[The DNA nucleotide composition of bacteria oxidizing diethylene glycols]. / Nukleotidnyi sostav DNK bakterii, okisliaiushchikh diétilenglikol'.

Nucleotide composition of DNA has been analyzed in bacteria composing the diethylene glycol-oxidizing association. It is shown that the microorganisms under study are representatives of genera Alkaligenes and Achromobacter.

Photophysics of metalloazurins.

The fluorescence lifetimes of Cu(II), Cu(I), Ag(I), Hg(II), Co(II), and Ni(II) azurin Pae from Pseudomonas aeruginosa and Cu(II), Cu(I), and Hg(II) azurin Afe from Alcaligenes faecalis were measured at 295 K by time-correlated single-photon counting. In addition, fluorescence lifetimes of Cu(II) azurin Pae were measured between 30 and 160 K and showed little change in value. Ultraviolet absorption difference spectra between metalloazurin Pae and apoazurin Pae were measured, as were the fluorescence spectra of metalloazurins. These spectra were used to determine the spectral overlap integral required for dipole-dipole resonance calculations. All metalloazurins exhibit a reduced fluorescence lifetime compared to their respective apoazurins. Forster electronic energy transfer rates were calculated for both metalloazurin Pae and metalloazurin Afe derivatives; both enzymes contain a single tryptophyl residue which is located in a different position in the two azurins. These azurins have markedly different fluorescence spectra, and electronic energy transfers occur from these two tryptophyl sites with different distances and orientations and spectral overlap integral values. Intramolecular distances and orientations were derived from an X-ray crystallographic structure and a molecular dynamic simulation of the homologous azurin Ade from Alcaligenes denitrificans, which contains both tryptophyl sites. Assignments were made of metal-ligand-field electronic transitions and of transition dipole moments and directions for tryptophyl residues, which accounted for the observed fluorescence quenching of Hg(II), Co(II), and Ni(II) azurin Pae and Cu(II) and Hg(II) azurin Afe. The fluorescence of azurin Pae is assigned as a 1Lb electronic transition, while that of azurin Afe is 1La. The marked fluorescence quenching of Cu(II) azurin Pae and Cu(I) azurin Pae and Afe is less well reproduced by our calculations, and long-range oxidative and reductive electron transfer, respectively, are proposed as additional quenching mechanisms. This study illustrates the application of Forster electronic energy transfer calculations to intramolecular transfers in structurally well characterized molecular systems and demonstrates its ability to predict observed fluorescence quenching rates when the necessary extensive structural, electronic transition assignment, and spectroscopic data are available. The agreement between Forster calculations and quenching rates derived from fluorescence lifetime measurements suggests there are limited changes in conformation between crystal structure and solution structures, with the exception of the tryptophyl residue of azurin Afe, where a conformation derived from a molecular simulation in water was necessary rather than that found in the crystal structure.

Numerical analysis of electrophoretic protein patterns of 'Achromobacter' group B, E and F strains from human blood.

Thirty-two clinical strains representing 'Achromobacter' groups B, E and F were characterized by one-dimensional SDS-PAGE of cellular proteins. All the strains were isolated from blood samples from hospital patients in the United Kingdom. The protein patterns, which contained 40 to 45 discrete bands, were highly reproducible and were used as the basis for a numerical analysis which included all the protein bands. The 32 'Achromobacter' strains formed two clusters at the 77% S level. The first, phenon 1, included the 28 group B and the two group E strains and the second, phenon 2, contained the two strains of group F. The strains in each phenon were characterized by a clearly distinct pattern of protein bands. Phenon 1 could be further divided at the 87% S level into three subphenons which correlated with differences in the principal bands found between 40.0 and 42.5 kD. Strains of group E clustered with group B strains from which they could not be distinguished by protein patterns. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides a useful method for the classification of this group of bacteria. Reference strains of each of the PAGE types identified are available from NCTC for inclusion in future studies.

Enhancement of polymorphonuclear leukocyte-mediated tumor cytotoxicity by serum factor(s).

It has been reported that beta-1,3-D-glucan isolated from Alcaligenes faecalis (TAK) promoted tumor cytolysis by mouse polymorphonuclear leukocytes (PMN). We investigated the effect of serum on mouse PMN tumor cytolysis induced by TAK and other PMN stimulators. Addition of fetal calf serum (FCS) to the cytolysis assay enhanced tumor cytolysis by PMN in a dose-dependent manner. Sera obtained from horses, mice, and rats were also effective enhancers of PMN tumor cytolysis. When FCS was added after the assay was under way, the enhancing effect decreased proportionally to the time elapsed. The enhancing activity was detected over a broad range of fractions with a peak at 170 kD by fractionation on a Superose 6 column. The responsible factor(s) in serum was stable after treatment at 60 degrees C, 30 min or after lowering the pH to 2. Mouse PMN stimulated with TAK increased production of hydrogen peroxide in the presence of FCS.

Biological effects of lipopolysaccharide from Achromobacter stenohalis on lymphocytes and macrophages.

The immunopotentiating activities of lipopolysaccharide from Achromobacter stenohalis (A-LPS) were examined. A-LPS was structurally atypical and gave no endotoxin shock in A-LPS-inoculated mice. Analysis in vitro showed that A-LPS was a potent activator of both macrophages and B-lymphocytes. After macrophage stimulation with A-LPS, interleukin-1 (IL-1) secretion, interferon (IFN) production and chemiluminescence (CL) response were induced. A-LPS was a potent mitogen for spleen lymphocytes. However, induction of interleukin-2 (IL-2) secretion in T lymphocytes was not observed. These activities of A-LPS were similar to or higher than that of enterobacterial LPS.

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.

1H nuclear magnetic resonance study of the protonation behaviour of the histidine residues and the electron self-exchange reaction of azurin from Alcaligenes denitrificans.

The proton nuclear magnetic resonance spectrum of azurin from Alcaligenes denitrificans at pH 6.0 and 309 K is reported. Proton signals from all methionine and histidine residues (among them the copper ligands) have been assigned. The data have been used to study the pH behaviour of His35 and to establish the electron self-exchange rate of the protein. His35 appears to be protonated at pH less than 4.5, possibly after rupture of a salt bridge. No effects of this protonation on the tertiary structure around the copper site are observed, however, contrary to the case of Pseudomonas aeruginosa azurin. The electron self-exchange rate amounts to 4 x 10(5) M-1 S-1 at pH 6.7 and 297 K. The data support the conclusion that the electron self-exchange takes place by way of the hydrophobic surface patch around His117, and that His35 is not involved in this reaction. Oxidation of azurin increases the acidity of the freely titrating His32 and His83 by 0.07 and 0.25 pKa units, respectively. The data can be used to test the theory of electrostatic interactions in proteins. The optical extinction coefficient at 625 nm was experimentally determined and amounts to 4.8(+/- 0.1) x 10(3) M-1 cm-1.

EPR of azurins from Pseudomonas aeruginosa and Alcaligenes denitrificans demonstrates pH-dependence of the copper-site geometry in Pseudomonas aeruginosa protein.

The X- and Q-band EPR spectra of Pseudomonas aeruginosa (63Cu)azurin and Alcaligenes denitrificans azurin have been measured at pH = 5.2 and 9.2, in the presence and absence of 40% glycerol. The EPR spectra of both proteins could properly be simulated by taking into account a spread in the tetrahedral angle of the copper site. The change in the EPR spectrum of Pseudomonas aeruginosa (63Cu)azurin that is observed upon an increase of the pH from 5.2 to 9.2 is consistent with a small decrease of the average tetrahedral angle from 61 degrees to 60 degrees. This geometrical change is consistent with the interpretation of earlier NMR and EXAFS observations. No pH effect is observed for Alcaligenes denitrificans azurin, in agreement with predictions based on crystallographic evidence. Glycerol has only a marginal effect on the appearance of the EPR spectra, and does not alleviate the "g-strain."

The crystal structure of pseudoazurin from Alcaligenes faecalis S-6 determined at 2.9 A resolution.

The three-dimensional structure of pseudoazurin, a single copper-containing protein from Alcaligenes faecalis strain S-6, has been determined at 2.9 A resolution by X-ray crystallography. The sequences of two other pseudoazurins from Pseudomonas AM1 and Achromobacter cycloclastes may also be accommodated in this structure. The structure, an eight-stranded beta-barrel, resembles closely those of plastocyanin and azurin. It possesses two extra alpha-helices at the C-terminus, whereas azurins have an alpha-helical flap in the middle of their sequences.

Differentiation of Bordetella avium and related species by cellular fatty acid analysis.

The fatty acids of 18 strains of Bordetella avium, 3 strains of Alcaligenes faecalis, 5 strains of Bordetella bronchiseptica, and 12 strains of a B. avium-like organism were examined by gas chromatography-mass spectrometry. The presence of a significant amount of the acid 2-OH C14:0 characterized B. avium and the B. avium-like organism. B. avium and the B. avium-like organism differed in their relative concentrations of C16:1 and 3-OH C14:0 acids. B. bronchiseptica and A. faecalis were distinguishable by comparison of the relative concentrations of C18:0 and C18:1 acids.

A note on the isoprenoid quinone content of Bordetella avium and related species.

The isoprenoid quinone content of isolates of Bordetella avium (four strains), Alcaligenes faecalis (one strain), Bordetella bronchiseptica (one strain) and a Bordetella avium-like organism (four strains) was determined by reverse-phase high-performance liquid chromatography. All the isolates contained ubiquinones with eight isoprene units as the major component. No menaquinones were detected.

Properties and electron transfer specificity of copper proteins from the denitrifier "Achromobacter cycloclastes".

A blue copper protein (Mr 12,000) was purified from cells of "Achromobacter cycloclastes" grown as a denitrifier. When reduced, the blue copper protein transferred electrons to the copper protein nitrite reductase purified from the same cells, whereas a variety of cytochromes from denitrifiers failed to do so. Inclusion of a protease inhibitor, phenylmethylsulfonyl fluoride, in the buffers employed during preparation yielded purified blue copper protein with 18 more amino acid residues and two times more specific enzyme activity than other researchers have found.

The amino acid sequence of the blue copper protein of Alcaligenes faecalis.

The complete amino acid sequence of a blue copper protein from Alcaligenes faecalis S-6 has been determined. This protein is clearly homologous to pseudoazurins in Achromobacter cycloclastes and Pseudomonas AM1, more distantly related to plant plastocyanins, and markedly different from the azurin of Pseudomonas aeruginosa. Yet all of these proteins bind copper, and analogous ligands appear to be involved.

Contribution to the taxonomy of the turkey coryza agent: cellular fatty acid analysis of the bacterium.

Gas-liquid chromatography was used to analyze bacterial cellular fatty acids to elucidate the relatedness of the turkey coryza (TC) bacterium to Alcaligenes spp., Bordetella spp., and other gram-negative bacteria. The results indicated that the TC bacterium is not closely related to Alcaligenes faecalis or any of the reference strains of Alcaligenes and Bordetella studied. Most urease-positive bacterial isolates obtained from the upper respiratory tract of turkeys were identified as Bordetella bronchiseptica. It is suggested that Bordetella avium is a suitable designation for the TC bacterium formally called Bordetella-"like" and A. faecalis type I. It is also suggested that the nonpathogenic bacterium previously identified as type II A. faecalis be designated B. avium-like until further taxonomic studies are available. Furthermore, it is proposed that the term turkey coryza be used to refer to the disease induced by this bacterium.

Proton NMR spectroscopy of Alcaligenes cytochrome c556.

A high molecular-weight c-type cytochrome was purified from Alcaligenes faecalis ATCC 8750. Its weight was 40,000 daltons by sodium dodecyl sulfate-gel electrophoresis. Heme content was determined to be one heme per 40,000 daltons. Proton nuclear magnetic resonance-NMR-spectroscopy determined that the ferrous form is low spin. The detection of a methyl resonance at -3 ppm in the ferrous form indicated that methionine is a heme ligand in this state. The NMR spectrum of the ferric form at pH 7.2 revealed hyperfine shifted methyl resonances at 67.79, 63.17, 57.71, and 50.46 ppm. The large downfield shifts observed are indicative of high spin character. The ferric spectrum was pH-sensitive, indicating two pH-linked structural transitions with estimated pKs at 6.0 and 10.5. The first is interpreted as due to the ionization of a heme propionate. The second is interpreted as the acquisition of a strong field ligand and the subsequent conversion to a low spin ferric form. The ferricytochrome did not form complexes with cyanide, azide, or fluoride at pH 5.2 or 7.9.

Evidence for plasmid DNA in Alcaligenes faecalis.

A 10.6 megadalton plasmid was isolated from a virulent strain (NC-D) of Alcaligenes faecalis. Virulence and antibiotic sensitivity of this strain were compared with those characteristics of a mutant plasmid-free derivative, strain NC-D1. Strain NC-D1 was avirulent and lacked the streptomycin and sulfonamide resistances of the parent strain.

Ornithine-containing lipids in Thiobacillus A2 and Achromobacter sp.

20 bacterial strains (corresponding to 16 species) were screened for ornithine lipids. Only two species (Thiobacillus A2 and Achromobacter sp.) turned out to contain ornithine lipids (2.71 mmol/100 g and 0.38 mmol/100 g bacterial dry weight, respectively). In both ornithine lipids, a 3-hydroxy fatty acid was amide-linked to the alpha-amino group of ornithine, a normal fatty acid was ester-linked to the 3-hydroxy group of the former. The predominant fatty acids were 18:1(11) and 3-hydroxy-20:1(13) in Thiobacillus A2, 16:0 and 3-hydroxy-18:1(11) in Achromobacter sp. All monounsaturated fatty acids (with one exception) belonged to the (n-7) family. 11, 12-Epoxy octadecanoic acid was identified among the ester-linked fatty acids of Thiobacillus A2. Phosphatidylcholine was the principal phospholipid in both bacterial species.

A cleavage product of mouse C3 adsorbable to (1----3)-beta-D-glucan from Alcaligenes faecalis var. myxogenes IFO 13140.

By using affinity chromatography of TAK, a gel-forming antitumor (1----3)-beta-D-glucan produced by Alcaligenes faecalis var. myxogenes IFO 13140 also known as an activator of the alternative complement pathway (ACP), we obtained from mouse serum a cleavage product of C3 (tentatively designated as C3X). Binding between C3X and TAK seemed to be noncovalent. In contrast to C3, C3X had an intact beta chain (70,000 daltons) and instead of an alpha chain, two peptide chains (40,000 and 30,000 daltons) linked by disulfide bonds. C3X retained a portion of C3 antigenicity. By trypsinolysis C3 yielded a fragment similar to C3X on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that C3X is C3c. Since C3X was obtained by elution with a high salt solution from TAK incubated with ethylenediaminetetraacetate (EDTA)-serum (serum containing EDTA) but not from TAK incubated with EDTA-plasma, C3X could already be present in the serum before the activation of ACP by TAK.

Structure of azurin from Alcaligenes denitrificans at 2.5 A resolution.

The structure of the blue copper protein, azurin, from Alcaligenes denitrificans has been determined from an electron density map at a nominal resolution of 3.0 A. Four isomorphous heavy-atom derivatives, prepared with KAu(CN)2, uranyl acetate, Hg(NH3)2Cl2 and KAu(CN)2 + uranyl acetate (a double derivative) were used to calculate phases by the method of isomorphous replacement. The overall figure of merit was 0.61. The two molecules in the asymmetric unit are related by an approximate 2-fold axis. Independent interpretations of the density were made for the two molecules, and the structures have since been partially refined. After 12 refinement cycles, using the Hendrickson-Konnert restrained least-squares program, the R factor is 0.318 for data to 2.5 A resolution and there are no major conformational differences between the two molecules. Refinement is continuing. Eight extended strands of the polypeptide chain form a beta-barrel structure whose topology is the same as that of plastocyanin and the alternative folding proposed for Pseudomonas aeruginosa azurin. As in the latter two proteins, the copper atom forms three short bonds, with His-46 N delta 1, His117 N delta 1 and Cys112 S gamma, and one longer bond, with Met121 S delta, these four ligands forming a very distorted tetrahedron. A possible additional interaction, between copper and the carbonyl oxygen of Gly45, cannot be discounted at the present stage of the analysis. A surface hydrophobic patch, around the edge of the imidazole ring of His117 appears the most likely electron transfer locus. The sequences of azurin and plastocyanin have been aligned and the homology between the two proteins is discussed.