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.     

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.     

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.     

A 2.0-A structure of the blue copper protein (cupredoxin) from Alcaligenes faecalis S-6

The structure of a blue copper protein, cupredoxin, from the potent denitrifying bacterium Alcaligenes faecalis S-6, has been determined and refined against 2 A x-ray diffraction data. The agreement between observed and calculated structure factors is 0.159, and estimated errors in coordinates are 0.09-0.15 A. The protein folds in a beta sandwich similar to plastocyanin and azurin and includes features such as a "kink" and a "tyrosine loop" which have been noted previously for these proteins as well as immunoglobulins. The copper is bound by four ligands, in a distorted tetrahedral arrangement, with Cu-S gamma = 2.07 A (Cys-78), Cu-N delta 1 = 2.10 and 2.21 for His-40 and His-81, and Cu-S delta = 2.69 A (Met-86). Two of the ligands are further oriented by hydrogen bonds either to other side chains (Asn-9 to His-40), backbone atoms (NH...S) or a water molecule (to His-40). The methionine ligand has no extra constraints. The C-terminal loop containing three of the ligands is hydrogen-bonded to the strand containing His-40 by hydrogen bonds between the conserved residues Thr-79 and Asn-41. The pronounced dichroism of the crystal is a result of the orientation of the normal to the C beta-S gamma-Cu plane parallel to the crystallographic 6-fold axis.     

The blue copper protein gene of Alcaligenes faecalis S-6 directs secretion of blue copper protein from Escherichia coli cells.

The gene encoding a blue copper protein (a member of the pseudoazurins) of 123 amino acid residues, containing a single type I Cu2+ ion, was cloned from Alcaligenes faecalis S-6. The nucleotide sequence of the coding region, as well as the 5'- and 3'-flanking regions, was determined. The deduced amino acid sequence after Glu-24 coincided with the reported sequence of the blue protein, and its NH2-terminal sequence of 23 residues resembled a typical signal peptide. The cloned gene was expressed under the control of the tac promoter in Escherichia coli, and the correctly processed blue protein was secreted into the periplasm. The blue protein produced in E. coli possessed the activity to transfer electrons to the copper-containing nitrite reductase of A. faecalis S-6 in vitro.     

Two isofunctional nitric oxide reductases in Alcaligenes eutrophus H16.

Two genes, norB and norZ, encoding two independent nitric oxide reductases have been identified in Alcaligenes eutrophus H16. norB and norZ predict polypeptides of 84.5 kDa with amino acid sequence identity of 90%. While norB resides on the megaplasmid pHG1, the norZ gene is located on a chromosomal DNA fragment. Amino acid sequence analysis suggests that norB and norZ encode integral membrane proteins composed of 14 membrane-spanning helices. The region encompassing helices 3 to 14 shows similarity to the NorB subunit of common bacterial nitric oxide reductases, including the positions of six strictly conserved histidine residues. Unlike the Nor enzymes characterized so far from denitrifying bacteria, NorB and NorZ of A. eutrophus contain an amino-terminal extension which may form two additional helices connected by a hydrophilic loop of 203 amino acids. The presence of a NorB/NorZ-like protein was predicted from the genome sequence of the cyanobacterium Synechocystis sp. strain PCC6803. While the common NorB of denitrifying bacteria is associated with a second cytochrome c subunit, encoded by the neighboring gene norC, the nor loci of A. eutrophus and Synechocystis lack adjacent norC homologs. The physiological roles of norB and norZ in A. eutrophus were investigated with mutants disrupted in the two genes. Mutants bearing single-site deletions in norB or norZ were affected neither in aerobic nor in anaerobic growth with nitrate or nitrite as the terminal electron acceptor. Inactivation of both norB and norZ was lethal to the cells under anaerobic growth conditions. Anaerobic growth was restored in the double mutant by introducing either norB or norZ on a broad-host-range plasmid. These results show that the norB and norZ gene products are isofunctional and instrumental in denitrification.     

Molecular basis for nitric oxide dynamics and affinity with Alcaligenes xylosoxidans cytochrome c

The bacterial heme protein cytochrome ? from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman time-resolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level. We found that after NO dissociation from the heme iron, the structure of the proximal heme pocket of AXCP confines NO close to the iron so that an ultrafast (7 ps) and complete (99 +/- 1%) geminate rebinding occurs, whereas the proximal histidine does not rebind to the heme iron on the timescale of NO geminate rebinding. The distal side controls the initial NO binding, whereas the proximal heme pocket controls its release. These dynamic properties allow the trapping of NO within the protein core and represent an extreme behavior observed among heme proteins.     

Alcaligenes faecalis kw-a biofilm for denitrification of nitrate-rich effluent

Alcaligenes faecalis kw-A selected for possessing good denitrification efficiency was used for biofilm development. The biofilm could be developed on a glass surface within 12 hr when 5%, Ix 10(8) cells/ml was used as inoculum. The microcolonies were seen in 6 hr and glycocalyx in 9 hr stage. At 24 hr the biofilm was developed fully and hence was visualised as dense mass. The biofilm protein content showed 48.5% increase in shake flask than in static condition. The exopoplymer is produced in larger amounts in biofilm as compared to the suspended cells. Also, its amount was more by 43% in the biofilm produced in shake flask condition than in static condition. The biofilm could remove 95% nitrate from nitrate-rich effluent in a bench-scale process in 36 hr. The attached growth technique demonstrated here can be utilised to study the effect of favourable as well as adverse conditions on the denitrification efficiency of a culture. The ultimate application of a denitrifying biofilm would be in attached growth or biofilm reactor.     

Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis

Nitrous oxide (N₂O), a greenhouse gas, is emitted during autotrophic and heterotrophic ammonia oxidation. This emission may result from either coupling to aerobic denitrification, or it may be formed in the oxidation of hydroxylamine (NH₂OH) to nitrite (NO₂ ⁻). Therefore, the N₂O production during NH₂OH oxidation was studied with Alcaligenes faecalis strain TUD. Continuous cultures of A. faecalis showed increased N₂O production when supplemented with increasing NH₂OH concentrations. ¹⁵N-labeling experiments showed that this N₂O production was not due to aerobic denitrification of NO₂ ⁻. Addition of ¹⁵N-labeled NH₂OH indicated that N₂O was a direct by-product of NH₂OH oxidation, which was subsequently reduced to N₂. These observations are sustained by the fact that NO₂ ⁻ production was low (0.23 mM maximum) and did not increase significantly with increasing NH₂OH concentration in the feed. The NH₂OH-oxidizing capacity increased with increasing NH₂OH concentrations. The apparent V _max and K _m were 31 nmol min⁻¹ mg dry weight⁻¹ and 1.5 mM respectively. The culture did not increase its growth yield and was not able to use NH₂OH as the sole N source. A non-haem hydroxylamine oxidoreductase was partially purified from A. faecalis strain TUD. The enzyme could only use K₃Fe(CN)₆ as an electron acceptor and reacted with antibodies raised against the hydroxylamine oxidoreductase of Thiosphaera pantotropha. Received: 1 September 1998 / Received revision: 5 November 1998 / Accepted: 7 November 1998     

粪产碱杆菌青霉素G酰化酶的分离提取

比较研究了几种破碎大肠杆菌细胞的方法,如渗透压法、超声波法、玻珠震碎法、玻珠研磨法、有机溶剂法、冻融法以及盐酸胍/EDTA法等,以确定出一种简单、快速、高效的破碎重组大肠杆菌细胞的方法获得粪产碱杆菌青霉素G酰化酶(AfPGA)用于后续试验。结果表明玻珠震碎法、超声波法和渗透压法是较优的细胞破碎方法,活力回收率分别为99.7%、78.4%、60.7%,其他方法均低于22%。而比活力以渗透压法为最高,达到4.40 U/mg。     

粪产碱菌（Alcaligenes faecalis）吸收氢和同化CO2的特性

应用同位素氚(T_2)和~13C(~13CO_2),证明了水稻联合固氮菌——粪产碱菌A—15是一种含有吸氢酶的兼性化能自养细菌,具有较强的吸氢能力,吸氨酶活性可达到13.11μmol H_2 ml~(-1) cultureh~(-1);同时,它还可利用H_2为能源同化CO_2营化能自养生活,其RuBPC活性为24.65 nmolCO_2 mg~(-1) protein min~(-1)。无论在自养还是异养条件下,H_2都支持、并促进固氮活性。粪产碱菌培养在N_2条件下比在NH_4~ 条件下能积累更多的多聚-β羟基丁酸(PHB)。     

Nitrosative stress in Escherichia coli: reduction of nitric oxide

The ability of enteric bacteria to protect themselves against reactive nitrogen species generated by their own metabolism, or as part of the innate immune response, is critical to their survival. One important defence mechanism is their ability to reduce NO (nitric oxide) to harmless products. The highest rates of NO reduction by Escherichia coli K-12 were detected after anaerobic growth in the presence of nitrate. Four proteins have been implicated as catalysts of NO reduction: the cytoplasmic sirohaem-containing nitrite reductase, NirB; the periplasmic cytochrome c nitrite reductase, NrfA; the flavorubredoxin NorV and its associated oxidoreductase, NorW; and the flavohaemoglobin, Hmp. Single mutants defective in any one of these proteins and even the mutant defective in all four proteins reduced NO at the same rate as the parent. Clearly, therefore, there are mechanisms of NO reduction by enteric bacteria that remain to be characterized. Far from being minor pathways, the currently unknown pathways are adequate to sustain almost optimal rates of NO reduction, and hence potentially provide significant protection against nitrosative stress.     

Denitrification and nitric oxide reduction in an aerobic toluene-treating biofilter

The presence of significant denitrification activity in an aerobic toluene-treating biofilter was demonstrated under batch and flow-through conditions. N2O concentrations of 9.2 ppmv were produced by denitrifying bacteria in the presence of 15% acetylene, in a flow-through system with a bulk gas phase O2 concentration of >17%. The carbon source for denitrification was not toluene but a byproduct or metabolite of toluene catabolism. Denitrification conditions were successfully used for the reduction of 60 ppmv nitric oxide to 15 ppmv at a flow rate of 3 L min-1 (EBRT of 3 min) in a fully aerated, 17% v/v O2 (superficially aerobic) biofilter. Higher NO removal efficiency (97%) was obtained by increasing the toluene supply to the biofilter.     

Molecular cloning, genetic organization of gene cluster encoding phenol hydroxylase and catechol 2,3-dioxygenase in Alcaligenes faecalis IS-46

Alcaligenes faecalis IS-46 can utilize phenol as the sole carbon and energy source at concentration up to 1000 mg/l. In this report we created a cosmid library of this strain and the two clones specifying the whole L-46d type of phenol hydroxylase gene cluster were identified and characterized. Sequence analysis revealed that although the overall gene organization of the clusters was quite similar, few coding sequences differed or were found to have two copies compared with other source organisms. One of these coding sequences showed a good protein sequence similarity to a hypothetical protein and one matched with a regulatory protein of the LysR system. Their putative role in phenol degradation was discussed. Bioinformatic analysis suggested tentative phylogenetic assignments of the retrieved clusters. This work described for first time the complete nucleotide sequence and genetic organization of the whole phenol hydroxylase gene cluster in A. faecalis species.