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Applied and Environmental Microbiology, December 2007, p. 7740-7743, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01079-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

TiO2 Photocatalysis Causes DNA Damage via Fenton Reaction-Generated Hydroxyl Radicals during the Recovery Period{triangledown}

Gaëtan Gogniat and Sam Dukan*

Laboratoire de Chimie Bactérienne, UPR 9043, Université de la Méditerranée, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France

Received 15 May 2007/ Accepted 1 October 2007


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ABSTRACT
 
Here, we show that resistance of Escherichia coli to TiO2 photocatalysis involves defenses against reactive oxygen species. Results support the idea that TiO2 photocatalysis generates damage which later becomes deleterious during recovery. We found this to be partly due to DNA attack via hydroxyl radicals generated by the Fenton reaction during recovery.


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INTRODUCTION
 
Studies in the past few years have revealed that classical disinfection by chlorine or ozonation can generate carcinogenic and mutagenic by-products, thereby boosting research into alternative methods, such as photocatalysis (2, 26, 28). This process is based on the ability of a semiconductive catalyst (TiO2) to kill bacteria upon illumination in aqueous solution (1, 12, 15, 16, 30). However, the basis for the bactericidal effect of photocatalysis is not well established.

Active TiO2 in anatase crystalline form behaves as a classical semiconductor. The bactericidal effect of photocatalysis with TiO2 could be due to the presence of reactive oxygen species (ROS), such as superoxide (O2·–), hydrogen peroxide (H2O2), and hydroxyl radical (HO·), generated either by illuminated TiO2 or by the illumination (mainly UV) of the cells. Most studies have concluded that HO·, directly generated by this process, is the main cause of the bactericidal effect of photocatalysis (5, 19).

To prevent the harmful effects of ROS generated during the normal course of aerobic metabolism, especially that of the extremely reactive HO· able to damage DNA (18), bacteria like Escherichia coli are equipped with defenses, including catalases (KatG and KatE) and superoxide dismutases (SodA, SodB, and SodC) (4, 17). These defenses decrease H2O2 and O2·– steady states and consequently limit the formation of HO·, for which no defense exists (22). Previous reports have shown that HO· is generated via a Fenton reaction (H2O2 + Fe2+ -> HO· + HO– + Fe3+) and that regulating iron uptake by the transcriptional repressor Fur (3, 7) permits maintenance of low-level HO· production.

Here, we aimed to investigate the resistance of E. coli to TiO2 photocatalysis. Cells were grown in Luria-Bertani broth at 37°C on a rotary shaker (160 rpm) to an absorbance at 600 nm of 0.5. We then washed the cells twice with sodium phosphate (0.05 mol/liter, pH 7, 4°C) and resuspended them in sodium phosphate (0.05 mol/liter, pH 7) solution to a concentration of 2 x 107 CFU/ml. As previously described (14), culture plates were illuminated from 310 nm to 800 nm with a xenon lamp in a Hanau Suntest system (AM1) at 550 W/m2 light intensity with a filter cutting off wavelengths below 310 nm. Stopped bacterial growth in the exponential phase followed by incubation in phosphate buffer causes starvation and induction of the RpoS regulon, involved in resistance towards many environmental stresses (7, 13). With this in mind, we used a mutant strain of E. coli to test whether the induction of the RpoS regulon protects cells against photocatalysis (Degussa P25, 20% rutile 80% anatase crystalline form; Degussa AG, Switzerland). As depicted in Fig. 1, we observed a drastic increase in sensitivity to photocatalysis in the rpoS::Tn10 mutant strain following just 20 min of illumination. This sensitivity was not observed with inactive TiO2 (Huntsman TR 92, 100% rutile crystalline form; Tioxide Europe Ltd., England), suggesting that light (UV) alone has no effect on bacterium culturability between strains.


Figure 1
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  		FIG. 1. Survival ratios of wild-type E. coli MG1655 (x) and rpoS::Tn10 ({blacksquare}), dps::kan (Figure 1), katE::Tn10 katG::Tn10 ({blacktriangleup}), and katE::Tn10 katG::Tn10 dps::kan ({Delta}) mutants with 60 min of illumination with active TiO2 (Degussa P25, 1 g/liter). Data points indicate the mean values and standard deviations of three or more independent experiments.

The ability of TiO2 photocatalysis to generate ROS led us to investigate whether RpoS-controlled functions protecting against ROS also participate in the RpoS-dependent protection against TiO2 photocatalysis. The catalases (HphI and HphII) and Dps protein, all involved in resistance to H2O2, are induced during starvation under RpoS control (13). While we observed no differences in terms of sensitivity with inactive TiO2 (data not shown), dps::kan, katE::Tn10 katG::Tn10, and dps::kan katE::Tn10 katG::Tn10 mutant strains showed high sensitivity to TiO2 photocatalysis (Fig. 1), indicating the involvement of Dps and catalases in resistance to the toxic effects of TiO2-mediated production of ROS.

Since RpoS-dependent resistance observed in TiO2 photocatalysis depends in turn on genes involved in the resistance to H2O2, we wished to test the involvement of other defenses against ROS on resistance to TiO2 photocatalysis. To this end, we tested the sensitivity of the {Delta}sodA sodB::MudIIPR3 mutant strain, deficient in cytosolic superoxide dismutases, and also that of the {Delta}fur mutant. As indicated in Fig. 2, we found dramatic sensitivity of both of these mutants to photocatalysis (active TiO2). Moreover, we detected no difference in culturability after illumination with inactive TiO2 (data not shown). The concomitant sensitivity of all of these mutants suggests the occurrence of a Fenton reaction, enhanced by iron overload in the presence of active TiO2. With this in mind, we wanted to test whether DNA damage could be detected after TiO2 photocatalysis. As depicted in Fig. 3A, we found a sensitivity in the {Delta}recA srl::Tn10 mutant similar to that of the wild type, incapable of SOS induction and homologous recombination, thus rendering it unable to repair DNA strand breaks (20). The dps::kan {Delta}recA srl::Tn10 mutant, however, showed a high sensitivity to TiO2 photocatalysis, suggesting a synergistic effect between dps and recA mutations on DNA, although part of this sensitivity was due to a light-only effect (UV) (Fig. 3B). Finally, we verified that the srl::Tn10 mutation had no effect on {Delta}recA srl::Tn10 or dps::kan {Delta}recA srl::Tn10 mutant sensitivity (data not shown). Taken together, these results provide evidence in favor of a Fenton reaction occurring, leading to the formation of a hydroxyl radical and consequently DNA damage.


Figure 2
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  		FIG. 2. Sensitivities of wild-type E. coli MG1655 (x) and {Delta}fur::kan ({square}), katE::Tn10 katG::Tn10 ({blacktriangleup}), and {Delta}sodA sodB::MudIIPR3 (*) mutants to TiO2 photocatalysis (Degussa P25, 1 g/liter). Data points indicate the mean values and standard deviations of three or more independent experiments.


Figure 3
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  		FIG. 3. Sensitivities of wild-type E. coli MG1655 (x) and dps::kan (Figure 3), {Delta}recA srl::Tn10 ({blacktriangledown}), and dps::kan {Delta}recA srl::Tn10 (Figure 3) mutants to (A) TiO2 photocatalysis (Degussa P25, 1 g/liter) and (B) light plus inactive TiO2 (Huntsman, 1 g/liter). Data points indicate the mean values and standard deviations of three or more independent experiments.

We have shown that resistance of E. coli to TiO2 photocatalysis is mediated largely by genes involved in ROS resistance. Interestingly, either an increase in ROS concentration when plating cells onto a solid growth medium or an exogenous supply of ROS may provoke stress via an imbalance between ROS concentration and the defense mechanisms (6). Since the 1950s, reports have shown that apparently dead cells could be reactivated on addition of ROS scavengers such as catalase or pyruvate to agar plates (8-11, 23-25, 27, 29). As a result of cumulative cellular damage, these so-called "injured cells" are in a transient state, reversible under appropriate conditions to enable resumed growth. We therefore decided to incubate in parallel the illuminated cells with active or inactive TiO2, with or without adding 20,000 U of catalase to the petri dish. As depicted in Fig. 4A, we observed no increase in the survival of the wild type or the katE::Tn10 katG::Tn10 and katE::Tn10 katG::Tn10 dps::kan mutants when plated with catalase. Interestingly, however, the survival rates of the {Delta}fur and rpoS::Tn10 mutants increased more than 1,000-fold when plated with catalase, and these survival rates were identical to that of the wild-type strain during the first 20 or 30 min of illumination (Fig. 4B). Finally, the survival rates of dps::kan and dps::kan {Delta}recA srl::Tn10 mutants increased up to 10,000 times when plated with catalase, and these were the same (or similar) survival rates as that of the wild-type strain (Fig. 4C). Interestingly, adding catalase to the plate restored the light-alone effect on the dps::kan {Delta}recA srl::Tn10 mutant (Fig. 4D). These results demonstrate that TiO2 photocatalysis generates damage that becomes deleterious during recovery from TiO2 photocatalytic stress, especially for mutants sensitive to ROS species.


Figure 4
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  		FIG. 4. Sensitivities after incubation either with (dashed lines) or without (plain lines) 20,000 U catalase. Sensitivities are shown for (A) E. coli MG1655 (x) and katE::Tn10 katG::Tn10 ({blacktriangleup}) and katE::Tn10 katG::Tn10 dps::kan ({Delta}) mutants, (B) {Delta}fur::kan ({square}) and rpoS::Tn10 ({blacksquare}) mutants, and (C) dps::kan (Figure 4) and dps::kan {Delta}recA srl::Tn10 (Figure 4) mutants to TiO2 photocatalysis (Degussa P25, 1 g/liter) or, (D) with inactive TiO2 (Huntsman, 1 g/liter), the dps::kan {Delta}recA srl::Tn10 mutant ({circ}), the dps::kan mutant with catalase (Figure 4), and the dps::kan {Delta}recA srl::Tn10 mutant with catalase (*). Data points indicate the mean values and standard deviations of three or more independent experiments.


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ACKNOWLEDGMENTS
 
We thank M. Chippaux, D. Brynes, and P. Moreau, Laboratoire de Chimie Bactérienne, Marseille, France, for their helpful comments on the manuscript and D. Touati for the generous gifts of strains.

This work was supported by ACI Jeunes Chercheurs.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Chimie Bactérienne, UPR 9043, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Phone: 33-(0)491 164 459. Fax: 33-(0)491 718 914. E-mail: sdukan{at}ibsm.cnrs-mrs.fr Back

{triangledown} Published ahead of print on 12 October 2007. Back


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Applied and Environmental Microbiology, December 2007, p. 7740-7743, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01079-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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