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PubMed1981

Purification and properties of a copper-containing nitrite reductase from a denitrifying bacterium, Alcaligenes faecalis strain S-6.

A copper-containing nitrite reductase was purified and crystallized from a potent denitrifying bacterium, Alcaligenes faecalis strain S-6. The enzyme was composed of 4 subunits with a molecular weight of about 30,000, each containing 1 atom of Cu2+. Nitric oxide was identified as a main reduction product from nitrite in the enzyme-catalyzed reaction. The enzyme activity was inhibited strongly by KCN but only slightly by sulfhydryl reagents such as p-chloromercuribenzoate and N-ethylmaleimide.

Alcaligenes↗
PubMed1981

A blue protein as an inactivating factor for nitrite reductase from Alcaligenes faecalis strain S-6.

A blue protein with a molecule weight of 12,000 containing 1 atom of type I Cu2+ was purified and crystallized from a denitrifying bacterium, Alcaligenes faecalis strain S-6, as an inactivating factor for copper-containing nitrite reductase of the same organism. Inactivation of the enzyme occurred when the enzyme was incubated aerobically with a catalytic amount of the blue protein in the presence of reducing agents such as cysteine and ascorbate. The blue protein acts as a direct electron donor for the enzyme to catalyze the reduction of nitrite, but in the absence of nitrite, the enzyme-reduced blue protein system reacts with oxygen to produce H2O2. A suicide inactivation mechanism of the enzyme due to this H2O2 production is proposed.

Alcaligenes↗
PubMed1984

Characterization of nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015. A novel copper protein.

A copper-containing dissimilatory nitrite reductase [nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1] was purified from a denitrifier, Alcaligenes sp. NCIB 11015, by ion-exchange chromatography on CM-cellulose, gel filtration on Sephadex G-150, and adsorption on hydroxyapatite. The preparation was homogeneous by SDS-polyacrylamide gel electrophoretic criteria, and its enzymatic activity increased considerably by freezing (at -20 degrees C) and thawing. The enzyme consists of two subunits with a molecular weight of 37,000, and the isoelectric point and redox potential are 8.4 and +260 mV (pH 7.2), respectively. The EPR spectrum and copper analysis clearly indicated that the enzyme contains two type I copper atoms per molecule but no other types of copper. This is the first blue copper protein that exhibits catalytic activity despite possessing only type I copper.

Alcaligenes↗
PubMed1985

Effects of freezing on purified nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015.

The effects of freezing on Alcaligenes sp. nitrite reductase [nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1] dissolved in sodium phosphate (pH 7.2) were investigated. The nitrite reductase was gradually activated with time in the frozen state, resulting in an increase in its activity of 2.5-4.5 times. The final freezing temperature influenced the enzyme activation, maximal activation being observed at around -20 degrees C. All the enzymatic activities that the nitrite reductase is known to catalyze were enhanced by freeze-thawing. The activation was followed by neither association-dissociation nor any gross conformational change of the enzyme molecule, but was accompanied by an increase in the fluorescence intensity of 2-p-toluidinonaphthalene-6-sulfonate used as a hydrophobic probe. The results are consistent with the hypothesis that the activation of the NiR is due to a limited conformational change of the enzyme molecule, particularly in the hydrophobic region. The mechanism of the activation of NiR by freeze-thawing is discussed, in comparison with the mechanisms of inactivation by freeze-thawing of many enzymes reported by previous workers.

Alcaligenes↗
PubMed1992

Site-directed mutagenesis of pseudoazurin from Alcaligenes faecalis S-6; Pro80Ala mutant exhibits marked increase in reduction potential.

Pseudoazurin (a blue copper protein or cupredoxin) of a denitrifying bacterium Alcaligenes faecalis S-6 is a direct electron carrier for a Cu-containing nitrite reductase (NIR) of the same organism. Site-directed mutagenesis of the pseudoazurin was carried out using an Escherichia coli expression system. Replacement of Tyr74 by Phe to remove an internal hydrogen bond in the beta-barrel caused a slight decrease in heat stability as well as a requirement for a higher concentration of Cu2+ for production in the E. coli host. Exchange of Ala for Pro80 adjacent to His81, one of the four ligands binding a type I Cu atom, caused a marked increase in reduction potential by 139 mV without change in the optical absorption spectrum. The ability of the pseudoazurin to transfer electrons to NIR was markedly diminished but the apparent Km of NIR for pseudoazurin was not affected by the mutation. X-ray diffraction data collected on the oxidized and reduced forms of the Pro80Ala mutant show that a water molecule occupies the pocket created by the absent side chain. This observation suggests that the increase in reduction potential may be caused due to the increased solvent accessibility to the Cu atom. The electron density difference maps on these structures (at 2.0 A) show that this water moves during the change in oxidation state, and that there are small, but localized, conformational changes greater than 6.5 A from the copper site, as well as movement of both the Cu2+ and the cysteinate sulfur.

Alcaligenes↗
PubMed1995

Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6.

Pseudoazurin, a low molecular weight protein containing a single type I copper, functions as an electron donor to a copper-containing nitrite reductase (NIR) in a denitrifying bacterium Alcaligenes faecalis S-6. To elucidate the protein-protein interaction between these two copper-containing proteins, each of nine out of 13 lysine residues on the surface of pseudoazurin were independently replaced by alanine or aspartate, and the effects of the mutations on the interaction with NIR, as well as the physicochemical properties of pseudoazurin, were analyzed. All of the mutated pseudoazurins showed optical spectra and oxidation-reduction potentials almost identical to those of wild-type pseudoazurin, suggesting that none of the replacements of these lysine residues affected the environment around the type I copper site. Kinetic analysis of electron transfer between mutated pseudoazurins and NIR reveals that the lysine mutations have very little effect on the rate of electron transfer to NIR, but substitution at residues 10, 38, 57 and 77, all close to the copper site, substantially decreases the affinity of pseudoazurin for NIR. This suggests that pseudoazurin interacts with NIR through the region close to the type I copper site. The refined X-ray structures of Lys38Asp and Lys10Asp/Lys38Asp show that the molecular structure has indeed changed little. A new space group is observed for the Lys109Ala mutant crystal. Crystal packing interactions change for the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Asp and Lys59Ala mutants.

Alanine↗
PubMed2005

Pancreatic abscess secondary to Alcaligenes faecalis.

We report a patient with pancreatic abscesses and necrosis secondary to Alcaligenes faecalis infection. He initially presented with alcohol-induced acute pancreatitis. Twenty days after the initial presentation, he re-presented with increasing pain and was found to have pancreatic necrosis and abscesses. Treatment was initiated with meropenem. Because of persistent fevers, computed tomography-guided drainage was performed. The fluid grew A faecalis resistant to meropenem and the patient continued to be febrile. He recovered only after adequate surgical intervention and appropriate antibiotic coverage. Although this is the first case of A faecalis reported to cause pancreatic abscess, we believe selection of this organism occurred because surgical drainage was delayed while the patient was on the recommended treatment with meropenem. This case emphasizes the need for early surgical drainage of pancreatic abscesses to avoid the selection of such resistant pathogens.

Abscess↗
PubMed2002

Postkeratoplasty endophthalmitis by Alcaligenes faecalis: a case report.

PURPOSE: To describe a postkeratoplasty endophthalmitis owing to Alcaligenes faecalis that resolved with medical management. METHODS: Case report and review of literature. RESULTS: To best of our knowledge, postkeratoplasty endophthalmitis caused by A. faecalis has not been reported in literature. The source of infection was found to be the donor tissue. The organism isolated was resistant to gentamicin, which was the only antibiotic present in the storage medium (McCarey and Kaufman medium). The patient was treated aggressively with topical, systemic and intravitreal antibiotics. The endophthalmitis resolved completely within two weeks. CONCLUSION: A. faecalis is a gram negative, non-spore forming rod that can cause endophthalmitis if donor tissue is contaminated with the organism. Clinical suspicion, review of bacterial culture and sensitivity report from donor tissue, anterior chamber and vitreous taps, along with early therapy with appropriate antibiotics can lead to resolution of infection.

Aged↗
PubMed2005

Recurrent Alcaligenes xylosoxidans keratitis.

PURPOSE: To describe a case of postkeratoplasty recurrent Alcaligenes xylosoxidans keratitis. METHODS: A 33-year-old man with a history of penetrating keratoplasty developed corneal infiltrate with intact epithelium and then was treated with broad-spectrum antibiotics. Corneal scraping was taken for microbiologic study. RESULTS: The culture result identified A. xylosoxidans, sensitive to piperacillin and ceftazidime. There were 3 recurrent episodes within 2 months after each apparent resolution achieved after the instillation of topical piperacillin (10 mg/mL). During the fourth attack, the lesion responded poorly to piperacillin and ceftazidime (25 mg/mL), so therapeutic penetrating keratoplasty was performed to eradicate the recalcitrant infection. CONCLUSIONS: A. xylosoxidans has probably been underreported as a cause of ocular infection. It does not respond to conventional antibiotic therapy and may be difficult to eradicate. Therapeutic penetrating keratoplasty might be necessary if medical treatment fails.

Abscess↗
PubMed1978

Isolation and characterization of polysheaths, phage tail-like defective bacteriophages of Alcaligenes eutrophus H 16.

Polysheaths were spontaneously formed inside cells of the hydrogen bacterium Alcaligenes eutrophus H 16. These particles are long tube-like structures of 24 nm diam. belonging to the phage tail-like defective bacteriophages (Lotz, 1976). In mid log-phase fermenter-grown cells, polysheaths were observed in about 20% of all cells sectioned. Evidence is provided for an inhibition of cell fission by polysheaths. Polysheaths were isolated by differential centrifugation and precipitation techniques using PEG and antibodies. The morphology of polysheaths was investigated electron microscopically by negative staining, ultrathin sectioning and metal shadowing. A surface lattice of the polysheath was derived from light optical diffraction data. The particles were also characterized by their biochemical and biophysical features: mol. wt. of the subunit determined by SDS-gel electrophoresis (58 000), amino acid composition, isoelectric point (4.4), u.v. absorbance spectrum indicating the absence of nucleic acid, buoyant density (1.258), and stability against denaturants and proteolytic enzymes.

Alcaligenes↗
PubMed1983

The effects of nalidixic acid on respiratory activity of asynchronous and synchronous cultures of Alcaligenes eutrophus.

Nalidixic acid inhibited DNA synthesis and cell division during asynchronous growth of Alcaligenes eutrophus but treated cells continued to grow as monitored by A550, respiratory activity and cell volume. Differential effects on cell division were seen when the antibiotic was added at different times during synchronous growth. The earlier in the cell cycle the time of addition the greater the inhibition of cell division. These results suggests that chromosome replication is confined to the first half of the cell cycle in A. eutrophus grown at 30 degrees C. Respiration rates of cells selected by continuous-flow centrifugation and immediately treated with nalidixic acid increased in a stepwise fashion. These steps were similar to those observed in untreated control cells and imply that the DNA-division cycle is not the causal factor for the periodicities in respiration rates and ATPase activity that have been previously reported in this bacterium.

Alcaligenes↗
PubMed1993

Cloning and characterization of a nitrite reductase gene from Alcaligenes faecalis and its expression in Escherichia coli.

The gene (nir) encoding the copper-containing nitrite reductase (NIR) of a denitrifying bacterium, Alcaligenes faecalis S-6, was cloned by a synthetic oligonucleotide-probing method. The nucleotide sequence of the cloned DNA fragment revealed the primary structure of the NIR precursor containing the N-terminal signal sequence for secretion. A nucleotide sequence, possibly recognized by a transcriptional regulator resembling FNR was found upstream of the structural gene. When the cloned gene was expressed in Escherichia coli under the control of the lac promoter at 37 degrees C, NIR was produced as large inclusion bodies and little activity was detected. When cultivation was at 20 degrees C, most of the NIR was detected in the soluble fraction and a significant portion of the protein was translocated into the periplasmic space, accompanied by removal of its signal sequence.

Alcaligenes↗
PubMed1997

Anaerobic taurine oxidation: a novel reaction by a nitrate-reducing Alcaligenes sp.

Enrichment cultures were prepared under strictly anoxic conditions in medium representing fresh water and containing an organosulfonate as electron donor and carbon source, and nitrate as electron acceptor. The inoculum was from the anaerobic digestor of two communal sewage works. The natural organosulfonates 2-aminoethanesulfonate (taurine), DL-2-amino-3-sulfopropionate (cysteate) and 2-hydroxyethanesulfonate (isethionate) all gave positive enrichments, whereas unsubstituted alkanesulfonates, such as methanesulfonate and arenesulfonates, gave no enrichment. Two representative enrichments were used to obtain pure cultures, and strains NKNTAU (utilizing taurine) and NKNIS (utilizing isethionate) were isolated. Strain NKNTAU was examined in detail. Out of 18 tested organosulfonates, it utilized only one, taurine, and was identified as a novel Alcaligenes sp., a facultatively anaerobic bacterium. Carbon from taurine was converted to cell material and carbon dioxide. The amino group was released as ammonium ion and the sulfonate moiety was recovered as sulfate. Nitrate was reduced to nitrogen gas.

Alcaligenes↗
PubMed1999

The oxygenase component of the 2-aminobenzenesulfonate dioxygenase system from Alcaligenes sp. strain O-1.

Growth of Alcaligenes sp. strain O-1 with 2-aminobenzenesulfonate (ABS; orthanilate) as sole source of carbon and energy requires expression of the soluble, multicomponent 2-aminobenzenesulfonate 2,3-dioxygenase system (deaminating) (ABSDOS) which is plasmid-encoded. ABSDOS was separated by anion-exchange chromatography to yield a flavin-dependent reductase component and an iron-dependent oxygenase component. The oxygenase component was purified to about 98% homogeneity and an alpha2beta2 subunit structure was deduced from the molecular masses of 134,45 and 16 kDa for the native complex, and the alpha and beta subunits, respectively. Analysis of the amount of acid labile sulfur and total iron, and the UV spectrum of the purified oxygenase component indicated one [2Fe-2S] Rieske centre per alpha subunit. The inhibition of activity by the iron-specific chelator o-phenanthroline indicated the presence of an additional iron-binding site. Recovery of active protein required strictly anoxic conditions during all purification steps. The FAD-containing reductase could not be purified. ABSDOS oxygenated nine sulfonated compounds; no oxygen uptake was detected with carboxylated aromatic compounds or with aliphatic sulfonated compounds. Km values of 29, 18 and 108 microM and Vmax values of 140, 110 and 72 pkat for ABS, benzenesulfonate and 4-toluenesulfonate, respectively, were observed. The N-terminal amino acid sequences of the alpha- and beta-subunits of the oxygenase component allowed PCR primers to be deduced and the DNA sequence of the alpha-subunit was thereafter determined. Both redox centres were detected in the deduced amino acid sequence. Sequence data and biochemical properties of the enzyme system indicate a novel member of the class IB ring-hydroxylating dioxygenases.

Alcaligenes↗
PubMed1994

3-Sulphocatechol 2,3-dioxygenase and other dioxygenases (EC 1.13.11.2 and EC 1.14.12.-) in the degradative pathways of 2-aminobenzenesulphonic, benzenesulphonic and 4-toluenesulphonic acids in Alcaligenes sp. strain O-1.

Alcaligenes sp. strain O-1 utilizes three sulphonated aromatic compounds as sole sources of carbon and energy for growth in minimal salts medium-benzenesulphonate (BS), 4-toluenesulphonate (TS) and 2-aminobenzenesulphonate (2AS). The degradative pathway(s) in 2AS-grown cells are initiated with membrane transport, NADH-dependent dioxygenation and meta ring cleavage. The specific activity of the NADH-dependent dioxygenation(s) varied with the growth phase and was maximal near the end of exponential growth for each growth substrate. Cells were harvested at this point from BS-, TS- and 2AS-salts medium. Cells grown with each sulphonated substrate could oxygenate all three compounds, but only 2AS-grown cells consumed 2 mol O2 per mol 2AS or BS or TS. BS- and TS-grown cells consumed 2 mol O2 per mol BS or TS but failed to oxygenate the product of oxygenation of 2AS, 3-sulphocatechol (3SC). These observations were repeated with cell extracts and we concluded that there were two sets of desulphonative pathways in the organism, one for 2AS and one for BS and TS. We confirmed this hypothesis by separating the degradative enzymes from 2AS-, BS- or TS-grown cells. A 2AS dioxygenase system and a 3SC-2,3-dioxygenase (3SC23O) were detected in 2AS-grown cells only. In both BS- and TS-grown cells a dioxygenase system for BS and TS was observed as well as a principal catechol 2,3-dioxygenase (C23O-III), neither of which was present in 2AS-grown cells. The 3SC23O was purified to near homogeneity, found to be monomeric (M(r) 42,000), and to catalyse 2,3-dioxygenation to a product that decayed spontaneously to sulphite and 2-hydroxymuconate. The 2AS dioxygenase system could cause not only deamination of 2AS but also desulphonation of BS and TS. The BS dioxygenase could desulphonate BS and apparently either desulphonate or deaminate 2AS. Strain O-1 thus seems to contain two putative, independently regulated operons involving oxygenation and spontaneous desulphonation(s). One operon encodes at least the 2AS dioxygenase system and 3SC23O whereas the other encodes at least the BS/TS dioxygenase system and C23O-III.

Alcaligenes↗
PubMed1995

A gene from Alcaligenes denitrificans that confers albicidin resistance by reversible antibiotic binding.

Albicidin antibiotics specifically block prokaryote DNA replication. The albicidin resistance gene (albB) cloned from a soil isolate of Alcaligenes denitrificans encodes a 23 kDa protein capable of detoxifying albicidin by reversible binding. This mechanism operates intracellularly to protect DNA replication in albicidin-sensitive Escherichia coli expressing the cloned resistance gene, which can be induced fivefold in the presence of 1.5 micrograms albicidin ml-1 in the surrounding medium. The coding region of 621 bp has regions with partial DNA sequence homology to an albicidin resistance gene (albA) from Klebsiella oxytoca, but with rearrangements and frame-shifts resulting in loss of protein homology. There is a short region of N-terminal homology between the albicidin resistance (Albr) proteins from A. denitrificans and K. oxytoca, although the two genes use different codons for shared amino acids. The N-terminal homology suggested a common functional domain; this was confirmed by deletion analysis, translational fusions and albicidin binding by a synthetic oligopeptide. Antibiotic binding provides a high level of albicidin resistance in E. coli. The gene appears to be a useful candidate for transfer to plants to protect plastid DNA replication from inhibition by albicidin phytotoxins involved in sugarcane leaf scald disease.

Alcaligenes↗
PubMed1996

Flux limitations in the ortho pathway of benzoate degradation of Alcaligenes eutrophus: metabolite overflow and induction of the meta pathway at high substrate concentrations.

The growth behaviour of Alcaligenes eutrophus using various concentrations of benzoate was investigated. In batch culture, growth was exponential and growth rate (mu) and yields (Y) were high [mu = 0.51 h-1 and Yx/benzoate = 0.56 mol carbon (mol carbon)-1] when low concentrations of benzoate (< 5 mM) were used. These kinetic parameters were close to the maxima determined in a benzoate-limited chemostat [mu(max) = 0.55 h-1 and YX/benzoatemax = 0.57 mol carbon (mol carbon)-1] and the part of the energy for maintenance was limited (mATP = 4.3 +/- 2.2 mmol ATP g-1 h-1). When higher concentrations of benzoate were used (up to 40 mM), several metabolic limitations appeared. The specific rate of benzoate consumption was not altered, whereas growth was inhibited [Ki(benzoate) approximately 27 mM]. Furthermore, high concentrations of catechol together with some 1,2-dihydro-1,2-dihydroxybenzoate (DHB) transiently accumulated in the medium. The accumulation of catechol was attributed to limiting flux through catechol 1,2-dioxygenase estimated to be 5.2 mmol g-1 h-1, whereas that of DHB was provoked by an imbalance in the NADH/NAD+ intracellular content. The direct consequence of DHB accumulation was the induction of the meta pathway for the degradation of catechol, and this pathway contributed up to 20% of the total flux of catechol to the central metabolism. Finally, when very high concentrations of benzoate were used (55 mM), both growth and the specific rate of benzoate degradation were diminished due to a strong decrease in benzoate 1,2-dioxygenase specific activity.

Adenosine Triphosphate↗
PubMed2006

The sulfonated osmolyte N-methyltaurine is dissimilated by Alcaligenes faecalis and by Paracoccus versutus with release of methylamine.

Selective enrichments yielded bacterial cultures able to utilize the osmolyte N-methyltaurine as sole source of carbon and energy or as sole source of fixed nitrogen for aerobic growth. Strain MT1, which degraded N-methyltaurine as a sole source of carbon concomitantly with growth, was identified as a strain of Alcaligenes faecalis. Stoichiometric amounts of methylamine, whose identity was confirmed by matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry, and of sulfate were released during growth. Inducible N-methyltaurine dehydrogenase, sulfoacetaldehyde acetyltransferase (Xsc) and a sulfite dehydrogenase could be detected. Taurine dehydrogenase was also present and it was hypothesized that taurine dehydrogenase has a substrate range that includes N-methyltaurine. Partial sequences of a tauY-like gene (encoding the putative large component of taurine dehydrogenase) and an xsc gene were obtained by PCR with degenerate primers. Strain N-MT utilized N-methyltaurine as a sole source of fixed nitrogen for growth and could also utilize the compound as sole source of carbon. This bacterium was identified as a strain of Paracoccus versutus. This organism also expressed inducible (N-methyl)taurine dehydrogenase, Xsc and a sulfite dehydrogenase. The presence of a gene cluster with high identity to a larger cluster from Paracoccus pantotrophus NKNCYSA, which is now known to dissimilate N-methyltaurine via Xsc, allowed most of the overall pathway, including transport and excretion, to be defined. N-Methyltaurine is thus another compound whose catabolism is channelled directly through sulfoacetaldehyde.

Acetyltransferases↗