Application of gas-liquid chromatography to the routine identification of nonfermenting gram-negative bacteria in clinical specimens.

A total of 430 strains of glucose-nonfermenting gram-negative bacteria representing 35 species were analyzed for their cellular fatty acid composition by gas-liquid chromatography (GLC). On the basis of qualitative differences in their cellular fatty acid composition, these bacteria could be divided into 19 distinct chromatographic groups. Eight Pseudomonas species, Achromobacter xylosoxidans, group Vd, and Agrobacterium radiobacter were identified from their fatty acid compositions alone. The other glucose-nonfermenting gram-negative bacterial species studied here, classified within nine distinct GLC groups, were easily recognized by using the GLC fatty acid analysis supplemented with a limited number of conventional biochemical tests. The results support the hypothesis that bacterial fatty acid composition is rather specific and that qualitative GLC fatty acid analysis can be adapted in the clinical laboratory either to provide additional criteria for differentiation of closely related groups or to serve as a rapid and highly reproducible method for their routine identification.

Purification and some properties of a 2Fe ferredoxin in Pseudomonas ovalis.

A [2Fe-2S] ferredoxin was found in Pseudomonas ovalis which was grown in a medium supplemented with glucose and ammonium sulfate. The molecular weight of the 2Fe ferredoxin was estimated to be 13,000. It contained 2.2 gramatoms of non-heme iron and 2.3 gramatoms of acid-labile sulfur per mole protein. The absorption and circular dichroism spectra were characteristic of those of [2Fe-2S] type ferredoxins, especially adrenodoxin and putidaredoxin. The electron paramagnetic resonance spectrum of the reduced protein showed an axial symmetry (g = 2.020, g = 1.939). The amino acid composition was determined.

Ornithine-containing lipids of some Pseudomonas species.

Ornithine-containing lipids purified by thin-layer chromatography were found to represent 2-15% of the total extractable cellular lipids in two or three strains each of four Pseudomonas species: P. aeruginosa, P. fluorescens, P. stutzeri and P. cepacia. The structures of the ornithine-containing lipids were elucidated by chemical analysis, thin-layer chromatography, gas-liquid chromatography, gas-liquid chromatography/mass spectrometry (electron impact or secondary ion) and infrared absorption spectroscopy. At least six molecular species of ornithine-containing lipids were present in common in all of the preparations of the four Pseudomonas species. The structure which was the most abundantly in P. fluorescens (about 60% of the total amount of the ornithine-containing lipid) was 3-hydroxyhexadecanoic acid amide-linked to ornithine and esterified to hexadecanoic acid. In addition to this structure, 3-hydroxyoctadecenoic acid amide-linked to ornithine and esterified to hexadecanoic acid was a dominant structure in the ornithine-containing lipids of P. aeruginosa, P. stutzeri or P. cepacia. In P. cepacia, another ornithine-containing lipids with a terminal polar fatty acid, 3-hydroxyhexadecanoic acid amide-linked to ornithine and esterified to 2-hydroxynonadecacyclopropanoic acid or 2-hydroxyoctadecenoic acid, was found; its content, which represented 8-11% of the total extractable cellular lipids, was higher than that of the ornithine-containing lipids with a terminal nonpolar fatty acid. These ornithine-containing lipids exhibited hemagglutinating activity. Additionally, it was very interesting that hydroxy fatty acids included in the ornithine-containing lipids were not found in the phospholipids which represented more than 80% of the total extractable cellular lipids.

Isolation of a new phenazine antibiotic, DOB-41, from Pseudomonas species.

A new phenazine antibiotic, DOB-41, was isolated from the culture broth of a Pseudomonas strain. The antibiotic obtained as yellow crystals showed UV maxima at 255 nm and 370 nm. A molecular formula, C19H18N2O6, was indicated by elemental analysis and mass spectrometry. The structure was elucidated by X-ray diffraction analysis. The antibiotic exhibited inhibitory activity against Gram-positive bacteria, and antitumor effect against leukemia P388 in mice.

Lipopolysaccharides of Pseudomonas spp. that stimulate plant growth: composition and use for strain identification.

The outer membrane proteins of a series of fluorescent, root-colonizing, plant-growth-stimulating Pseudomonas spp. having been characterized (L. A. de Weger et al., J. Bacteriol. 165:585-594, 1986), the lipopolysaccharides (LPSs) of these strains were examined. The chemical composition of the LPSs of the three best-studied plant-growth-stimulating Pseudomonas strains WCS358, WCS361, and WCS374 and of P. aeruginosa PAO1 as a reference strain was determined and appeared to differ from strain to strain. The 2,6-dideoxy-2-aminosugar quinovasamine was the most abundant compound in the LPS of strain WCS358. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified LPS and of proteinase K-treated cell envelopes revealed ladderlike patterns for most of these strains. These patterns were not substantially influenced by differences in culture conditions. Analysis of proteinase K-treated cell envelopes of 24 root-colonizing Pseudomonas spp. revealed a unique band pattern for each strain, suggesting a great variety in the LPS structures present in these root colonizers. Therefore, electrophoretic analysis of LPS can be used for characterization and identification of the fluorescent root-colonizing Pseudomonas strains.

The primary structures of Pseudomonas AM1 amicyanin and pseudoazurin. Two new sequence classes of blue copper proteins.

The amino acid sequences of two blue copper proteins from the pink facultative methylotroph Pseudomonas AM1 (N.C.I.B. 9133) were determined. They each consist of a single polypeptide chain and bind one copper atom. Amicyanin contains 99 and pseudoazurin 123 residues. Copper-binding sites, consisting of the side chains of two histidine, one cysteine and one methionine residues, can be recognized in each protein by analogy with azurin and plastocyanin, but the spacings of the ligand residues are different, and other sequence similarity is limited. Proteins that are in the pseudoazurin sequence class can be recognized in some strains of Alcaligenes, and probably also in Paracoccus denitrificans. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50130 (23 pp.) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1985) 225, 5.

Phospholipid metabolism in Pseudomonas BAL-31 infected with lipid-containing bacteriophage PM2.

Infection of Pseudomonas BAL-31 with the lipid-containing bacteriophage PM2 resulted in no detectable change in the rate of phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) biosynthesis. An increase in the PG content of infected cultures was not seen until the cultures began to lyse, and this increase was in fact only a relative increase resulting from the extensive turnover of PE at the onset of culture lysis. Turnover studies revealed that the glycerol, phosphorus fatty acid, and ethanolamine moieties of PE turned over simultaneously at the time of lysis, and therefore made it unlikely that there was a PE to PG conversion during the latent period of the phage. The lipid found in the bacteriophage did not reflect a preferential selection for lipid synthesized before or after infection, but in fact reflected the composition of the host membrane at the time the phage were assembled. The use of a modified medium that allowed the cultivation of Pseudomonas BAL-31 as a prototroph and resulted in reliable lysis times of infected cultures led us to the conclusion that PM2 infection effects little change in host phospholipid metabolism, and that there is sufficient PG in the host cytoplasmic membrane to account for a full burst of phage. As a result of the reliable lysis times that we have achieved, we concluded that certain metabolic events, i.e., PE turnover, are lytic phenomena and must not be confused with events relevant to the biosynthesis and maturation of the phage.

Localization of selenium in bacterial cells using TEM and energy dispersive X-Ray analysis.

Bacteria isolated from lake sediment samples reduced sodium selenite to elemental selenium. Finestructural observations were made on a number of different bacterial species cultured in the presence of sodium selenite. Examination of Escherichia coli and a Pseudomonas species revealed electron-dense deposits of irregular shape, composed of smaller units, within the cytoplasm but not on the cell wall and cell membrane. Cells of Aeromonas and Flavobacterium species exhibited conspicuous intranuclear fibrillary aggregates and different electron-dense inclusions. It appeared that the membrane structures were somewhat more easily stained in some bacterial cells after growth on agar plates containing sodium selenite. The deposits and fibrillary accumulations were interpreted to contain selenium on the basis of energy dispersive X-ray analysis. Control preparations and cells grown in the presence of sodium selenate were void of any fine-structural abnormalities. Alterations in fine structure are discussed in relation to the metabolism of selenium by bacterial cells and possible sites of inhibition.

Heterogeneity in lipid composition of the outer membrane and cytoplasmic membrane and cytoplasmic membrane of Pseudomonas BAL-31.

The outer membranes and cytoplasmic membranes of the marine bacterium Pseudomonas BAL-31 were separated by washing the cells three times in 0.5 M NaCl and twice in 0.5 M sucrose. Electron microscopy during the removal of membranes revealed that the outer membranes fragmented in a regular manner to give rise to fairly uniform vesicles measuring approximately 140 nm in diameter. Isolated outer membranes had a buoyant density in sucrose of 1.230 g per cm(3), whereas the cytoplasmic membranes had a density of 1.194 g per cm(3). The removal of the outer membrane during the application of this procedure was monitored by measuring the release of 2-keto-3-deoxyoctulosonic acid and phospholipid. The cells lost 85.5% of their 2-keto-3-deoxyoctulosonic acid and 47.3% of their phospholipid during this treatment. Complete recovery of outer membrane material could be achieved. The removal of 25.5% of the 2-keto-3-deoxyoctulosonic acid and 0.9% of the phospholipid rendered the cells sensitive to lysis with Triton X-100. The phospholipid composition of the outer membrane was calculated to be 78.9% phosphatidylethanolamine and 16.1% phosphatidylglycerol. The phospholipid composition of the cytoplasmic membrane proved to be 71.5% phosphatidylethanolamine and 23.5% phosphatidylglycerol. The fatty acid composition was also found to be quantitatively heterogeneous between the two membranes.

The amino acid sequences of cytochromes c-551 from three species of Pseudomonas.

The amino acid sequences of the cytochromes c-551 from three species of Pseudomonas have been determined. Each resembles the protein from Pseudomonas strain P6009 (now known to be Pseudomonas aeruginosa, not Pseudomonas fluorescens) in containing 82 amino acids in a single peptide chain, with a haem group covalently attached to cysteine residues 12 and 15. In all four sequences 43 residues are identical. Although by bacteriological criteria the organisms are closely related, the differences between pairs of sequences range from 22% to 39%. These values should be compared with the differences in the sequence of mitochondrial cytochrome c between mammals and amphibians (about 18%) or between mammals and insects (about 33%). Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50015 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

Production of bacterial cells from methane.

A mixed methane-oxidizing bacterial culture capable of stable and predictable growth in continuous culture was isolated. The culture consisted of two types of gram-negative nonsporulating rods resembling pseudomonads. The culture grew well at 45 C on an inorganic medium without asepsis. Specific metal requirements for Ca(2+), Cu(2+), MoO(4) (2-), Zn(2+), Mn(2+), Mg(2+), and Fe(3+) (or Fe(2+)) were shown. The cells grown in continuous culture contained 11.7 to 12.1% total nitrogen. From an animal nutrition standpoint, the distribution of amino acids was satisfactory. The continuous fermentation was operated over a range of steady-state dilution rates from 0.085 to 0.301 hr(-1). The maximum specific growth rate for the culture, mu(max), was 0.303 hr(-1) (doubling time 2.29 hr). The average yield for all fermentations analyzed was 0.616 g (dry weight of cells per g of methane used and 0.215 g (dry weight) of cells per g of oxygen used. The yields on both methane and oxygen were higher for the oxygen-limited than for the methane-limited fermentations. The maximum productivity attained in the fermentor was 2.39 g (dry weight) of cells per hr per liter at a dilution rate of 0.187 hr(-1) and a cell concentration of 12.8 g (dry weight) of cells per liter. The limit on maximum cell productivity was determined only by the mass transfer rate of oxygen in the fermentor. The simultaneous volumetric mass-transfer coefficients (k(L)a in hr(-1)) for oxygen and methane were determined. The results appear to indicate an oxygen to methane mass-transfer coefficient ratio of approximately 1.4.

Biosynthesis of ubiquinone in non-photosynthetic gram-negative bacteria.

1. The polyprenylphenol and quinone complements of the non-photosynthetic Gram-negative bacteria, Pseudomonas ovalis Chester, Proteus mirabilis and ;Vibrio O1' (Moraxella sp.), were investigated. 2. Ps. ovalis Chester and Prot. mirabilis were shown to contain 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-2-polyprenyl-1,4-benzoquinones, 5-demethoxyubiquinones, ubiquinones, an unidentified 1,4-benzoquinone [2-polyprenyl-1,4-benzoquinone (?)] and ;epoxyubiquinones'. ;Vibrio O1' was shown to contain only 5-demethoxyubiquinones, ubiquinones and ;epoxyubiquinones'. 3. It was established that in Ps. ovalis Chester 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-2-polyprenyl-1,4-benzoquinones, 5-demethoxyubiquinones and 2-polyprenyl-1,4-benzoquinones (?) are precursors of ubiquinones. 4. Intracellular distribution studies showed that in Ps. ovalis Chester ubiquinone and its prenylated precursors are localized entirely on the protoplast membrane. 5. Investigations into the oxygen requirements for ubiquinone biosynthesis by Ps. ovalis Chester showed that the organism could not convert p-hydroxybenzoic acid into ubiquinone in the absence of oxygen, although it could convert a limited amount into 2-polyprenylphenols. 6. Attempts were made to prepare cell-free preparations capable of synthesizing ubiquinone. Purified protoplast membranes of Ps. ovalis Chester were found to be incapable of carrying out this synthesis, even when supplemented with cytoplasm. With crushed-cell preparations of Ps. ovalis Chester, organism PC4 (Achromobacter sp.) and Escherichia coli, synthesis was observed, although this was attributable in part to a small number of intact cells present in the preparations.