease. This was supported by the fact that steady state conditions could not be maintained in the culture at D values below 0.02 when the substrate supply rate decreased below 45 mg glucose per gram cell weight per hour.Material contained in this paper was submitted as a thesis in partial fulfillment of the requirements for the Ph. D. degree of Dr. R. S. Lipe.     

Evaluation of the phytic acid effect on the labeling of blood elements with technetium-99m and on the survival of a strain of Escherichia coli treated with stannous fluoride

The labeling of red blood cells with technetium-99m(^99mTc) depends on a reducing agent and stannous ions, as chloride or fluoride, are widely utilized. This labeling may also be altered by drugs. Moreover, some authors have reported that the survival of Escherichia coli (E. coli) cultures decreases in presence of stannous ions. Phytic acid is present in the daily diet and we evaluated its influence on: (i) the labeling of blood elements with ^99mTc and (ii) on the survival of an E. coli strain treated with stannous fluoride. Heparinized whole blood was withdrawn from Wistar rats and it was incubated with stannous chloride and with ^99mTc, as sodium pertechnetate, centrifuged and plasma (P) and blood cells (BC) were isolated. Samples of P and BC were also precipitaded with trichloroacetic acid, centrifuged and soluble (SF) and insoluble fractions (IF) isolated. E. coli culture was treated with stannous fluoride in presence of phytic acid. As phytic acid altered the fixation of ^99mTc on BC, on IF-P and on IF-BC and, moreover, it abolished the lethal effect of stannous fluoride on the E. coli culture, we can suggest that, probably, phytic acid would have chelating properties to the stannous ions.