Initiation of ovalbumin synthesis in chicken oviduct magnum: Transient labeling of the NH2-terminus by methionine

The incorporation of [³⁵S]methionine into ovalbumin, a protein containing NH₂-terminal N-acetylglycine, has been studied in chicken oviduct magnum cells. The purification of [³⁵S]methionine-labeled ovalbumin from total oviduct proteins was accomplished by dialysis of a crude extract at pH 3.6 followed by chromatography on carboxymethyl cellulose. The radioactive ovalbumin eluted from the column in three peaks (P₀, P₁, and P₂-containing 0, 1, and 2 moles of phosphate, respectively, per mole of ovalbumin). The kinetics of labeling of peaks P₀ and P₁ showed that the ratio of radioactivity in NH₂-terminal methionine to total incorporation was greater at 2 min of labeling than at later times. The transient labeling of the NH₂-terminus of ovalbumin with methionine indicates that methionine is the initiator amino acid for the synthesis of this protein, which in its mature form contains NH₂-terminal N-acetylglycine.     

Ovalbumin-related Protein X Is a Heparin-binding Ov-Serpin Exhibiting Antimicrobial Activities

Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys³⁶⁷-His³⁶⁸ is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.     

Thiol and disulphide contents of hen ovalbumin. C-Terminal sequence and location of disulphide bond

1. The thiol and disulphide contents of hen ovalbumin were investigated by p-chloromercuribenzoate titration, by determination of cysteic acid content after performic acid oxidation, by measurement of uptake of radioactive iodoacetic acid, and by assay of S-aminoethylcysteine after reaction with ethyleneimine. All results showed that ovalbumin had 6 half-cystine residues. Experiments with and without reducing agents demonstrated that there were 4 thiol groups and 1 disulphide bond. 2. A peptide containing equimolar amounts of S-carboxymethyl-cysteine, serine, valine and proline, but no lysine or arginine, was obtained by radioactive labelling of the cysteine residues with iodo[¹⁴C]acetic acid followed by electrophoretic and chromatographic separation of tryptic digests. It was concluded that the C-terminal sequence of ovalbumin is -Cys-Val-Ser-Pro. 3. The location of the disulphide bond was studied by using a double-labelling technique. It was shown that one end of the disulphide was located in this C-terminal peptide.     

Regulation of the reconstituted chloroplast phosphate translocator by an H+ gradient

This report describes the influence of ΔpH on the transport of phosphate, triose phosphate and 3-phosphoglycerate catalyzed by the phosphate translocator in a reconstituted system. The H⁺ gradient across the liposome membrane is adjusted by the addition of external buffer solution and maintained for several minutes. The following results are obtained: (1) An inward directed H⁺ gradient leads to an increase of 3-phosphoglycerate transport and to a decrease of phosphate and triose phosphate transport. (2) An H⁺ gradient in the opposite direction results in a restriction of 3-phosphoglycerate influx whereas the influx of phosphate and triose phosphate is enhanced. (3) The magnitude of the pH effect depends on the internal substrate. Compared to the homoexchange mode, the effect of applied ΔpH is more pronounced in the heteroexchange mode. (4) Transport of phosphate and 3-phosphoglycerate is influenced by ΔpH in a different manner. In the case of phosphate and triose phosphate transport the observed effects are associated with changes in the apparent K_m values whereas in the case of 3-phosphoglycerate transport the application of a pH gradient is linked to a change of V_max. (5) In competition experiments with both substrates in the external medium, ΔpH influences the effect of phosphate as a competitive inhibitor of 3-phosphoglycerate transport whereas the effect of 3-phosphoglycerate on phosphate transport is not affected by a pH gradient. (6) The measured apparent K_m and V_max values under the influence of ΔpH can be used for the calculation of substrate fluxes across the envelope during illumination. It can be demonstrated that the increase of stromal pH in the light gives rise to a considerable change in the ratio of the substrates transported. Under conditions without pH gradient, the species transported out is mainly 3-phosphoglycerate and the species transported in is mainly triose phosphate. These fluxes are reversed when a pH gradient is applied (light conditions).     

3-O-Methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca-ATPase

3-O-Methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca²⁺-ATPase in the absence of Ca²⁺, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca²⁺, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca²⁺ concentration produces a bell-shaped curve with a maximum at 50 μM Ca²⁺. At optimal Ca²⁺ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca²⁺, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca²⁺ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca²⁺-ATPases.     

Ovalbumin labeling with p-hydroxymercurybenzoate: The effect of different denaturing agents and the kinetics of reaction

The aim of our study was to investigate how denaturing agents commonly used in protein analysis influence the labeling between a reactive molecule and proteins. For this reason, we investigated the labeling of ovalbumin (OVA) as a globular model protein with p-hydroxymercurybenzoate (pHMB) in its native state (phosphate buffer solution) and in different denaturing conditions (8 mol L⁻¹ urea, 3 mol L⁻¹ guanidinium thiocyanate, 6 mol L⁻¹ guanidinium chloride, 0.2% sodium dodecyl sulfate, and 20% methanol). In addition to chemical denaturation, thermal denaturation was also tested. The protein was pre-column simultaneously denatured and derivatized, and the pHMB-labeled denatured OVA complexes were analyzed by size exclusion chromatography (SEC) coupled online with chemical vapor generation–atomic fluorescence spectrometry (CVG–AFS). The number of –SH groups titrated greatly depends on the protein structure in solution. Indeed, we found that, depending on the adopted denaturing conditions, OVA gave different aggregate species that influence the complexation process. The results were compared with those obtained by a common alternative procedure for the titration of –SH groups that employs monobromobimane (mBBr) as tagging molecule and molecular fluorescence spectroscopy as detection technique.     

Resolution of some glycopeptides of hen ovalbumin by reverse-phase high-pressure liquid chromatography

Reverse-phase high-pressure liquid chromatography has been shown to be useful in fractionation of oligosaccharides containing N-acetyl amino sugars on the basis of stereochemical differences. Data in this paper show this same stereochemical selectivity can be obtained for glycopeptides. Using dilute aqueous phosphate for elution, three major high-mannose glycopeptides from ovalbumin can be separated on the analytical scale in a few minutes and the same method can also be used for preparation of the glycopeptides on the milligram scale. High-field proton NMR spectroscopy is used to identify two of the compounds as the mannose-5 and -6 glycopeptides (E3 and D3) and the third as the mannose-6 glycopeptide substituted by two N-acetyl glucosamine (GlcNAc) residues (C3B). The NMR spectra and analytical chromatograms of the glycopeptides show high purity and freedom from contaminants previously identified in ovalbumin glycopeptides.     

Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily^1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya⁶. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila^7-14. An acetate kinase which can only utilize PP_i but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus^15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann^17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD⁺ by the enzymes pyruvate kinase and lactate dehydrogenase^21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine²³. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP⁺ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase²⁴.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PP_i.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

Phosphohistidine as a stoichiometric intermediate in reactions catalyzed by isoenzymes of wheat germ acid phosphatase.

Burst titration experiments conducted on a highly purified isoenzyme of wheat germ acid phosphatase under conditions where [S]_o > K_m indicate that there is one titratable active site per molecule of enzyme of molecular weight 59,000. The enzyme is labeled to only a small extent with inorganic [³²P]phosphate ion. Incubation of wheat germ acid phosphatase with ³²P-labeled substrates such as p-nitrophenyl phosphate or inorganic pyrophosphate followed by quenching in alkali results in the stoichiometric trapping of a base-stable, acid-labile phosphorylated protein. The extent of ³²P incorporation parallels the degree of purity of the enzyme and corresponds to the incorporation of 1 mol of phosphate per mole of enzyme. The incorporation is eliminated by the simultaneous presence of excess unlabeled phosphate ion (a competitive inhibitor) and is not observed when a noncatalytic protein (such as bovine serum albumin) is substituted for the enzyme. Complete alkaline hydrolysis of the labeled protein results in the recovery of an 85% yield of τ-phosphohistidine, identified by ion-exchange chromatography, high-voltage paper electrophoresis, and comparison with a synthetic sample. A ³²P-labeled tryptic tetradecapeptide was isolated following hydrolysis of the labeled, reduced, and carboxymethylated protein with trypsin at pH 8.3, separation of the labeled peptide, and purification by two methods including a novel variant of a diagonal electrophoresis technique. The end groups and composition of the peptide are reported. The data are consistent with the interpretation that a phosphohistidine-enzyme intermediate is formed as an obligatory intermediate in the catalytic reaction involving this enzyme.     

Energy-linked Sulfate Uptake by Corn Mitochondria via the Phosphate Transporter

Corn shoot mitochondria possess an energy-linked transport system for sulfate uptake as demonstrated by osmotic swelling and [³⁵S]SO₄²⁻ accumulation. Maximum uptake is secured in the presence of Mg²⁺ and oligomycin with sucrose for osmotic support. Neither phosphate nor dicarboxylate anions are required. When added simultaneously, millimolar concentrations of phosphate block [³⁵S]SO₄²⁻ uptake after the initial minute. Mersalyl, N-ethylmaleimide, and 2,4-dinitrophenol are strong inhibitors of sulfate uptake; n-butylmalonate is a weak inhibitor. These inhibitors act in the same fashion on phosphate uptake. It is concluded that sulfate uptake in the absence of phosphate is by the phosphate transporter.     

Evidence for a novel Entamoeba histolytica lectin activity that recognises carbohydrates present on ovalbumin

Entamoeba histolytica, an intestinal amoeba that causes dysentery and liver abscesses, acquires nutrients by engulfing bacteria in the colonic lumen and phagocytoses apoptotic cells during tissue invasion. In preliminary studies to identify ligands that stimulate amoebic phagocytosis, we used ovalbumin immobilized on latex particles as a potential negative control protein. Surprisingly, ovalbumin strongly stimulated E. histolytica particle uptake. Experiments using highly purified ovalbumin confirmed the specificity of this finding. The mechanism of particle uptake was actin-dependent, and the Entamoeba phagosome marker amoebapore A localised to ovalbumin-bead containing vacuoles. The most well described amoebic receptor is a Gal/GalNAc-specific lectin, but d-galactose had no effect on ovalbumin-stimulated phagocytosis. Ovalbumin has a single N-glycosylation site (Asn₂₉₂) and is modified with oligomannose and hybrid-type oligosaccharides. We used both trifluoromethanesulfonic acid and N-glycanase to deglycosylate ovalbumin and tested the effect. Both methods substantially reduced the stimulatory effect of ovalbumin. Biotinylated ovalbumin bound the surface of fixed E. histolytica trophozoites saturably; furthermore, denatured ovalbumin and native ovalbumin both specifically inhibited ovalbumin-biotin binding, but deglycosylated ovalbumin had no effect. Collectively, these data suggest that E. histolytica has a previously unrecognised surface lectin activity that binds to carbohydrates on ovalbumin and stimulates phagocytosis.     

Proline biosynthesis in Escherichia coli kinetic and mechanistic properties of glutamate semialdehyde dehydrogenase

The kinetics of the NADP⁺- and phosphate-dependent oxidation of glutamic acid 5-semialdehyde are consistent with a rapid-equilibrium random order mechanism. The K_m for dl-pyrroline-5-carboxylic acid is 2.5 mM, for NADP⁺ is 0.05 mM and for phosphate is 0.35 mM. TheV_max is approx. 8.0 units per mg protein. The reaction is highly specific for the dl-pyrroline-5-carboxylic acid and NADP⁺, but a number of divalent anions can substitute for phosphate. NADPH is competitive with respect to all three substrates and an analog of γ-glutamyl phosphate, 3-(phosphonoacetylamido)-l-alanine, is competitive with respect to dl-pyrroline-5-carboxylic acid and non-competitive with respect to NADP⁺ and phosphate, suggesting dead-end complex formation.     

New structures and composition of cell wall teichoic acids from Nocardiopsis synnemataformans,Nocardiopsis halotolerans,Nocardiopsis composta and Nocardiopsis metallicus: a chemotaxonomic value

The structures of the cell wall teichoic acids (TA) from some species of the genus Nocardiopsis were established by chemical and NMR spectroscopic methods. The cell walls of Nocardiopsis synnemataformans VKM Ac-2518^T and Nocardiopsis halotolerans VKM Ac-2519^T both contain two TA with unique structures—poly(polyol phosphate-glycosylpolyol phosphate)—belonging to the type IV TA. In both organisms, the minor TA have identical structures: poly(glycerol phosphate-N-acetyl-β-galactosaminylglycerol phosphate) with the phosphodiester bond between C-3 of glycerol and C-4 of the amino sugar. This structure is found for the first time. The major TA of N. halotolerans has a hitherto unknown structure: poly(glycerol phosphate-N-acetyl-β-galactosaminylglycerol phosphate), the N-acetyl-β-galactosamine being acetalated with pyruvic acid at positions 4 and 6. The major TA of N. synnemataformans is a poly(glycerol phosphate-N-acetyl-β-galactosaminylglycerol phosphate) with the phosphodiester bond between C-3 of glycerol and C-3 of the amino sugar. The cell walls of Nocardiopsis composta VKM Ac-2520 and N. composta VKM Ac-2521^T contain only one TA, namely 1,3-poly(glycerol phosphate) partially substituted with N-acetyl-α-glucosamine. The cell wall of Nocardiopsis metallicus VKM Ac-2522^T contains two TA. The major TA is 1,5-poly(ribitol phosphate), each ribitol unit carrying a pyruvate ketal group at positions 2 and 4. The structure of the minor TA is the same as that of N. composta. The results presented correlate well with the phylogenetic grouping of strains and confirm the species and strain specific features of cell wall TA in members of the genus Nocardiopsis.     

Models of prebiological phosphorylation

The hypothesis that contemporary metabolic pathways evolved from analogous chemical reaction sequences on the primitive Earth leads to a reexamination of models of prebiological phosphorylation. Present-day phosphate uptake by algae and bacteria seems to involve two transport systems: (a) An active transport process occurring at low external phosphate concentrations (as in unpollusive) process at higher phosphate concentrations (>10⁻⁶ M) (as in the interstitial water of reducing sediments). Laboratory model experiments are described for the reaction of reducing sugars with orthophosphate in the presence of cyanogen, producing glycosyl phosphates. These reactions proceed with appreciable yields only at high phosphate concentrations (>10⁻³ M), and may thus possibly serve as simulations of prebiological phosphorylation with diffusive transport, as it may have occurred in the interstitial water of reducing sediments.     

Conditional Deletion of Fgfr1 in the Proximal and Distal Tubule Identifies Distinct Roles in Phosphate and Calcium Transport

A postnatal role of fibroblast growth factor receptor-1 (FGFR1) in the kidney is suggested by its binding to α-Klotho to form an obligate receptor for the hormone fibroblast growth factor-23 (FGF-23). FGFR1 is expressed in both the proximal and distal renal tubular segments, but its tubular specific functions are unclear. In this study, we crossed Fgfr1^flox/flox mice with either gamma-glutamyltransferase-Cre (γGT-Cre) or kidney specific-Cre (Ksp-Cre) mice to selectively create proximal tubule (PT) and distal tubule (DT) Fgfr1 conditional knockout mice (designated Fgfr1^PT-cKO and Fgfr1^DT-cKO, respectively). Fgfr1^PT-cKO mice exhibited an increase in sodium-dependent phosphate co-transporter expression, hyperphosphatemia, and refractoriness to the phosphaturic actions of FGF-23, consistent with a direct role of FGFR1 in mediating the proximal tubular phosphate responses to FGF-23. In contrast, Fgfr1^DT-cKO mice unexpectedly developed hypercalciuria, secondary elevations of parathyroid hormone (PTH), hypophosphatemia and enhanced urinary phosphate excretion. Fgfr1^PT-cKO mice also developed a curly tail/spina bifida-like skeletal phenotype, whereas Fgfr1^DT-cKO mice developed renal tubular micro-calcifications and reductions in cortical bone thickness. Thus, FGFR1 has dual functions to directly regulate proximal and distal tubule phosphate and calcium reabsorption, indicating a physiological role of FGFR1 signaling in both phosphate and calcium homeostasis.     

Phosphate transport in membrane vesicles from Escherichia coli

Escherichia coli strain AN710 possesses only the PIT system for phosphate transport. Membrane vesicles from this strain, which contain phosphate internally, perform exchange and active transport of phosphate. The energy for active transport is supplied by the respiratory chain with ascorbate-phenazine methosulphate as electron donor. To a lesser extent also the oxidation of d-lactate energizes phosphate transport; the oxidation of succinate is only marginally effective. Phosphate transport is driven by the proton-motive force and in particular by the pH gradient across the membrane. This view is supported by the observation that phosphate transport is stimulated by valinomycin, inhibited by nigericin and abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Neither inhibitor affects phosphate exchange. The phosphate analogue arsenate inhibits both the exchange reaction and active transport. Both processes are stimulated by K⁺ and Mg²⁺, the highest activities being observed with both ions present.Membrane vesicles have also been isolated from Escherichia coli K10, a strain which possesses only a functional PST phosphate transport system. These vesicles perform neither exchange nor active transport of phosphate, although active transport of amino acids is observed in the presence of ascorbate-phenazine methosulphate or d-lactate.     

Regulation of inorganic polyphosphate is required for proper vacuolar proteolysis in fission yeast

Regulation of cellular proliferation and quiescence is a central issue in biology that has been studied using model unicellular eukaryotes, such as the fission yeast Schizosaccharomyces pombe. We previously reported that the ubiquitin/proteasome pathway and autophagy are essential to maintain quiescence induced by nitrogen deprivation in S. pombe; however, specific ubiquitin ligases that maintain quiescence are not fully understood. Here we investigated the SPX-RING-type ubiquitin ligase Pqr1, identified as required for quiescence in a genetic screen. Pqr1 is found to be crucial for vacuolar proteolysis, the final step of autophagy, through proper regulation of phosphate and its polymer polyphosphate. Pqr1 restricts phosphate uptake into the cell through ubiquitination and subsequent degradation of phosphate transporters on plasma membranes. We hypothesized that Pqr1 may act as the central regulator for phosphate control in S. pombe, through the function of the SPX domain involved in phosphate sensing. Deletion of pqr1⁺ resulted in hyperaccumulation of intracellular phosphate and polyphosphate and in improper autophagy-dependent proteolysis under conditions of nitrogen starvation. Polyphosphate hyperaccumulation in pqr1⁺-deficient cells was mediated by the polyphosphate synthase VTC complex in vacuoles. Simultaneous deletion of VTC complex subunits rescued Pqr1 mutant phenotypes, including defects in proteolysis and loss of viability during quiescence. We conclude that excess polyphosphate may interfere with proteolysis in vacuoles by mechanisms that as yet remain unknown. The present results demonstrate a connection between polyphosphate metabolism and vacuolar functions for proper autophagy-dependent proteolysis, and we propose that polyphosphate homeostasis contributes to maintenance of cellular viability during quiescence.     

Phosphate concentration alters the effective bacterial quorum in the symbiosis of Medicago truncatula-Sinorhizobium meliloti

The symbiosis of Medicago truncatula-Sinorhizobium meliloti is affected by phosphate (P) deficiency in the environment. Quorum sensing (QS) is a regulatory pathway in S. meliloti that controls various functions of free-living and symbiotic bacteria in response to phosphate availability and regulation is mediated by a periplasmic protein PstS, and also bacterial density. The quorum sensing pathway of S. meliloti, involves three genes named sinI, sinR and expR and also some bacterial auto-inducers such as N-acyl homoserine lactones (AHLs). In the current study, the expression of the different genes of quorum sensing and pstS were evaluated under 0.1, 0.5 and 2 mM P. The qRT-PCR results showed an increased expression of pstS and also the quorum sensing genes sinI and sinR but not expR, following phosphate starvation. Indeed, the enhanced level of sinR induces the expression of sinI that is responsible for the N-acyl homoserine lactones (AHL) production in S. meliloti. The different response of expR may be due to its negative control on sinR expression. In the symbiosis of M. truncatula-S. meliloti, it was shown that the concentration of phosphate in the medium alters the effective inoculating bacterial quorum (density). By increasing the phosphate concentration in the medium from 0.1 to 0.5 and 2 mM, considering the optimal plant growth and pink nodule (nitrogen-fixing) formation, the effective inoculating bacterial densities were 10⁵, 10⁷ and 10⁹ CFU ml^?1, respectively. Therefore, low phosphate concentrations can compensate for a low bacterial density by inducing the quorum sensing pathway and establishing a symbiosis. Conversely, bacterial density plays the main role in the formation of symbiosis at high phosphate concentrations.