Bacterial siderophores: structures of pyoverdins Pt, siderophores of Pseudomonas tolaasii NCPPB 2192, and pyoverdins Pf, siderophores of Pseudomonas fluorescens CCM 2798. Identification of an unusual natural amino acid.

Pyoverdins were isolated and characterized respectively from the cultures of Pseudomonas tolaasii NCPPB 2192 (pyoverdins Pt, Pt A, and Pt B) and Pseudomonas fluorescens CCM 2798 (Pyoverdins Pf/1, Pf/2, Pf, Pf/3/1, and Pf/3/2) each grown in iron-deficient conditions. Their structures were established by using FAB-MS, NMR, and CD techniques. These siderophores are chromopeptides, and all but one (pyoverdin Pf/3/3) possess at the N-terminal end of their peptide chain the same chromophore that has been reported in pyoverdin Pa from Pseudomonas aeruginosa ATCC 15692 [Wendenbaum, S., Demange, P., Dell, A., Meyer, J. M., & Abdallah, M. A. (1983) Tetrahedron Lett. 24, 4877-4880] and pseudobactin B 10 from Pseudomonas B10 [Teintze, M., Hossain, M. B., Barnes, C. L., Leong, J., & Van der Helm, D. (1981) Biochemistry 20, 6446-6457] which is derived from 2,3-diamino-6,7-dihydroxyquinoline. In pyoverdins Pt this chromophore is bound to a linear peptide chain D-Ser-L-Lys-L-Ser-D-Ser-L-Thr-D-Ser-L-OHOrn-L-Thr-D-Ser-D-OHOrn (cyclic) which has its C-terminal end blocked by cyclic D-N delta-hydroxyornithine. In pyoverdins Pf, the peptide chain is also linear, SerCTHPMD-Gly-L-Ser-D-threo-OHAsp-L-Ala-Gly-D-Ala-Gly-L-O HOrn(cyclic), and contains an unusual natural amino acid which is the result of the condensation of 1 mol of serine and 1 mol of 2,4-diaminobutyric acid, forming a cyclic amidine. The pyoverdins Pt differ only in substituent bound to the nitrogen on C-3 of the chromophore, which is succinic acid in pyoverdin Pt A, succinamide in pyoverdin Pt, and alpha-ketoglutaric acid bound to the chromophore by its C-5 carbon atom in pyoverdin Pt B. Similarly, pyoverdin Pf/1, pyoverdin Pf/2, pyoverdin Pf (the major compound), and pyoverdin Pf/3/2 are substituted respectively by L-malic acid, succinic acid, L-malic amide, and succinamide. Pyoverdin Pf/3/3 has the same chromophore as azotobactin, the peptidic siderophore of Azotobacter vinelandii. These pyoverdins are very similar to pseudobactin B 10, the siderophore of Pseudomonas B10: they are linear peptides containing three bidentate groups strongly chelating Fe(III) and blocked at their N-terminal end by the catecholic chromophore and at their C-terminal end by cyclic N delta-hydroxyornithine. They differ therefore from other pyoverdins such as those from P. aeruginosa ATCC 15692 which contain a partly cyclic peptide [Briskot, G., Taraz, K., & Budzikiewicz, H. (1989) Liebigs Ann. Chem., 375-384].

Polymorphism of a lipid extract from Pseudomonas fluorescens: structure analysis of a hexagonal phase and of a novel cubic phase of extinction symbol Fd--.

The phase diagram of the Pseudomonas fluorescens lipid extract is unusual, in the sense that it displays a cubic phase straddled by a hexagonal phase. The hexagonal phase was studied over an extended concentration range, and the reflections were phased on the assumption that the structure contains circular cylinders of known radius. The cubic phase, whose extinction symbol is Fd--, was analyzed by reference to space group No. 227 (Fd3m). The phases of the reflections were determined by using a novel pattern recognition approach, based upon the notion that the average fourth power of the electron density contrast mean value of (delta r)4 is dependent on chemical composition but not on physical structure, provided that the function delta r(r) satisfies the constraints mean value of (delta r) = 0 and mean value of (delta r)2 = 1. As a further constraint, a shape normalization is used, in the form of a Gaussian apodization of the intensities, which has the effect of normalizing the curvature of the autocorrelation function at the origin. We analyzed two cubic samples of different composition: for each of them we generated all the phase combinations compatible with the X-ray scattering data and we searched for those whose mean value of (delta r)4 best agrees with the hexagonal phase. Taking advantage of the favorable properties of the phase diagram, we carefully explored the effects of various parameters; we concluded that the chemical composition of the phases being compared must be identical, that the X-ray scattering data should not be truncated artificially, and that the apodization must be mild so that the curvature takes a value intermediate between those corresponding to the raw data of the two phases. When all these precautions were taken, mean value of (delta r)4 was found to be remarkably invariant; this conclusion is important in view of the possible usefulness of the novel technique in tackling ab initio--and at very low resolution--structural problems of more general interest. The structure of the cubic phase consists of a 3D network of rods joined tetrahedrally 4 by 4 according to a diamond lattice and of a family of quasi-spherical disjointed micelles; the core of the rods and of the micelles is polar, and the interstices are filled by the hydrocarbon chains (structure of type II). All the dimensions (diameter of rods and micelles, area per chain at the polar/apolar interface) are consistent with the chemical properties of the system.(ABSTRACT TRUNCATED AT 400 WORDS)

[Fatty acid composition of lipid A in Pseudomonas fluorescens]. / Zhirno-kislotnyi sostav lipida A u Pseudomonas fluorescens.

The fatty acid composition of lipid A was studied using gas-liquid chromatography (GLC) and GLC-mass spectrometry in Pseudomonas fluorescens strains of biovars A, B, C, i, F and G, the type strain ATCC 13525 (biovar A) inclusive. The following fatty acids were identified as predominant in the composition of lipid A in the strains representing biovars A, B, C, i, F and G: 3-hydroxydecanoic (3-OH C10:0), 2-hydroxydodecanoic (2-OH C12:0), 3-hydroxydodecanoic (3-OH C12:0), dodecanoic (C12:0), hexadecanoic (C16:0), octadecanoic (C18:0), hexadecenoic (C16:1) and octadecenoic (C18:1) acids. Lipid A of a biovar G strain differed noticeably from other strains in its fatty acid composition. Its main components were as follows: 3-hydroxytetradecanoic (3-OH C14:0), 3-hydroxypentadecanoic (3-OH C15:0) and dodecanoic (C12:0) fatty acids. The coefficients of similarity were determined for lipid A specimens isolated from the studied strains of P. fluorescens by calculating their fatty acid composition with a computer.

Highly sensitive determination and characterization of intact cellular ester-linked phospholipids using liquid chromatography-plasma spray mass spectrometry.

Liquid chromatographic class separations of common cellular phospholipids combined with plasma spray ionization of the effluents were investigated. Comparison with true thermospray ionization involving ammonium acetate buffering revealed a gain in total ionization in the plasma spray of a factor of approximately 10 using a cation-exchange column and a solvent mixture consisting of acetonitrile-methanol-water (400:100:15, v/v). Plasma spray ionization studies of bovine brain polyphosphoinositides interrelated by the phosphate content in the inositol moiety showed almost identical monoglyceride and diglyceride ion clusters, indicating possibilities of studying the biochemical turnover of such phospholipids. Plasma spray ionization liquid chromatography-mass spectrometry of bacterial membrane phospholipids (Pseudomonas fluorescens) revealed possibilities of obtaining indications of individual fatty acid compositions from the spectra of the phosphatidylinositol and phosphatidylethanolamine fractions present. Conventional gas chromatographic fatty acid analysis agreed with the direct mass spectrometric structure elucidations. Interestingly, the two phospholipid classes had different relative fatty acid compositions with a significantly higher degree of cyclic fatty acids in the phosphatidyl ethanolamines. Plasma spray ionization yielded linear dose-response curves for both the monoglyceride and diglyceride fragment signals in the selected-ion monitoring mode. The detection limit for the monoglyceride and diglyceride species of phosphatidylcholine under the chromatographic and mass spectrometric conditions used was found to be in the picogram range.

Structure of the pseudomonad fungal antibiotic phenazine-1-carboxylic acid.

C13H8N2O2, Mr = 224.2, monoclinic, Cc, a = 3.955 (1), b = 19.278 (4), c = 13.468 (1) A, beta = 98.90 (2) degrees, V = 1015 (2) A3, Z = 4, D chi = 1.468 Mg m-3, lambda (Mo K alpha) = 0.7107 A, mu = 0.061 mm-1, F(000) = 464, T = 293 (2) K, R = 0.047 for 571 observed reflections. The crystal-structure determination of the title compound, a phenazine antibiotic from Pseudomonas fluorescens 2-79 (NRRL B-15132), confirms its structure as phenazine-1-carboxylic acid. The molecular packing is described by discrete stacks of molecules parallel to the a axis with the distance between the essentially planar molecules being ca 3.96 A; there are no significant intermolecular contacts in the lattice.

Immunological characterization of ice nucleation proteins from Pseudomonas syringae, Pseudomonas fluorescens, and Erwinia herbicola.

Antibodies were raised against the InaW protein, the product of the ice nucleation gene of Pseudomonas fluorescens MS1650, after protein isolation from an Escherichia coli clone. On Western blots (immunoblots), these antibodies recognized InaW protein and InaZ protein (the ice nucleation gene product of Pseudomonas syringae S203), produced by both E. coli clones and the source organisms. The InaZ protein appeared in P. syringae S203 during stationary phase; its appearance was correlated with the appearance of the ice nucleation-active phenotype. In contrast, the InaW protein occurred at relatively constant levels throughout the growth phases of P. fluorescens MS1650; the ice nucleation activity was also constant. Western analyses of membrane preparations of P. syringae PS31 and Erwinia herbicola MS3000 with this antibody revealed proteins which were synthesized with development of the nucleating phenotype. In these species the presence or absence of the nucleating phenotype was controlled by manipulation of culture conditions. In all nucleation-positive cultures examined, cross-reacting low-molecular-weight bands were observed; these bands appeared to be products of proteolytic degradation of ice nucleation proteins. The proteolysis pattern of InaZ protein seen on Western blots showed a periodic pattern of fragment sizes, suggesting a highly repetitive site for protease action. A periodic primary structure is predicted by the DNA sequence of the inaZ gene.

Purification and characterization of adhesive exopolysaccharides from Pseudomonas putida and Pseudomonas fluorescens.

In this study, the adhesive exopolysaccharides of strains of Pseudomonas putida and P. fluorescens, both isolated from freshwater epilithic communities, were examined with regard to their chemical composition, biosynthesis, and their role in adhesion. Electron microscopy showed that both strains were enrobed in fibrous glycocalyces and that these structures were involved in attachment of the cells to a solid surface and as structural matrices in the microcolony mode of growth. In batch culture experiments most of the extracellular polysaccharide of both strains was found to be soluble in the growth medium rather than being associated with bacterial cells. Exopolysaccharide was synthesized during all phases of growth, but when growth was limited by exhaustion of the carbon source, exopolysaccharide synthesis ceased whereas exopolysaccharide synthesis continued for some time after cessation of growth in nitrogen-limited cultures. Exopolysaccharide from both strains was isolated and purified. Pseudomonas putida synthesized an exopolysaccharide composed of glucose, galactose, and pyruvate in a ratio of 1:1:1; the P. fluorescens polymer contained glucose, galactose, and pyruvate in a ratio of 1:1:0.5, respectively. Polymers from both strains were acetylated to a variable degree.

Luciferin-luciferase assay of adenosine triphosphate from bacteria: a comparison of dimethylsulphoxide (DMSO) and acetone with other solvents.

The extraction of adenosine triphosphate (ATP) from six strains of bacteria, chosen for differences in cell-wall composition and habitat, was performed. The solvents were two in common use, Tris-ethylenediaminetetraacetate (Tris-EDTA) and trichloroacetic acid (TCA), and two promising, though less utilized solvents, dimethylsulphoxide (DMSO) and acetone. ATP was determined by the luciferin-luciferase reaction. Of the solvents used, DMSO and acetone were the most effective considering the different kinds of bacteria tested and, of these two, DMSO was the most convenient to use. Tris-EDTA was not as effective as the other solvents tested and TCA, which was effective with most strains, gave low yields when used with cultures grown in artificial seawater broth. Internal standards were used to determine if there were substances present that could inhibit the reaction of released ATP with the luciferin-luciferase reagent. Extracts of ATP, stored at -20 degrees C, were stable for up to 3 weeks.

Monomer sequence and acetylation pattern in some bacterial alginates.

The sequential structures and acetylation patterns of alginates from several strains of Azotobacter vinelandii and Pseudomonas species, including P. aeruginosa, P. putida, P. fluorescens, and P. mendocina, have been studied by 1H-n.m.r. spectroscopy. O-Acetyl groups were exclusively associated with the D-mannuronic acid residues and the degree of acetylation varied in the range 4-57%, depending upon the proportion of this acid in the polymer. 1H-N.m.r. spectroscopy of a naturally occurring and an artificially acetylated D-mannuronan made it possible to determine the degrees of acetylation at O-2, O-3, and O-2,3. The most conspicuous difference between alginates from A. vinelandii and the four Pseudomonas species was the complete absence of consecutive L-guluronic acid residues in the latter.

Levels of water-soluble vitamins in methanogenic and non-methanogenic bacteria.

The levels of seven water-soluble vitamins in Methanobacterium thermoautotrophicum, Methanococcus voltae, Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Bacteroides thetaiotaomicron were compared by using a vitamin-requiring Leuconostoc strain. Both methanogens contained levels of folic acid and pantothenic acid which were approximately two orders of magnitude lower than levels in the nonmethanogens. Methanobacterium thermoautotrophicum contained levels of thiamine, biotin, nicotinic acid, and pyridoxine which were approximately one order of magnitude lower than levels in the nonmethanogens. The thiamine level in Methanococcus voltae was approximately one order of magnitude lower than levels in the nonmethanogens. Only the levels of riboflavin (and nicotinic acid and pyridoxine in Methanococcus voltae) were approximately equal in the methanogens and nonmethanogens. Folic acid may have been present in extracts of methanogens merely as a precursor, by-product, or hydrolysis product of methanopterin.

Siderochromes from Pseudomonas fluorescens. I. Isolation and characterization.

Several iron-binding pigments (siderochromes) produced by Pseudomonas fluorescens have been isolated and partially characterized. They include ferribactin and various forms of pyoverdine, as well as some previously unreported compounds. In particular, the existence of ferribactin has been independently confirmed for the first time. Column and thin layer chromatographic procedures have been developed to fractionate, purify, and identify the siderochromes. We find ferribactin to contain nine amino acids, one residue each of glutamine, tyrosine, and glycine, and two each of serine, lysine, and N-hydroxyornithine, rather than 10 as earlier reported. Pyoverdine is a peptide with the same composition as ferribactin except for the absence of glutamine and the substitution of a fluorescent chromophore for tyrosine. Paper electrophoresis reveals an extra ionizable group in ferric pyoverdine relative to pyoverdine or ferribactin which provides that complex a definite cathodic mobility at pH 3. Optical spectra of the pyoverdine fluorescent component indicate that, in conjunction with the two hydroxamate groups, it is involved in the metal ion coordination, conferring on pyoverdine a dramatically increased affinity for Fe(III) relative to ferribactin.

Siderochromes from Pseudomonas fluorescens. II. Structural homology as revealed by NMR spectroscopy.

Ferribactin and the pyoverdines, siderochromes that are obtained from liquid cultures of Pseudomonas fluorescens cells, have been studied and compared by 1H and 13C NMR spectroscopy. The proton spectra of the iron-free compounds show that the pyoverdines share with ferribactin a common feature, formyl hydroxamic acid groups, that previously had only been observed in hadacidin, and antitumor antibiotic produced by Penicillium frequentans. The 1H and 13C NMR data confirm that ferribactin is a nonapeptide that contains two residues each of lysine and N6-formyl-N6-hydroxyornithine. This corrects an earlier report (Maurer, B., Müller, A., Keller-Schierlein, W., and Zähner, H. (1968) Arch. Mikrobiol. 60, 326-339) ascribing two acetyl hydroxamic acid groups and three lysyl residues to ferribactin. Similarly, the spectroscopic data show that pyoverdine lacks the Glx and Tyr residues present in ferribactin. On the basis of the compositional analogy exhibited by pyoverdine and ferribactin, it is suggested that the two siderochromes may be metabolically related. The 13C NMR spectra of pyoverdine indicate that its fluorescent component is a nine-carbon aromatic heterocycle, probably identical with an o-dihydroxyquinoline chromophore found in pseudobactin, a fluorescent siderophore produced by Pseudomonas B10.

Multiparameter appraoch to the "fingerprinting" of fluorescent Pseudomonads.

A recent study has shown that an emission-excitation matrix could be used to provide a unique fingerprint for the selective identification and characterization of certain species of Pseudomonas. This paper describes the results of a systematic study of the variables that contribute to the uniqueness of the fingerprint. Additional insight concerning the nature of the fluorescent pigments of these species is gained by analysis with a high-performance liquid chromatograph/video fluorometer combination. While providing information about the analytical variables contributing to the analysis, this combination also added the variable of chromatographic retention time for unambiguous fingerprinting.