Characterization of the copper resistance mechanism and bioremediation potential of an Acinetobacter calcoaceticus strain isolated from copper mine sludge.

Bioremediation is one of the most effective ways for removal of heavy metals and restoration of contaminated sites. This study investigated the copper (Cu) resistance mechanism and bioremediation potential of an Acinetobacter calcoaceticus strain KW3 isolated from sludge of Cu mine. The effect of Cu concentrations on the bacterial growth, biomass, and adsorption capacity, as well as the effect of contact time on the adsorption process was evaluated in a batch biosorption test. The strain exhibited strong tolerance of Cu, and the minimal inhibitory concentration was around 400 mg Cu2+ L-1, at which the maximum adsorption capacity was 14.1 mg g-1 dry cell mass. Cell walls and intracellular soluble components adsorbed 51.2% and 46.6% of Cu2+, respectively, suggesting that the strain not only adsorbed Cu2+ on the surface but also metastasized ions into cells. The adsorption and kinetic data were well fitted with Freundlich isotherm and Pseudo-second-order models, suggesting that cell surface had a high affinity for Cu2+ and the chemisorption could be the main adsorption mechanism. Scanning electron microscope and Fourier transform infrared spectroscopy analysis revealed that hydroxyl, carboxylic, amide, sulfate, and phosphate on cell walls might be involved in the biosorption process. Two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry revealed that some oxidoreductases, in particular Cu resistance protein A (CopA) expression levels, were upregulated. Antioxidant defense and P1B-type ATPases CopA efflux might play a crucial role in maintaining cellular homeostasis and intracellular detoxification. To our knowledge, this is the first time that Cu resistance mechanisms, especially intracellular enzymatic mechanisms, were identified in an A. calcoaceticus KW3 strain.

Xanthine oxidase of Acinetobacter calcoaceticus RL2-M4: Production, purification and characterization.

Xanthine oxidase (EC 1.17.3.2) is a key enzyme of purine metabolism and has potential applications in food and pharmaceutical industries. In the present study, a new bacterial source of xanthine oxidase i.e. Acinetobacter calcoaceticus RL2-M4 with high oxidase activity was isolated from soil. The culture conditions were optimized with one variable at a time (OVAT) and response surface methodology (RSM) approaches included: a minimal salt medium (MSM) of pH 7.0 supplemented with 0.8% yeast extract, 8.5 mM xanthine and incubation at 30 °C for 24 h. Under these culture conditions 11.57 fold increase in the production of this enzyme was achieved. The enzyme was purified from A. calcoaceticus RL2-M4 using anion exchange chromatography to 8.18 fold with 31% yield and specific activity of 4.58 U/mg protein. SDS-PAGE analysis of the purified enzyme revealed that it was homodimer of 95 kDa and its native molecular mass was estimated to be 190 kDa. This enzyme was found to be stable at 35 °C for 5 h. The purified xanthine oxidase of A. calcoaceticus RL2-M4 had Km 0.3 mM and Vmax 5.8 U/mg protein using xanthine as substrate. The activity and stability characteristic of xanthine oxidase of A. calcoaceticus RL2-M4 makes it a potentially good enzyme for industrial applications.

Differentiation of Taxonomically Closely Related Species of the Genus Acinetobacter Using Raman Spectroscopy and Chemometrics.

In recent years, several efforts have been made to develop quick and low cost bacterial identification methods. Genotypic methods, despite their accuracy, are laborious and time consuming, leaving spectroscopic methods as a potential alternative. Mass and infrared spectroscopy are among the most reconnoitered techniques for this purpose, with Raman having been practically unexplored. Some species of the bacterial genus Acinetobacter are recognized as etiological agents of nosocomial infections associated with high rates of mortality and morbidity, which makes their accurate identification important. The goal of this study was to assess the ability of Raman spectroscopy to discriminate between 16 Acinetobacter species belonging to two phylogroups containing taxonomically closely related species, that is, the Acinetobacter baumannii-Acinetobacter calcoaceticus complex (six species) and haemolytic clade (10 species). Bacterial spectra were acquired without the need for any sample pre-treatment and were further analyzed with multivariate data analysis, namely partial least squares discriminant analysis (PLSDA). Species discrimination was achieved through a series of sequential PLSDA models, with the percentage of correct species assignments ranging from 72.1% to 98.7%. The obtained results suggest that Raman spectroscopy is a promising alternative for identification of Acinetobacter species.

Design of Ultrasensitive Protein Biosensor Strips for Selective Detection of Aromatic Contaminants in Environmental Wastewater.

Phenol and its derivatives constitute a class of highly toxic xenobiotics that pollute both river and groundwater. Here, we use a highly stable enzyme-based in vitro biosensing scaffold to develop a chip-based environmental diagnostic for in situ accurate, direct detection of phenol with selectively down to 10 ppb. Mesoporous silica nanoparticles (MCM41) having a pore diameter of 6.5 nm was screened and found to be the optimal solid support for creation of a robust immobilized protein based sensor, which retains stability, enzyme activity, sensitivity, and selectivity at par with solution format. The sensor strip exhibits minimal cross reactivity in simulated wastewater, crowded with several common pollutants. Moreover, this design is competent towards detection of phenol content with 95% accuracy in real-time environmental samples collected from local surroundings, making it a viable candidate for commercialization. The enzyme has been further modified via evolution driven mutagenesis to generate an exclusive 2,3-dimethylphenol sensor with equivalent selectivity and sensitivity as the native phenol sensor. Thus, this approach can be extended to generate a battery of sensors for other priority aromatic pollutants, highlighting the versatility of the biosensor unit. This novel biosensor design presents promising potential for direct detection and can be integrated in a device format for on-site pollutant monitoring.

Using Vitek MALDI-TOF mass spectrometry to identify species belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex: a relevant alternative to molecular biology?

Acinetobacter baumannii belongs to the Acinetobacter calcoaceticus-baumannii complex (Acb) containing 2 other pathogenic species: Acinetobacter pittii and Acinetobacter nosocomialis. Identification of these bacteria remains problematic despite the use of matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF MS). Here, we enriched the SARAMIS™ database of the Vitek MS® plus mass spectrometer to improve the identification of species of the Acb complex. For each species, we incremented reference spectra. Then, a SuperSpectrum was created based on the selection of 40 specific masses. In a second step, we validated reference spectra and SuperSpectra with 100 isolates identified by rpoB gene sequencing. All the isolates were correctly identified by MALDI-TOF MS with the database we created as compared to the identifications obtained by rpoB sequencing. Our database enabled rapid and reliable identification of the pathogen species belonging to the Acb complex. Identification by MALDI-TOF MS with our database is a good alternative to molecular biology.

MALDI-TOF MS and chemometric based identification of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex species.

MALDI-TOF MS is becoming the technique of choice for rapid bacterial identification at species level in routine diagnostics. However, some drawbacks concerning the identification of closely related species such as those belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex lead to high rates of misidentifications. In this work we successfully developed an approach that combines MALDI-TOF MS and chemometric tools to discriminate the six Acb complex species (A. baumannii, Acinetobacter nosocomialis, Acinetobacter pittii, A. calcoaceticus, genomic species "Close to 13TU" and genomic species "Between 1 and 3"). Mass spectra of 83 taxonomically well characterized clinical strains, reflecting the breadth of currently known phenetic diversity within the Acb complex, were achieved from intact cells and cell extracts and analyzed with hierarchical cluster analysis (HCA) and partial least squares discriminant analysis (PLSDA). This combined approach lead to 100% of correct species identification using mass spectra obtained from intact cells. Moreover, it was possible to discriminate two Acb complex species (genomic species "Close to 13TU" and genomic species "Between 1 and 3") not included in the MALDI Biotyper database.

Discrimination of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex species by Fourier transform infrared spectroscopy.

The main goal of this work was to assess the ability of Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) to discriminate between the species of the Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex, i.e. A. baumannii, A. nosocomialis, A. pittii, A. calcoaceticus, genomic species "Between 1 and 3" and genomic species "Close to 13TU". A total of 122 clinical isolates of the Acb complex previously identified by rpoB sequencing were studied. FTIR-ATR spectra was analysed by partial least squares discriminant analysis (PLSDA) and the model scores were presented in a dendrogram form. This spectroscopic technique proved to be effective in the discrimination of the Acb complex species, with sensitivities from 90 to 100%. Moreover, a flowchart aiming to help with species identification was developed and tested with 100% correct predictions for A. baumannii, A. nosocomialis and A. pittii test isolates. This rapid, low cost and environmentally friendly technique proved to be a reliable alternative for the identification of these closely related Acinetobacter species that share many clinical and epidemiological characteristics and are often difficult to distinguish. Its validation towards application on a routine basis could revolutionise high-throughput bacterial identification.

[Antiadhesive properties of the surfactants of Acinetobacter calcoaceticus IMB B-7241, Rhodococcus erythropolis IMB Ac-5017, and Nocardia vaccinii IMB B-7405].

Attachment of the cells of some bacteria, yeasts, and micromycetes to various surfaces (catheters, dentures, plastic, polyvinyl chloride, tiles, and steel) treated with the surfactants fromAcinetobacter calcoace- ticus IMB B-7241, Rhodococcus erythropolis IMB Ac-5017, and Nocardia vaccinii IMB B-7405 was studied. Adhesion of microorganisms to all the studied surfaces depended on the surfactant concentration and purity, kind of surface, and the test culture. Treatment with the surfactants from N. vaccinii IMB B-7405 (0.005- 0.05 mg/mL), A. calcoaceticus IMB B-7241 (0.003-0.036 mg/mL), and R. erythropolis IMB Ac-5017 (0.03- 0.12 mg/mL) resulted in adhesion decreased respectively by 35-75, 60-75, and 25-90% for bacteria (Es- cherichia coli IEM-1, Bacillus subtilis BT-2, etc.), by 80-85, 55-90, and 15-60% for yeasts Candida albicans D-6, and by 40-50, 35-45, and 10-20% for micromycetes (Aspergillus niger P-3 and Fusarium culmorum T-7).

[Overexpression, purification and characterization of phospholipase C from Acinetobacter calcoaceticus].

OBJECTIVE: In this study, we constructed two recombinant Escherichia coli strains to produce phospholipase C (PLC) from Acinetobacter calcoaceticus. The recombinant enzymes were purified to homogeneity and characterized. [Methods] We cloned the PLC encoding gene plc1, plc2 from genome DNA of A. calcoaceticus ATCC17902. The amplified fragments were inserted into pET28a(+ to obtain expression plasmids. E. coli BL21 (DE3) harboring the above plasmids were cultivated and induced with isopropyl-beta-D-thiogalactopyranoside to express PLCs. The recombinant PLCs were purified by affinity chromatography and their catalytic properties were characterized. RESULTS: Two PLCs from A. calcoaceticus were cloned and functional expressed in E. coli. The recombinant enzymes have activities of 31,160 +/- 418 U/mg for PLC1 and 13640 +/- 354 U/mg for PLC2, when using p-nitrophenyl phosphorycholine as substrate. The purified PLC1 and PLC2 exhibited optimum temperature at 65 degrees C and 50 degrees C, respectively. Their optimal pH were 8 and 7.5, respectively. PLC2 was stable under 40 degrees C and pH at 8, whereas the residual activity of PLC1 was less than 25% in the same condition. Mg2+ and Ca2+ stimulated two enzymes activity, whereas Zn2. stimulated PLC1 and inhibited PLC2. PLC1 and PLC2 hydrolyzed phosphatidylinositol. CONCLUSION: It is the first time to express and characterize the PLC gene from A. calcoaceticus ATCC17902. These research results provide reference for the study of food-safety microbiological PLC.

Isolation and structural characterization of a (Kdo-isosteric) D-glycero-α-D-talo-oct-2-ulopyranosidonic acid (Ko) interlinking lipid A and core oligosaccharide in the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305.

In Acinetobacter calcoaceticus NCTC 10305 and A. haemolyticus NCTC 10305 lipopolysaccharide (LPS) a Kdo (3-deoxy-D-manno-oct-2-ulosonic acid)-related octulosonic acid (Ko) interlinks the lipid A with the core-oligosaccharide. This Ko replaces the first Kdo (Kdo(I)) attached to the lipid A backbone in the LPS. The only structural difference between Kdo and Ko is the 3-hydroxylation. After the discovery of the final step in Ko-biosynthesis it is now generally accepted that Ko is structurally related to Kdo, although a final proof so far is lacking. In the present paper we describe the stereochemical determination of the natural Ko isolated from the LPS of A. calcoaceticus NCTC 10305 by chemical, mass spectrometry (MS), and (1)H and (13)C NMR spectroscopy. Our results show that in A. calcoaceticusd-glycero-α-D-talo-oct-2-ulopyranosonic acid (DgαDt-Kop) represents the Kdo-related sugar interlinking the core-oligosaccharide and the lipid A backbone.

[Effect of surface-active substances of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017, and Nocardia vaccinii K-8 on phytopathogenic bacteria].

The effect of surface-active substances (SAS's) of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017, and Nocardia vaccinii K-8 on phytopathogenic bacteria has been studied. It was shown that the survival of cells (10(5)-10(7) in a milliliter) of the Pseudomonas and Xanthomonas phytopathogenic bacteria was found to be 0-33% after treatment with SAS preparations of the IMV Ac-5017 and IMV B-7241 strains for 2 h (0.15-0.4 mg/mL). In the presence of N. vaccinii K-8 SAS preparations (0.085-0.85 mg/mL), the number of cells of the majority of the studied phytopathogenic bacteria decreased by 95-100%. These data show prospects for using microbial SAS's for the construction of ecologically friendly drugs for regulating the number of phytopathogenic bacteria.

[Chemiluminescence analysis of oil oxidizing bacteria Actinetobacter calcoaceticus extracts: effects of the extracts on pSoxS-lux biosensor].

A comparative H2O2-luminol- and Fe(II)-induced chemiluminescence analysis of extracts of two strains of marine oil oxidizing bacteria Actinetobacter calcoaceticus cultivated either in the presence or absence of oil was carried out. Effects of these extracts on E. coli MG1655 biosensor (pSoxS-lux) were studied. Activation of H2O2-induced chemiluminescence in the presence of oil was observed. This suggests activation of free radical lipid peroxidation. Aqueous extracts of microorganisms cultivated in the presence of oil were shown to activate reactive oxygen species production (ROS) in Fe(II)-induced chemiluminescence reaction mixture. Acetone-ethanol extracts induced antioxidative systems of both strains. Chemiluminescence analysis in a biological system carried utilizing E. coli MG1655 (pSoxS-lux) revealed that aqueous extracts of the strains cultivated in the absence of oil contained potential antioxidants.

Direct quantification of inorganic polyphosphate in microbial cells using 4'-6-diamidino-2-phenylindole (DAPI).

Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in microbial cultures and environmental samples. A major drawback is the need to extract polyP from cells prior to analysis. Due to extraction inefficiencies this can lead to an underestimation of both intracellular polyP levels and its environmental pool size: we observed 23-58% loss of polyP using standard solutions and current protocols. Here we report a direct fluorescence based DAPI assay system which removes the requirement for prior polyP extraction before quantification. This increased the efficiency of polyP detection by 28-55% in microbial cultures suggesting quantitative measurement of the intracellular polyP pool. It provides a direct polyP assay which combines quantification capability with technical simplicity. This is an important step forward in our ability to explore the role of polyP in cellular biology and biogeochemical nutrient cycling.

[Effect of biosurfactants Acinetobacter calcoaceticus K-4 and Rhodococcus erythropolis EK-1 on some microorganisms].

It has been established that the preparations of biosurfactants Rhodococcus erythropolis EK-1 (0.61 -2.1 mg/ml) and Acinetobacter calcoaceticus K-4 (0.15 - 0.22 mg/ml) in a form of supernatants of the cultural liquid show antimicrobial effect in respect of a number of microorganisms (Bacillus subtilis BT-2, Escherichia coli IEM-1, Candida tropicalis BT-5, Candida albicans D6, Candida utilis BVC-65, Saccharomyces cerevisiae OB-3). No inhibiting effect of biosurfactant preparations of R. erythropolis EK-1 on the cells of S. cerevisiae OB-3 and E. coli IEM-I and antifungal effect of the both studied surfactants on Aspergillus niger P-3 and Fusarium culmorum T-7 were revealed. The survival of microbe cells depend on biosurfactants concentration in the preparations, on exposure time as well as on physiological state of test-cultures. It has been established that the surfactant preparations of A. calcoaceticus K-4 had a higher effect on the spores of B. subtilis BT-2, than on vegetative cells, thus decreasing the spore culture survival by 75% in 2 h of exposure.

Biodegradation of shellfish wastes and production of chitosanases by a squid pen-assimilating bacterium, Acinetobacter calcoaceticus TKU024.

Two chitosanases (CHSA1 and CHSA2) were purified from the culture supernatant of Acinetobacter calcoaceticus TKU024 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHSA1 and CHSA2 determined by SDS-PAGE were approximately 27 and 66 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHSA1 and CHSA2 were (pH 6, 50°C, pH 4-10, <90°C) and (pH 7, 60°C, pH 6-11, <70°C), respectively. CHSA1 and CHSA2 had broad pH and thermal stability. CHSA1 and CHSA2 were both inhibited by EDTA and were inhibited completely by 5 mM Mn(2+). CHSA1 and CHSA2 degraded chitosan with DD ranging from 60 to 98%, and also degraded some chitin. The most susceptible substrate was 60% deacetylated chitosan. Furthermore, TKU024 culture supernatant (1.5% SPP) incubated for 5 days has the most reducing sugars (0.63 mg/ml). With this method, we have shown that shellfish wastes may have a great potential for the production of bioactive materials.

Structural and immunochemical analysis of the lipopolysaccharide from Acinetobacter lwoffii F78 located outside Chlamydiaceae with a Chlamydia-specific lipopolysaccharide epitope.

Chemical analyses, NMR spectroscopy, and mass spectrometry were used to elucidate the structure of the rough lipopolysaccharide (LPS) isolated from Acinetobacter lwoffii F78. As a prominent feature, the core region of this LPS contained the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo=3-deoxy-d-D-manno-oct-2-ulopyranosonic acid), which so far has been identified only in chlamydial LPS. In serological investigations, the anti-chlamydial LPS monoclonal antibody S25-2, which is specific for the epitope alpha-Kdo-(2-->8)-alpha-Kdo, reacted with A. lwoffii F78 LPS. Thus, an LPS was identified outside Chlamydiaceae that contains a Chlamydia-specific LPS epitope in its core region.

Determination of organophosphorus aromatic nitro insecticides and p-nitrophenol by microbial-cell respiratory activity.

We examined the possibility of measuring the organophosphorus aromatic nitro insecticides metaphos and sumithion as well as their hydrolysis product p-nitrophenol (PNP) by the specific respiratory activity (SRA) of Pseudomonas putida C-11, P. putida BA-11, and Acinetobacter calcoaceticum A-122. The plots of cellular SRA against the two insecticides and PNP were linear over the ranges of 0.5-2.5 microM for P. putida C-11 and BA-11 and 0.5-1.0 microM for A. calcoaceticum A-122. P. putida BA-11 showed the greatest respiratory-response selectivity in the determination of the test substrates. We made comparison studies of the SRA of cells immobilised by two methods: carrier-surface adsorption and inclusion in various gels. We discuss the feasibility of developing a microbial sensor system for the determination of metaphos, sumithion, and PNP in aqueous media.

Effect of aliphatic alcohols on growth and degree of saturation of membrane lipids in Acinetobacter calcoaceticus.

The adaptive responses of the bacterium Acinetobacter calcoaceticus to different aliphatic alcohols on the level of the membrane fatty acids were studied in detail. The toxicity of the aliphatic alcohols increased with an increasing hydrophobicity. As alcohols are known to increase the fluidity of the membrane they consequently should cause the same adaptive effect on membrane level. Yet, cells of A. calcoaceticus react completely different to the alcohols: in the presence of long-chained alcohols they increase their degree of saturation, while in the presence of short-chained alcohols they decrease the degree of saturation. So, there are no observable differences in the adaptive responses of bacteria with the so-called anaerobic pathway, like Escherichia coli and Pseudomonas putida, and the bacterium carrying the so-called aerobic pathway like A. calcoaceticus. These results strongly indicate a physico-chemical difference in the membrane effect of both the partitioning and localisation of the different alcohols into the membrane and the membrane adaptive responses of the bacteria to these effects.

Purification and characterization of lipase from psychrophilic Acinetobacter calcoaceticus LP009.

A lipase-producing bacterium, Acinetobacter calcoacetius LP009, was isolated from raw milk. The optimum conditions for growth and lipase production by A. calcoaceticus LP009 were 15 degrees C with shaking at 200 rpm in LB supplemented with 1.0% (v/v) Tween 80. The crude lipase was purified to homogeneous state by ultrafiltration and gel filtration chromatography on Sephadex G-100. Its molecular weight determined by SDS-PAGE was 23 kDa and it exhibited maximum activity at pH 7.0 and 50 degrees C. It was stable over the pH range of 4.0 to 8.0 and at temperatures lower than 45 degrees C. It was a metalloenzyme that is positionally non-specific and had the ability to improve fat hydrolysis in soybean meal and in premixed animals feed.