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At least 1,099 records · Page 61Linked to original sources
PubMed1996

Production, characterization, and reconstitution of recombinant quinoprotein glucose dehydrogenase (soluble type; EC 1.1.99.17) apoenzyme of Acinetobacter calcoaceticus.

Soluble, periplasmic quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus (sGDH; EC 1.1.99.17) was produced in good yield in the apoenzyme form (without the cofactor pyrroloquinoline quinone, PQQ) by an Escherichia coli recombinant strain provided with a plasmid containing the gene under control of a lac promoter. Structural analysis of the purified apoenzyme revealed that the E. coli strain used produces the correct mature protein. Titration of the apoenzyme with PQQ in the presence of Ca2+ showed that a linear relation exists between the amount of added PQQ and activity observed, and that the subunit and PQQ associate in a molar ratio of 1:1. Based on spectral and enzymatic criteria, it is concluded that the present holoenzyme preparation has a better quality than the previously described preparations of authentic holoenzyme. As isolated here, the recombinant apoenzyme was in the dimeric form. Partial monomerization occurred upon gel filtration in a buffer with chelator and the process could be reversed with Ca2+. PQQ binds to the dimer in the presence of chelator, not to the monomer. However, the PQQ-containing dimer was not active and showed an unusual absorption spectrum which was slowly converted into a PQQH2-like spectrum when glucose was added. Full restoration of activity was achieved upon addition of Ca2+ and the spectra were immediately converted into those of normal holoenzyme in the oxidized and reduced form, respectively. Addition of chelator to holoenzyme did not lead to inactivation or monomerization. It is concluded, therefore, that Ca2+ has a dual role in this enzyme, being required for dimerization of the subunits as well as for functionalization of the bound PQQ, and that it is more firmly attached to the holoenzyme than to the apoenzyme.

Acinetobacter calcoaceticus↗
PubMed1995

Elucidation of the region responsible for EDTA tolerance in PQQ glucose dehydrogenases by constructing Escherichia coli and Acinetobacter calcoaceticus chimeric enzymes.

We constructed various chimeric PQQ glucose dehydrogenases (PQQGDHs) from an EDTA-sensitive PQQGDH from Escherichia coli and an EDTA-tolerant PQQGDH from Acinetobacter calcoaceticus by homologous recombination of their structural genes. The EDTA tolerance of the resulting chimeric enzymes was investigated. Our results demonstrated that EDTA tolerance of PQQGDHs can be completely altered by substituting each corresponding region. The EDTA tolerance of A. calcoaceticus PQQGDH is mostly within a region composed of about 90 amino acid residues located between 45 and 56% of the distance from the N-terminal region.

Acinetobacter calcoaceticus↗
PubMed2001

Molecular characterization of the 56-kDa CYP153 from Acinetobacter sp. EB104.

CYP153 a cytochrome P450 from Acinetobacter sp. EB104 catalyzes the hydroxylation of unsubstituted n-alkanes. We have decided to use the CYP153 system as a model for mechanistic studies on regioselective n-alkane oxidation and the interaction of hydrophobic substrates with soluble enzymes. Here the molecular cloning of the CYP153 gene is reported. Single specific primer PCR was applied to yield the whole gene sequence via chromosomal walks. CYP153 consists of 497 amino acids (M(r) = 56 kDa) and thus represents an unusually long bacterial P450, containing all P450 typical structural elements. It constitutes the new P450 family CYP153. The prolonged N-terminus of about 90 amino acids does not contain a so far known membrane-anchoring sequence but a 28-amino acid long amphipathic helix. The relevance of the remarkably long N-terminus and of other sequence motives like the hydrophobic F-G loop is discussed with respect to substrate binding and recognition.

Acinetobacter↗
PubMed1993

Crystallization and preliminary crystallographic investigations of the soluble glucose dehydrogenase from Acinetobacter calcoaceticus.

Single crystals of the soluble glucose dehydrogenase (GDH) from Acinetobacter calcoaceticus have been grown by the vapour diffusion method. These crystals diffract to beyond 2.1 A and are suitable for X-ray crystallography. The space group was determined to be P2(1) with unit cell parameters a = 55.5 A, b = 104.5 A, c = 86.5 A and beta = 99.8 degrees. One asymmetric unit contains a dimer of the GDH molecule.

Acinetobacter calcoaceticus↗
PubMed1994

Crystallization and preliminary X-ray analysis of protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus.

X-ray quality single crystals of protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus were obtained by the hanging drop method. The intradiol dioxygenase crystallizes in the cubic space group I23 with unit cell dimensions a = b = c = 145.5 A. The dodecahedral crystals diffract to beyond 2.5 A resolution. The asymmetric unit contains one twelfth of the enzyme (alpha beta Fe+3)12 complex.

Acinetobacter calcoaceticus↗
PubMed1999

The 1.7 A crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat.

The crystal structure of a dimeric apo form of the soluble quinoprotein glucose dehydrogenase (s-GDH) from Acinetobacter calcoaceticus has been solved by multiple isomorphous replacement followed by density modification, and was subsequently refined at 1. 72 A resolution to a final crystallographic R-factor of 16.5% and free R-factor of 20.8% [corrected]. The s-GDH monomer has a beta-propeller fold consisting of six four-stranded anti-parallel beta-sheets aligned around a pseudo 6-fold symmetry axis. The enzyme binds three calcium ions per monomer, two of which are located in the dimer interface. The third is bound in the putative active site, where it may bind and functionalize the pyrroloquinoline quinone (PQQ) cofactor. A data base search unexpectedly showed that four uncharacterized protein sequences are homologous to s-GDH with many residues in the putative active site absolutely conserved. This indicates that these homologs may have a similar structure and that they may catalyze similar PQQ-dependent reactions.A structure-based sequence alignment of the six four-stranded beta-sheets in s-GDH's beta-propeller fold shows an internally conserved sequence repeat that gives rise to two distinct conserved structural motifs. The first structural motif is found at the corner of the short beta-turn between the inner two beta-strands of the beta-sheets, where an Asp side-chain points back into the beta-sheet to form a hydrogen-bond with the OH/NH of a Tyr/Trp side-chain in the same beta-sheet. The second motif involves an Arg/Lys side-chain in the C beta-strand of one beta-sheet, which forms a bidentate salt-bridge with an Asp/Glu in the CD loop of the next beta-sheet. These intra and inter-beta-sheet hydrogen-bonds are likely to contribute to the stability of the s-GDH beta-propeller fold.

Acinetobacter calcoaceticus↗
PubMed1993

Molecular characterization of an aberrant mercury resistance transposable element from an environmental Acinetobacter strain.

We present the complete nucleotide sequence of a mer operon located on a 60-kb conjugative plasmid pKLH2 from an environmental bacterium, Acinetobacter calcoaceticus, isolated from a mercury mine. The pKLH2 mer operon has essentially the same gene organization as that of Tn21 and Tn501 from clinical bacteria. The pKLH2 mer operon nucleotide sequence shows 85.5% identity with the Tn501 and 80.9% identity with the Tn21 sequences. Vestigial sequences have been found at the ends of the pKLH2 mer operon, indicating that the pKLH2 mer operon was once a part of a Tn21-like transposon, which had committed suicide by an aberrant resolution event.

Acinetobacter↗
PubMed1996

Utilization and degradation of an ester-based synthetic lubricant by Acinetobacter lwoffi.

An oil-degrading bacterium, Acinetobacter lwoffi, isolated by elective culture from the Medway estuary, utilized an ester-based synthetic lubricating oil EMKARATE DE 155 as sole carbon and energy source. Analysis of culture supernatants by gas chromatography showed the accumulation of a nondegradable metabolite 1,1,1 Tris (hydroxymethyl)propane in addition to two metabolizable fatty acids, octanoic and decanoic acids as products of the synthetic oil degradation. Esterase activities were subsequently demonstrated in oil and acetate-grown cells. The synthetic oil therefore appears to be partially biodegradable in the environment.

Acinetobacter↗
PubMed1994

Structure of acinetoferrin, a new citrate-based dihydroxamate siderophore from Acinetobacter haemolyticus.

From low-iron cultures of Acinetobacter haemolyticus ATCC 17906, a new hydroxamate siderophore was purified by XAD-7 adsorption followed by preparative thin layer chromatography. The siderophore, named acinetoferrin, released citric acid, 1,3-diaminopropane and (E)-2-octenoic acid upon hydrolysis with HCl, reductive hydrolysis with HI and oxidation with periodate, respectively. Structure elucidation by a combination of NMR spectroscopy and positive fast atom bombardment mass spectrometry revealed that acinetoferrin is a derivative of citric acid, both of its terminal carboxyl groups being symmetrically amide-linked with the 1-amino-3-(N-hydroxy-N-2-octenylamino)propane residues. The (E)-2-octenoic acid is novel as a component of the siderophores.

Acinetobacter↗
PubMed1990

Expression of an Erwinia sp. gene encoding diphenyl ether cleavage in Escherichia coli and an isolated Acinetobacter strain PE7.

An Acinetobacter strain PE7 with the ability to grow on salicyclic acid and to degrade diphenyl ethers was isolated from a petroleum waste pit in Louisiana. A cloned Erwinia sp. dpe gene encoding diphenyl ether cleavage was introduced into PE7 in order to enhance its degradative ability. A broad-host-range expression plasmid, pDPE2388, was constructed by inserting an SspI-HpaI fragment from a dpe gene-containing plasmid, pDPE7321, into the kanamycin resistance gene of plasmid pKT230. The DNA fragment contained the dpe gene flanked between sp6 and T7 promoters. Transconjugants of pDPE2388 plasmid into PE7 were isolated. Expression of the dpe gene in Escherichia coli or PE7 displayed a degradative ability to cleave the following diphenyl ethers: 4-chlorodiphenyl ether, 4-nitrodiphenyl ether, and 4-hydroxydiphenyl ether.

Acinetobacter↗
PubMed1994

Plasmid mediated silver resistance in Acinetobacter baumannii.

Acinetobacter baumannii BL88, an environmental isolate, was resistant to 13 metals and 10 antibiotics. Plumbagin cured resistance to silver, cadmium, antimony, streptomycin and ampicillin at varying frequencies. However, only silver resistance transferred (1 x 10(-6) recepient-1) to Escherichia coli K12 during conjugation. Correspondingly there was transfer of a 54 kb plasmid (pUPI199) from A. baumannii BL88. The plasmid transformed E. coli DH5 alpha cells at a frequency of 1 x 10(-8) recepient-1. The growth rate of E. coli DH5 alpha (pUPI199) was slower as compared with E. coli DH5 alpha. Plasmid pUPI199 was 76 and 9.6% stable in the host A. baumannii BL88 in the presence and absence of selection pressure, respectively. A. baumannii BL88 was found to accumulate and retain silver whereas E. coli DH5 alpha (pUPI199) effluxed 63% of the accumulated silver ions.

Acinetobacter↗
PubMed1992

Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater.

The natural transformation of Acinetobacter calcoaceticus BD413 (trpE27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp+) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca2+. With CaCl2 solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl2 and MgCl2. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80 degrees C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.

Acinetobacter calcoaceticus↗
PubMed1992

Purification of a periplasmic insulin-cleaving proteinase from Acinetobacter calcoaceticus.

Cells of Acinetobacter calcoaceticus contain a constitutive periplasmic metalloproteinase showing similar properties as the periplasmic metalloproteinase of Escherichia coli. The periplasmic proteinase of A. calcoaceticus was purified, starting from periplasm, by ammonium sulfate precipitation, hydrophobic interaction chromatography and chromatofocusing up to the homogeneity of the enzyme in SDS-electrophoresis with a yield of 6.7% and a purification factor of 417. The enzyme has a molecular mass of 108,000 (gel filtration) or 112,000 (native electrophoresis), and consists of four identical subunits with a molecular mass of 27,000 (SDS-electrophoresis). The purified enzyme degrades preferentially polypeptides such as glucagon and insulin. Larger proteins are accepted as substrates to a considerably lower extent. All tested synthetic substrates with trypsin, chymotrypsin, elastase and thermolysin specificity were not cleaved. Therefore, the described enzyme was designated "insulin-cleaving proteinase" (ICP).

Acinetobacter calcoaceticus↗
PubMed1994

Isolation and structure elucidation of acinetobactin, a novel siderophore from Acinetobacter baumannii.

A novel siderophore, called acinetobactin, with both catecholate and hydroxamate functional groups was isolated from low-iron cultures of Acinetobacter baumannii ATCC 19606. The structure was elucidated by chemical degradation, fast-atom bombardment mass spectrometry and 1H and 13C NMR spectroscopy. Acinetobactin was composed of omega-N-hydroxyhistamine, threonine and 2,3-dihydroxybenzoic acid, the last two components forming an oxazoline ring. Acinetobactin was structurally related to anguibactin, a plasmid-encoded siderophore of Vibrio anguillarum. The only difference was that acinetobactin possessed an oxazoline ring instead of a thiazoline ring. Four of 12 other clinical A. baumannii strains examined produced acinetobactin, indicative of strain-to-strain variation in the ability to produce acinetobactin. In addition, a relatively small amount of acinetobactin was also detected in A. haemolyticus ATCC 17906.

Acinetobacter↗
PubMed1990

Identification and nucleotide sequence of the Acinetobacter calcoaceticus encoded trpE gene.

The trpE gene from Acinetobacter calcoaceticus encoding the anthranilate synthase component I was cloned, identified by deletion analysis and sequenced. It encodes a predicted polypeptide of 497 amino acids with a calculated molecular weight of 55,323. Its primary structure shows 49% identical amino acids with the enzyme from Clostridium thermocellum, 45% with that of Thermus thermophilus and only 35% with that of Escherichia coli. The codon usage of the trpE genes encoding the most homologous enzymes differs greatly indicating selection for amino acid maintainance. The homologies are clustered in the C-terminal 200 amino acids of the sequences indicating that this part is important for enzymic activity.

Acinetobacter↗
PubMed1988

Flow cytometric screening and isolation of Escherichia coli clones which express surface antigens of the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1.

Flow cytometry (FCM) in conjunction with immunocytochemical-labeling was used to analyze and screen a population of Escherichia coli clones containing a genomic library from the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1 surface antigens. Reconstruction experiments using mixed populations indicated that RAG-1 cells could be clearly distinguished at a ratio of one RAG-1 cell to 500 Escherichia coli cells. Using this technique two clones, WM143 and WM191, were isolated and shown by restriction endonuclease cleavage and Southern hybridization to contain plasmids carrying inserts of RAG-1 DNA of 9.4 and 9.8 kb respectively.

Acinetobacter↗
PubMed1975

Microbial assimilation of hydrocarbons. I. The fine-structure of a hydrocarbon oxidizing Acinetobacter sp.

1. The fine-structure analysis of the hydrocarbon oxidizing microorganism, Acinetobacter sp., demonstrated a cytoplasmic modification resulting from growth on paraffinic and olefinic hydrocarbons. 2. Intracytoplasmic hydrocarbon inclusions were documented by electron microscopy with chemical identifications obtained by gas chromatography and X-ray diffraction. 3. These results demonstrate the ability of a microorganism to accumulate hydrocarbon substrates intracellularly which, in turn, indicates the transport across the cell membrane.

Acinetobacter↗