Achromobacter Cycloclastes Research Articles

Crystallization of nitrite reductase from Achromobacter cycloclastes

Crystals of a green copper-containing nitrite reductase from Achromobacter cycloclastes, which diffract to high resolution, belong to the cubic space group P2 13, with a = b = c = 98.4 A ̊ . Crystals of a nitrite reductase from Alcaligenes faecalis S-6 have been made, and belong to space group P2 12 12 1, with a = 77.3 A ̊ , b = 94.6 A ̊ and c = 141 A ̊ . Crystals of the blue copper protein from Ac. cycloclastes have also been obtained: these belong to space group P2 12 12, with cell dimensions a = 34.9 A ̊ , b = 91.1 A ̊ and c = 36.7 A ̊ (1 Å = 0.1 nm).

Trapping of nitric oxide produced during denitrification by extracellular hemoglobin.

A spectrophotometric method has been developed that uses extracellular hemoglobin (Hb) to trap nitric oxide (NO) released during denitrification as nitrosyl hemoglobin (HbNO). The rate of complexation of NO with Hb is about at the diffusion controlled limit for protein molecules and the product, HbNO, is essentially stable. Hb was added to an anaerobic bacterial suspension and denitrification was initiated with either KNO2 or KNO3. HbNO formation was observed for six species of denitrifying bacteria and showed isosbestic points at 544, 568, and 586 nm. Cellular NO production, presumably by nitrite reductase, was kinetically distinct from the much slower chemical reaction of Hb with KNO2 to form methemoglobin and HbNO. The rate of HbNO formation was proportional to cell density, essentially independent of pH from 6.8 to 7.4, nearly zero order in [Hb] and, at least with Paracoccus denitrificans, strongly inhibited by rotenone and antimycin A. The Cu chelator, diethyldithiocarbamate, had no effect on HbNO formation by Pa. denitrificans, but abolished that by Achromobacter cycloclastes which uses a Cu-containing nitrite reductase known to be inactivated by the chelator. HbNO formation did not occur with non-denitrifying bacteria. The stoichiometry at high [Hb] for conversion of Hb to HbNO was 1.3-1.8 KNO2 per Hb for Pa. denitrificans, Pseudomonas aeruginosa, and A. cycloclastes and about 3.4 for Pseudomonas stutzeri. The former range of values corresponds to a partition of about 2 N atoms in 3 toward trapping and 1 in 3 toward reduction on the pathway to N2. Nitrogen not trapped appeared largely as N2O in presence of acetylene. The results are consistent with a model in which NO is a freely diffusible intermediate between nitrite and N2O, providing that nitric oxide reductase is or nearly is a diffusion controlled enzyme.

Use of a chemiluminescence detector for quantitation of nitric oxide produced in assays of denitrifying enzymes

We have developed a closed-flow system that continuously sweeps away gases evolved in enzyme assay mixtures into a commercially available oxides of nitrogen (NO x) analyzer for the quantitation of any nitric oxide present in these gases. The system enabled us to study both the stoichiometry and the kinetics of NO production by a copper-containing nitrite reductase (EC 1.7.99.3) purified from Achromobacter cycloclastes IAM 1013. In addition to its much greater sensitivity in comparison with standard gas chromatographic (GC) techniques, the method offers the advantage that NO, a very reactive free radical, is immediately swept away and quantitated, obviating the necessity for periodic manipulations and disturbances of the reaction mixture characteristic of other GC quantitations. The characteristics of the system are discussed and its utility in studies of the kinetics and stoichiometry of NO production from nitrite is confirmed by comparison with results obtained using manometric and GC techniques.

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

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

Kinetic studies of the copper nitrite reductase from [formula omitted] and its interaction with a blue copper protein

Transient state, burst and steady state kinetics of reactions of the blue copper nitrite reductase (NIR) and blue copper protein from Achromobacter cycloclastes are investigated. The two copper-containing species are reacted with each other and where possible with dithionite, ascorbate and nitrite. Both copper proteins are fully reduced by dithionite with both S 2O 4 2− and SO 2 • species active. NIR is only partially reduced by ascorbate in an unusual biphasic reaction consistent with complete reduction of type-one copper followed by partial reduction of type-two copper. The rate of reduction of the type-one copper is accelerated using phenazine methosulfate as mediator. Nitrite can oxidize dithionite-reduced NIR but cannot reduce oxidized NIR. Rate constants were determined for all observed reactions.

Detergent inhibition of nitric-oxide reductase activity

Gas chromatography revealed that exposure of extracts of the denitrifiers ‘Achromobacter cycloclastes’, Paracoccus denitrificans, Pseudomonas aeruginosa and Pseudomonas perfectomarina to Triton X-100 inhibited reduction of NO to N 2O, and thus concomitantly inhibited reduction of NO 2 − to N 2O. After exposure of extracts to Triton X-100, the ratio of H + consumed to NO 2 − added decreased from approx. 2.0 (for untreated extracts) to approx. 1.5, which indicated that NO 2 − was reduced to NO by the treated extracts. Addition of a CHAPS-soluble extracts (devoid of nitrite reductase activity but rich in nitric-oxide reductase activity) to the Triton X-100-treated extract of P. denitrificans restored capacity for reduction of NO 2 − on to N 2O. Exposure to either the NO that accumulated from reduction of NO 2 − or to enthetic NO transiently inhibited rates of NO 2 − reduction in Triton X-100-treated extracts. Use of an Oxides of Nitrogen analyzer indicated that only 5–33% of NO 2 − reduced by untreated extracts appeared in the stripping gas as NO, whereas 80–95% of NO 2 − reduced by Triton X-100-treated extracts was recovered as NO.

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.

Differentiation ofc,d1cytochrome and copper nitrite reductase production in denitrifiers

Abstract Gas chromatographic analyses revealed that rates of release of nitrous oxide from nitrite or nitric oxide in extracts of the c , d 1 cytochrome nitrite reductase-producing denitrifiers, Paracoccus denitrificans and Pseudomonas perfectomarina , were unaffected by preincubation with the metal chelator, diethyldithiocarbamate (DDC). In contrast, preincubation with DDC completely inhibited generation of nitrous oxide from nitrite in extracts of copper protein nitrite reductase-producing denitrifiers, “ Achromobacter cycloclastes ” and Rhodopseudomonas sphaeroides forma species denitrificans . Pre-exposure to DDC lessened but did not completely inhibit nitric oxide reduction in extracts of the copper protein nitrite reductase-producing denitrifiers. Proton consumption values resulting from pulsing with nitrite were similarly completely inhibited by preincubation with DDC of extracts of the two copper protein-producing denitrifiers. Uptake values related to pulsing with nitric oxide were also lessened but not completely inhibited by prior exposure to DDC. As anticipated, proton consumption was not affected by preincubation with DDC in extracts of P. denitrificans pulsed with nitrite or nitric oxide. Differential sensitivity of copper protein nitrite reductase activity to DDC could provide the simple assay method needed for determination of the distribution of two types of nitrite reductase producers among populations of denitrifiers in nature.

Achromobacter cycloclastes Nitrite Reductase The Function of Copper, Amino Acid Composition, and ESR Spectra1

1. Dialysis against cyanide at pH 7 of Achromobacter cycloclastes nitrite reductase [EC 1.7.99.3] of a dissimilatory type led to the removal of about 50% of the copper from the enzyme molecule, with a concomitant decrease of the enzymatic activities. It was inferred that enzyme-bound copper atoms play an essential role in the catalytic activities of the enzyme. 2. The amino acid composition of the enzyme was determined after acid hydrolysis. 3. ESR spectra of the frozen solution and lyophilized powder of the nitrite reductase predominantly showed the presence of two kinds of copper: Type 1 Cu2+, which had narrow and sharp hyperfine splitting, and Type 2 Cu2+, which had broader hyperfine splitting. The bond between the oxidized enzyme and nitrite seems to be ionic.

Purification and some properties of Achromobacter cycloclastes azurin.

Purification and some properties of Achromobacter cycloclastes azurin.

Incorporation of 14C-Labelled Glycerol and γ-Aminobutyric Acid into Vitamin B6 in a Cell-suspension of Achromobacter cycloclastes

Incorporation of 14C from various 14C-labelled compounds into vitamin B6 (abbreviate as B6) in a cell-suspension of B6-producing bacteria, Achromobacter cycloclastes A.M.S. 6021, was studied by using a newly designed purification procedure of the radioactive B6. The procedure consisted of Sephadex G–25 gel filtration, Dowex 50W–X8 column chromatography, Amberlite CG–50 column adsorption, powdered cellulose partition column chromatography, crystallization and sublimatography. It was observed that the labels both from 1,3- and from 2-labelled glycerol were clearly incorporated into B6 and the label of 14C-labelled γ-aminobutyric acid was also incorporated. The incorporation of 14C from 14C-labelled glycerol or γ-aminobutyric acid was affected by the addition of cold γ-aminobutyric acid or glycerol. The implication of these compounds as precursors of B6 was discussed.

Distribution Patterns of 14C in the Labelled Vitamin B6 Prepared with the Cell-suspension of Achromobacter cycloclastes

Distribution Patterns of 14C in the Labelled Vitamin B6 Prepared with the Cell-suspension of Achromobacter cycloclastes

Studies on the Biosynthesis of Vitamin B6

The production of vitamin B6 (B6) compounds with a cell-suspension of Achromobacter cycloclastes A.M.S. 6201 under various conditions were examined. An obvious accumulation of B6 compounds in the incubation medium with a cell-suspension of the organism harvested at the middle to later part of exponential phase of growth was observed. γ-Aminobutyric acid or β-alanine was found to stimulate the B6 production markedly, when they were added to the incubation mixture together with glycerol. Some discussion on the implication of these compounds as precursors of B6 was presented.

Studies on the Biosynthesis of Vitamin B6

The production of vitamin B6 (B6) compounds with a cell-suspension of Achromobacter cycloclastes A.M.S. 6201 under various conditions were examined. An obvious accumulation of B6 compounds in the incubation medium with a cell-suspension of the organism harvested at the middle to later part of exponential phase of growth was observed. γ-Aminobutyric acid or β-alanine was found to stimulate the B6 production markedly, when they were added to the incubation mixture together with glycerol. Some discussion on the implication of these compounds as precursors of B6 was presented.

Studies on the Utilization of Hydrocarbons by Microorganisms

Taxonomical studies on ten strains of hydrocarbon-utilizing bacteria reported in previous paper, which produced various kinds of amino acid, were carried out. They were Achromobacter cycloclastes, Achromobacter delmarvae, Bacillus species, Corynebacterium species, Micrococcus species. Many of them were not identical with the species which are described in Bergey's Manual of 7th Edition.