Dec 8, 2015 - sitivity to UV-C or affecting spore structure was evaluated and compared to resistance to continuous UV-C and moist heat, in order to reveal ...
Photochemistry and Photobiology, 2016, 92: 301–307
Inactivation by Pulsed Light of Bacillus subtilis Spores with Impaired Protection Factors a1,2, Ge re my Clair1,2 and Fre de ric Carlin*1,2 Julia Esbelin1,2, Sabine Malle 1 2
curite et Qualite des Produits d’Origine Ve ge tale, INRA, Avignon, France UMR408 Se curite et Qualite des Produits d’Origine Ve ge tale, Avignon Universite , Avignon, France UMR408 Se
Received 29 July 2015, accepted 8 December 2015, DOI: 10.1111/php.12568
ABSTRACT
systems such as the excision repair system (NER, encoded by uvr genes) (9). The universal homologous recombination (recA) has a central role in protection as repair system of DNA doublestrand breaks, interstrand cross-links and collapsed replication forks. These functions are essential for maintaining genomic stability (10). Coat proteins may be also implicated in UV resistance. The absence of CotA, a copper-dependent laccase that participates in the biosynthesis of a brown spore pigment, results in the production of nonpigmented spores less resistant to UV-A and UV-B compared to wild-type spores (11). In contrast, despite major roles in synthesis and morphogenesis of spore coat proteins, and in coat structures, spores lacking CotE or GerE are as resistant to 254 nm UV-C as wild-type parental strain, suggesting that spore coat layers are not major determinants of spore UV-C resistance (12–15). PL is expected to have photochemical effects quite similar to the ones of continuous UV-C, in particular on DNA. However, PL might also have still unknown effects (16) because of a potential localized heating of microorganisms, of the high energy in each pulse of light (more than 1000 W cm 2 with PL, 1 9 10 3 W cm 2 with low pressure mercury lamps), or a wide spectrum ranging from UV-C to infrared radiation (IR). Consequently the PL resistance of spores of strain B. subtilis 168 and of strains with mutations increasing their sensitivity to UV-C or affecting their structure was evaluated. The same spores were also tested for their resistance to UV-C and moist heat. We assumed that differences in the relative importance of the mutations in resistance to PL, UV-C and moist heat may reveal original mechanism(s) of inactivation by PL.
The resistance to pulsed light (PL) of spores of Bacillus subtilis strain 168 and of strains with mutations increasing sensitivity to UV-C or affecting spore structure was evaluated and compared to resistance to continuous UV-C and moist heat, in order to reveal original mechanisms of inactivation by PL. Spores of B. subtilis strain 168 (1A1) and eight mutant strains (sspA, sspB, sspAB, cotA, gerE, cotE, uvrA and recA) were exposed to PL (up to 1.77 J cm 2), continuous UV-C (up to 147 mJ cm 2) and moist heat at 90°C. Spores of the strains lacking proteins linked to coat formation or structure (cotA, gerE and cotE) were markedly more sensitive to PL than 1A1, while their sensitivity to continuous UV-C or to moist heat was similar to the one of strain 1A1. Coat proteins had a major contribution to the resistance of B. subtilis spores to PL irradiation characterized by short-time and high-energy pulses of white light in the wavelengths 200–1100 nm. In contrast the role of coat proteins to UV-C or to moist heat resistance was marginal or null.
INTRODUCTION Pulsed light (PL) consists of short intense light flashes with wavelengths between 200 nm and 1100 nm generated by xenon lamps or other inert gases. UV-C radiations play a major role in decontamination by PL and the technology is therefore also named “pulsed UV light.” PL is increasingly applied to food contact materials or to liquid foods for the inactivation of microorganisms, including spore-forming bacteria (1–4). Microbiological inactivation by PL has been attributed to high UV emission causing DNA damages and preventing cell proliferation (2,5). Mechanisms of microorganism resistance to UV treatments are well documented, in particular on the model bacterium Bacillus subtilis, and involve prevention or repair of UV-C–induced lesions. For instance the small, acid-soluble proteins (SASPs) of the a/b type participate to the resistance of dormant spores to UV light. These SASPs bind to double-stranded DNA and prevent chemical and enzymatic cleavage caused by UV-C exposition (6,7). The DNA of dormant spores irradiated with UV-C accumulates spore photoproduct (SP), a thymine dimer (8). In B. subtilis, this potentially lethal lesion in DNA is eliminated by
MATERIALS AND METHODS Bacillus subtilis strains used and preparation of spores. Bacillus subtilis strain 1A1, wild-type background initially B. subtilis 168 and derived mutants strains (Table 1) were obtained from the Bacillus Genetic Stock center (Columbus, Ohio). The strains were routinely cultivated on LuriaBertani (LB) agar supplemented with the antibiotic concentrations recommended by BGSC to ensure strain purity (17). Then, one colony was plated on double-strength Schaeffer sporulation (2xSG) agar without antibiotics (18) and placed for 7 days at 30°C before spore harvest. At least three independent spore suspensions of each strain were prepared and the resistance of each suspension to PL, UV-C and moist heat was tested. Spores were purified by repeated washing as previously described (19). Spore preparations were checked to be free ( 0.1). The cotA and cotE mutant strains tended to be more sensitive to moist heat than the 1A1 strain, but differences in resistance parameters d and R60 were not significant (P > 0.05). Influence of B. subtilis spore decoating on PL sensitivity Spores of B. subtilis strain 168 (1A1) were chemically treated to remove coat proteins. The sensitivity to PL and UV-C was compared to the one of intact 1A1 spores and of the coat defective mutant cotE (Fig. 4). Although the inactivation of decoated spores was overall less pronounced than the inactivation of spores of the mutant strain cotE, inactivation at the maximal tested fluence was not significantly different (P > 0.1). Moreover and similarly to the cotE strain, decoated spores were markedly more sensitive to PL than intact 1A1 spores; Rmax values were significantly different (P < 0.01) (Fig. 4, Table 2). In contrast, there was no significant difference between intact and decoated B. subtilis 1A1 spores in continuous UV-C inactivation.
Effect of defect in proteins involved in coat formation and structure
Importance of UV-C in the sensitivity of strain cotE to PL
Spores of cotE, gerE and cotA mutant strains (1S105, 1L45 and 1A184 respectively) with defects in proteins involved in coat formation and structure were tested for their resistance to PL. The
UV-C removal from the emitted PL resulted in a tremendous reduction in sensitivity of spores of the mutant strain cotE to PL (Fig. 5).
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Figure 1. Inactivation rates of Bacillus subtilis 1A1 (●) and B. subtilis sspA (□), sspB (Δ) and sspAB (○) mutant strains exposed to pulsed light (A), continuous UV-C (B) and moist heat at 90°C (C). Each experimental point is the mean of log10 reduction obtained with n = 3 independent spore preparations (n = 4 for 1A1). Bars represent standard error (SE). UV-C fluences are expressed as a.u. (arbitrary units) and are comparable, whether delivered by PL or by the continuous UV-C source.
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Table 2. Parameters of resistance to pulsed light, continuous UV-C and moist heat at 90°C of spores of Bacillus subtilis 168, of derivative mutant strains and of WT decoated spores. Parameters of resistance to:‡ Strains 1A1 sspA (1S109) sspB (1S110) sspAB (1S111) cotA (1A184) gerE (1L45) cotE (1S105) uvrA (1A345) recA (1A43) 1A1, decoated
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Moist heat at 90°C d (min)†
Moist heat at 90°C R60 (log10)†
1.59 0.62**§ 1.05 6.0*** 3.45 4.15* 5.14** 4.21* 5.09** 5.02**
0.82 0.38*** 0.65 6.0** 3.96 4.68 5.20* 3.84 5.40* 4.90
34.8 9.4* 19.1 2.36** 29.2 37.1 24.9 39.0 43.7 nt
1.97 5.72** 4.20* >5.0*** 2.97 2.16 3.36 1.77 1.67 nt
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F3: fluence to 3 log10 reduction (a.u., arbitrary units); Rmax: log10 reduction at the maximal tested fluence; d: time to first decimal reduction; R60: log10 reduction after 60 min; nt: not tested; ‡n ≥ 3. §Significant differences between 1A1 and other strains were indicated by ***P < 0.001, **P < 0.01 or *P < 0.05.
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Figure 2. Inactivation rates of Bacillus subtilis 1A1 (●) and B. subtilis uvrA (Δ) and recA (○) mutant strains exposed to pulsed light (A), continuous UV-C (B) and moist heat at 90°C (C). Each experimental point is the mean of log10 reduction obtained with n = 3 independent spore preparations (n = 4 for 1A1). Bars represent SE. UV-C fluences are expressed as a.u. (arbitrary units) and are comparable, whether delivered by PL or by the continuous UV-C source.
DISCUSSION Proteins involved in bacterial spore protection were investigated for their contribution to the resistance to pulsed light, an emerging decontamination technology based on light emission. Because of a high proportion of UV-C in PL and of its major role in PL inactivation (1,3), factors implicated in UV-C and PL resistance were expected to be similar. In a previous work the inactivation of spores of B. subtilis, B. atrophaeus and B. cereus sensu lato by comparable UV-C fluences, whether delivered by PL or a continuous low pressure UV source, was similar (1). This was not the case with the 1A1 strain in the present work. Consequently the importance of a protein defect was evaluated, for each physical treatment, by the significance of the relative difference between 1A1 and the mutant strain rather by compared sensitivity of each tested strain to PL and UV-C. UV-C radiation causes several types of damages to DNA and the formation of the spore photoproduct in bacterial spores (8,23).
Some of these damages are prevented by the presence of SASPs, which affects the production of detrimental photoproducts in spores exposed to UV (24). The spores of derivative strains with deletion in SASP-coding genes were much more sensitive to PL than 1A1, as they were to UV-C here and in previous works (6,7,24) (Table 3). Systems allowing repair of damages to DNA are also implicated in resistance to continuous UV-C. Spores of B. subtilis strains mutated in the genes coding for the spore photoproduct lyase or the protein UvrA participating to the nucleotide excision repair reaction were reported to be more sensitive to UV-C (23,24). RecA has a central role in homologous recombination and is essential for repair of DNA double-strand breaks, interstrand cross-links and collapsed replication forks, and overall for maintenance of genome stability (10). In the present work, spores of the uvrA mutant strain exposed to PL were markedly more sensitive than spores of 1A1 strain, while their inactivation with continuous UV-C was not significantly different (Fig. 2,
Photochemistry and Photobiology, 2016, 92
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Figure 3. Inactivation rates of Bacillus subtilis 1A1 (●) and B. subtilis cotA (Δ), cotE (○) and gerE (□) mutant strains exposed to pulsed light (A), continuous UV-C (B) and moist heat at 90°C (C). Each experimental point is the mean of log10 reduction obtained with n = 3 independent spore preparations (n = 4 for 1A1). Bars represent SE. UV-C fluences are expressed as a.u. (arbitrary units) and are comparable, whether delivered by PL or by the continuous UV-C source.
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Figure 4. Inactivation rates of intact (●) and decoated (Δ) spores of Bacillus subtilis 1A1 and of B. subtilis cotE (○) mutant strain exposed to pulsed light (A) and continuous UV-C (B). Each experimental point is the mean of log10 reduction obtained with n = 3 independent spore preparations (n = 4 for 1A1). Bars represent SE. Inactivation curves of 1A1 and B. subtilis cotE are the same as the ones shown in Fig. 3A and B. UV-C fluences are expressed as a.u. (arbitrary units) and are comparable, whether delivered by PL or by the continuous UV-C source.
Table 3). A higher induction of DNA damages by PL, such as pyrimidine dimer formation requiring the involvement of NER, could therefore be considered. Spores of the recA mutant strain were markedly more sensitive than 1A1 strain when exposed to PL, and also to UV-C confirming previous observations (25,26). Some previous results suggest that PL inactivation is not the only consequence of UV-C emission (1). A photothermal effect is often mentioned as possibly involved in PL inactivation (1,3). The resistance to moist heat at 90°C was therefore also examined to detect whether PL inactivation patterns could be explained by differences in resistance to moist heat. SASP defective mutants have the same sensitivity pattern after exposure to UV-C, PL and moist heat, and no conclusion about the balance between photothermal and photochemical effect can be drawn from our
experiments. Mutations in repair mechanisms did not affect resistance to moist heat (Fig. 2, Table 3), as expected because moistheat killing is likely not caused by DNA damages (27,28). Consequently the differences in patterns of PL and continuous UV-C sensitivity, mainly observed in this work for mutant strain uvrA, are due for a major part to the different emission spectra of the two irradiation systems. Our experiments are not fully conclusive, but a large photothermal effect can be excluded and, if present, this effect will likely be small. Coats are implicated in spore protection against chemicals, but likely play a minor role in resistance to moist heat (29,30), and our results tend to confirm this (Fig. 3, Table 2). Whatever the underlying mechanism of inactivation by PL could be (photochemical or photothermal effect), proteins participating to the
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mutant strain. The importance of the non-UV-C part of the PL spectrum in this spore coat protective effect was also studied. Without UV-C, inactivation of cotE spores by PL is tremendously reduced, as it is the case for the 1A1 strain (data not shown) and B. subtilis DSM402 (Fig. 5) (1). This low, but observable, inactivation may be a contribution of UV-A to UVB, or IR radiation, which also shows sporicidal effects (23,34). However, lethal effects of IR on spores are commonly associated with temperature greater than 100°C (34). In the present work local temperature increase was only by a few °C (data not shown), which is consistent with treatments at doses much higher than the ones applied here (35). UV-C radiation has by far the strongest inactivation effect. The role of coat proteins in PL resistance may have revealed an original mechanism of spore inactivation due to the specific way of delivery of these radiations (short-time pulses with high energy).
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Figure 5. Inactivation rates of Bacillus subtilis cotE mutant strain exposed to a full spectrum of pulsed light (○) (wavelengths in the range 200–1100 nm) and UV-C–free pulsed light (□) (wavelengths in the range 280–1100 nm). Bars represent SE.
Acknowledgements—This work was supported by OSEO (now Banque publique d’investissement, bpifrance) (Maisons-Alfort, France) under contract Beata Lux number 10810001W coordinated by CLARANOR SA (Avignon, France), the company supplying the technology and the equipment used in this work. Thanks are due to the Bacillus Genetic Stock Center (Columbus, Ohio) for supply of the strains tested in this work.
Table 3. Role of some factors in resistance of Bacillus subtilis spores to moist heat, UV radiations and pulsed light.
SUPPORTING INFORMATION
Fluence (J.cm-2)
Effect on resistance of spore components Treatment Pulsed light UV radiations Moist heat
a/b type SASP
Repair
Spore coats
?/++* ++/++ ++/++
?/++ ++/+† /
?/++ / /‡
*Left symbol: importance as proposed by Nicholson et al. (30); right symbol: importance evaluated in this work. Symbols as proposed by Nicholson et al. (30): ++, major importance; +, some importance but not a major factor; , minor importance but has some effect; , no effect.; ?, no data available. †Only recA mutation led to a significant effect. ‡A tendency to higher heat sensitivity caused by cotE mutation.
formation of, or composing coats, are highly implicated in resistance to PL, while their implication in resistance to continuous UV-C or to moist heat was low (for mutant strain cotE, and to some extent for mutant strain gerE, for example) or null (Fig. 3, Table 2). In particular the cotE mutant strain that lacks the outer coat structure (31,32) is among one of the most affected tested strains by PL. This suggests that the intact coat structure may be necessary for full PL resistance. This hypothesis is strengthened by the high PL sensitivity of chemically decoated spores (Fig. 4). The gerE strain has also modified coat structure (14). The GerE protein binds to DNA and activates genes coding for spore coat components (13), but it is also both a positive and negative regulator of many other genes (33). GerE is certainly involved in PL resistance, but the present results do not allow drawing any clear conclusion about its actual role. The cotA mutant strain could have been quite sensitive to PL because this strain lacks a pigment protective against UV-A and UV-B, also delivered with the used PL device (11). In our work the cotA mutant strain is marginally and not significantly affected in UV-C and PL resistance. CotA presence in outer coat is CotE dependent (31) and the lack of CotA cannot explain alone the higher sensitivity of the cotE
Additional Supporting Information may be found in the online version of this article: Figure S1. Emission spectrum of the flash lamp implemented in the PL equipment.
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