Differentiation of Mesophilic and Thermophilic ...

Differentiation of Mesophilic and Thermophilic Bacteria with Fourier Transform Infrared Spectroscopy SEBNEM GARIP, FARUK BOZOGLU, and FERIDE SEVERCAN* Department of Biology, Middle East Technical University, 06531 Ankara, Turkey (S.G., F.S.); and Department of Food Engineering, Middle East Technical University, 06531 Ankara, Turkey (F.B.)

In the present study the characterization and differentiation of mesophilic and thermophilic bacteria were investigated by using Fourier transform infrared (FT-IR) spectroscopy. Our results showed significant differences between the FT-IR spectra of mesophilic and thermophilic bacteria. The protein-to-lipid ratio was significantly higher for thermophiles compared to mesophiles. The absorption intensity of the CH3 asymmetric stretching vibration was higher in thermophilic bacteria, indicating a change in the composition of the acyl chains. The higher intensity/area observed in the CH2 symmetric stretching mode at 2857 cm1, and the CH2 bending vibration band at 1452 cm1, indicated a higher amount of saturated lipids in thermophilic bacteria. The lipid C¼O stretching vibration at 1739 cm1, which was observed in the mesophilic group, was not observed clearly in the thermophilic group, indicating a difference in packing that is presumably due to the decreased proportion of unsaturated acyl chains in thermophilic bacteria. In addition, the carbonyl groups become hydrogen bonded and the cellular DNA content was lower in thermophilic bacteria. Moreover, in the 1000–400 cm1 frequency region, the spectra of each bacterial species belonging to both the mesophilic and thermophilic bacterial groups, showed characteristic differences that were discriminated via dendrogram using cluster analysis. The curent study implies that FT-IR spectroscopy could be succesfuly applied for the rapid comparison of bacterial groups and species to establish either similarities or discrepencies, as well as to confirm biochemical or physiological characteristics. Index Headings: Mesophilic; Bacteria; Differentiation; Fourier trasnform infrared spectroscopy; FT-IR spectroscopy; Thermophilic.

INTRODUCTION Identification of bacteria is very important in clinical microbiology and in food analysis because of an increasing prevalence of infectious diseases and food poisoning.1 Bacterial classification and differentiation techniques are used in various fields such as clinical, environmental, and food microbiology.2 There are some traditional methods for the identification of bacterial species such as 16S rDNA sequencing analysis.3 Besides 16S rDNA sequencing analysis, some other techniques have been used for differentiation of different bacterial species such as denaturing gradient gel electrophoresis and sequencing4 and PCR with DNA–DNA hybridization analysis.5 For the identification of gram (þ) lactic acid bacteria, the technique called RAPD-PCR has been used.6 Another technique that has been used for the identification of bacteria is florescence in situ hybridization (FISH). The most commonly used method for the differentiation and identification of bacteria is using selective and differential media. Identification of Enterobacter sakazakii with a chromogenic medium (Druggan-Forsythe-Iversen agar, DFI) is an example of this method in the literature.7 Received 18 February 2006; accepted 20 November 2006. * Author to whom correspondence should be sent. E-mail: feride@metu. edu.tr.

186

Volume 61, Number 2, 2007

Differing from all of these traditional techniques, Fourier transform infrared (FT-IR) spectroscopy is used as an analytical tool for the identification and differentiation of bacteria because it is a rapid and easy method.2 Temperature is one of the most important environmental factors affecting the activity and evolution of living organisms. Since all the processes of growth depend on the biochemical reactions that are affected by temperature, it has a significant role in the growth of microorganisms.8 It is possible to distinguish four groups of bacteria in relation to their temperature optima; psychrophiles (10–25 8C), mesophiles (20–45 8C), thermophiles (50–70 8C), and hyperthermophiles (75 8C). In the current study, four thermophilic bacterial species (Thermoanaerobacter ethanolicus, Clostridium thermohydrosulfuricum, Thermobrachium celere, and Geobacillus caldoxylosilyticus) and four mesophilic bacterial species (Lactobacillus plantarum, Escherichia coli, Pseudomonas aeruginosa, and Micrococcus luteus) were investigated. Thermophilic microorganisms have a biotechnological significance in food, chemical, and pharmaceutical industries and in environmental biotechnology.9 Lactic acid bacteria play a significant role in the food industry, as they aid in various food processes and possess unique characteristics that allow them to be potential probiotic organisms.10 They are used industrially for the production of yogurt, sauerkraut, pickles, and other fermented foods, such as silage.11 Escherichia coli has found extensive use as a vehicle for the preparation of biological polymers, including polypeptide hormones, proteins, carbohydrates, etc.12 E. coli can also cause enteric infections, extraintestinal infections, and animal infections.13,14 Pseudomonas aeruginosa has medical importance and causes human infections, such as urinary tract infections, cystic fibrosis, and nosocomical infections.15 Micrococcus luteus is useful for the economic production of long-chain (C21-C34) aliphatic hydrocarbons, which few bacteria other than micrococci produce. These may be useful as lubricating oils and may be substitutes for equivalent petroleum products.16 M. luteus has also been used as a test organism for the assay of antibiotics in body fluids, animal feeds, milks, and pharmaceuticals. This study aims to investigate the similarities and differences between thermophilic and mesophilic bacteria groups and between bacterial species with FT-IR spectroscopy. Although there are several bacterial identification studies using FT-IR spectroscopy,17 to the best of our knowledge, there is no study reported in the literature on differentiation of thermophilic and mesophilic bacteria.

MATERIALS AND METHODS Organisms. Thermophilic bacteria Thermoanaerobacter ethanolicus (JW200), Clostridium thermohydrosulfuricum (DSM 2247), and Thermobrachium celere were provided by

0003-7028/07/6102-0186$2.00/0 Ó 2007 Society for Applied Spectroscopy

APPLIED SPECTROSCOPY

TABLE I. General band assignments of bacteria in the literature.a

FIG. 1. A general representative FT-IR spectrum of a bacteria.

Sedat Do¨nmez (Ankara University, Food Engineering). The other thermophilic bacterium, Geobacillus caldoxylosilyticus (HBB-D4), was provided by Halil Bıyık (Adnan Menderes University, Biology). Mesophilic bacteria Escherichia coli, Micrococcus luteus, and Pseudomonas aeruginosa were obtained from Refik Saydam Hıfzıssıhha Merkezi (ANKARA). Lactobacillus plantarum was provided by Candan Gu¨rakan (METU, Food Engineering). Growth Medium. Mesophilic microorganisms were grown in 200 mL nutrient broth at 37 8C in an incubator overnight. Four thermophilic microorganisms were obtained separately in 60 mL TR1 media in four 100 mL serum bottles. One milliliter (1 mL) samples from each culture with visible turbidity caused by massive growth were taken and transferred to Eppendorf tubes. Sample Preparation. Bacterial cells were collected from these liquid inoculations at their late exponential phase by centrifugation (Sorvall) at 10 000 rpm (SS34 Rotor) for 10 minutes. After removing the supernatants, pellets were washed two times with phosphate buffer (pH 7; 1 mM Na2HPO4/ NaH2PO4 buffer). After a second wash in phosphate buffer, samples were lyophilized (Labconco FreeZonet, Model 77520). Sample Preparation for Fourier Transform Infrared Spectroscopy Studies. The samples were first ground into fine particles using a mortar and pestle. One milligram (1 mg) of each sample was then mixed with 100 mg KBr, which was extensively dried in microfuge tubes using a lyophilizer. This mixture was then lyophilized in the same microfuge tubes. KBr based pellets were prepared by establishing pressure of 100 kg/cm2 (1200 psi) for about 6 minutes. Also, 1 mg phosphate buffer was dried and mixed with 100 mg KBr to prepare a pellet. Fourier Transform Infrared Spectroscopy and Data Analysis. Infrared spectra were obtained by scanning the prepared pellets with a Perkin Elmer Spectrum One Spectrometer (Norwalk, CT). The spectrum of air was recorded as background and subtracted automatically using the Spectrum One software program. Atmospheric vapor was also automatically subtracted. FT-IR spectra of bacterial samples were recorded in the 4000–400 cm1 region at room temperature. One hundred scans were taken for each interferogram at 4 cm1 resolution. Recording and analysis of the spectral data were performed using the Spectrum One software from Perkin Elmer. First the phosphate buffer’s spectrum was recorded to show where the phosphate absorption occurs. Then this spectrum was subtracted from all bacterial spectra to prevent absorptions

a

Peak numbers

Wavenumbers (cm1)

1

3307

2

2959

3

2927

4

2876

5

2857

6 7 8

1739–1744 1657 1541

9 10

1452 1391

11

1236

12

1152

13

1080

14

969

Definition of the spectral assignment N–H and O–H stretching vibration: polysaccharides, proteins CH3 asymmetric stretch: methyl groups in fatty acids CH2 asymmetric stretch: methylene groups in fatty acids CH3 symmetric stretch: methyl groups in fatty acids. CH2 symmetric stretch: methylene groups in fatty acids Ester C¼O stretch: lipid, triglycerides Amide I (protein C¼O stretching): a helices Amide II (protein N–H bend, C–N stretch): a helices CH2 bending: lipids COO symmetric stretch: aminoacid side chains, fatty acids PO2 asymmetric stretching: mainly nucleic acids with the little contribution from phospholipids CO–O–C asymmetric stretching: glycogen and nucleic acids PO2 symmetric stretching: nucleic acids and phospholipids; C–O stretch: glycogen C–Nþ–C stretch: nucleic acids

See Refs. 18–27.

from the inorganic phosphate. The band positions were measured according to center of weight. The averages of the spectra belonging to the same experimental groups, baseline correction, normalization, and the band areas were obtained by using the same software program. The average spectra and normalization process were applied only for visual representation of the differences; however, for the determination of the spectral parameters and calculation of mean values and statistical analysis, each baseline-corrected original spectrum was taken into consideration. Finally, cluster analysis was applied that classifies objects, via a tree diagram (dendrogram) calculated using the Ward’s algorithm. Constructed with the OPUS 5.5 software (Bruker Optics), the dendrogram graphically represents the cluster analysis groups. Statistics. The results were expressed as mean 6 standard deviation values. The differences in the means of the thermophilic and mesophilic bacteria were compared using the Mann–Whitney U-test. A p value of less than 0.05 was considered significant (p , 0.05*, p , 0.01**, p , 0.001***).

RESULTS We have carried out FT-IR spectroscopic studies on thermophilic and mesophilic bacterial groups and compared the spectral differences and similarities between both the groups and the bacterial species. A general representative FT-IR spectrum of a bacterium is shown in Fig. 1. As seen from the spectrum, it is a complex spectrum containing several bands. The band assignments are given in Table I. Figure 2A shows the average FT-IR spectra of four thermophilic bacteria in 4000–1000 cm1 region. As seen from this figure, these four thermophilic bacteria show identical FT-IR spectra so we used the average spectra of these microorganisms when comparing with mesophiles in this


187

FIG. 2. (A) The average spectra of (a) T. celere (n ¼ 20), (b) T. ethanolicus (n ¼ 20), (c) C. thermohydrosulfuricum (n ¼ 20), and (d) G. caldoxylosilyticus (n ¼ 20) samples in the 4000–1000 cm1 region. (B) The average spectra of (a) E. coli (n ¼ 20), (b) M. luteus (n ¼ 20), (c) L. plantarum (n ¼ 20), and (d) P. aeruginosa (n ¼ 20) samples in the 4000–1000 cm1 region.

region. Also as seen from Fig. 2B, four mesophilic bacteria show identical FT-IR spectra so the average spectra of these bacteria was used in the comparison of thermophiles and mesophiles in the 4000–1000 cm1 region. On the other hand, in the 1000–400 cm1 frequency region, all the thermophilic and the mesophilic bacterial species show different spectra from each other and are differentiated by cluster analysis (Fig. 3). Comparison of Mesophilic and Thermophilic Bacterial Groups. Figure 4 shows the normalized infrared spectra of thermophilic bacteria and mesophilic bacteria samples in the 3000–2800 cm 1 region. The bands centered at 2927 cm1 and 2857 cm1 correspond to the stretching mode of asymmetrical and symmetrical CH2 vibrations due to methylene groups in fatty acids.22,24–27 The band centered at 2959 cm1 and 2876 cm1 corresponds to the stretching mode of asymmetrical and symmetrical CH3 vibrations due to methyl groups in fatty acids.27 The frequency and intensity values of both thermohiles and mesophiles are listed in Table II. As seen from the table, the wavenumber of the CH3 asymmetric stretching vibration at 2959 cm1 of thermophilic bacteria (2962.01 6 0.30) was significantly lower than mesophilic bacteria (2962.51 6 0.80) (p , 0.001). However, the absorption intensity of this band was significantly higher for thermophilic bacteria (0.38 6 0.12) compared to mesophilic bacteria (0.25 6 0.05) (p , 0.001). Moreover, the absorption intensity of the CH2 asymmetric stretching band at 2927 cm1 was higher (p , 0.01) for the thermophilic group (0.38 6 0.24) than for the mesophilic group (0.26 6 0.04), as shown in Table II. As seen from Fig. 4 and Table II, the wavenumber of the CH3

188


symmetric stretching band at 2876 cm1 is significantly higher (2875.30 6 0.22) for thermophilic bacteria than mesophilic bacteria (2872.20 6 0.98) (p , 0.001). Also, the intensity of this band is higher (p , 0.01) for the thermophilic group (0.33 6 0.12) compared to the mesophilic group (0.24 6 0.05). It is also observed that the intensity of the band at 2857 cm1, which is assigned as CH2 symmetric stretching, showed a higher value (p , 0.01) for thermophilic bacteria. The frequency of this band was significantly higher (p , 0.001) for thermophilic bacteria (2854.29 6 0.60) compared to mesophilic bacteria (2853.24 6 0.40), indicating an increase in the number of gauche conformers, e.g., an increase in disorder status of the acyl chains.22,24,26–28 The signal intensity and, more accurately, the area under the peaks give information about the concentration of the functional groups responsible for the corresponding band.26,27,29–32 From the FT-IR spectrum, a precise protein-to-lipid ratio can be derived by calculating the ratio of the areas of the bands arising from lipids and proteins whose amounts in the membranes are an important factor affecting the membrane structure and dynamics.33 It is seen from Table III that the ratio of the sum area of the CH2 and CH3 symmetric and asymmetric bands in the 3000–2800 cm1 region to the area of amide I þ amide II gives information about the protein-to-lipid ratio,27 which was significantly higher (p , 0.01) for the thermophilic group (3.20 6 0.05) than for the mesophilic group (2.75 6 0.06). Figure 5 shows the 1800–400 cm1 region of the normalized FT-IR spectra of thermophilic and mesophilic bacteria. The band centered at 1739 cm1 is mainly assigned to the .C¼O ester stretching vibration in phospholipids.22,24 As is illustrated in Fig. 3, the C¼O ester stretching vibration band at 1739 cm1 was not observed in the thermophilic group, which was different from that of the mesophilic one, indicating a difference in packing of the ester groups.21 The bands centered at 1657 cm1 corresponding to the stretching C¼O and bending C–N (amide I) vibrational modes of the polypeptide and protein backbone and at 1541 cm1 are assigned to the bending N–H and stretching C–N (amide II).24 The amide I region is useful for determination of protein secondary structure. The frequency of vibration is very sensitive to changes in the nature of hydrogen bonds in different types of protein secondary structures.34 As can be seen from Fig. 5 and Table II, the frequency value of the amide I band is lower (1653.22 6 0.53) for the thermophilic group compared to the mesophilic group (1656.05 6 0.41) (p , 0.001). However, the intensity of amide I was higher (p , 0.001) for the thermophilic group (0.59 6 0.07) than for the mesophilic group (0.29 6 0.05). Both the absorbance and the bandwidth of the amide II band (at 1541 cm1) was significantly higher (p , 0.001) for the thermophilic group compared to the mesophilic group, as seen from Table IV. Moreover, the frequency of the amide II band was lower for the thermophilic group (1536.09 6 0.01) compared to the mesophilic group (1540.23 6 0.07) (p , 0.001). The intense band at 1452 cm1 is assigned to the CH2 bending mode of lipids.24,35 As seen from Fig. 5, a higher value of the intensity for thermophilic bacteria (0.34 6 0.12) than mesophilic bacteria (0.23 6 0.05) (p , 0.01) at 1452 cm1 is due to the bending vibration of CH2 in the lipids. It can be seen from Table II that there is a lower frequency of this band for the thermophilic group (1452.89 6 0.77) compared to the mesophilic group (1455.72 6 0.80) (p , 0.001). The band

FIG. 3. Hierarchical cluster analysis performed on the first-derivative spectra of bacterial samples and resulting from Ward’s algorithm. The study was conducted in the 1000–400 cm1 spectral region.


189

FIG. 5. The average spectra of the mesophilic (n ¼ 80) and thermophilic (n ¼ 80) bacteria samples in the 1800–400 cm1 region (the spectra were normalized with respect to the amide I band). FIG. 4. The average spectra of the mesophilic (n ¼ 80) and thermophilic (n ¼ 80) bacteria samples in the 3000–2840 cm1 region (the spectra were normalized with respect to the CH2 asymmetric stretching band).

at 1391 cm1 is due to COO symmetric stretching vibration of amino acid side chains and fatty acids.21,24 In the thermophilic spectrum (0.35 6 0.11) the intensity of the COO symmetric stretching vibration band at 1395 cm1 was higher (p , 0.01) compared to the mesophilic group (0.23 6 0.03). In addition, the frequency value of this band was significantly lower for thermophiles (1396.55 6 0.29) compared to mesophiles (1402.73 6 0.44) (p , 0.001) (Table II). The relatively strong bands at 1236 and 1080 cm1 are mainly due to the asymmetric and symmetric stretching modes of phosphodiester groups in nucleic acids rather than in phospholipids, respectively.20,24 Study of these bands revealed significant differences in the infrared spectra between thermophilic and mesophilic bacteria. The intensity of the phosphate asymmetric stretching band was significantly higher (p , 0.01) for thermophilic bacteria (0.35 6 0.12) compared to mesophilic bacteria (0.24 6 0.03). Furthermore, the frequency of this band was also significantly higher for thermophiles (1236.70 6 1.26) compared to mesophiles (1232.64 6 0.86) (p , 0.001), as shown in Fig. 5. The frequency of the phosphate symmetric stretching band also showed a higher value for thermophilic bacteria (1077.13 6 1.20) than for mesophilic bacteria (1073.85 6 1.91) (p , 0.001) (Table II). The peak area ratio of bands at 1087 cm1 and 1540 cm1 (A1087/A1540) is often used to illustrate the change of the DNA/ protein content of the cells.36–39 A higher value of this ratio implies a higher DNA content.36–39 The results shown in Table III suggest that the cellular DNA content was lower for

thermophilic bacteria (0.30 6 0.10) compared to mesophilic bacteria (1.16 6 0.50) (p , 0.001). Comparison of Thermophilic Bacterial Species. Thermophilic bacterial species show different FT-IR spectra in the 1000–400 cm1 frequency region. As seen from Fig. 6, there were characteristic bands for each species in this region. For three thermophilic species there were two bands, at 964 and 914 cm1, but in G. caldoxylosilyticus, instead of these two bands, there was another band at 925 cm1. Moreover, there were characteristic peaks at 851 and 529 cm1 that were only seen in G. caldoxylosilyticus. Another difference was the peak at 798 cm1, which was observed in three thermophiles except for T. ethanolicus (Fig. 6). Comparison of Mesophilic Bacterial Species. Mesophilic bacterial species show different FT-IR spectra in the 1000–400 cm1 frequency region. There were characteristic peaks for both Lactobacillus plantarum and Pseudomonas aeruginosa, as seen from Fig. 7. The band at 966 cm1 was observed in E. coli and P. aeruginosa species, while it was not seen in the other two mesophilic bacteria. Moreover, the band at 935 cm1 was sharper in P. aeruginosa than in the other bacterial species. There were characteristic bands for L. plantarum at 505 and 420 cm1 (Fig. 7).

DISCUSSION High temperatures lead to an increase in the fluidity of the cellular membrane and to maintain optimal membrane fluidity, the composition of the membrane is altered in some cells.40 As can be seen from Fig. 2 and Table II, the absorption intensity of the CH3 asymmetric stretching vibration at 2959 cm1 was

TABLE II. The band frequencies and intensity values of various functional groups in the mesophilic (n ¼ 80) and thermophilic (n ¼ 80) bacterial samples. Frequency Functional groups CH3 asymmetric stretching CH2 asymmetric stretching CH3 symmetric stretching CH2 symmetric stretching Amide I Amide II CH2 bending COO symmetric stretching PO2 asymmetric stretching PO2 symmetric stretching

190

Mesophilic (n ¼ 80) 2962.50 2928.52 2872.20 2853.24 1656.05 1540.23 1455.72 1402.73 1232.64 1073.85

6 6 6 6 6 6 6 6 6 6

0.80 1.00 0.98 0.40 0.41 0.07 0.80 0.44 0.86 1.91


Intensity

Thermophilic (n ¼ 80) 2962.01 2926.44 2875.30 2854.29 1653.22 1536.09 1452.89 1396.55 1236.70 1077.13

6 6 6 6 6 6 6 6 6 6

0.30 0.30 0.22 0.60 0.53 0.01 0.77 0.29 1.26 1.20

p values ,0.001*** ,0.001*** ,0.001*** ,0.001*** ,0.001*** ,0.001*** ,0.001*** ,0.001*** ,0.001*** ,0.001***

Mesophilic (n ¼ 80) 0.25 0.26 0.24 0.24 0.29 0.26 0.23 0.23 0.24

6 6 6 6 6 6 6 6 6

0.05 0.04 0.05 0.05 0.05 0.04 0.05 0.03 0.03

Thermophilic (n ¼ 80)

p values

6 6 6 6 6 6 6 6 6

,0.001*** ,0.01** ,0.01** ,0.01** ,0.001*** ,0.001*** ,0.01** ,0.01** ,0.01**

0.38 0.38 0.33 0.32 0.59 0.49 0.34 0.35 0.35

0.12 0.24 0.12 0.12 0.07 0.20 0.12 0.11 0.12

TABLE III. The band area ratios of some functional groups in the thermophilic (n ¼ 80) and mesophilic (n ¼ 80) samples. Ratio of peak areas

Functional groups

Mesophilic (n ¼ 80)


p values

Protein/lipid DNA/protein

Amide I þ Amide II/CH3 Sym.þCH3 Asym.þCH2 Sym.þ CH2 Asym. PO2 Symmetric/Amide II

2.75 6 0.06 1.16 6 0.50

3.20 6 0.05 0.30 6 0.10

,0.01** ,0.001***

higher in thermophilic bacteria, indicating a change in the composition of acyl chains in the thermophilic group.19,24,26 Growth-temperature-dependent changes of the lipid composition of the cytoplasmic membranes of bacteria were also reported previously.41–43 As seen from Table II, the higher value of the frequency of the CH2 symmetric stretching mode at 2857 cm1 for the thermophilic group (p , 0.001) indicated a reorganization of the membrane in an ordered direction to an average bilayer structure.44,24 In addition to this difference, the intensity of this band was higher (p , 0.01) in thermophilic bacteria compared to mesophilic bacteria. These results indicate a higher concentration of saturated fatty acyl chains in thermophilic bacteria. This was confirmed by the higher value of the intensity of the CH2 bending vibration band at 1452 cm1 in thermophilic bacteria (Fig. 4 and Table II). These results were confirmed by the study of Chan et al.,45 which suggested that notable differences were that the thermophiles contained a higher content of saturated straight- and branched-chain fatty acids. Moreover, the lipid C¼O stretching vibration at 1739 cm1, which was observed in the mesophilic group, was not clearly observed in the thermophilic group. This band suggests an increased concentration and difference in packing of the ester groups in mesophilic bacteria21 (Fig. 5), which is in agreement with Chan et al.45 The melting points of saturated straightchain and iso-fatty acids range from 51 to 107 8C, whereas unsaturated, anteiso fatty acids have melting points ranging from below 0 to 40 8C.45 According to our results, the thermophiles contain a preponderance of higher melting fatty acids. The general pattern indicated that organisms grown at higher temperatures contain a higher percentage of saturated acids with comparatively higher melting points.45 Vossenberg et al.43 investigated the phase transition behavior of the lipids derived from mesophilic B. subtilis and thermophilic B. subtilis cells grown at 13 and 50 8C by differential scanning calorimetry (DSC) and the viscosity of the membranes by TMA-DPH fluorescence anisotropy (r) and lifetime (sh) measurements. They indicated that the most significant effect of growth temperature was a drastic decrease in the anteiso fatty acids and an increase in the iso-branched fatty acids that was in accordance with our results. It is seen from Table III that the protein-to-lipid ratio was higher (p , 0.01) in the thermophilic group compared to the mesophilic group. Generally, the higher value of this ratio suggests a high protein content or low lipid content or both.24,46 In our case, the high protein content in thermophilic bacteria was supported by the higher value of the area of the amide I and amide II bands, while the low lipid content was

supported by the lower value of the area of the CH2 and the CH3 asymmetric stretching vibration band in the thermophilic bacterial samples. In thermophilic microorganisms, thermostability is acquired by thermostable proteins. The amino acid composition of proteins from mesophilic and thermophilic organisms is commonly assumed to reflect the mechanism of molecular adaptation to extremes of physical conditions.48 Thermostability seems to be a property acquired by a protein through many small structural modifications obtained with the exchange of some aminoacids.49–51 The advantages of the amino acid exchanges most frequently reported in thermophilic proteins are that a higher number of hydrogen bonds may be formed.49 From the comparative analysis of the X-ray structures available for several families of proteins, including at least one thermophilic structure in each case, it appears that thermal stabilization is accompanied by an increase in hydrogen bonds.49,52,53 Our results confirmed these studies since we found a significant decrease in the frequency of the carbonyl group, indicating an increase in the number of hydrogen bonds. The lower DNA/ protein ratio might suggest that the cellular DNA content was lower for thermophilic bacteria36–39 (Table III). In the comparison of the thermophilic bacterial species, in the 1000–400 cm1 region they showed different FT-IR spectra. There were characteristic bands for each species in this region and they can be used for the discrimination of these bacteria (Fig. 6). The bands at 964 and 914 cm1, which are due to the vibration of C–O–C ring deoxyribose,54,55 were seen in three thermophilic species, while in G. caldoxylosilyticus, instead of these two bands, there was another deoxyribose band55,56 at 925 cm1. There were characteristic peaks at 851 and 529 cm1 for G. caldoxylosilyticus. The peak at 798 cm1, which is due to adenine, was not observed in T. ethanolicus.56 In the comparison of the mesophilic bacterial species, they also showed different FT-IR spectra in the 1000–400 cm1 frequency region (Fig. 7). There were characteristic peaks for Lactobacillus plantarum at 505 and 420 cm1, and these bands can be used for the discrimination of this mesophilic bacteria. The band at 966 cm1 that is due to the vibration of C–O–C

TABLE IV. Changes in the bandwidth of various functional groups in the thermophilic (n ¼ 80) and mesophilic (n ¼ 80) samples. Functional groups: bandwidth

Mesophilic (n ¼ 80)


p values

Amide II

3.8 6 0.5

4.3 6 0.4

,0.001***

FIG. 6. The average spectra of T. celere (n ¼ 20), T. ethanolicus (n ¼ 20), C. thermohydrosulfuricum (n ¼ 20), and G. caldoxylosilyticus (n ¼ 20) samples in the 1000–400 cm1 region (the spectra were normalized with respect to the amide I band).


191

FIG. 7. The average spectra of E. coli (n ¼ 20), M. luteus (n ¼ 20), L. plantarum (n ¼ 20), and P. aeruginosa (n ¼ 20) samples in the 1000–400 cm1 region (the spectra were normalized with respect to the amide I band).

ring deoxyribose was only observed in the E. coli and P. aeruginosa species.54,55 Moreover, another deoxyribose band at 935 cm1 was sharper in P. aeruginosa than in the other mesophilic species.54

CONCLUSION In conclusion, our results show that there were significant spectral differences between mesophilic and thermophilic bacteria, indicating that the protein concentration was high; however, lipid concentration, the level of triglycerides, and the unsaturated acyl chains were low in thermophilic bacteria compared to the mesophilic bacteria studied. Moreover, it was also found that the number of hydrogen bonds was high in thermophiles. In addition to that, there were characteristic peaks for both thermophilic and mesophilic species that can be used for the discrimination of these different bacterial species. ACKNOWLEDGMENTS This study was supported by Middle East Technical University (METU) research fund BAP-2005-07-02-00-12. 1. D. Naumann, ‘‘Infrared Spectroscopy in Microbiology’’, in Encyclopedia of Analytical Chemistry, R. A. Meyers, Ed. (2002), pp. 102–131. 2. D. Naumann, ‘‘FT-IR and FT-NIR Raman Spectroscopy in Biomedical Research’’, in ‘‘Fourier Transform Spectroscopy: 11th International Conference’’ AIP Conference Proceedings, J. A. Haseth, Ed. (Woodbury, New York, 1998), 430, pp. 96–109. 3. C. Teyssier, E. Jumas-Bilak, H. Marchandin, H. Jean-Pierre, J. L Jeannot, G. Dusart, V. Foulongne, and M. Simeon de Buochberg, Pathologie Biologie 51, 5 (2003). 4. J. F. Siqueira, Jr., I. N. Roˆc¸as, and A. S. Rosado, J. Endodontics 31, 775 (2005). 5. P. Scheldeman, L. Herman, J. Goris, P. De vos, and M. Heyndrickx, J. Appl. Microbiol. 92, 983 (2002). 6. F. B. Elegado, M. A. R. V. Guerra, R. A. Mayacan, H. A. Mendoza, and M. B. Lirazan, Int. J. Food Microbiol. 95, 11 (2004). 7. C. Iversen, P. Druggan, and S. Forsythe, Int. J. Food Microbiol. 96, 133 (2004). 8. J. Tortora, R. B. Gerard, and C. L. Case, Microbiology. An Introduction (The Benjamin Cummings Publishing Company, San Francisco, CA, 1995), 5th ed., pp. 125–127. 9. F. Niehaus, C. Bertoldo, M. Ko¨hler, and G. Antranikian, Appl. Microbiol. Biotechnol. 51, 711 (1999). 10. R. F. Vogel and M. Ehrmann, Biotechnol. Annu. Rev. 2, 123 (1996). 11. C. N. Jacobsen, V. R. Nielsen, A. E. Haydorf, P. L. Moller, K. F. Michaelsen, A. Paerregaard, B. Sandstrom, M. Tvede, and M. Jakobsen, Appl. Environ. Microbiol. 65, 4949 (1999). 12. J. R. Birch and Y. Onakunle, Methods. Mol. Biol. 308, 1 (2005).

192


13. F. Qadri, A. M. Svennerholm, A. S. Faruque, and R. B. Sack, Clin. Microbiol. Rev. 18, 465 (2005). 14. E. B. Breitschwerdt, C. DebRoy, A. M. Mexas, T. T. Brown, and A. K. Remick, J. Am. Vet. Med. Assoc. 226, 2016 (2005). 15. A. B. Aguilar, J. Perpinan, P. Ramos, M. Peris, L. Lorente, and A. Zuniga, J. Hosp. Infect. 6, (2006) [Epub ahead of print]. 16. E. Bieszkiewich, H. Boszczyk-Maleszak, A. Wlodarczyk, and M. Horoch, Acta Microbiol. Pol. 51, 285 (2002). 17. L. Mariey, J. P. Signolle, C. Amiel, and J. Travert, Vib. Spectrosc. 26, 151 (2001). 18. D. Naumann, D. Helm, and H. Labischinski, Nature (London) 351, 81 (1991). 19. H. Takahashi, S. M. French, and P. T. T. Wong, Clin. Exp. Res. 15, 219 (1991). 20. P. T. T. Wong, E. D. Papavassiliou, and B. Rigas, Appl. Spectrosc. 45, 1563 (1991). 21. M. Jackson, B. Ramjiawan, M. Hewko, and H. H. Mantsch, Cell Mol. Biol. 44, 89 (1998). 22. A. M. Melin, A. Perromat, and G. Deleris, Bioploymers (Biospectroscopy) 57, 160 (2000). 23. L. Chiriboga, H. Yee, and M. Diem, Appl. Spectrosc. 54, 480 (2000). 24. G. Cakmak, I. Togan, C. Ug˘uz, and F. Severcan, Appl. Spectrosc. 57, 835 (2003). 25. N. Toyran and F. Severcan, Chem. Phys. Lipids 123, 165 (2003). 26. G. Cakmak, I. Togan, and F. Severcan, Aquatic Toxicol. 77, 53 (2006). 27. J. Kneipp, M. Beekes, P. Lasch, and D. Naumann, J. Neurosci. 22, 2989 (2002). 28. C. Schultz and D. Naumann, FEBS Lett. 294, 43 (1991). 29. T. J. Bruno, Appl. Spectrosc. Rev. 34, 91 (1999). 30. A. Femenia, S. Sanchez, and C. Rosello, J. Agric. Food Chem. 46, 271 (1998). 31. N. Toyran, F. Zorlu, G. Donmez, K. Oge, and F. Severcan, Eur. Biophys. J. 33, 549 (2004). 32. A. Dogan, G. Siyakus, and F. Severcan, Food Chem. 100, 1106 (2007). 33. B. Szalontai, Y. Nishiyama, Z. Gombos, and N. Murata, Biochim. Biophys. Acta 1509, 409 (2000). 34. P. I. Haris and F. Severcan, J. Mol. Catal. B: Enzymatic 7, 207 (1999). 35. R. Manoharan, J. J. Baraga, P. R. Rava, R. R. Dasari, M. Fitzmaurice, and M. S. Feld, Atherosclerosis 103, 181 (1993). 36. J. R. Mourant, Y. R. Yamada, S. Carpenter, L. R. Dominique, and J. P. Freyer, Biophys. J. 85, 1938 (2003). 37. A. Perromat, A. Melin, C. Lorin, and G. Deleris, Biopolymers (Biospectroscopy) 72, 207 (2003). 38. J. Zhou, Z. Wang, S. Sun, M. Liu, and H. Zhang, Biotechnol. Appl. Biochem. 33, 127 (2001). 39. E. Taillendier and J. Liquier, Methods Enzymol. 211, 307 (1992). 40. L. J. Rothschild and R. L. Mancinelli, Nature (London) 409, 1092 (2001). 41. R. Garu and D. de Mendoza, Mol. Microbiol 8, 535 (1993). 42. J. Svobodova and P. Svoboda, Folia Microbiol. (Praha.) 33, 161 (1988). 43. J. L. C. M. Vossenberg, A. J. M. Driessen, M. S. Costa, and W. N. Konings, Biochim. Biophys. Acta 1419(9), 97 (1999). 44. E. N. Lewis, I. W. Lewin, and C. J. Steer, Biochim. Biophysics. Acta 986, 161 (1989). 45. M. Chan, R. H. Himes, and J. M. Akagi, J. Bacteriol. June, 876 (1971). 46. M. Jackson, M. G. Sowa, and H. H. Mantsch, Biophys. Chem. 68, 109 (1997). 47. K. F. M. Fung, M. Senterman, P. Eid, W. Faught, Z. N. Mikhael, and P. T. T. Wong, Gynocol. Oncol. 66, 10 (1997). 48. G. Bo¨hm and R. Jaenicke, Int. J. Pept. Protein Res. 43, 97 (1994). 49. R. Scandurra, V. Consalvi, R. Chiaraluce, L. Peliti, and P. C. Engel, Biochimie 80, 993 (1998). 50. P. Argos, M. G. Rossman, U. M. Grau, H. Zuber, G. Frank, and J. D. Tratschin, Biochemistry 18, 5698 (1979). 51. G. L. Warren and G. A. Petsko, Protein Eng. 8, 905 (1995). 52. E. Querol, J. A. Perez-Pons, and A. Mozo-Villarias, Protein Eng. 9, 265 (1996). 53. G. Vogt, S. Woell, and P. Argos, J. Mol. Biol. 269, 631 (1997). 54. M. Ghomi, R. Lettellier, J. Liquier, and E. Taillandier, Int. J. Biochem. 22, 691 (1990). 55. G. I. Dovbeshko, O. P. Gnatyuk, V. I. Chegel, Y. M. Shirshov, D. V. Kosenhov, E. A. Andreev, H. A. Tajmir-Riahi, and P. M. Lytvyn, Semicond. Phys., Quant. Electron. Optoelectron. 7, 318 (2004). 56. E. Taillandier, J. Liquier, and J. A. Taboury, Adv. Infrared Raman Spectrosc. 65 (1985).