Discrimination of enterobacterial repetitive intergenic consensus PCR types of Campylobacter coli and Campylobacter jejuni by Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FT-IR) has been used together with pattern recognition methodology to study isolates belonging to the species Campylobacter coli and Campylobacter jejuni and to compare FT-IR typing schemes with established genomic profiles based on enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Seventeen isolates were cultivated under standardized conditions for 2, 3, and 4 days to study variability and improve reproducibility. ERIC-PCR profiles and FT-IR spectra were obtained from strains belonging to the species Campylobacter coli and C. jejuni, normalized, and explored by hierarchical clustering and stepwise discriminant analysis. Strains could be differentiated by using mainly the first-derivative FT-IR spectral range, 1,200 to 900 cm(-1) (described as the carbohydrate region). The reproducibility index varied depending on the ages of the cultures and on the spectral ranges investigated. Classification obtained by FT-IR spectroscopy provided valuable taxonomic information and was mostly in agreement with data from the genotypic method, ERIC-PCR. The classification functions obtained from the discriminant analysis allowed the identification of 98.72% of isolates from the validation set. FT-IR can serve as a valuable tool in the classification, identification, and typing of thermophilic Campylobacter isolates, and a number of types can be differentiated by means of FT-IR spectroscopy.     

Whole-organism fingerprinting of the genus Carnobacterium using Fourier transform infrared spectroscopy (FT-IR)

Sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a six month period were also investigated. The results were analysed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies. Taxonomic correlations were observed between the FT-IR data and these studies. Culture age was observed to have little effect on the spectra obtained. The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period. It was concluded that FTIR is a reliable method for investigating carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification.     

Chemical characterization of airborne bacteria using X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIRS)

In this study, the chemical characterization of airborne bacteria is explored using X-ray photoelectron spectrometry (XPS) and Fourier transform infrared spectroscopy (FTIRS). Samples were taken by using a 6-stage Andersen impactor. Airborne bacteria were collected on nutrient media in Petri dishes located on all impactor stages. On the basis of the obtained XPS spectra the ratios of N/C, O/C, and P/C were calculated for 11 Gram-positive and 4 Gram-negative strains. The results agree well with the data obtained by others for the test microorganisms/stock cultures. It is concluded that XPS analysis might be applied for identification of airborne bacterial strains and species but rather in conjunction with different techniques. Especially, a complementary method to XPS seems to be FTIRS.     

Production of inulinase by Xanthomonas campestris pv phaseoli using onion (Allium cepa) and garlic (Allium sativum) peels in solid state cultivation

AIMS: To access inulinase production by Xanthomonas campestris pv phaseoli using the submerged and solid state cultivation (SSC) methods. METHODS AND RESULTS: Various carbon sources, inulin-rich solid substrates and pure synthetic inulin were tested for their efficiency in inulinase induction. The highest inulinase production (17.42 IU ml(-1)) in submerged cultures of X. campestris was observed with inulin as a carbon source with an initial pH, temperature and agitation of 7.0, 37 degrees C and 150 rev min(-1) respectively. Among the various substrates, garlic peels (117 IU gds(-1)) and onion peels (101 IU gds(-1)) were found to be the best for inulinase production. CONCLUSION: The inulinase production level of X. campestris was 6.7-fold higher in garlic and 5.8-fold in onion, under optimized SSC conditions compared with the submerged culture. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on inulinase production from garlic and onion peels by X. campestris using SSC. SSC is an efficient method for inulinase production by X. campestris for commercial applications.     

Quantification of micro-organisms in binary mixed populations by Fourier transform infrared (FT-IR) spectroscopy

Fourier Transform Infrared (FT-IR) spectroscopy was used for the first time to determine the ratios of different microorganisms in mixtures. Exemplarily, systems composed of two food-associated yeast species (Saccharomyces cerevisiae/Hanseniaspora uvarum) and two yoghurt lactic acid bacteria (Lactobacillus acidophilus/Streptococcus salivarius ssp. thermophilus) were investigated. Determination of the cell number ratio in the lactic acid bacteria system was possible with a minimal prediction accuracy of +/- 16 ratio percentage points while the minimum accuracy of prediction in the yeast two-component system was +/- 4% (both at a 95% confidence level). These results show that FT-IR spectroscopy is potentially a rapid method for the quantification of cell ratios in mixtures of two different microorganisms, provided that the cell ratio does not drop below a certain, system-specific threshold.     

Molecular rearrangements of thylakoids after heavy metal poisoning, as seen by Fourier transform infrared (FTIR) and electron spin resonance (ESR) spectroscopy

The specific effects exerted by different heavy metals on both the function and the structure of the photosynthetic apparatus were addressed. The functional analysis performed via the fluorescence induction kinetics revealed that the applied toxic heavy metals can be classified into two groups: Cd and Ni had no significant effect on the photosynthetic electron transport, while Cu, Pb and Zn strongly inhibited the Photosystem II (PS II) activity, as evidenced by the dramatic decreases in both the variable (F_v) and the maximal (F_m) fluorescence. The structural effects of the heavy metal ions on the thylakoid membranes were considered in three relations: (1) lipids, (2) proteins — studied by Fourier transform infrared (FTIR) spectroscopy, and (3) lipid—protein interactions — investigated by electron spin resonance (ESR) spectroscopy using spin-labeled probe molecules. The studied heavy metal ions had only a non-specific rigidifying effect on the thylakoid lipids. As regards proteins, Cd and Ni had no effect on the course of their heat denaturation. The heat denaturation of the proteins was accompanied by a decrease in the -helix content (1656 cm^-1), a parallel increase in the disordered segments (1651 cm^-1), a decrease in the intramolecular -sheet (1636 cm^-1) content and the concomitant appearance of an intermolecular -structure (1621 cm^-1). In contrast with Cd and Ni, Cu and Zn blocked the appearance of the intermolecular -structure. Pb represented an intermediate case. It seems that these heavy metals alter the native membrane structure in such a way that heat-induced aggregation becomes more limited. The ESR data revealed that certain heavy metals also affect the lipid—protein interactions. While Cd and Ni had hardly any effect on the solvation fraction of thylakoid lipids, Cu, Pb and Zn increased the fraction of lipids solvating the proteins. On the basis of the FTIR and ESR data, it seems that Cu, Pb, and Zn increase the surfaces available for lipid—protein interactions by dissociating membrane protein complexes, and that these lipidated proteins have a smaller chance to aggregate upon heat denaturation. The data presented here indicate that the damaging effects of poisonous heavy metals are element-specific, Cu, Pb and Zn interact directly with the thylakoid membranes of the photosynthetic apparatus, while Cd and Ni interfere rather with other metabolic processes of plants.     

Evidence for specific complex formation between alpha-melanocyte stimulating hormone and 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin using near infrared Fourier transform Raman spectroscopy

The cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)) and its 2 and 4 electron oxidation products 7,8-dihydro-L-biopterin and L-biopterin have been shown to form 1:1 complexes with the thirteen amino acid peptide alpha-melanocyte stimulating hormone (alpha-MSH). Hydrogen bonding to the pyrimidine ring of the cofactor has been established for glu(5) and his(6) of the hormone using Near Infrared Fourier Transform Raman spectroscopy. Binding of these pterins primarily involves the pyrimidine ring, although with the reduced pterins, 7,8-dihydro-L-biopterin and 6BH(4), there is evidence for pi orbital interaction with the pyrazine ring. It is proposed that this pi orbital interaction with the reduced biopterins and alpha-MSH could provide the basis for the observed stability of these pterins to oxidation by either molecular oxygen or photooxidation by UVB (290-320 nm) light. Our results suggest that the formation of the alpha-MSH/6BH(4) complex could play a major role in the control of all 6BH(4) dependent processes.     

Direct discrimination of different plant populations and study on temperature effects by Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy was used to characterise highland and lowland populations of Polygonum minus Huds. grown in different controlled environments. A thermal perturbation technique of two-dimensional correlation infrared spectroscopy (2D-IR) correlation spectra was applied to establish differences between the populations. The absorption peaks at 3,480 cm^?1 (hydroxyl group), 2,927 cm^?1 (methyl group), 1,623 cm^?1 (carbonyl group), and 1,068 cm^?1 (C–O group) were particularly powerful in separating the populations. These peaks, which indicate the presence of carbohydrate, terpenes, amide and flavonoids were more intense for the highland populations than lowland populations, and increased in environments with a higher temperature. Wavenumbers (1,634, 669 cm^?1) and (1,634, 1,555 cm^?1) in the 2D-IR correlation spectra provided fingerprint signals to differentiate plants grown at different temperatures. This study demonstrates that IR fingerprinting, which combines mid-IR spectra and 2D-IR correlation spectra, can directly discriminate different populations of P. minus and the effects of temperature.     

Integration of Raman microscopy, differential interference contrast microscopy, and attenuated total reflection Fourier transform infrared spectroscopy to investigate chlorhexidine spatial and temporal distribution in Candida albicans biofilms.

Two spectroscopic techniques, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and Raman microscopy (RM), were used to characterize transport of chlorhexidine digluconate (CHG) in Candida albicans (CA) biofilms. Different (volumetric) regions of the biofilm are sampled by these two vibrational spectroscopies making them complementary techniques. Simple mathematical models were developed to analyze ATR-FTIR and RM data to obtain an effective diffusion coefficient describing transport through CA biofilms. CA biofilms were composed primarily of yeast and hyphal forms, with some pseudohyphae. Upper regions of biofilms that had become confluent, (i.e., biofilms that completely covered the germanium (Ge) substratum) were composed primarily of a tangled mass of hyphae with openings between germtubes about 10 to 50 microm across. Quantitative analysis of ATR-FTIR kinetic data curves indicated that the effective diffusion coefficient for transport of CHG through confluent biofilms about 200-microm thick was reduced 0.1 to 0.3 times compared to the diffusion coefficient for CHG in water. Effective diffusion coefficients obtained from analysis of RM data were consistently higher than those indicated by ATR-FTIR data suggesting that transport is more hindered in regions near the base of the biofilm than in the outer layers. Analysis of both ATR-FTIR and RM data obtained from thicker films indicated that adsorption of CHG to biofilm components was responsible for a substantial portion of the transport limitation imposed by the biofilm. Comparison of ATR-FTIR and RM data for both types of biofilms indicated that sites of CHG adsorption were more concentrated in the interfacial region than in the bulk biofilm. Comparison of results for ATR-FTIR and RM measurements suggests that these relatively thick CA biofilms can be modeled, for purposes of predicting transport, approximately as a homogeneous thin planar sheet. Thus, these biofilms offer a relatively tractable model system for initial investigations of the relation between antimicrobial transport and kinetics of antimicrobial action.     

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases.     

Hyperaccumulation of cadmium by roots, bulbs and shoots of garlic (Allium sativum L.)

The effects of cadmium chloride concentration on root, bulb and shoot growth of garlic (Allium sativum L.), and the uptake and accumulation of Cd2+ by garlic roots, bulbs and shoots were investigated. The range of cadmium chloride (CdCl2 x 2.5H2O) concentrations was 10(-6) - 10(-2) M. Cadmium stimulated root length at lower concentrations (10(-6) - 10(-5) M) significantly (P < 0.005) during the entire treatment period. The seedlings exposed to 10(-3) - 10(-2) M Cd exhibited substantial growth reduction (P < 0.005), but did not develop chlorosis. Garlic has considerable ability to remove Cd from solutions and accumulate it. The Cd content in roots of garlic increased with increasing solution concentration of Cd2+. The roots in plants exposed to 10(-2) M Cd accumulated a large amount of Cd. approximately 1,826 times the control. The Cd contents in roots of plants treated with 10(-3), 10(-4), 10(-5) and 10(-6) M Cd were approximately 114, 59, 24 and 4 times the control, respectively. However, the plants transported only a small amount of Cd to their bulbs and shoots and concentrations in these tissues were low.     

Detection of pathological molecular alterations in scrapie-infected hamster brain by Fourier transform infrared (FT-IR) spectroscopy

In this report a new approach for the identification of pathological changes in scrapie-infected Syrian hamster brains using Fourier transform infrared microspectroscopy is discussed. Using computer-based pattern recognition techniques and imaging, infrared maps with high structural contrast were obtained. This strategy permitted comparison of spectroscopic data from identical anatomical structures in scrapie-infected and control brains. Consistent alterations in membrane state-of-order, protein composition, carbohydrate and nucleic acid constituents were detected in scrapie-infected tissues. Cluster analysis performed on spectra of homogenized medulla oblongata and pons samples also reliably separated uninfected from infected specimens. This method provides a useful tool not only for the exploration of the disease process but also for the development of rapid diagnostic and screening techniques of transmissible spongiform encephalopathies.     

Inhibition and partial reactions of Na,K-ATPase studied by Fourier transform infrared difference spectroscopy

Reaction-induced infrared (IR) difference spectroscopy with caged ATP and Na,K-ATPase allows us to differentiate unambiguously between phosphorylated and unphosphorylated states of the enzyme as well as of its ouabain complex. The IR spectral changes upon phosphoenzyme formation are characterized and interpreted. Our results show clearly that high Na(+) concentrations prevent the binding of ouabain with high affinity, which is consistent with the results of a corresponding kinetic study employing spectrofluorimetry and calorimetric titrations. This unexpected antagonism leading to low ouabain affinity is assumed related to a conformation of the protein, induced by low affinity binding of the third Na(+) ion. We thus conclude that not the free enzyme but a phosphorylated state of the reaction cycle preferentially binds ouabain and leads to the loss of hydrolytic activity.     

Subcellular localization of copper in the root cells of Allium sativum by electron energy loss spectroscopy (EELS)

Ultrastructural investigation of the root cells of Allium sativum L. exposed to three different concentrations of Cu (1, 10 and 100 microM) for 9 days was carried out using transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS). The results presented here indicate that excess Cu induces ultrastructural changes such as strong vacuolization, condensed nuclear chromatin, decreased endoplasmic reticulum (ER) and ribosome and serious plasmolysis. EELS analysis indicated that electron dense granules containing Cu appeared in the cells after Cu treatment. The vacuoles of the root tip cells were the main Cu-accumulation site. Small amounts of copper were also localized to cytoplasmic vesicles or cell walls of cortical cells. The results of the present investigation have significant importance in further understanding the mechanisms of absorption, transportation and accumulation of heavy metals in plants grown in polluted soil.     

Component analysis and growth process of nasopharyngeal calculus as revealed by Fourier transform infrared (FT-IR) spectroscopy.

A quite rare case of nasopharyngeal calculus in a woman in her twenties associated with the nasal discharge of pseudomonas infection was reported. As the substance was irregularly large in size, we extracted it partially by piecemeal resection using forceps and also by cracking technique using the holmium yttrium-aluminum-garnet (YAG) laser, under saline irrigation and stereotactic microscopic navigator (SMN) system under endoscopic observation. The substance was firmly fixed to the pharyngeal tonsil bed. The final extract was a small piece of singly folded bandage, which is probably the focal background for calculus formation. In a cross section of calculus specimen removed during surgery, Fourier transform infrared (FT-IR) analysis revealed that a) signal ratio of methylene group (organic substance) to amide I (protein) was 21.6% at the nasal cavity side, gradually decreased toward nasal mucous membrane showing approximate 50%, b) signal ratio of amide I to P04(3-) (inorganic substance) ranged between 17.7% and 26.7% at the different sites and inside the calculus, the protein content was approximate 1/5 of the inorganic substance, and c) signal ratio of the methylene group to amide I at the nasal cavity site showed that their contents were almost equal. The quantity of the organic substance was estimated at approximate 1/2 quantity of the protein at both the central part and the part contacted with the mucous membrane. From these results, it seems that throughout the course of calculus growth, both inorganic substance and protein remain almost constant inside the calculus, while organic substance is released from the internal part of the calculus being probably formed at an early stage.     

The structure of tri-proline in water probed by polarized Raman, Fourier transform infrared, vibrational circular dichroism, and electric ultraviolet circular dichroism spectroscopy

Tripeptidesserve as model systems for understanding the so-called random-coil state of peptides and proteins. While it is well known that polyproline or proline-rich polypeptides adopt the very regular polyproline-II (PPII) or left-handed 3(1)-helix conformation, it was thus far not clear whether this is also the predominant structure adopted by proline-containing tripeptides. To clarify this issue, we have investigated the amide I' band profile in the ir, isotropic, and anisotropic Raman, and vibrational circular dichroism (VCD) spectrum of cationic and zwitterionic tri-proline in D(2)O. The data were analyzed by modifying a recently developed algorithm, which allows one to obtain the central dihedral angles of tripeptides from the amide I' band intensities (R. Schweitzer-Stenner, Biophysical Journal, 2002, Vol. 83, pp. 523-532). Our analysis revealed that the peptide adopts a nearly canonical PPII structure in water with psi and phi values in the range of 175 degrees -165 degrees and -70 degrees -(-80 degrees ), respectively. This is fully confirmed by the respective electronic ultraviolet-CD spectra. Our result indicates that the strong PPII propensity of trans proline results from local interactions between the pyrrolidine ring and the backbone and is not due to any long-range interactions.     

Carbonmonoxy dopamine beta-hydroxylase. Structural characterization by Fourier transform infrared, fluorescence, and x-ray absorption spectroscopy

The carbon monoxide complex of ascorbate-reduced dopamine beta-hydroxylase has been prepared and characterized by Fourier transform infrared, fluorescence, and x-ray absorption spectroscopies. CO has previously been shown to be a competitive inhibitor with respect to O2, and binds to only one of the two copper atoms/active site (Blackburn, N. J., Pettingill, T. M., Seagraves, K. S., and Shigeta, R. T. (1990) J. Biol. Chem. 265, 15383-15386). Thus, it acts as an excellent probe of the O2-binding site. A single C-O infrared absorption band is observed at 2089 cm-1, shifting by 46 cm-1 to lower energy on substitution with either 13C16O or 12C18O. The 13C isotope shift is reversed to the position expected for 12CO upon vacuum flushing with 12CO gas, indicating that formation of the CO adduct is a fully reversible process. Binding of the substrate tyramine does not eliminate the infrared peak but causes a 3-cm-1 shift to lower energy. On the other hand, binding of a bifunctional inhibitor which cross-links the substrate and O2-binding site does eliminate the CO peak. These data, in conjunction with the competitive nature of CO binding with respect to O2, identify the CO-binding site as the O2-binding site, and place it in close proximity to the substrate-binding site. CO-dopamine beta-hydroxylase exhibits no luminescence in the visible region, suggesting a structure different from carbonmonoxy hemocyanin, and in all probability mononuclear. Analysis of extended x-ray absorption spectroscopy data is most consistent with an average coordination per Cu of 2-3 histidines, 0.5 CO, and 0.5 S atoms as ligands, and absorption edge comparisons indicates pseudo-4 coordination as the most likely geometry at each Cu(I) center. The results can be interpreted by a model involving inequivalent 4-coordination at each Cu(I) center in the CO adduct with CuAHis3S...CuBHis2CO-X as the coordination most consistent with all of the data.     

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to Q(B) in Rhodobacter sphaeroides reaction centers

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q(A-)Q(B)-->Q(A)Q(B-) (k(AB)(1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k(AB)(1)), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q(B) [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (-), 1601 (-) and 1467 (+) cm(-1), characteristic of Q(A) reduction. The time evolution of the spectra shows reoxidation of Q(A-) and concomitant reduction of Q(B) with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q(B-) occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm(-1) [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q(B) in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q(A-) to Q(B) and the identification of Glu-L212 as the main proton acceptor in the state Q(A)Q(B-).