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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The purpose of this study was to explore the possible link between metals and UV-B-induced damage in bacteria. The effect of growth in the presence of enhanced concentrations of different transition metals (Co, Cu, Fe, Mn and Zn) on the UV-B sensitivity of a set of bacterial isolates was explored in terms of survival, activity and oxidative stress biomarkers (ROS generation, damage to DNA, lipid and proteins and activity of antioxidant enzymes). Metal amendment, particularly Fe, Cu and Mn, enhanced bacterial inactivation during irradiation by up to 35.8%. Amendment with Fe increased ROS generation during irradiation by 1.2–13.3%, DNA damage by 10.8–37.4% and lipid oxidative damage by 9.6–68.7%. Lipid damage during irradiation also increased after incubation with Cu and Co by up to 66.8% and 56.5% respectively. Mn amendment decreased protein carbonylation during irradiation by up to 44.2%. These results suggest a role of Fe, Co, Cu and Mn in UV-B-induced bacterial inactivation and the importance of metal homeostasis to limit the detrimental effects of ROS generated during irradiation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

UV-B radiation (320–280 nm) is the most detrimental wavelength of the solar radiation spectrum. The severest biological consequence of UV-B radiation has been traditionally considered DNA damage, most notably the formation of DNA photoproducts [1]. However, UV-B-induced cell damage can also arise as the result of increased production of reactive oxygen species (ROS) during irradiation and subsequent oxidative damage to biomolecules [2]. Studies reporting the induction of antioxidant defenses during UV-B exposure of bacteria support the hypothesis of the contribution of oxidative stress to UV-B-induced damage [3, 4]. The cellular and biological consequences of ROS are strongly influenced by metal ion homeostasis [2]. Metal ions, most notably iron (Fe2+) and copper (Cu+), play a catalytic role in the generation of ROS through participation in Fenton and Haber–Weiss reactions that generate toxic hydroxyl radicals that can damage biomolecules and cause cell death [5]. As many of the respiratory chain proteins use iron as a cofactor, they are among the first targets of oxidative damage through the Fenton reaction [6]. The hydroxyl radicals generated in the process can in turn attack membrane lipids and other membrane proteins [7, 8]. On the other hand, cobalt (Co2+) can compete directly with iron, thereby affecting the synthesis of Fe–S clusters [9, 10], leading to decreased iron bioavailability and eventually oxidative stress [11].

Recently, a possible protective role of transition metal ions against ionizing radiation has been discussed. A high capacity for intracellular copper ion sequestration was detected in Kineococcus radiotolerans (Domain Bacteria, Phylum Actinobacteria) that provided protection against the damaging effects of ionizing radiation [12]. Manganese (Mn2+) ions also seem to play a role in the prevention of oxidative damage during exposure to different types of stress including UV-C radiation, gamma-irradiation, wet and dry heat and H2O2 [13-18]. Zinc (Zn2+) uptake is also a key component of the adaptive response to peroxide stress [19], protecting copper-treated Escherichia coli against superoxide killing [20] and countering the effects of oxidative stress in Lactococcus lactis [21].

Increased levels of proteins involved in iron homeostasis, induction of iron-sequestering protein-encoding genes and repression of genes involved in iron uptake have been observed in bacteria irradiated with UV-B [3, 4], suggesting the importance of regulation of the intracellular iron ion pool following UV-B exposure. However, whether certain transition metal ions play a physiological role in bacterial resistance to environmentally relevant UV radiation, most notably UV-B radiation, remain uncertain. This information is critical to understanding the effects of UV radiation on the ecological context of natural communities, where these metals can be particularly enriched when industries and agriculture facilities are located nearby [22]. Furthermore, understanding the role that metals play during UV-induced bacterial inactivation can help develop more efficient photocatalytic disinfection strategies [23]. Accordingly, the objectives of this study were to investigate the potential synergistic effects of transition metals and exposure to UV-B radiation in bacterial cells and to evaluate their potential protective role against structural and functional cellular damage elicited during UV-B exposure in a set of bacterial isolates.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Culture conditions and experimental layout

The bacterial strains used in this study were previously isolated from the surface waters of an estuarine system. The isolates are affiliated to different phylogenetic groups: Alphaproteobacteria (Paracoccus sp.), Gammaproteobacteria (Pseudomonas sp.), Bacilli (Staphylococcus sp.) and Actinobacteria (Micrococcus sp.) and display different UV-B sensitivities (Santos et al., 2011), which are presented in Table 1 as LD50 values (UV-B dose necessary for the inactivation of 50% of bacteria).

Table 1. Bacterial strains used in the experiments, their closest relatives, accession number and bacterial group and UV-B LD50 values. Nonsignificant differences in LD50 values of metal-amended treatments compared with unamended samples are indicated by n.s
StrainClosest relativeAccession NumberGroupLD50(kJ m−2)
UV-B+Co+Cu+Fe+Mn+Zn
NT25I3.2AA Micrococcus sp.GQ365196 Actinobacteria50.148.9n.s.42.043.140.142.5
NT25I3.1A Paracoccus sp.GQ365195 Alphaproteobacteria 40.335.629.835.429.139.9n.s.
NT5I1.2B Pseudomonas sp.GU084169 Gammaproteobacteria 49.342.027.930.129.040.5
NT25I2.1 Staphylococcus sp.GQ365197 Bacilli37.038.7n.s.32.530.733.839.0n.s.

Stock solutions (10 mM) of various transition metals (Co, Cu, Fe, Mn and Zn) were prepared in deionized, filter-sterilized water from the corresponding metallic salts: ferrous ammonium sulfate, manganese chloride, zinc sulfate, cupric sulfate and cobalt sulfate. To select the metal concentration that affected the least the growth curve of the different bacterial strains, and that was subsequently used for UV exposure assays, preliminary experiments were conducted with different metal concentrations (1, 10, 100 μM) in TGY medium (1.0% tryptone, 0.1% glucose, 0.5% yeast extract) [12]. Growth curves obtained in these conditions were compared with those obtained in the absence of added metals. Cultures were incubated for up to 72 h at 25°C with agitation (120 rpm). At predetermined intervals, triplicate aliquots were collected and the optical density (O.D.) at 600 nm was determined. From the results of the preliminary experiments, 1 μM was found to be the maximum noninhibitory concentration for all metals (data not shown) and used in the UV irradiation assays.

Liquid cultures of each isolate were grown to midexponential phase (O.D.600 of 0.2–0.3) in TGY or TGY amended with 1 μM of Co, Cu, Fe, Mn or Zn. Cells were harvested by centrifugation (3200 g for 15 min, Eppendorf Centrifuge 5415R, Hamburg, Germany) and the pellet was washed three times with 50 mM EDTA/ 10 mM phosphate buffered saline (PBS; pH 7.5) to remove cell surface adsorbed transition metals [12]. Cells were then resuspended in 10 mM PBS for determination of bacterial abundance by epifluorescence microscopy [24], and adjusted to 106 cells mL−1 with 10 mM PBS. A convenient volume of cell suspension was transferred to sterile 150 mm × 25 mm plastic tissue culture dishes (Corning Science Products, Corning, NY, USA) so that the depth of the liquid was <2 mm. The lid was removed during UV-B irradiation (Philips TL 100 W/01 lamps, main emission wavelength line of 302 nm). Metal-amended and unamended dark controls (covered in aluminum foil) treated in the same way as the irradiated samples were included in every experiment. Inactivation curves of bacterial strains under the different metal-amended and unamended regimes were determined by irradiating bacterial suspensions under UV-B for a total light dose of 60 kJ m−2 and collecting aliquots at predetermined light doses for CFU determination, as described below. UV intensities were measured with a monochromator spectro-radiometer placed at the sample level (DM 300, Bentham Instruments, Reading, UK). Inactivation curves were used to determine the LD50 in metal-amended and unamended conditions. In addition, aliquots of cell suspensions were collected before and after irradiation at the LD50 of unamended samples (50.1 kJ m−2 for Micrococcus sp., 40.3 kJ m−2 for Paracoccus sp., 49.3 kJ m−2 for Pseudomonas sp. and 37.0 kJ m−2 for Staphylococcus sp.) (Table 1) for biological and biochemical analysis. For protein carbonyls and antioxidant activity [catalase (CAT) and superoxide dismutase (SOD)] determinations, cells were immediately resuspended in cold 50 mM PBS (pH 7.8) containing 1 mM EDTA and sonicated in ice (Branson Instruments Co. Sonifier, Stamford, Conn.; 2 min, 30 s pulses, 1 min cooling). The extracts were centrifuged (10 000 g, 15 min) and the supernatant frozen at −80°C until analysis. All experiments were repeated in three independent assays. Parameters were always determined in a minimum of three analytical replicates. Positive (50 mM H2O2-treated) and negative (untreated) controls were always included and processed along experimental samples to ensure proper functioning of the procedures on all strains.

Colony-forming units (CFU)

Before and after irradiation, sample aliquots were serially diluted in 1 × PBS and spread plated in triplicate in agar plates (Difco, Detroit, MI). Colonies were counted after up to 7 days of incubation in the dark, at 25°C. The dilution and plating procedures were carried out under low-luminosity conditions to minimize photoreactivation.

Glucose uptake

The effects of UV radiation combined with the exposure to metals on bacterial energy metabolism were assessed from glucose uptake activity, using a protocol adapted from Harada et al. [25]. Triplicate 1.5 mL aliquots of bacterial suspension and a 5% (wt/vol) trichloroacetic acid-fixed control were incubated with 14C-glucose (Amersham Biosciences, Buckinghamshire, UK; SA 310 mCi mmol−1) at a saturating concentration of 150 μM. Samples were incubated in the dark at in situ temperatures for 1 h. Incubations were stopped by the addition of chilled 5% (wt/vol) trichloroacetic acid (Sigma, St. Louis, MO, USA) and samples were filtered through nitrocellulose filters (0.2 μm pore size; Millipore, Tokyo, Japan). Filters were then rinsed three times with chilled 5% trichloroacetic acid and transferred to eppendorfs. A volume of 1.5 mL of Universal liquid scintillation cocktail (ICN Biomedicals, USA) was added. The amount of radioactivity incorporated into bacterial cells was determined in a Beckman LS 6000 IC liquid scintillation counter (Beckman Instruments, Inc., Fullerton, CA).

In Vivo production of ROS

In vivo production of ROS was detected using the probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Cayman Chemical Co. Ltd., MI, USA) [26]. Control and irradiated samples were centrifuged and washed with 10 mM PBS (pH 7.0). After amendment with the probe (final concentration 10 μM), the mixture was incubated in the dark for 30 min. Afterward, cells were washed, sonicated and the cell extracts were mixed with potassium phosphate buffer. The fluorescence of the samples was measured with a Jasco FP-777 Fluorometer (Japan Spectroscopic Co., Ltd., Tokyo, Japan) at room temperature (λex 490 nm; λem 519 nm). The fluorescence intensity at 519 nm was corrected against the blank control (without cells) and then normalized to the biomass, determined as the amount of cellular protein according to the method of Bradford (Bradford, 1972). Unless otherwise specified, all reagents used for this and subsequent protocols were purchased from Sigma-Aldrich (St Louis, MO).

DNA strand breakage

UV-induced DNA damage was assessed using the quantification of DNA strand breaks (DSB) as a proxy, following a previously described procedure [27]. The method requires three sets of samples: test samples (so-called P-samples), samples not subjected to alkaline unwinding (T-samples) and samples subjected to complete alkaline unwinding (B-samples). Cells were collected by centrifugation (3,000 g, 15 min) and digested with lysozyme (4 mg mL−1 final concentration) and proteinase K (0.25 mg ml−1 final concentration). A volume of 300 μl of 0.1 M NaOH was added to the three sets of samples: [1] T-samples were immediately neutralized with 300 μl of 0.1 M HCl, incubated at room temperature for 30 min and sonicated for 15 s; [2] B-samples were sonicated for 2 min, incubated for 30 min at room temperature, neutralized with 300 μl of 0.1 M HCl and sonicated again for 15 s; and [3] P-samples were incubated for 30 min, neutralized with 300 μl of 0.1 M HCl and sonicated for 15 s.

A final concentration of 5 μM of Hoechst 33258 was added to all samples. After centrifugation, the supernatant was used for fluorescence measurements (λex 350 nm; λem 450 nm) in a Jasco FP-777 Fluorometer (Japan Spectroscopic Co., Ltd., Tokyo, Japan). The fraction of double-stranded DNA (dsDNA) was calculated as dsDNA = (P−B)/(T−B) × 100, where T, P and B were fluorescence intensities of T-, P- and B-samples normalized to the biomass respectively.

Lipid peroxidation

Lipid peroxidation was determined as the amount of thiobarbituric acid reactive substances (TBARS) as previously described [26]. Control and irradiated cells were centrifuged, washed and suspended in 50 mM PBS (pH 7.4) containing 0.1 mM butylated hydroxytoluene and 1 mM of the protease inhibitor phenylmethanesulfonyl fluoride. Afterward, cell suspensions were sonicated and centrifuged, and the soluble fraction was mixed with an equal volume of 20% trichloroacetic acid. Following another centrifugation step at 10 000 g for 5 min, supernatants were removed and mixed with 1 volume of 0.5% (w/v) thiobarbituric acid in 0.1 M HCl and 10 mM butylated hydroxytoluene. After incubation at 100°C for 1 h, aliquots were removed, cooled and mixed with 1.5 volumes of butanol. The mixture was centrifuged (4 000 g, 10 min) and the organic fraction was removed. The absorbance at 535 nm was read using a Thermo Spectronic Genesys 10 UV spectrophotometer and TBARS content was determined using an extinction coefficient of 156 mM−1 cm−1. Results were normalized to the cell biomass.

Protein oxidation

The carbonyl content in oxidized proteins was determined as previously reported [28]. Aliquots of sonicated cells were incubated with 10 mM diphenylhydrazine (DPNH) in 2 M HCl for 1 h at room temperature. In blanks, DPNH was omitted. To precipitate proteins, 20% trichloroacetic acid was added, after which suspensions were centrifuged (14 000 g, 5 min), and the pellet washed three times with 1:1 (vol/vol) ethanol-ethyl acetate. The final precipitate was dissolved in 6 M guanidine hydrochloride. The absorbance at 360 nm was determined against a blank of guanidine solution (6 M guanidine hydrochloride with 2 mM potassium phosphate), and a molar absorption coefficient of 22 mM−1 cm−1 was used to quantify the levels of protein carbonyls. Results were normalized to the biomass.

Antioxidant enzymatic activity

CAT activity was measured spectrophotometrically by monitoring the rate of decomposition of H2O2 [29]. One unit of CAT activity was defined as the amount of activity required to decompose 1 μmol of H2O2 per minute under the assay conditions. The strain Enterococcus faecalis was used as a negative control. A mixture of 18 mM hydrogen peroxide and sterile potassium phosphate buffer (1:5) was used as an additional negative control [30]. SOD activity was determined according to McCord and Fridovich [31], in which a xanthine–xanthine oxidase system is used to generate superoxide and nitroblue tetrazolium is used as an indicator. One unit of SOD activity was defined as the amount of SOD that resulted in 50% inhibition of the reduction of nitroblue tetrazolium. Potassium phosphate buffer was used as a blank. Results were normalized to the biomass.

Bacterial biomass

Bacterial biomass was determined from the protein concentration in cell homogenates according to the Bradford method using bovine serum albumin as a standard [32].

Statistical analysis

All experiments were repeated in three independent assays and biochemical and microbiological analysis were always conducted in triplicate. Differences between treatments were assessed by one-way ANOVA using the statistical software SPSS v.17. Levene test was used to assess homogeneity of variances. If variances were not homogeneous, the natural logarithm transformation was applied. If variances were still not homogenous, the nonparametric Mann–Whitney test was used to assess the overall effect of treatment. Stepwise multiple regression, used to identify groups of independent variables (ROS, DSB, TBARS and carbonyls levels as well as SOD and CAT activity) with the most significant contribution to the variability of the dependent variable (bacterial inactivation expressed as LD50), was conducted with SPSS v. 17.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Effects of UV irradiation on biological and biochemical parameters

To select metal concentrations that would not significantly affect either the growth curve of bacterial isolates or the level of oxidative stress markers on unirradiated cells, thereby minimizing any potential toxic effect of the metals during irradiation experiments, different metal concentrations were tested. A concentration of 1 μM affected the least the growth curve and did not significantly alter the levels of oxidative stress markers of cells (Table 2) and was therefore chosen for subsequent UV-assays.

Table 2. Absolute values of the different stress markers in unirradiated metal-amended and unamended cell suspensions. A.U.: arbitrary units. Nonsignificant differences (ANOVA,> 0.05) in the values of the different stress markers in metal-amended treatments compared with unamended samples are indicated by n.s
StrainTreatmentROS (DCF fluorescence, A. U.)DSB (Hoechst 33258 fluorescence, A.U.)TBARS (pmol/mg protein)Carbonyls (μmol/mg protein)CAT (μmol/min/mg protein)SOD (U/mg protein)
Micrococcus sp. NT25I3.2AA No metal8432.7 ± 846.4171.7 ± 16.423.3 ± 2.96.7 ± 0.641.8 ± 4.7266.7 ± 25.7
Co9023.0 ± 954.6n.s.189.2 ± 18.1n.s.26.1 ± 2.9n.s.7.3 ± 0.6n.s.44.8 ± 4.7n.s.289.3 ± 29.7n.s.
Cu7715.9 ± 853.6n.s.157.1 ± 17.7n.s.20.3 ± 2.8n.s.6.1 ± 0.9n.s.37.5 ± 5.3n.s.248.0 ± 26.4n.s.
Fe8390.5 ± 831.5n.s.172.6 ± 18.7n.s.23.3 ± 2.7n.s.6.7 ± 0.7n.s.41.4 ± 4.2n.s.268.0 ± 28.8n.s.
Mn8977.8 ± 851.3n.s.188.5 ± 20.2n.s.25.8 ± 2.6n.s.7.9 ± 0.7n.s.47.9 ± 5.1n.s.290.8 ± 29.0n.s.
Zn7677.3 ± 850.5n.s.173.1 ± 18.2n.s.20.5 ± 3.0n.s.6.6 ± 0.6n.s.40.1 ± 3.9n.s.249.2 ± 23.5n.s.
Paracoccus sp. NT25I3.1A No metal12870.0 ± 1115.3110.9 ± 10.217.2 ± 2.06.7 ± 0.834.8 ± 4.1172.0 ± 21.5
Co14414.4 ± 1609.2n.s.122.3 ± 11.7n.s.18.7 ± 2.3n.s.7.7 ± 0.8n.s.39.7 ± 4.1n.s.194.4 ± 21.5n.s.
Cu11196.9 ± 1608.8n.s.103.1 ± 10.6n.s.16.2 ± 2.3n.s.6.0 ± 1.4n.s.31.5 ± 4.1n.s.151.4 ± 21.8n.s.
Fe12827.1 ± 1584.9n.s.112.1 ± 13.0n.s.17.4 ± 2.1n.s.6.8 ± 1.1n.s.35.3 ± 4.6n.s.172.6 ± 21.6n.s.
Mn14366.4 ± 1603.8n.s.114.9 ± 13.8n.s.18.4 ± 1.3n.s.7.7 ± 1.1n.s.35.2 ± 5.7n.s.195.0 ± 21.6n.s.
Zn11159.6 ± 862.3n.s.113.8 ± 11.9n.s.15.7 ± 1.8n.s.6.8 ± 1.0n.s.35.9 ± 4.7n.s.151.9 ± 24.7n.s.
Pseudomonas sp. NT5I1.2B No metal12020.0 ± 1365.9121.5 ± 12.714.3 ± 1.26.8 ± 0.850.3 ± 5.0143.3 ± 17.9
Co13582.6 ± 1502.9n.s.130.0 ± 12.7n.s.15.3 ± 1.1n.s.7.5 ± 0.8n.s.55.9 ± 5.0n.s.160.5 ± 17.9n.s.
Cu10577.6 ± 1502.5n.s.108.7 ± 11.4n.s.13.1 ± 1.1n.s.6.0 ± 1.2n.s.45.8 ± 8.3n.s.124.7 ± 17.7n.s.
Fe12060.1 ± 1525.3n.s.120.1 ± 13.6n.s.14.3 ± 1.5n.s.6.8 ± 1.0n.s.50.7 ± 6.5n.s.142.9 ± 17.9n.s.
Mn13627.9 ± 1507.9n.s.139.1 ± 14.6n.s.16.1 ± 1.8n.s.7.4 ± 1.0n.s.52.0 ± 6.6n.s.160.0 ± 17.9n.s.
Zn10612.9 ± 1034.4n.s.116.3 ± 12.2n.s.12.5 ± 1.1n.s.6.6 ± 0.9n.s.50.8 ± 5.6n.s.124.3 ± 17.8n.s.
Staphylococcus sp. NT25I2.1 No metal15193.3 ± 1384.0157.9 ± 16.637.0 ± 3.25.3 ± 0.434.8 ± 3.7240.0 ± 22.2
Co16560.7 ± 1705.6n.s.171.3 ± 16.0n.s.42.1 ± 5.0n.s.5.7 ± 0.4n.s.37.2 ± 3.7n.s.262.8 ± 22.2n.s.
Cu13750.0 ± 1405.4n.s.141.3 ± 16.3n.s.32.2 ± 4.0n.s.4.9 ± 0.7n.s.31.8 ± 4.1n.s.218.4 ± 22.4n.s.
Fe15168.0 ± 1491.7n.s.156.8 ± 17.4n.s.37.1 ± 4.8n.s.5.3 ± 0.5n.s.34.6 ± 4.2n.s.240.4 ± 22.1n.s.
Mn16533.1 ± 1603.2n.s.155.9 ± 18.9n.s.41.8 ± 4.6n.s.5.2 ± 0.5n.s.39.8 ± 4.1n.s.263.2 ± 22.0n.s.
Zn13727.1 ± 1294.8n.s.153.3 ± 16.6n.s.32.5 ± 4.6n.s.5.3 ± 0.4n.s.34.1 ± 3.9n.s.218.8 ± 22.3n.s.

UV-B inactivation curves of bacterial isolates grown with and without added concentrations of metals are represented in Fig. 1. Dark controls for every metal treatment were also performed but not included in the figure for simplicity as significant differences between dark controls of unamended and metal-amended samples were not observed (> 0.05).

image

Figure 1. Influence of transition metals on UV-B inactivation curves of (A) Micrococcus sp., (B) Paracoccus sp., (C) Pseudomonas sp. and (D) Staphylococcus sp. Each point corresponds to the average of three independent experiments, conducted with triplicate replicas (= 9). Error bars are standard deviation of the mean. Where error bars are not visible, they are smaller than the graph symbol. D.C.: dark control. L.C.: light control (without metal).

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Compared with unamended samples, metal amendment generally reduced the LD50 by up to 21.4 kJ m−2. This effect was particularly significant for the Cu, Fe and Mn treatments and variable depending on the bacterial isolate (Table 1). The effects of different transition metals on survival and activity during UV-B exposure are represented in Fig. 2. Amendment with Cu, Fe and Mn significantly (< 0.05) decreased survival following exposure to UV-B by 10.8–27.7%, 16.5–35.8% and 13.1–31.0%, respectively, in all isolates tested (Fig. 2A). Depending on the strain, the effects of metal treatment were not significant, protective or detrimental to glucose incorporation during irradiation (Fig. 2B).

image

Figure 2. Variation in (A) cell survival and (B) glucose incorporation upon UV-B exposure in cell suspensions unamended and amended with 1 μM of the different transition metals. Results are shown as the percentage of the initial value, calculated as Ni/N0 × 100, where Ni is the value of the parameter after irradiation and N0 is the value of the parameter at time 0, for each condition tested. Each bar corresponds to the average of three independent experiments, conducted with triplicate replicas (= 9). Error bars are standard deviation of the mean. Some error bars are hidden behind the graph symbol. The significance of the differences between metal-amended and nonamended samples is presented: *< 0.05, **< 0.01, ***< 0.005, D.C.: dark control. L.C.: light control (without metal).

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Treatment with Co and Zn resulted in either an increase or attenuation of ROS generation, depending on the strains. Cu and Fe treatments were responsible for a net increase in ROS generation during UV-B exposure by up to 9.8% and 13.3% respectively. Amendment with Mn significantly attenuated ROS generation during irradiation (up to 20.5%; < 0.05) in all strains (Fig. 3A). Treatment with Co, Cu, Mn and Zn had variable effects on DNA damage (assessed as DSB) during UV-B exposure, depending on the strain, while amendment with Fe significantly increased DNA damage by up to 37.4% in all strains (Fig. 3B). Cultures grown with additional concentrations of Co, Cu and Fe displayed increased TBARS formation during irradiation by up to 56.5%, 66.8% and 68.7% respectively. Treatment with Zn either had no effect or increased TBARS generation during irradiation by up to 53.5%, while Mn either attenuated or increased UV-induced TBARS formation depending on the strain (Fig. 3C). Amendment with Co, Cu, Fe and Zn either attenuated or increased the formation of protein carbonyls during UV-B irradiation depending on the strain, while modification with Mn attenuated carbonyl formation in all strains by up to 44.2% (Fig. 3D).

image

Figure 3. Variation in (A) ROS, (B) DSB, (C) TBARS and (D) carbonyl levels upon UV-B exposure in cell suspensions unamended and amended with 1 μM of the different transition metals. Results are shown as the percentage of the initial value, calculated as Ni/N0 × 100, where Ni is the value of the parameter after irradiation and N0 is the value of the parameter at time 0, for each condition tested. Each bar corresponds to the average of three independent experiments, conducted with triplicate replicas (= 9). Error bars are standard deviation of the mean. Some error bars are hidden behind the graph symbol. The significance of the differences between metal-amended and nonamended samples is presented: *< 0.05, **< 0.01, ***< 0.005. D.C.: dark control. L.C.: light control (without metal).

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Effects of metal treatment on CAT and SOD activity were more variable depending on the strain (Figs. 4A, B). In Micrococcus sp., metal-amended suspensions showed greater CAT activity compared with unamended controls by up to 61.6%, whereas in Paracoccus sp. and Pseudomonas sp., irradiation in the presence of added metal decreased CAT activity compared with metal-free cell suspensions by up to 50.2% and 70.1% respectively. In Staphylococcus sp., the presence of different metals either increased or decreased CAT activity compared with unamended samples. SOD activity was decreased in metal-amended suspensions of Micrococcus sp. by up to 70.4% and enhanced in Pseudomonas sp. by up to 40.3%, compared with unamended samples. In Paracoccus sp. and Staphylococcus sp., effects of metal amendment on SOD activity during irradiation depended on the metal added.

image

Figure 4. Variation in (A) CAT activity and (B) SOD activity upon UV-B exposure in cell suspensions unamended and amended with 1 μM of the different transition metals. Results are shown as the percentage of the initial value, calculated as Ni/N0 × 100, where Ni is the value of the parameter after irradiation and N0 is the value of the parameter at time 0, for each condition tested. Each bar corresponds to the average of three independent experiments, conducted with triplicate replicas (= 9). Error bars are standard deviation of the mean. Some error bars are hidden behind the graph symbol. The significance of the differences between metal-amended and nonamended samples is presented: *< 0.05, **< 0.01, ***< 0.005, D.C.: dark control, L.C.: light control (without metal).

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Multiple stepwise linear regression

To obtain further insights on the influence of metals on UV-induced inactivation, a multiple stepwise linear regression analysis was applied to the dataset using LD50 as a dependent variable and oxidative stress markers (ROS levels, DSB, TBARS, protein carbonyls, CAT and SOD activity) as predictor variables (Table 3). In controls not treated with metals, 99.4% of the variability of LD50 was accounted for by DSB levels, TBARS and CAT activity. In cultures supplemented with Co, 96.5% of the variation in LD50 was predicted by the contributions of SOD, carbonyls and CAT. In Cu-treated samples, variations in LD50 were not significantly explained by any of the independent variables used. In the Fe treatment, 99.3% of the variability in LD50 was explained by ROS, TBARS and DSB, whereas in Mn treatments, 32.7% of the variation in LD50 was explained by TBARS. In the Zn treatment, 99.8% of the variations in LC50 were explained by CAT, carbonyls and ROS.

Table 3. Multiple stepwise regression analysis used to infer the parameters contributing to bacterial inactivation (expressed as LD50) under the different treatments. DSB: DNA strand breaks. CAT: catalase. ROS: reactive oxygen species. TBARS: thiobarbituric acid reactive substances. β: standardized coefficient. P: probability. R2: coefficient of correlation
 Adjusted R2 of the model (P)Predictor variableβ P
UV0.994 (0.001)DSB0.6040.000
TBARS0.2850.023
CAT0.1860.001
UV + Co0.965 (0.005)SOD−0.8010.000
Carbonyls2.1150.000
CAT−1.4220.005
UV + Cu
UV + Fe0.993 (0.000)ROS0.8540.000
TBARS0.3970.000
DSB0.2960.000
UV + Mn0.327 (0.030)TBARS0.6230.030
UV + Zn0.998 (0.020)CAT−0.2040.004
Carbonyls1.0540.000
ROS0.6280.000

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Research conducted with several biological systems has highlighted the role of transition metal ions in protecting against the detrimental effects of gamma-radiation, UV-C radiation, wet and dry heat and H2O2 [12, 33-35]. However, the role of transition metals on the susceptibility of bacteria to environmentally relevant UV radiation remains unknown. This lack of information impairs the realistic understanding of UV effects in bacterial communities in natural environments where metals are frequently present, seasonally variable and can represent additional stress factors [22].

In the present work, the effect of five transition metals on UV sensitivity and UV-induced cellular damage on a set of bacterial isolates was studied. The concentrations of metals used were generally in agreement with reported environmental concentrations in aquatic systems, particularly in the runoff of urban areas [36]. The importance of metals to UV-B-induced damage was evident from the shift in the descriptors that contributed the most to explain bacterial inactivation (expressed as LD50) following treatment with different metals, as revealed by linear regression.

Role of Co, Cu and Fe

Despite the observed variability in the effects of UV-induced damage in the different strains tested, amendment with metals generally decreased the dose necessary for inactivation by UV-B (LD50), thereby suggesting a synergistic effect of metals and UV-B radiation in bacterial cell damage.

The Fenton reactive metals Fe and Cu had a significant detrimental effect on bacterial survival during irradiation, potentially associated with enhanced ROS and TBARS generation following irradiation in the metal-treated bacterial suspensions. In the case of Fe, an increase in DNA damage compared with untreated controls was also observed and, together with ROS generation and TBARS, accounted for 99.3% of the variation in LD50, as shown by linear regression. Such effects are in line with the role of the divalent Fe ion (Fe2+) in promoting the generation of the powerful oxidant hydroxyl radical according to the following reaction:

  • display math

The hydroxyl radical can in turn cause damage to any biomolecule. The current study suggests the involvement of Fe in UV-B-induced oxidative damage and the importance of Fe homeostasis through the regulation of genes involved in Fe influx, storage and expression of Fe-containing enzymes [3, 4] in limiting the detrimental effects of exposure to UV-B.

Under aerobic conditions, Cu (similarly to Fe) can participate in Haber–Weiss reactions to mediate the generation of the hydroxyl radical [37]. In accordance with what was observed with Fe, treatment with Cu increased TBARS and ROS generation. However, as opposed to Fe, it did not significantly increase DNA damage during irradiation. This observation can be explained, at least in Gram-negative bacteria, by the compartmentalization of hydroxyl radicals generated by Cu in the periplasm of the cell, which limited its damage to the membrane and cell wall [38], thus accounting for the observed increase in TBARS in Cu-amended samples.

Irradiation of cell suspensions after treatment with Co also caused a significant increase in TBARS levels which, in Gram-negative bacteria, can be attributed to the ability of Co to react with H2O2 generated inside the cell by a Fenton-type reaction within the periplasmic space [39]. The resulting hydroxyl radicals are able to subsequently attack membrane and cell wall components [39, 40].

Together, these results suggest that the toxicity of ROS generated during UV exposure is mediated by intracellular metals, particularly by the more biologically relevant Fe and Cu, whose homeostasis plays a crucial role in limiting the detrimental effects of oxidative damage during UV-B irradiation.

Role of Mn

Mn was the only metal that reduced protein carbonyl levels during UV exposure in all strains tested. High levels of intracellular Mn have also been shown to contribute to resistance of bacteria to UV-C radiation and ionizing radiation by mitigating protein oxidation [33-35]. The protective effect of Mn against protein carbonylation seems to stem from its ability to metallate mononuclear enzymes containing Fe–S clusters, thereby replacing Fe ions and preventing the formation of damaging hydroxyl radicals [41]. In addition, Mn is also able to form complexes with metabolites that can scavenge superoxide, H2O2 and hydroxyl radicals [13]. Accordingly, cell suspensions added of Mn showed significantly lower (< 0.05) ROS generation, compared with the unamended counterparts. Surprisingly, the protective effect of Mn against protein carbonylation and ROS generation was accompanied by a statistically significant reduction in survival during exposure to UV-B, compared with nonamended controls, and in three of the strains tested, a significant increase in DNA damage was observed. The reasons for such effects remain still unclear, in face of current knowledge. Recently, a transcriptomic study surveying UV-C effects on Deinococcus gobiensis reported an over two-fold reduction in the levels of the Mn transporter mntA upon irradiation, thus suggesting inhibition of Mn influx during UV-C exposure [42]. It has been suggested that, by replacing Fe ions in the Fe–S clusters of proteins, Mn can selectively protect the function of metabolic pathways (e.g. pentose phosphate pathway), which may or may not be favorable for survival depending on the stressful condition [43, 44]. Such replacement could have also affected the correct functioning of the biochemical pathways on which these proteins participate, resulting in a detrimental effect on survival during UV exposure.

The intracellular Mn/Fe concentration ratio is also crucial in determining the protective effect of manganese against ionizing radiation [14, 33]. For example, Neisseria gonorrhoeae accumulates Mn2+, but is sensitive to ionizing radiation due to its high requirement for Fe2+ [45]. It should be noted that the concentrations of Mn used in this study were chosen so that no significant effects on bacterial survival, activity and markers of oxidative stress were observed in the respective dark controls, to minimize the possible confounding effects of metal toxicity in the UV response. As a result, concentrations used were up to 25 times lower than those reported in similar studies [33-35], which could have hindered the detection of a potential protective role of Mn in UV-B-induced damage that might require higher concentrations of manganese. Further studies are required to elucidate the role of Mn in photooxidative stress in bacteria.

Experimental limitations on the determination of metal oxidation status

The oxidation status of transition metals is important in determining their biological effects. The cycling between oxidation states is involved in oxidative stress, as in the case of Cu and Fe. When Cu2+ enters the cell, it is readily reduced to the toxic Cu+ form which establishes complexes with thiol groups and certain amino acid residues, such as cysteine or ascorbic acid [46]. In Escherichia coli, the CueO protein in the periplasm converts Cu+ into the less permeable and thus less toxic Cu2+ [47]. Metal iron, on the other hand, can be readily acquired by bacteria through the reduction of insoluble Fe3+ to soluble and potentially more toxic Fe2+, followed by transport of Fe2+ to the cytoplasm [48, 49].

Cycling between oxidation states is also involved in the protective effect of Mn against oxidative stress. Quenching of the superoxide radical and peroxyl radical species by Mn2+ results in the formation of Mn3+ species, which are stabilized by coordination ligands such as pyrophosphate or carboxylic acids, thus lowering the redox potential of the Mn2+–Mn3+ couple [50].

However, the ability of metal ions to undergo redox cycling poses some complications in terms of experimental work. For example, while free Cu2+ ions are stable in air-exposed neutral aqueous solutions, free Cu+ can only be maintained at very acidic pH or in complexed forms [51]. In the case of iron, the presence of oxygen at a pH of ≥7, readily converts Fe2+ into almost completely insoluble Fe3+ [52].

Another difficulty in the study of the interaction between cells and metals is the ability of metals to form complexes with biological molecules, which compromises the determination of the actual concentration of free, bioavailable metal in a biological system [51]. Accordingly, using a complex growth medium like TGY it is likely that some of the metal added to the bacterial cultures could have complexed with the components of the medium and it is therefore virtually impossible to predict the metallic species present in the system. However, it is unlikely that in natural aquatic systems these metals are freely available in solution in their cationic form, which makes our experimental conditions more similar to what probably occurs in the environment, than using, for instance, solution buffers with low metal-binding properties.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Metal amendment generally acted synergistically with UV-B exposure to promote bacterial inactivation. Fe and Cu amendment exacerbated oxidative damage during UV-B exposure, highlighting the importance of regulating the availability of these metals in the cell in response to irradiation. Mn attenuated the UV-induced formation of protein carbonyls but had a negative impact on survival, suggesting that displaced Fe ions might promote oxidative damage in other targets of the cell. Furthermore, the detection of some differences in the responses of Gram-negative and Gram-positive bacteria to the synergistic effects of metal amendment and irradiation might suggest that the presence of metals in the environment could influence the effects of UV exposure on bacterial community composition.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Acknowledgments are due to Prof. Dr. Amparo Faustino (Chemistry Department, University of Aveiro) for carefully reviewing the preliminary version of the manuscript and providing access to the spectrofluorimeter. We also thank the anonymous reviewers and editor for their helpful criticism. Financial support for this work was provided by CESAM (Centre for Environmental and Marine Studies, University of Aveiro) and the Portuguese Foundation for Science and Technology (FCT) in the form of a PhD grant to A.L. Santos (SFRH/BD/40160/2007) and a post-Doctoral grant to I. Henriques (SFRH/BPD/63487/2009).

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  6. Discussion
  7. Conclusions
  8. Acknowledgements
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