The rapid identification of Acinetobacter species using Fourier transform infrared spectroscopy

AIMS: Fourier transform infrared (FT-IR) was used to analyse a selection of Acinetobacter isolates in order to determine if this approach could discriminate readily between the known genomic species of this genus and environmental isolates from activated sludge. METHODS AND RESULTS: FT-IR spectroscopy is a rapid whole-organism fingerprinting method, typically taking only 10 s per sample, and generates 'holistic' biochemical profiles (or 'fingerprints') from biological materials. The cluster analysis produced by FT-IR was compared with previous polyphasic taxonomic studies on these isolates and with 16S-23S rDNA intergenic spacer region (ISR) fingerprinting presented in this paper. FT-IR and 16S-23S rDNA ISR analyses together indicate that some of the Acinetobacter genomic species are particularly heterogeneous and poorly defined, making characterization of the unknown environmental isolates with the genomic species difficult. CONCLUSIONS: Whilst the characterization of the isolates from activated sludge revealed by FT-IR and 16S-23S rDNA ISR were not directly comparable, the dendrogram produced from FT-IR data did correlate well with the outcomes of the other polyphasic taxonomic work. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe it would be advantageous to pursue this approach further and establish a comprehensive database of taxonomically well-defined Acinetobacter species to aid the identification of unknown strains. In this instance, FT-IR may provide the rapid identification method eagerly sought for the routine identification of Acinetobacter isolates from a wide range of environmental sources.     

Determination of the degree of esterification of pectinates with decyl and benzyl ester groups by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and curve-fitting deconvolution method

Pectinates with benzyl and decyl ester groups were prepared by alkylation of the tetrabutylammonium salt of pectic acid with benzyl and decyl bromides, respectively. The degree of esterification (DE) of the pectin derivatives was determined by diffuse reflectance infrared Fourier transform spectroscopy and the curve-fitting deconvolution method. A linear relationship between DE and the ratio of the peak area at 1745 cm⁻¹ to the sum of the peak areas at 1745 and 1608 cm⁻¹ was established with a high correlation coefficient 0.98. The deconvolution analysis using the curve-fitting method allowed the elimination of spectral interferences from pectin components and their degradation products. The limits of the method are given by DE 6 and 93%. The method was compared with chemical analysis and found to be equivalent in view of accuracy and repeatability (t-test, F-test). The method is applicable in analysis of natural or synthetic mixtures and/or crude pectin substances.     

Differentiation of outer membrane proteins from Salmonellaenterica serotypes using Fourier transform infrared spectroscopy and chemometrics

AIMS: To differentiate between outer membrane proteins (OMPs) from six Salmonellaenterica serotypes using a Fourier transform infrared (FTIR) spectroscopy method and chemometrics. METHODS AND RESULTS: The OMPs from Salmonella serotypes (Typhimurium, Enteritidis, Thomasville, Hadar, Seftenberg and Brandenburg) were isolated using a sarcosyl extraction method. OMP profiles on SDS-PAGE exhibited two or three bands between 48 and 54 kDa. Spectra of 10 microl of OMP preparations (5 mg ml(-1)) dried on a gold reflective slide were collected using 128 scans at 4 cm(-1) resolution and units of log (1/R) and analyzed using canonical variate analysis (CVA) and linear discriminant analysis (LDA). The CVA of Salmonella OMP spectra in the 1800-1500 cm(-1) region separated the serotypes and LDA provided a 100% correct classification. CONCLUSIONS: The use of a FTIR method combined with chemometrics provided better differentiation of Salmonella OMPs than the OMP pattern analysis by SDS-PAGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that spectra of OMP extracts from Salmonella serotypes can be used for 100% correct classification of the serotypes studied.     

Fourier transform infrared spectroscopy as a new tool to determine rosmarinic acid in situ

A new procedure has been developed for the in situ FT-IR determination of rosmarinic acid (RA) in suspension cultures of Lavandula officinalis. The method involves sample preparation on ZnSe crystals or microplates from silicon, and measuring absorbance spectra between 4000 and 700 cm(-1). First derivative spectra were analysed after normalisation using partial least square (PLS) algorithm. The correlation between spectral analysis and HPLC measurements of cell extracts shows that the FT-IR procedure is suitable for qualitative and quantitative analyses of RA in cell suspension cultures.     

Chronic hypoperfusion alters the content and structure of proteins and lipids of rat brain homogenates: a Fourier transform infrared spectroscopy study

Arteriovenous malformations (AVMs), masses of abnormal blood vessels which grow in the brain, produce high flow shunts that steal blood from surrounding brain tissue, which is chronically hypoperfused. Hypoperfusion is a condition of inadequate tissue perfusion and oxygenation, resulting in abnormal tissue metabolism. Fourier transform infrared (FTIR) spectroscopy is used in this study to investigate the effect of hypoperfusion on homogenized rat brain samples at the molecular level. The results suggest that the lipid content increases, the protein content decreases, the lipid-to-protein ratio increases, and the state of order of the lipids increases in the hypoperfused brain samples. FTIR results also revealed that, owing to hypoperfusion, not only the protein synthesis but also the protein secondary structure profile is altered in favor of -sheets and random coils. These findings clearly demonstrate that, FTIR spectroscopy can be used to extract valuable information at the molecular level so as to have a better understanding of the effect of hypoperfusion on rat brain.     

Molecular interactions of the redox-active accessory chlorophyll on the electron-donor side of photosystem II as studied by Fourier transform infrared spectroscopy

A Fourier transform infrared (FTIR) difference spectrum upon photooxidation of the accessory chlorophyll (Chl_z) of photosystem II (PS II) was obtained at 210 K with Mn-depleted PS II membranes in the presence of fericyanide and silicomolybdate. The observed Chl_z⁺/Chl_z spectrum showed two differential bands at 1747/1736 and 1714/1684 cm⁻. The former was assigned to the free carbomethoxy C = 0 and the latter to the keto C = 0 that is hydrogen-bonded or in a highly polar environment. Also, the negative 1614 cm⁻ band assignable to the macrocycle mode indicated 5-coordination of the central Mg. The negative 1660 cm⁻¹ band, possibly due to the strongly hydrogen-bonded keto C = 0, may suggest oxidation of one more Chl_z, although an alternative assignment, the amide I mode of proteins perturbed by Chl_z oxidation, is also possible.     

Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase

This study demonstrates the use of Fourier transform infrared (FTIR) spectroscopy for monitoring both synthesis and hydrolysis reactions catalyzed by a recombinant amidase (EC 3.5.1.4) from Pseudomonas aeruginosa. The kinetics of hydrolysis of acetamide, propionamide, butyramide, acrylamide, benzamide, phenylalaninamide, alaninamide, glycinamide, and leucinamide were determined. This revealed that very short-chain substrates displayed higher amidase activity than did branched side-chain or aromatic substrates. In addition, on reducing the polarity and increasing the substrates' bulkiness, a reduction of the amidase affinity for the substrates took place. Using FTIR spectroscopy it was possible to monitor and quantify the synthesis of several hydroxamic acid derivatives and ester hydrolysis products. These products may occur simultaneously in a reaction catalyzed by the amidase. The substrates used for the study of such reactions were ethyl acetate and glycine ethyl ester. Hydroxylamine was the nucleophile substrate used for the synthesis of acetohydroxamate compounds. Results presented in this article demonstrate the usefulness of FTIR spectroscopy as an important tool for understanding the enzyme structure-activity relationship because it provides a simple and rapid real-time assay for the detection and quantification of amidase hydrolysis and synthesis reactions in situ.     

Dehydrating phospholipid vesicles measured in real-time using ATR Fourier transform infrared spectroscopy

According to the water replacement hypothesis, trehalose stabilizes dry membranes by preventing the decrease in spacing between adjacent phopspholipid headgroups during dehydration. Alternatively, the water-entrapment hypothesis postulates that in the dried state sugars trap residual water at the biomolecule sugar interface. In this study, Fourier transform infrared spectroscopy with an attenuated total reflection accessory was used to investigate the influence of trehalose on the dehydration kinetics and residual water content of egg phosphatidylcholine liposomes in real time under controlled relative humidity conditions. In the absence of trehalose, the lipids displayed a transition to a more ordered gel phase upon drying. The membrane conformational disorder in the dried state was found to decrease with decreasing relative humidity. Even at a relative humidity as high as 94% the conformational disorder of the lipid acyl chains decreased after evaporation of the bulk water. The presence of trehalose affects the rate of water removal from the system and the lipid phase behavior. The rate of water removal is decreased and the residual water content is higher, as compared to drying in the absence of trehalose. During drying, the level of hydrogen bonding to the head groups remains constant. In addition, the conformational disorder of the lipid acyl chains in the dried state more closely resembles that of the lipids in the fully hydrated state. We conclude that water entrapment rather than water replacement explains the effect of trehalose on lipid phase behavior of phosphatidylcholine lipid bilayers during the initial phase of drying.     

Conformational mapping of the N-terminal peptide of HIV-1 gp41 in membrane environments using C-enhanced Fourier transform infrared spectroscopy

The N-terminal domain of HIV-1 glycoprotein 41?000 (FP; residues 1-23; AVGIGALFLGFLGAAGSTMGARSCONH₂) participates in fusion processes underlying virus-cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, ¹²C-Fourier transform infrared (FTIR) spectroscopy indicated the following α-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes∼hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). ¹²C-FTIR spectra also showed disordered FP structures in these environments, along with substantial β-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with ¹³C-carbonyl at multiple sites. Combining these ¹³C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: α-helix (residues 3-16) and random and β-structures (residues 1-2 and residues 17-23). Additional ¹³C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the α-helix extends over residues 1-16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, ¹³C-FTIR spectroscopy showed α-helical conformations for the central core of FP (residues 5-15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel β-structure. FP at low loading in ghosts probably inserts deeply as an α-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, β-sheet. In future studies, ¹³C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.     

Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of β-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of β-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon β-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding β-sheet structure as evidenced by the appearance of a strong band at 1614 cm⁻¹ at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of β-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular β-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of β-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular β-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.     

Rapid strain classification and taxa delimitation within the edible mushroom genus Pleurotus through the use of diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy

Fourier transform infrared (FT-IR) spectroscopy has been successfully applied for the identification of bacteria and yeasts, but only to a limited extent for discriminating specific groups of filamentous fungi. In the frame of this study, 73 strains - from different associated hosts/substrates and geographic regions - representing 16 taxa of the edible mushroom genus Pleurotus (Basidiomycota, Agaricales) were examined through the use of diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. A binary matrix, elaborated on the basis of presence/absence of specific absorbance peaks combined with cluster analysis, demonstrated that the spectral region 1800-600 cm(-1) permitted clear delimitation of individual strains into Pleurotus species. In addition, closely related species (e.g., Pleurotus ostreatus and Pleurotus pulmonarius) or taxa of the subgenus Coremiopleurotus demonstrated high similarity in their absorbance patterns, whereas genetically distinct entities such as Pleurotus dryinus, Pleurotus djamor, and Pleurotus eryngii provided spectra with noteworthy differences. When specific regions (1800-1700, 1360-1285, 1125-1068, and 950-650 cm(-1)) were evaluated in respect to the absorbance values demonstrated by individual strains, it was evidenced that this methodology could be eventually exploited for the identification of unknown Pleurotus specimens with a stepwise process and with the aid of a dichotomous key developed for this purpose. Moreover, it was shown that the nature of original fungal material examined (mycelium, basidiomata, and basidiospores) had an effect on the outcome of such analyses, and so did the use of different mycelium growth substrates. In conclusion, application of FT-IR spectroscopy provided a fast, reliable, and cost-efficient solution for the classification of pure cultures from closely related mushroom species.     

Evaluation of the biological stability of waste during landfill stabilization by thermogravimetric analysis and Fourier transform infrared spectroscopy

This study seeks to assess the biological stability of landfilled municipal solid waste (MSW) based on the changes in organic matter, as revealed by thermogravimetric analysis and Fourier transform infrared (FTIR) spectroscopy. Derivate thermogravimetry profiles (DTG) showed a reduction in peak intensity at 200-350 °C (DTG2), while an increase in peak intensity and a shift towards higher temperature at 400-600 °C (DTG3). The decrease in the peak intensity of the aliphatic methylene at 2920 and 2850 cm(-1), and the increase of aromatic substances and polysaccharide at 1640 cm(-1) in the FTIR spectra also confirm the changes. Well-fitted correlations of the peak intensity ratio (2920/1640) and peak area ratio (DTG2/DTG3) to C/N ratio were also established, confirming that the 2920/1640 and the DTG2/DTG3 ratios can be considered as reliable parameters for tracking the biological stability of MSW during landfill stabilization.     

Generation of a substructure library for the description and classification of protein secondary structure. II. Application to spectra-structure correlations in Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy has become well known as a sensitive and informative tool for studying secondary structure in proteins. Present analysis of the conformation-sensitive amide I region in protein infrared spectra, when combined with band narrowing techniques, provides more information concerning protein secondary structure than can be meaningfully interpreted. This is due in part to limited models for secondary structure. Using the algorithm described in the previous paper of this series, we have generated a library of substructures for several trypsin-like serine proteases. This library was used as a basis for spectra-structure correlations with infrared spectra in the amide I' region, for five homologous proteins for which spectra were collected. Use of the substructure library has allowed correlations not previously possible with template-based methods of protein conformational analysis.     

Liposome-based formulations for the antibiotic nonapeptide Leucinostatin A: Fourier transform infrared spectroscopy characterization and in vivo toxicologic study

Leucinostatin-A is a nonapeptide isolated fromPaecilomyces marquandii Paecilomyces lilacinus A257, andAcremonium sp., exerting remarkable phytotoxic, antibacterial (especially against Gram-positive) and antimycotic activities. With the aim to find alternative formulation for in vivo administration, a number of Leucinostatin-A—loaded liposomal formulations have been prepared and characterized. Both large unilamellar vesicles and multilamellar vesicles consisting of synthetic and natural lipids were evaluated. In addition, to determine the nature of peptide-membrane interactions and the stability of liposomes loaded with Leucinostatin-A, a Fourier Transform Infrared Spectroscopy study was performed. The results suggest that the mode of interaction of the peptide is dependent on its concentration, on bilayer fluidity, and on liposome type. Finally, the LD₅₀ of both free and liposome-delivered Leucinostatin-A was determined in mice. These results suggest that the incorporation of Leucinostatin-A into liposomes may result in decreased Leucinostatin-A toxicity, as the intraperitoneal administration of Leucinostatin-A—loaded liposomes reduced the LD₅₀ of Leucinostatin-A 15-fold.     

Freezing and desiccation tolerance in the moss Physcomitrella patens: An in situ Fourier transform infrared spectroscopic study

In situ Fourier transform infrared spectroscopy (FTIR) was used in order to obtain more insights in the underlying protective mechanisms upon freezing and drying of ABA-treated tissues of the moss Physcomitrella patens. The effects of different treatments on the membrane phase behaviour, glassy state, and overall protein secondary structure were studied. We found that growth on ABA resulted in the accumulation of sucrose: up to 22% of the tissue on a dry weight basis, compared to only 3.7% in non-ABA-treated tissues. Sucrose functions as a protectant during freezing and drying, but accumulation of sucrose alone is not sufficient for survival. ABA-treated tissue survives a freeze–thaw cycle down to −80 °C only after addition of an additional cryoprotectant (DMSO). Survival correlates with preservation of membrane phase behaviour. We found that ABA-treated P. patens can survive slow but not rapid drying down to water contents as low as 0.02 g H₂O per g DW. Rapidly and slowly dried ABA-treated tissues were found to have similar sugar compositions and glass transition temperatures. The average strength of hydrogen bonding in the cytoplasmic glassy matrix, however, was found to be increased upon slow drying. In addition, slowly dried tissues were found to have a higher relative proportion of α-helical structures compared to rapidly dried tissues.     

The Three-Dimensional Structure of [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hildenborough: A Hydrogenase without a Bridging Ligand in the Active Site in Its Oxidised, “as-Isolated” State

Hydrogen is a good energy vector, and its production from renewable sources is a requirement for its widespread use. [NiFeSe] hydrogenases (Hases) are attractive candidates for the biological production of hydrogen because they are capable of high production rates even in the presence of moderate amounts of O₂, lessening the requirements for anaerobic conditions. The three-dimensional structure of the [NiFeSe] Hase from Desulfovibrio vulgaris Hildenborough has been determined in its oxidised “as-isolated” form at 2.04-Å resolution. Remarkably, this is the first structure of an oxidised Hase of the [NiFe] family that does not contain an oxide bridging ligand at the active site. Instead, an extra sulfur atom is observed binding Ni and Se, leading to a SeCys conformation that shields the NiFe site from contact with oxygen. This structure provides several insights that may explain the fast activation and O₂ tolerance of these enzymes.     

Entrapment of human flavin-containing monooxygenase 3 in the presence of gold nanoparticles: TEM,FTIR and electrocatalysis

Background

Nanosized particles of gold are widely used as advanced materials for enzyme catalysis investigations. In some bioanalytical methods these nanoparticles can be exploited to increase the sensitivity by enhancing electron transfer to the biological component i.e. redox enzymes such as drug metabolizing enzymes.

Methods

In this work, we describe the characterization of human flavin-containing monooxygenase 3 (hFMO3) in a nanoelectrode system based on AuNPs stabilized with didodecyldimethylammonium bromide (DDAB) on glassy carbon electrodes. Once confirmed by FTIR spectroscopy that in the presence of DDAB-AuNPs the structural integrity of hFMO3 is preserved, the influence of AuNPs on the electrochemistry of the enzyme was studied by cyclic voltammetry and square wave voltammetry.

Results

Our results show that AuNPs improve the electrochemical performance of hFMO3 on glassy carbon electrodes by enhancing the electron transfer rate and the current signal-to-noise ratio. Moreover, the electrocatalytic activity of hFMO3-DDAB-AuNP electrodes which was investigated in the presence of two well known substrates, benzydamine and sulindac sulfide, resulted in K_M values of 52 μM and 27 μM, with V_max of 8 nmol min^− 1 mg^− 1 and 4 nmol min^− 1 mg^− 1, respectively, which are in agreement with data obtained with the microsomal enzyme.

Conclusions

The immobilization of hFMO3 protein in DDAB stabilized AuNP electrodes improves the bioelectrochemical performance of this important phase I drug metabolizing enzyme.

General significance

This bio-analytical method can be considered as a promising advance in the development of new techniques suitable for the screening of novel hFMO3 metabolized pharmaceuticals.     

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Dihydropyrimidinase is involved in the reductive pathway of pyrimidine degradation, catalysing the reversible hydrolysis of the cyclic amide bond (–CO–NH–) of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl-β-amino acids. This enzyme is an attractive candidate for commercial production of D-amino acids, which are used in the production of semi-synthetic β-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides. We have obtained the crystal structure of the dihydropyrimidinase from Sinorhizobium meliloti (SmelDhp) in the presence of zinc ions, but we have not been able to obtain good diffracting crystals in its absence. Then, the role of the ion in the structure of the protein, and in its stability, remains to be elucidated. In this work, the stability and the structure of SmelDhp have been studied in the absence and in the presence of zinc. In its absence, the protein acquired a tetrameric functional structure at pH ∼ 6.0, which is stable up to pH ∼ 9.0, as concluded from fluorescence and CD. Chemical-denaturation occurred via a monomeric intermediate with non-native structure. The addition of zinc caused: (i) an increase of the helical structure, and changes in the environment of aromatic residues; and, (ii) a higher thermal stability. However, chemical-denaturation still occurred through a monomeric intermediate. This is the first hydantoinase whose changes in the stability and in the secondary structure upon addition of zinc are described and explained, and one of the few examples where the zinc exclusively alters the secondary helical structure and the environment of some aromatic residues in the protein, leaving unchanged the quaternary structure.