Structure of the glycerol phosphate-containing O-specific polysaccharide and serological studies on the lipopolysaccharides of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14)

The O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14) and found to contain D-glucose, D-glucosamine and glycerol-1-phosphate in molar ratios 2 : 2 : 1. Based on methylation analysis and 1H and 13C nuclear magnetic resonance spectroscopy data, it was established that the O-specific polysaccharides from both strains have the identical branched tetrasaccharide repeating unit with 3,6-disubstituted GlcNAc, followed by 2,4-disubstituted Glc residues carrying at the branching points lateral residues of Glc and GlcNAc at positions 6 and 2, respectively. Glycerol-1-phosphate is linked to position 6 of the chain Glc. All sugars have a beta configuration, except for the side-chain Glc, which is alpha. Serological studies revealed a close relatedness of the lipopolysaccharides of C. werkmanii PCM 1548 and PCM 1549, both belonging to serogroup O14. In immunoblotting, anti-C. werkmanii PCM 1548 serum showed no cross-reactivity with the O-polysaccharide bands of the lipopolysaccharides of Citrobacter youngae PCM 1550 (serogroup O16) and Hafnia alvei PCM 1207, also containing a lateral glycerol phosphate residue.     

Composition of O-antigenic lipopolysaccharides from Enterobacter cloacae

Analyses have been carried out on lipopolysaccharides (LPS) from 14 strains of Enterobacter cloacae representing different O serotypes. All of the products appeared to have a composition and architecture typical of enterobacterial LPS, but points of interest include the absence of phosphate residues from the core oligosaccharide, the presence of both L-glycero-D-mannoheptose and D-glycero-D-mannoheptose (ratio usually about 4:1), and the presence in lipid A of small amounts of fatty acids with odd numbers of carbon atoms (mainly C13) in addition to tetradecanoic acid and 3-hydroxytetradecanoic acid. Monosaccharides identified as components of polymeric fractions from the LPS were glucose, galactose, mannose, rhamnose, glucosamine, galactosamine, fucosamine, and galacturonic acid. Most polymeric fractions also probably contained an O-acetyl substituent. Closely similar chemotypes found for the polymeric fractions from the LPS of cross-reacting serotypes support the view that these fractions contain the O-antigenic determinants and represent the side chains of the LPS.     

The metabolism and structure of phosphoglycoproteins in rat brain

Glycoproteins that contain phosphohexosyl groups were found to be present in the myelin- and synaptosomal-enriched fractions as well as in the microsomes of rat brain. The kinetics of flow of intraperitoneally injected [³²P]phosphate suggests that the phosphate is enzymatically added in structures found in the microsomal fraction. The newly synthesized phosphoglycoproteins then appear in the soluble fraction of the synaptosomes and in the cytosol, prior to incorporation into the membranes of the synaptosomes and myelin. Phosphoglycopeptides recovered from the phosphoglycoprotein contain 3 Mannose units per N-acetylglucosamine residue; one of the mannose residues is phosphorylated. [¹³C]NMR studies indicate that the phosphoglycopeptides contain a chitobiose group and more than four sugar residues. Thus, the phosphomannoglycopeptides from rat brain contain an average of 2 N-acetylglucosamine, 6 mannose, and two phosphate moieties per oligosaccharide chain. Enzymatic treatment with -mannosidase failed to remove the phosphomannose, although some mannose residues were released. Thus, the phosphorylated mannose is not removed by the glycosidase and terminal nonphosphorylated mannose residues are present in the oligosaccharide. The phosphate residues are removed by treatment with alkaline phosphatase.     

Studies on the structure of lipopolysaccharides of Salmonella minnesotta and Salmonella typhimurium R strains

1. The composition of the lipopolysaccharides and the corresponding lipid-free polysaccharides from four R-mutants of Salmonella has been studied. All the lipopolysaccharides, from RI and RII serotypes contained d-glucose, d-galactose, heptose, N-acetylglucosamine and 3-deoxy-2-oxo-octonate. The polysaccharide obtained from the RII lipopolysaccharides also contained all these sugars. The polysaccharides from RI lipopolysaccharides lacked N-acetylglucosamine. 2. From partial hydrolysates of the lipopolysaccharides, a number of oligosaccharides have been isolated and partially characterized. Oligosaccharides containing N-acetylglucosamine or glucosamine were obtained only from RII lipopolysaccharides. Several oligosaccharides composed of glucose and galactose were common to RI and RII preparations. 3. A structural unit, based on the oligosaccharides found, is proposed for the RII lipopolysaccharide. It contains the sequence: alpha-N-acetylglucosaminyl- alpha-glucosyl-alpha-galactosyl-glucosyl.... A second alpha-galactosyl residue is bound to position 6 of the last glucosyl group. The complete unit is believed to to be attached to a polyheptose phosphate backbone in the RII antigen. 4. The RI lipopolysaccharide of Salmonella minnesota contains an analogous structure lacking the terminal N-acetylglucosamine residue. 5. A basal structure common to the lipopolysaccharides of several Salmonella species is proposed.     

Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans

Histatins 1, 3, and 5 from human parotid secretion were isolated by gel filtration on Bio-Gel P-2 and reverse phase high performance liquid chromatography. The complete amino acid sequences of histatins determined by automated Edman degradation of the proteins, Staphylococcus aureus V8 protease, and tryptic peptides, are as follows: (Sequence: see text). Histatins 1, 3, and 5 contain 38, 32, and 24 amino acid residues, have molecular weights of 4929, 4063, and 3037, respectively, and contain 7 residues of histidine. Histatin 1 contains 1 mol of phosphate/mol of protein; histatins 3 and 5 lack phosphate. With the exception of Glu (residue 4) and Arg (residue 11) in histatin 1, the first 22 amino acid residues of all three histatins are identical, and the carboxyl-terminal 7 residues of histatins 1 and 3 are also identical. The sequence, -Glu-Phe-Pro-Phe-Tyr-Gly-Asp-Tyr-Gly- (residues 23-29), in histatin 1 is absent in histatin 3; and the sequence, -Gly-Tyr-Arg- (residues 23-25), in histatin 3 is absent in histatin 1. The complete sequence of histatin 5 is contained within the amino terminal 24 residues of histatin 3. The structural data suggest that histatins 1 and 3 are derived from different structural genes, whereas histatin 5 is a proteolytic product of histatin 3. All three histatins exhibit the ability to kill the pathogenic yeast, Candida albicans.     

The immunochemistry of Shigella flexneri lipopolysaccharides. A quantitative analysis of their monosaccharide constituents

1. The lipopolysaccharides of a representative selection of Shigella flexneri serotypes all contain the same constituents as Salmonella chemotype VII, namely, aldoheptose phosphate, 3-deoxy-2-oxo-octonate, O-phosphorylethanolamine, d-galactose, d-glucose, N-acetyl-d-glucosamine and l-rhamnose. 2. The presence of all the Salmonella basal sugars in Sh. flexneri lipopolysaccharides is consistent with the view that the latter contain a basal structure or core which is similar to the common basal structure of Salmonella lipopolysaccharides. 3. Although the Sh. flexneri lipopolysaccharides belong to one chemotype, there appear to be quantitative differences in the composition of their O-specific side chains. The repeating units of Sh. flexneri serotypes 1a, 2a, 3a, 4a, and variant X contain d-glucose, N-acetyl-d-glucosamine and l-rhamnose in the proportions 1:1:2 respectively. The analogous repeating units of serotypes 5a and 6 contain an additional mole of d-glucose and d-galactose respectively and that of variant Y 1 mole of d-glucose less.     

Sudies of O-specific polysaccharide chains of Pseudomonas solanacearum lipopolysaccharides consisting of structually different repeating units.

The structure of the O-antigenic ploysaccharide chains of lipopolysaccharides of a number of Pseudomonas solanacearum strains were elucidated mainly with the help of methylation analysis and ¹³CNMR spectroscopy, including a computer-assisted ¹³CNMR-based analysis. Six structually distinct but related polysaccharides were identified. They have a backbone which is built up of three -rhammoyranosyl resides and one 2-acetamido-2-deoxy- -glucopyranosyl residue, and is unsubtituted or substituted with a residue of -xylopyranose or -rhamnopyranose as a monosaccharide side chain. The lipopolysaccharides of most of the strains contain polysaccharide chains consisting of at least two structually different types of repeating units. Three of the polysaccharides are common to more than one strain.     

Ribitol-containing lipopolysaccharides from Proteus mirabilis and their serological relationship.

Ribitol phosphate was recently identified as a constituent of lipopolysaccharides obtained from 'proteus mirabilis strain D52 giving 1:4-anhydroribitol during acid hydrolysis (Gmeiner, 1975). Two other Proteus mirabilis strains belonging to serogroups O16 and O33 were shown previously to contain an unknown compoound X as lipopolysaccharide constituent (Kotelko et al., 1975). In this report the identification of compound X as 1:4-anhydroribotol by gas-liquid chromatography, mass spectrometry and mass fragmentography is described. Serological investigations using passive hemagglutination, hemagglutination inhbition and semi-quantitative precipitin reactions indicate strongly that ribitol plays a role in the serological specificity of the respective lipopolysaccharides.     

Immunologic and structural studies of lipopolysaccharides from Pseudomonas cepacia

O-serotyping of 30 Pseudomonas cepacia strains isolated from the soil and rhizosphere of different plant species in the territory of the USSR has been performed using 15 O-typing antisera according to the Heidt and Nakamura schemes. It is suggested to introduce two new O-serogroups (serogroups K and L) into the available P. cepacia classification scheme. They are most often met among the P. cepacia strains in different geographical areas of the USSR simultaneously with serogroups 2 (G) and 1 (D). To elucidate the molecular principles of serological inhomogeneity of the species the immunochemical studies of lipopolysaccharides of a number of P. cepacia strains have been conducted and the structure has been determined for repeating links of O-specific polysaccharides of P. cepacia strains attributed to 4 Nakamura serogroups, 3 Heidt serogroups, to serogroups K and L, as well as for certain strains from the collection of the Institute of Microbiology and Virology of the Ukr. SSR Academy of Sciences.     

The linkage of sugar phosphate polymer to peptidoglycan in walls of Micrococcus sp. 2102.

1. Protein-free walls of Micrococcus sp. 2102 contain peptidoglycan, poly-(N-acetylglucosamine 1-phosphate) and small amounts of glycerol phosphate. 2. After destruction of the poly-(N-acetylglucosamine 1-phosphate) with periodate, the glycerol phosphate remains attached to the wall, but can be removed by controlled alkaline hydrolysis. The homogeneous product comprises a chain of three glycerol phosphates and an additional phosphate residue. 3. The poly-(N-acetylglucosamine 1-phosphate) is attached through its terminal phosphate to one end of the tri(glycerol phosphate). 4. The other end of the glycerol phosphate trimer is attached through its terminal phosphate to the 3-or 4-position of an N-acetylglucosamine. It is concluded that the sequence of residues in the sugar 1-phosphate polymer-peptidoglycan complex is: (N-acetylglucosamine 1-phosphate)24-(glycerol phosphate)3-N-acetylglucosamine 1-phosphate-muramic acid (in peptidoglycan). Thus in this organism the phosphorylated wall polymer is attached to the peptidoglycan of the wall through a linkage unit comprising a chain of three glycerol phosphate residues and an N-acetylglucosamine 1-phosphate, similar to or identical with the linkage unit in Staphylococcus aureus H.     

Complete structural characterization of the lipid A fraction of a clinical strain of B. cepacia genomovar I lipopolysaccharide

Burkholderia cepacia, a Gram-negative bacterium ubiquitous in the environment, is a plant pathogen causing soft rot of onions. This microorganism has recently emerged as a life-threatening multiresistant pathogen in cystic fibrosis patients. An important virulence factor of B. cepacia is the lipopolysaccharide (LPS) fraction. Clinical isolates and environmental strains possess LPS of high inflammatory nature, which induces a high level production of cytokines. For the first time, the complete structure of the lipid A components isolated from the lipopolysaccharide fraction of a clinical strain of B. cepacia is described. The structural studies carried out by selective chemical degradations, MS, and NMR spectroscopy revealed multiple species differing in the acylation and in the phosphorylation patterns. The highest mass species was identified as a penta-acylated tetrasaccharide backbone containing two phosphoryl-arabinosamine residues in addition to the archetypal glucosamine disaccharide [Arap4N-l-beta-1-P-4-beta-D-GlcpN-(1-6)-alpha-D-GlcpN-1-P-1-beta-L-Arap4N]. Lipid A fatty acids substitution was also deduced, with two 3-hydroxytetradecanoic acids 14:0 (3-OH) in ester linkage, and two 3-hydroxyhexadecanoic acids 16:0 (3-OH) in amide linkage, one of which was substituted by a secondary 14:0 residue at its C-3. Other lipid A species present in the mixture and exhibiting lower molecular weight lacked one or both beta-L-Arap4N residues.     

Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobacterium tuberculosis

The recent availability (Hunter, S.W., Gaylord, H., and Brennan, P.J. (1986) J. Biol. Chem. 261, 12345-12351) of the well known arabinomannan of Mycobacterium leprae and Mycobacterium tuberculosis as the pure native lipoarabinomannan has resulted in its implication in key aspects of the immunopathogenesis of leprosy and tuberculosis. We had indicated that the lipid moiety of lipoarabinomannan is probably based on a diacylglycerol unit in that glycerol and the two fatty acids, hexadecanoate and 10-methyloctadecanoate, were identified. In addition, lipoarabinomannan was also shown to contain myo-inositol 1-phosphate. Evidence is now presented, based on selective radiolabeling and analysis of various cleavage fragments, that the inositol phosphate exists as both an alkalilable phosphodiester and as part of a phosphatidylinositol "membrane anchor." The mannan of M. tuberculosis was also isolated as the native lipomannan. It also apparently contains a phosphatidylinositol unit but is devoid of the alkali-labile inositol phosphate residues. These lipopolysaccharides are apparently multiglycosylated versions of the well known myocobacterial mannosyl phosphatidylinositols and are prokaryotic versions of the growing list of phosphatidylinositol-anchored macromolecules. Immunogold labeling demonstrates that lipoarabinomannan is a true antigenic capsular or extracellular product of M. tuberculosis. The presence of a phosphatidylinositol residue on lipoarabinomannan may explain its interaction with macrophage membranes and role in mycobacterial pathogenesis.     

Lipid a component of Salmonella typhimurium carrying the derepressed Col Ib plasmids

The structure of the lipid A from S. typhimurium harboring the derepressed plasmids Col Ib is very similar: i, 1,4'-bis-phosphorylated-beta-1',6-linked glucosamine disaccharide forms a backbone of the lipid; ii, lipid preparations contain four residues of 3-hydroxytetradecanoic acid at positions C3, C3' and the amide linked at C2, C2' and two free hydroxyl groups at positions C4 and C6'. Differences concern: i, substitution of phosphoryl groups by 4-amino-4-deoxy-L-arabinopyranose and phosphorylethanolamine in S. typhimurium with Col Ib plasmids; ii, the degree of acylation of hydroxyl groups of 3-hydroxytetradecanoic acid by myristic, lauric and palmatic acids; iii, presence of tridecanoic acid bound to hydroxyl of 3-hydroxy-tetradecanate residue in S. typhimurium with Col Ibdrd2 plasmid. Lipopolysaccharides from the plasmid mutant strains express several times higher lethal toxicity in chick embryos compared to lipopolysaccharides from the strain with the wild type Col Ib.     

L-serine binds to arginine-148 of the beta 2 subunit of Escherichia coli tryptophan synthase

Inactivation of the beta 2 subunit and of the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of one arginyl residue per beta monomer. The substrate L-serine protects the holo beta 2 subunit and the holo alpha 2 beta 2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by both the holo beta 2 subunit and the holo alpha 2 beta 2 complex indicate that L-serine and phenylglyoxal both bind to the same essential arginyl residue in the holo beta 2 subunit. The apo beta 2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by either pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo beta 2 subunit binds pyridoxal phosphate and the alpha subunit but cannot bind L-serine or L-tryptophan. We conclude that the alpha-carboxyl group of L-serine and not the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the beta 2 subunit. The specific arginyl residue in the beta 2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal has been identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains three other basic residues (lysine-137, arginine-141, and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148, and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium, and yeast supports a functional role for these three residues in anion binding. The location and role of the active-site arginyl residues in the beta 2 subunit and in two other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.     

Serospecific antigens of Legionella pneumophila.

Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria.     

Evidence of tyrosine kinase activity in the photosynthetic bacterium Rhodospirillum rubrum

The photosynthetic bacterium Rohodospirillum rubrum evidenced tyrosine protein phosphorylation under photoautotrophic conditions in the presence of [32P]Pi. The stability to alkaline treatment of the [32P] bound to the cell-free extract proteins suggested that tyrosine residues were carrying the labelling. One- and two-dimensional high voltage paper electrophoresis analysis revealed that such extracts do contain [32P]-phosphotyrosine residues. Furthermore, the association of alkali stable [32P] bound to specific proteins of the cell-free extract was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with KOH treatment of the gel. A definite argument in favor of protein kinase(s) phosphorylating tyrosine residues in R.rubrum proteins was obtained by partial purification of a tyrosine kinase activity from cell-free extract capable of phosphorylating synthetic peptides that only contain a single tyrosine residue as phosphate acceptor.     

Phosphorylation of the lipid A region of meningococcal lipopolysaccharide: identification of a family of transferases that add phosphoethanolamine to lipopolysaccharide

A gene, NMB1638, with homology to the recently characterized gene encoding a phosphoethanolamine transferase, lpt-3, has been identified from the Neisseria meningitidis genome sequence and was found to be present in all meningococcal strains examined. Homology comparison with other database sequences would suggest that NMB1638 and lpt-3 represent genes coding for members of a family of proteins of related function identified in a wide range of gram-negative species of bacteria. When grown and isolated under appropriate conditions, N. meningitidis elaborated lipopolysaccharide (LPS) containing a lipid A that was characteristically phosphorylated with multiple phosphate and phosphoethanolamine residues. In all meningococcal strains examined, each lipid A species contained the basal diphosphorylated species, wherein a phosphate group is attached to each glucosamine residue. Also elaborated within the population of LPS molecules are a variety of "phosphoforms" that contain either an additional phosphate residue, an additional phosphoethanolamine residue, additional phosphate and phosphoethanolamine residues, or an additional phosphate and two phosphoethanolamine residues in the lipid A. Mass spectroscopic analyses of LPS from three strains in which NMB1638 had been inactivated by a specific mutation indicated that there were no phosphoethanolamine residues included in the lipid A region of the LPS and that there was no further phosphorylation of lipid A beyond one additional phosphate species. We propose that NMB1638 encodes a phosphoethanolamine transferase specific for lipid A and propose naming the gene "lptA," for "LPS phosphoethenolamine transferase for lipid A."     

Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate carboxylase/oxygenase modified by the affinity label N-bromoacetylethanolamine phosphate

We reported earlier (Schloss, J. V., and Hartman, F. C. (1977) Biochem. Biophys. Res. Commun. 77, 230-236) that N-bromoacetylethanolamine phosphate is an affinity label for spinach ribulosebisphosphate carboxylase/oxygenase. We now show inactivation to be correlated directly with the alkylation either of a single lysyl residue (in the presence of Mg2+) or of 2 different cysteinyl residues (in the absence of Mg2+), consistent with the likelihood that these residues are located in the active site region. This proposition is further supported by the demonstration that the residues are protected from alkylation by substrate, a competitive inhibitor, or the transition state analog 2-carboxyribitol bisphosphate. Tryptic peptides that contain the modified residues have been isolated and sequenced. One of the 2 cysteinyl residues that are subject to alkylation is only 3 residues distant in sequence from the lysyl residue modified by bromoacetylethanolamine phosphate. This lysyl residue is identical with 1 of the 2 lysyl residues alkylated by the previously described affinity label, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate (Stringer, C. D., and Hartman, F. C. (1978) Biochem. Biophys, Res. Commun. 80, 1043-1048).     

Hydrophobic substitution mutations in the S4 sequence alter voltage-dependent gating in Shaker K+ channels

Voltage-activated Na+, Ca2+, and K+ channels contain a common motif, the S4 sequence, characterized by a basic residue at every third position interspersed mainly with hydrophobic residues. The S4 sequence is proposed to function as the voltage sensor and to move in response to membrane depolarization, triggering conformational changes that open the channel. This hypothesis has been tested in previous studies which revealed that mutations of the S4 basic residues often shift the curve of voltage dependence of activation along the voltage axis. We find that comparable or larger shifts are caused by conservative substitutions of hydrophobic residues in the S4 sequence of the Shaker K+ channel. We suggest that the S4 structure plays an essential role in determining the relative stabilities of the closed and open states of the channel.     

Synergistic action of both Aspergillus niger and Burkholderia cepacea in co-culture increases phosphate solubilization in growth medium

Co-inoculation of the fungus Aspergillus niger and the bacterium Burkholderia cepacia was undertaken to understand the interaction between different species of phosphate-solubilizing microorganisms (PSM). PSM were inoculated in a single or mixed (A. niger-B. cepacia) culture. During 9 days of incubation, microbial biomass was enhanced, accompanied with increases in the levels of soluble phosphate and titratable acidity, as well as increased acid phosphatase activity. Production of acids and levels of phosphate solubilization were greater in the co-culture of A. niger-B. cepacia than in the single culture. The quantity of phosphate solubilized by the co-culture ranged from 40.51 ± 0.60 to 1103.64 ± 1.21 μg PO(4) 3- mL(-1) and was 9-22% higher than single cultures. pH of the medium dropped from 7.0 to 3.0 in the A. niger culture, 3.1 in the co-culture, and 4.2 in the B. cepacia culture. On the third day of postinoculation, acid production by the co-culture (mean 5.40 ± 0.31 mg NaOH mL(-1)) was 19-90% greater than single cultures. Glucose concentration decreased almost completely (97-99% of the starting concentration) by the ninth day of the incubation. These results show remarkable synergism by the co-culture in comparison with single cultures in the solubility of CaHPO(4) under in vitro conditions. This synergy between microorganisms can be used in poor available phosphate soils to enhance phosphate solubilization.