Effects of UV Radiation on the Lipids and Proteins of Bacteria Studied

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Effects of UV Radiation on the Lipids and Proteins of Bacteria Studied by Mid-Infrared Spectroscopy Ana L. Santos,*,† Catarina Moreirinha,†,‡ Diana Lopes,†,§ Ana Cristina Esteves,† Isabel Henriques,† Adelaide Almeida,† M. Rosário M. Domingues,§ Ivonne Delgadillo,‡ António Correia,† and Â ngela Cunha† †

Department of Biology and CESAM, ‡Department of Chemistry and QOPNA, and §Mass Spectrometry Centre, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal S Supporting Information *

ABSTRACT: Knowledge of the molecular effects of UV radiation (UVR) on bacteria can contribute to a better understanding of the environmental consequences of enhanced UV levels associated with global climate changes and will help to optimize UV-based disinfection strategies. In the present work, the effects of exposure to UVR in different spectral regions (UVC, 100−280 nm; UVB, 280−320 nm; and UVA, 320−400 nm) on the lipids and proteins of two bacterial strains (Acinetobacter sp. strain PT5I1.2G and Pseudomonas sp. strain NT5I1.2B) with distinct UV sensitivities were studied by mid-infrared spectroscopy. Exposure to UVR caused an increase in methyl groups associated with lipids, lipid oxidation, and also led to alterations in lipid composition, which were confirmed by gas chromatography. Additionally, mid-infrared spectroscopy revealed the effects of UVR on protein conformation and protein composition, which were confirmed by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE), oxidative damage to amino acids, and changes in the propionylation, glycosylation and/or phosphorylation status of cell proteins. Differences in the targets of UVR in the two strains tested were identified and may explain their discrepant UV sensitivities. The significance of the results is discussed from an ecological standpoint and with respect to potential improvements in UV-based disinfection technologies.

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cause cell death.13,14 UVC radiation is the most detrimental to living cells because it is directly absorbed by DNA, resulting in the formation of cyclobutane dimers and single-strand breaks in the sugar−phosphate backbone of DNA.15 UVB radiation produces both direct and indirect damage.1 DNA has been the most investigated target of UVR to date (e.g., refs 16−19). However, the observation of comparable levels of DNA photoproduct accumulation in bacteria displaying different UV sensitivities20 and the fact that DNA damage alone cannot account for the inhibition of bacterial activity21 suggest that damage to other biomolecules may contribute to the inhibitory effects of UVR on bacteria. Accordingly, lipids and proteins have emerged in recent years as potentially important targets of ROS generated during UV exposure.11,22−24 Bacterial membranes seem to be particularly susceptible to UV-induced oxidative damage because they contain large amounts of iron, which catalyzes ROS generation.25 However, the specific types of protein and lipid modifications induced by UVR are currently unknown. Identification of the targets of UVA and UVB in bacteria will help to further understand the effects of UVR on bacterial

INTRODUCTION Ultraviolet radiation (UVR) is an important stress factor for aquatic bacterial communities.1 UVR can be divided into three spectral regions: UVC (100−280 nm), UVB (280−320 nm), and UVA (320−400 nm). The majority of environmental photobiology studies have focused on the effects of UVB on bacteria.2−5 On a photon basis, UVA radiation contains less energy than UVB radiation; however, the fraction of solar UVR in the UVA region (95%) is far greater than that in the UVB region (5%). Thus, UVA can potentially cause substantial biological damage,6,7 a property which has been exploited in solar disinfection applications.8 Unlike UVA and UVB, UVC radiation does not penetrate the Earth’s atmosphere, but it is a convenient experimental tool to assess UV sensitivity in bacteria that are highly tolerant or insensitive to high doses of UVB.9 Furthermore, the ability of UVC to inactivate bacteria is widely recognized.10 The biological effects of the different UV spectral regions differ in their preferred cellular targets and the mechanisms by which they induce damage. The effects of UVA are considered to be mostly indirect, that is, mediated by reactive oxygen species (ROS) formed via photodynamic reactions involving intracellular or extracellular photosensitizers.11,12 These ROS can react with cellular constituents, most notably proteins and lipids, leading to altered membrane permeability and/or disruption of transmembrane ion gradients that can eventually © XXXX American Chemical Society

Received: February 12, 2013 Revised: May 16, 2013 Accepted: May 21, 2013

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dx.doi.org/10.1021/es400660g | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

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Figure 1. Difference spectra for lipid extracts of Acinetobacter sp. under (A) UVA, (B) UVB, and (C) UVC and Pseudomonas sp. under (D) UVA, (E) UVB, and (F) UVC. Main bands are identified. A 13-point smooth was applied to the difference spectra. The region between 2400 and 2200 cm−1, corresponding to the CO2 region, was removed from the spectra.

PT5I1.2G, NCBI accession number GQ365202 and Pseudomonas sp. strain NT5I1.2B, NCBI accession number GU084169) were isolated from the surface waters of the estuarine system of Ria de Aveiro (Portugal).27 Two Gammaproteobacteria strains were selected because this group is dominant in the UVexposed surface layers of water bodies and several of its members are considered UV-resistant.2,28,29 The Pseudomonas strain used is UV-resistant, recovering efficiently from UVinduced damage, while the Acinetobacter strain is more UV sensitive and less able to recover from UV-induced damage.24,27 Bacterial isolates were prepared as described in Supporting Information. Cell suspensions of each bacterial isolate (106 cells·mL−1) were placed in sterile Petri dishes (150 × 25 mm; Corning) and irradiated (without the lid) with UVA (Philips TL 100 W/10R lamps; Philips, Eindhoven, The Netherlands;

community structure and function and may provide insights into the biogeochemical effects of enhanced UV fluxes resulting from global climate changes.26 Additionally, integrated information on the effects of UVR of different wavelengths on bacteria may contribute to the design of more efficient and ecologically friendly UV-based disinfection strategies applicable either in the industrial setting or in a solar disinfection context. The objective of this work was to study the modifications induced by exposure to UVA, UVB, and UVC radiation on the lipids and proteins of two bacterial strains displaying different UV sensitivities by use of mid-infrared (IR) spectroscopy.

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MATERIALS AND METHODS Bacterial Strains and Irradiation Conditions. The bacterial strains used in this study (Acinetobacter sp. strain B



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control) were calculated by use of OPUS version 6.5 software (Bruker, Germany). In the resultant difference spectra, upwardmoving bands correspond to bands that appear (or increase in intensity) after irradiation, while downward-moving bands correspond to bands that disappear (or decrease in intensity) upon irradiation. For principal component analysis (PCA), the original spectra were transferred via JCAMP.DX format into the data analysis software package described in Barros.34

main emission line at 365 nm), UVB (Philips TL 100 W/01 lamps; Philips, Eindhoven, The Netherlands; main emission line at 311 nm) and UVC (low pressure mercury lamp NN 8/ 15; Heraeus, Berlin, Germany; main emission line at 254 nm), (spectra provided in Figure S1) under magnetic agitation at 25 °C. For each spectral region tested, the samples were irradiated to their LD50 (UV dose resulting in 50% bacterial inactivation), which can be found elsewhere.24 UV sources were placed 20 cm from the sample. UV intensities were measured with a monochromator spectroradiometer placed at the sample level (DM 300, Bentham Instruments, Reading, U.K.). The intensities of UVA, UVB, and UVC during irradiation were 50, 2.3, and 0.66 W·m−2, respectively. The UV dose (joules per square meter) was calculated by multiplying the intensity by the irradiation time (in seconds). The UVA and UVB doses used are in the range of the values detected in south Europe in clear summer days.30 Aliquots of cell suspensions were collected before and after irradiation, washed with ultrapure water, and immediately used for lipid and protein extraction. Experiments were repeated in three independent assays. Lipid Extraction. Total lipid extracts were obtained according to standard protocols.31 Briefly, irradiated and unirradiated cell suspensions were centrifuged (13000g for 10 min, 4 °C), and the pellet was washed with pure desalted water. The suspensions were centrifuged again and the supernatant was discarded. Chloroform (125 μL) and methanol (250 μL) were added to the pellet and the mixture was vortexed vigorously. Then, 6 M HCl (8.4 μL) and chloroform (125 μL) were added and the mixture was vortexed again. Finally, pure desalted water (125 μL) was added, and the solution was vortexed and centrifuged (20 min at 300g, 4 °C). Total lipid extract was collected in the lower phase and immediately used for spectra acquisition. All reagents were purchased from Sigma (St. Louis, MO). Protein Extraction. Proteins were extracted as previously described.32 Briefly, irradiated and unirradiated cell suspensions were centrifuged and the pellets were resuspended in 10 mM Tris-HCl (pH 8.0). The suspensions were sonicated in an ice bath four times for 5 s (Branson 450, Danbury, CT) and centrifuged (15000g, 1 h, 4 °C). The pellet was resuspended in 10 mM Tris-HCl (pH 8.0) and sarcosyl (1.5%, v/v). After incubation at room temperature (20 min), the protein extract was collected by centrifugation (15000g, 90 min, 4 °C) and suspended in ultrapure water. All reagents were purchased from Sigma (St. Louis, MO). Spectra Acquisition and Analysis. Lipid and protein extracts were spread over the horizontal diamond crystal of a Perkin-Elmer Spectrum BX System 2000 (Perkin-Elmer Corp., Norwalk, CT) equipped with a GoldenGate single-reflection attenuated total reflectance (ATR) system with a deuterated triglycine sulfate (DTGS) detector and dried under a gentle cold airflow. Measurements were recorded over the wavelength range 4000−700 cm−1 with a spectral resolution of 8 cm−1. The final spectra of the samples were achieved by averaging 32 scans. Three spectra of each assay conducted were acquired, yielding nine replicates for each irradiation condition in each strain. Spectra were normalized and baseline-corrected. Pearson’s correlation coefficient was calculated for the midinfrared range between 3000 and 2800 cm−1 and between 1800 and 700 cm−1 to assess spectral reproducibility.33 The six most similar spectra were identified and used for subsequent chemometric analysis. Difference spectra (irradiated minus

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RESULTS AND DISCUSSION The original mid-infrared spectra of the extracted lipids and proteins of Acinetobacter sp. PT5I1.2G and Pseudomonas sp. NT5I1.2B (henceforth referred as Acinetobacter sp. and Pseudomonas sp., respectively) are shown in Figure S2 (Supporting Information). Difference spectra are shown in Figures 1 and 3. UV Effects on Lipids. Exposure to different UV wavelengths resulted in similar overall effects on the lipids of the two strains tested, including drastic decreases in the intensity of the bands assigned to lipoproteins and/or nitrogen-containing lipids (≈3290, ≈1650, and ≈1540 cm−1) (Figure 1). As lipoproteins are susceptible to oxidative damage,35 reducing the amount of lipoproteins in the cell could be a mechanism to minimize oxidative damage to these molecules. Alternatively, it is possible that photosensitized reactions occurring in the protein fraction of the molecule could have interfered with lipid−protein interactions, resulting in dissociation of the lipid and protein moieties.35 As a result, proteins previously associated with lipids could have been lost during the process of lipid extraction. Exposure to UVR of different wavelengths also resulted in increased intensity of methyl bands (≈2950, ≈2870, ≈1460, and ≈1380 cm−1) (Figure 1), particularly in UVC-treated Acinetobacter sp. (Figure 1C). Changes in the phospholipid composition of the cell, consistent with the observed changes in the band at 1740 cm−1 assigned to esters of fatty acids,36 could explain the increase in methyl bands in the irradiated samples. Modifications in the phospholipid fatty acid composition of cells were confirmed by gas chromatography (GC) (Table S1, Supporting Information). Irradiation also caused a change in the intensity and frequency of methylene bands (≈2920 and ≈2850 cm−1), observed in the original and derivatized spectra (Figures S2 and S4, Supporting Information), thus denoting radiation-induced structural changes in membrane lipids.37 This result is supported by the observed UV-induced change in the membrane fluidity index (FI), calculated from the ratio of unsaturated (UFA) to saturated fatty acids (SFA) (Table S1, Supporting Information). Altered membrane fluidity has been associated with stress adaptation in Oenococcus oeni38 and the activation of protection mechanisms following stress exposure in plants.39 Whether such a mechanism could be involved in the bacterial response to UVR remains to be determined. In most cases, UV irradiation also caused oxidative damage to lipids, denoted by the increased intensity of the bands at 1018 cm−1 (assigned to O−O bonds of hydroperoxides),40 972 cm−1 (assigned to trans C=C bonds),41 1402 cm−1 (assigned to carboxyl groups formed during the oxidation process),42 and 888 cm−1 (assigned to CH2 and CH3 rocking modes of oxidized lipids).42 In some cases, the formation of lipid hydroperoxides could also be confirmed via the FOX2 assay43 (Table S2, Supporting Information). In the case of UVCtreated Acinetobacter sp., an increase in the intensity of the 1720 C



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Figure 2. Scores and loading plots for PCA analysis of lipid extracts of (A, B) Acinetobacter sp. and (C, D) Pseudomonas sp. irradiated with different UV spectral regions. Numbers on the axis labels of score plots indicate the percentage of the total variation in the data that was explained by the first and second principal components (PC1 and PC2).

cm−1 band was also observed (Figure 1C), indicating the possible formation of aldehydes due to lipid peroxidation,41 which was previously reported.24 In Acinetobacter sp. (Figure 1A−C) the effects of irradiation on oxidative damage marker bands were most prominent after exposure to UVC, while in Pseudomonas sp. oxidation seemed to occur as a result of exposure to UVR in all spectral regions (Figure 1D−F). Further evidence of lipid peroxidation was obtained from the analysis of second-derivative spectra, as previously described37 (Figure S3, Supporting Information). Increases in the intensity of several bands were observed in the region between 1630 and 1720 cm−1, comprising carbonyl stretching vibrations of unsaturated aldehydes resulting from the breakdown of lipid hydroperoxides.37 The occurrence of lipid oxidation was also inferred from the decrease in the relative abundance of UFA and corresponding increase in SFA,44 as determined by GC (Table S1, Supporting Information), particularly in UVA- and UVB-treated samples. PCA scores and loadings plots from the lipid spectra of Acinetobacter sp. are presented in Figure 2A,B. In the PCA score plot, the control samples (negative PC1) were separated from irradiated samples, particularly UVB- and UVC-irradiated samples (positive PC1), by the higher intensity of the bands assigned to ester carbonyls from phospholipids and triglycerides (1740 cm−1), amide I and amide II of lipoproteins (1654, 1546 cm−1), and phospholipids and/or glycolipids (1230, 1074

cm−1). The irradiated samples displayed an enhanced intensity of methyl bands (2954, 1460 cm−1) and bands associated with lipid peroxidation products (970, 888 cm−1). The different UV treatments were discriminated mostly along PC2. UVBirradiated samples were characterized by higher intensities of bands assigned to methylene groups (2914, 2846 cm−1), ester groups of phospholipids and triglycerides (1740 cm−1), and nitrogen-containing lipids and/or lipoproteins (1654, 1546 cm−1), while UVC-treated samples showed higher intensities of bands assigned to phospholipids and/or glycolipids (1214, 1158 cm−1) and oxidation products (888 cm−1). In contrast, PCA analysis of the lipids of Pseudomonas sp. did not show a clear separation between controls and irradiated samples (Figure 2C,D). The UVB-treated samples were located together with the control samples in negative PC1, separated from the UVA-treated samples located in positive PC1. The control and UVB-treated samples showed enhanced intensity of bands assigned to carbonyl ester groups of phospholipids and triglycerides (1740 cm−1), N−H stretching vibrations of amide and amine (3300, 1656 cm−1), and phospholipids and glycolipids (1216, 1180 cm−1), while UVA-treated samples showed enhanced intensity of CH3 bands (2940 cm−1) and bands associated with lipid oxidation products (888 cm−1). PC2 basically separated UVC- from UVA-irradiated samples and controls. The separation of these sets of samples was mostly associated with bands assigned to CH bonds (2940, 2926, D



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Figure 3. Difference spectra for protein extracts of Acinetobacter sp. under (A) UVA, (B) UVB, and (C) UVC and Pseudomonas sp. under (D) UVA, (E) UVB, and (F) UVC. Main bands are identified. A 13-point smooth was applied to the difference spectra. The region between 2400 and 2200 cm−1, corresponding to the CO2 region, was removed from the spectra.

2896, 2842, 1458, 1378 cm−1), esters of phospholipids (1740 cm−1), and lipoproteins and/or nitrogen-containing lipids (1656 and 1544 cm−1). Taken together, the PCA results identified several targets of UVR, namely, lipoproteins and/or nitrogen-containing lipids and lipid CH groups. Additionally, variations in bands assigned to phospholipids and/or glycolipids were observed, denoting irradiation-induced changes in bacterial lipid composition that were confirmed by GC (Table S1, Supporting Information). The altered lipid composition could be a result of UV-induced damage to enzymes involved in lipid synthesis or could reflect a bacterial metabolic strategy to cope with UVR. For instance, the observed general decrease in the relative abundance of phospholipids and glycolipids in UV-treated samples could be

explained by the high susceptibility of these lipid categories to oxidative damage.45 Decreased phospholipid and glycolipid content was also reported in UV-irradiated cyanobacteria and was attributed to oxidative damage of the membrane.46 UVinduced changes in the allocation of elements to biomolecules could also account for the observed changes in the relative proportion of phospholipids and glycolipids. For example, a reduction in phosphorus directed to membranes could be a mechanism elicited in order to ensure that enough phosphate supply is present for ATP-requiring repair strategies.47 Changes in the levels of transcripts of genes belonging to the rfa operon, which encodes for enzymes involved in lipid synthesis in the membrane of Escherichia coli, have also been observed in E



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Figure 4. Scores and loading plots for PCA analysis of protein extracts of (A, B) Acinetobacter sp. and (C, D) Pseudomonas sp. irradiated with different UV spectral regions. Numbers on the axis labels of scores plots indicate the percentage of the total variation in the data that was explained by the first and second principal components (PC1 and PC2).

response to UVR48 and thus contribute to explain the observed changes in lipid composition upon irradiation. UV Effects on Proteins. UV-irradiated samples were characterized by the increased intensity of methyl (≈2950 cm−1) and methylene (≈2920 and ≈2850 cm−1) bands associated with proteins and amino acids, particularly in Acinetobacter sp. (Figure 3A−C). The increased intensity of methylene bands could be attributed to UV-induced changes in protein composition, such as an increase in the proportion of CH2-rich amino acids including proline, lysine, isoleucine, glutamine, and glutamic acid. Accordingly, the synthesis of several amino acids (including isoleucine and glutamate) has been shown to be upregulated upon UV exposure in E. coli.49 UV-induced increase in proline concentration has also been observed in algae and associated with a stress-defense response.50 Changes in the protein profiles of bacteria with irradiation, particularly in the UV-sensitive strain Acinetobacter sp., were confirmed by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) (Figure S6, Supporting Information). Enhanced intensity of methyl and methylene bands could also reflect an increase in propionylation of lysine residues.51 However, lysine is not a strong infrared absorber, and its two main bands (≈1626 and ≈1526 cm−1) are hidden by the amide I and amide II bands, respectively,52 which limits further analysis. Propionylation is involved in the regulation of the

activity of some enzymes in bacteria;53 however, it is unknown whether this process plays a role in the bacterial response to UVR. A decrease in the intensity of bands assigned to aromatic amino acids, most notably tyrosine (1518 cm−1) and tryptophan (1452 cm−1), was also observed in all Acinetobacter sp. irradiated samples (Figure 3A−C) and in UVB-irradiated Pseudomonas sp. samples (Figure 3E). This decrease can be attributed to oxidative modifications of amino acid side chains.54,55 Additionally, it is known that in E. coli exposure to severe oxidative stress renders the cell unable to synthesize aromatic amino acids.56 Therefore, the decreased intensity of mid-IR bands associated with tyrosine and tryptophan could also be attributed to a UV effect on aromatic amino acid synthesis. UV exposure also caused a general decrease in the intensity of the main bands detected between 1200 and 900 cm−1. This region comprises vibrations of amino acids in addition to sugar and phosphate moieties associated with glycosylated and phosphorylated proteins; therefore, understanding the modifications represented in this region is relatively difficult. Possible explanations for the observed changes in this spectral region include decreased protein phosphorylation levels, which have been found to occur in Xanthomonas oryzae upon UVC exposure,57 and/or decreased protein glycosylation, a process that is involved in the regulation of protein activity in F



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band (1518 cm−1) and bands assigned to phosphorylated and/ or glycosylated proteins (1084, 1064 cm−1). In Pseudomonas sp., PCA analysis showed the separation of control and UVC-treated samples (negative PC1) from UVAand UVB-treated samples (positive PC1) (Figure 4C,D). Separation along PC1 was mostly due to bands assigned to CH2 groups (2924, 2854 cm−1), amide I (1656 cm−1), and carboxylate groups (1596 cm−1), which were enhanced in UVA- and UVB-treated samples. The control and UVC-treated samples showed enhanced intensity of amide II (1532 cm−1) and bands assigned to phosphorylated and/or glycosylated proteins (1218, 1042 cm−1). PC2 mostly separated UVA- and UVC-treated samples (negative PC2) from UVB-treated samples (positive PC2). Bands contributing to the separation along PC2 included the N−H (3294 cm−1), amide I (1656 cm−1), and phosphorylated and/or glycosylated protein bands (1222, 1048 cm−1), which were enhanced in UVB-treated samples. Bands assigned to CH2 groups (2924, 2854 cm−1) and carboxylate groups (1590, 1394 cm−1) were enhanced in UVAand UVC-treated samples. These results indicate that the effects of UVR on bacteria include changes in protein secondary structure and aromatic amino acids, potential formation of protein oxidation products, and changes in the levels of protein posttranslational modifications such as phosphorylation, glycosylation, and/or propionylation levels. Interspecific Variability in UV Effects on Lipids and Proteins. In general, exposure to each UV spectral region had similar effects on the two bacterial strains tested; however, substantial differences were also observed. For example, while the effects of UVA radiation on the lipids of the two strains tested were very similar, the effects of UVB differed considerably, particularly in the bands corresponding to lipoproteins and/or nitrogen-containing lipids (≈3300, ≈1650, and ≈1540 cm−1) and the CH region (3000−2800 cm−1). UVC also had differing effects on the two strains, particularly in the region between 1300 and 800 cm−1. Regarding proteins, differences between the two strains were mostly associated with the N−H band (≈3300 cm−1), carboxylate band (≈1590 cm−1), amide I component bands (≈1650 and ≈1624 cm−1), and tyrosine band (1518 cm−1). The significant differences in the effects of each UV spectral region in the two strains were confirmed by SDS−PAGE (Figure S6, Supporting Information) and may underlie their different UV sensitivities, in accordance with previous work.24,68 The observed differences in the effects of each UV spectral region on the two strains might be explained by differences in their original lipid and protein compositions, detected by midIR spectroscopy, GC analysis of fatty acids and SDS−PAGE of proteins (Table S1 and Figure S6, Supporting Information). For example, the relative abundance of SFA and UFA in the two strains may have influenced their sensitivity to UVR. An association between enhanced abundance of UFA and UV resistance was recently reported for Pseudomonas aeruginosa,69 potentially due to a role of UFA in ROS scavenging.70 Differences in the efficiency of antioxidant defense mechanisms24 may also have contributed to the observed divergence in the type of damage generated by the same spectral region in the different strains. Significance of the Study. The present study revealed profound UV-induced compositional and structural modifications in the lipids and proteins in two species of bacteria, as

bacteria.58 UV-induced changes in the levels of posttranslational modifications have been reported in eukaryotes,59 and a potential role of posttranslational modifications in the bacterial response to UVR was recently hypothesized.60 A substantial increase in intensity of the N−H bands around 3300 cm−1 and 1650 cm−1 was observed in Acinetobacter sp. exposed to UVA (Figure 3A) and Pseudomonas sp. exposed to UVB (Figure 3E), possibly indicating the accumulation of protein oxidation products such as kynurenine, resulting from the oxidation of tryptophan. This interpretation is also consistent with the observed decrease in intensity of the tryptophan band (1452 cm−1). Alternatively, ROS-mediated conversion of histidine to asparagine61 could also account for increased intensity of N−H bands. All irradiation treatments resulted in changes in the intensity and/or frequency of the component bands of amide I, which characterize the secondary structure of proteins, thus denoting irradiation-induced changes in protein conformation (Figure 3). The protein conformational changes were further confirmed by analysis of second-derivative and deconvoluted spectra of the extracted proteins, which showed major changes in the frequency, intensity, and bandwidth of the main component bands of amide I (≈1650 and ≈1624 cm−1) and in the abundance of α-helices (mean peak centered at ≈1650 cm−1) (Figure S5, Supporting Information). SDS−PAGE revealed the presence of high molecular weight protein aggregates in UVBand UVC-irradiated Acinetobacter sp., confirming the occurrence of UV-induced changes in protein conformation (Figure S6, Supporting Information). The accumulation of protein aggregates further supports the occurrence of protein oxidation suggested by mid-IR spectroscopy, particularly protein carbonylation,22 previously detected.24 Protein conformational changes may be elicited in order to protect sensitive amino acid residues from direct UV-induced damage62,63 or may be involved in coordinating stress response mechanisms and repair strategies.64 UV-induced synthesis of proteins with different conformations22,65 could also contribute to the observed changes in the amide I band. UVB- and UVC-treated Acinetobacter sp. also showed an increase in the intensity of carboxylate bands (1590 and 1390 cm−1), possibly reflecting the occurrence of UV-induced deamidation of asparagine and glutamine.66 In vivo deamidation of proteins can be used as a mechanism to initiate the production of specific proteins;67 however, whether such a mechanism is involved in the coordination of the bacterial response to UVR remains to be determined. PCA analysis of the protein spectra of Acinetobacter sp. showed a general separation between control and UVB-treated samples (negative PC1) from UVA- and UVC-treated samples (positive PC1) (Figure 4A,B). UVA- and UVC-treated samples were characterized by enhanced intensity of the N−H band (3294 cm−1) and amide I band (1652 cm−1), possibly denoting the accumulation of protein oxidation products. UVB-treated samples showed enhanced intensity of bands assigned to the aromatic amino acid tyrosine (1518 cm−1), CH3 deformation of amino acids (1380 cm−1), and phosphorylated and/or glycosylated proteins (1084, 1064 cm−1). PC2 separated UVC-treated samples from those treated with UVA and UVB; UVC-treated samples were differentiated by enhanced intensity of CH2 bands and carboxylate bands (1594, 1392 cm−1), whereas UVA- and UVB-treated samples showed enhanced intensity of the N−H band (3294 cm−1), tyrosine G



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community to different levels of ultraviolet-B radiation: A food web perspective. Microb. Ecol. 2001, 41 (1), 56−68. (4) Gustavson, K.; Garde, K.; Wängberg, S. A.; Selmer, J. S. Influence of UV-B radiation on bacterial activity in coastal waters. J. Plankton Res. 2000, 22 (8), 1501−1511. (5) Wängberg, S. Å.; Andreasson, K. I. M.; Gustavson, K.; Reinthaler, T.; Henriksen, P. UV-B effects on microplankton communities in Kongsfjord, Svalbard: A mesocosm experiment. J. Exp. Mar. Biol. Ecol. 2008, 365 (2), 156−163. (6) Frederick, J. E.; Lubin, D. The budget of biologically active ultraviolet radiation in the Earth-atmosphere system. J. Geophys. Res. 1988, 93 (D4), 3825−3832. (7) Moan, J.; Dahlback, A.; Setlow, R. B. Epidemiological support for an hypothesis for melanoma induction indicating a role for UVA radiation. Photochem. Photobiol. 1999, 70 (2), 243−247. (8) Wegelin, M.; Canonica, S.; Mechsner, K.; Fleischmann, T.; Pesaro, F.; Metzler, A. Solar water disinfection: Scope of the process and analysis of radiation experiments. J. Water Supply: Res. Technol. AQUA 1994, 43 (4), 154−169. (9) Sundin, G. W.; Jacobs, J. L. Ultraviolet radiation (UVR) sensitivity analysis and UVR survival strategies of a bacterial community from the phyllosphere of field-grown peanut (Arachis hypogeae L.). Microb. Ecol. 1999, 38 (1), 27−38. (10) Coohill, T. P.; Sagripanti, J.-L. Overview of the inactivation by 254 nm ultraviolet radiation of bacteria with particular relevance to biodefense. Photochem. Photobiol. 2008, 84 (5), 1084−1090. (11) Chamberlain, J.; Moss, S. H. Lipid peroxidation and other membrane damage produced in Escherichia coli K1060 by near-UV radiation and deuterium oxide. Photochem. Photobiol. 1987, 45 (5), 625−630. (12) Pattison, D. I.; Davies, M. J. Actions of ultraviolet light on cellular structures. EXS 2006, 96, 131−157. (13) Bose, B.; Chatterjee, S. N. Correlation between UVA-induced changes in microviscosity, permeability and malondialdehyde formation in liposomal membrane. J. Photochem. Photobiol. B 1995, 28 (2), 149−153. (14) Futsaether, C. M.; Kjeldstad, B.; Johnsson, A. Intracellular pH changes induced in Propionibacterium acnes by UVA radiation and blue light. J. Photochem. Photobiol. B. 1995, 31 (3), 125−131. (15) Pfeifer, G. P. Formation and processing of UV photoproducts: Effects of DNA sequence and chromatin environment. Photochem. Photobiol. 1997, 65 (2), 270−283. (16) Jagger, J. Solar UV Actions on Living Cells; Praeger Publishing: New York, 1985. (17) Harm, W. Biological Effects of Ultraviolet Radiation: Cambridge University Press: Cambridge, U.K., 1980. (18) Sinha, R. P.; Häder, D.-P. UV-induced DNA damage and repair: a review. Photochem. Photobiol. Sci. 2002, 1 (4), 225−236. (19) Ravanat, J. L.; Douki, T.; Cadet, J. Direct and indirect effects of UV radiation on DNA and its components. J. Photochem. Photobiol. Bem 2001, 63 (1−3), 88−102. (20) Matallana-Surget, S.; Meador, J. A.; Joux, F.; Douki, T. Effect of the GC content of DNA on the distribution of UVB-induced bipyrimidine photoproducts. Photoch. Photobiol. Sci. 2008, 7 (7), 794− 801. (21) Visser, P. M.; Poos, J. J.; Scheper, B. B.; Boelen, P.; Van Duyl, F. C. Diurnal variations in depth profiles of UV-induced DNA damage and inhibition of bacterioplankton production in tropical coastal waters. Mar. Ecol.Prog. Ser. 2002, 228, 25−33. (22) Bosshard, F.; Riedel, K.; Schneider, T.; Geiser, C.; Bucheli, M.; Egli, T. Protein oxidation and aggregation in UVA-irradiated Escherichia coli cells as signs of accelerated cellular senescence. Environ. Microbiol. 2010, 12 (11), 2931−2945. (23) Hoerter, J. D.; Arnold, A. A.; Kuczynska, D. A.; Shibuya, A.; Ward, C. S.; Sauer, M. G.; Gizachew, A.; Hotchkiss, T. M.; Fleming, T. J.; Johnson, S. Effects of sublethal UVA irradiation on activity levels of oxidative defense enzymes and protein oxidation in Escherichia coli. J. Photochem. Photobiol. B. 2005, 81 (3), 171−180.

assessed by mid-IR spectroscopy and confirmed by additional methods. From an ecological standpoint, our results indicate that, in addition to affecting aquatic trophic nets by reducing bacterial abundance, activity, and diversity,2−5 exposure to solar UVR (UVA + UVB) might also alter food quality for higher trophic levels by modifying the lipid and protein composition of bacterial cells, thus potentially affecting trophic interactions. Furthermore, by revealing the oxidizing potential of UVR on lipids and proteins, our observations also suggest that the efficiency of UV-based disinfection might be improved by increasing the oxidizing power of disinfection systems, for instance, by coupling UV irradiation to the presence of metals, like silver or iron.71,72 Accordingly, we have recently reported a synergistic effect of metals and UVB exposure in bacterial inactivation.73 In this study, mid-IR spectroscopy was proven to be a rapid tool to monitor UV-induced changes in bacterial composition. The use of complementary approaches to mid-IR spectroscopy, such as mass spectrometry-based lipidomics and proteomics analyses, will help to expand upon the findings of the present study in order to gain further insights into the mechanisms of UV-based bacterial inactivation.

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ASSOCIATED CONTENT

S Supporting Information *

Additional text, six figures, and two tables with details of experimental methodologies, UV lamp spectra, original and processed spectra of lipid and protein extracts for spectral regions of interest, SDS−polyacrylamide gels of proteins, lipid hydroperoxide levels detected, and GC results of fatty acid analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +351 234 372 594; fax: +351 234 426 408; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the three anonymous reviewers for their helpful comments. Financial support for this work was provided by CESAM, University of Aveiro (Project Pest-C/MAR/LA0017/ 2011) and the Portuguese Foundation for Science and Technology (FCT) in the form of grants SFRH/BD/40160/ 2007 (to A.L.S.), SFRH/BD/71512/2010 (to C.M.), SFRH/ BPD/38008/2007 (to A.C.E.) and SFRH/BPD/63487/2009 (to I.H.). Funding to QOPNA was provided by FCT and FEDER-COMPETE/QREN/EU (PEst UI 62-2011-2012).

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REFERENCES

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Effects of UV Radiation on the Lipids and Proteins of Bacteria Studied

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