Effect of Coat Layers in Bacillus Subtilis Spores Resistance to PhotoCatalytic Inactivation

Different water treatment processes (physical and chemical) exist to obtain safe water for human or food industry supply. The advanced oxidation technologies are rising as a new alternative to eliminate undesirable chemicals and waterborne diseases. In this work, we analyze the power of the photo-assisted Fenton process using Fe(II)/H2O2 and UV radiation (365 nm) to inactivate Bacillus subtilis spores, considered among the most resistant biological structures known. Different concentrations of Fe(II), H2O2 and UV radiation (365 nm) were used to inactivate wt and some coat spore mutants of B. subtilis. Wt spores of B. subtilis were inactivated after 60 min using this process. In general, all defective coat mutants were more sensitive than the wt spores and, particularly, the double mutant was 10 folds more sensitive than others being inactivated during the first 10 minutes using soft reaction conditions. Presence of Fe(II) ions was found essential for spore inactivating process and, for those spores inactivated using the Fe(II)/H2O2 under UV radiation process, it is suggested that coat structures are important to their resistance to the treatment process. The photo-assisted Fenton process using Fe(II), H2O2 and UV radiation (365 nm) can be used to inactivate any water


Introduction
The removal of microbial pathogens in water or food have been attempted by different emerging technologies, like Advanced Oxidation Technologies (AOTs).AOTs have been tested for their ability to inactivate several different pathogens [1,2].In particular, Fenton and Fenton-like processes are identified as effective alternatives for inactivating highly resistant microorganisms [3][4][5].Spores of various Bacillus species are metabolically dormant and extremely resistant to a variety of harsh treatments, including heat, radiation, and many toxic chemicals [6] (Table 1).This extreme resistance is the main reason that spores are major causative agents of food spoilage and food-borne disease and why spores of Bacillus anthracis are a potential biological warfare agent.Spore resistance is due to a variety of factors, being spore coat probably the most significant one against oxidant process.The coat is the outer layer of Bacillus species spores and consists primarily of protein.Particularly, the coat of Bacillus subtilis spores includes > 70 different individual proteins, cross-linked in most of the cases [7][8][9].Most of cross-linked proteins are components of the coat only, although a few coat proteins also have significant roles in coat assembly.The spore coats are organized into four distinct layers: a basement layer closely near to the cell membrane, the inner coat, outer coat and crust [10].
Assembly of the different coat layers depends on one major morphogenetic protein [10][11][12] to generate a semipermeable, generally allowing molecules < 5 kDa pass into interior layers [13] and protective barrier [14].Despite the precise protecting mechanism is unknown, coat proteins have been suggested containing enzymes (e.g., superoxide dismutase, catalase and CotA laccase), capable to assist in detoxification processes that contribute to spore resistance [13].The photo-assisted Fenton reaction have been tested in the past for the inactivation of highly resistant microorganisms [4,5] but few is known on the role that spore coat has on the efficiency of this AOT for spore inactivation.The goal of this research is using photo-assisted Fenton reaction to test the effect of different conditions of pH and concentrations of both Fe(II) and H 2 O 2 on wild type (wt) and coat mutant spores of Bacillus subtilis.The following issues are of particular interest for our research: (i) whether the reagent acts in the spore core or on some more external spore layer; and (ii) what spore features are important in protection against this inactivation process.were ACS grade and used as received without further purification.
Bacillus strains used: The B. subtilis wild-type strain (wt) used was PS533 [15], a derivative of the laboratory 168 strain, which carries plasmid pUB110, which encodes resistance to kanamycin (10 g mL -1 ).All other strains used were isogenic with strain PS533 but lacked plasmid pUB110 and were (i) PS3328 (cotE) [16], which has the majority of the gene encoding the spore coat morphogenetic protein CotE replaced with a tetracycline resistance (Tc r ; 10 g mL -1 ) cassette; (ii) PS4149 (gerE) [16] has the majority of the coding sequence of the gerE gene replaced by a spectinomycin resistance cassette (Sp r ; 100 g mL -1 ) (iii) PS4150 (cotE gerE) [13], which has the cotE mutation from PS3328 and also has the majority of the coding sequence of the gerE gene replaced by a spectinomycin resistance cassette (Sp r ; 100 g mL -1 ).

Preparation and purification of spores
B. subtilis spores were prepared as described elsewhere in literature [17,18].Briefly, the fresh culture of B. subtilis in a 2xYT was seeded on 250 mL 2xSG broth.The culture was incubated at 37 o C with mechanical shaking for 5 days, monitoring the sample under the microscope until the cells became spores.At the end of this time period, the culture was centrifuged at 10,000 rpm to collect the spores.The supernatant was discarded and the spores were re-suspended in cold water.
The spores were washed and sonicated until no vegetative cells or debris were observed under the microscope.Spores were finally suspended in 0.01 M phosphate buffer (pH 7.0) and the resulting stock had a spore density of 10 9 spore mL -1 .Spore number was counted by cell viability and the stock was refrigerated at 4 °C until used for experiments.

Spore inactivation experiments
All experiments were performed in sterilized distilled-deionized water (DI).Experiments designed to characterize the inactivation kinetics of B. subtilis spores wild strain and mutants with UV radiation were conducted in a bench-scale semi-batch reactor using a UV-A lamp photo reactor made of two low-pressure UV lamps (λ max = 365 nm, 15 W, GE F1578/BLB) with an OF-365AUV (black) Filter from Spectroline1 (cutting wavelength 365 nm) were used as a radiation source.
Semi batch reactors consisted on 25 mL glass bakers that contained 10 mL of spore suspension (10 9 spores) and were exposed to UV-A radiation (total radiation input 1.5 mW cm -2 ).Radiation intensity in the photo reactor was measured with a pyranometer (Solar light, Mod.PMA2141).
During the experiment, the spore suspensions were thoroughly mixed using a conventional stir plate and a magnetic bar [19].
Three different Fe(II) concentrations (0.0, 1.0 and 5 mM) were tested using the same initial concentration of hydrogen peroxide (100 mM) for wt and four different concentrations for the spore mutants (0.0, 1.0, 10.0 and 100 mM).For all experiments, the pH of the suspension was adjusted to 3.0 using 0.1 M H 2 SO 4 according the efficiency of previous reports [20a,24a], then the necessary amount of Fe(II) was added to the spore suspension in order to obtain the desired catalyst concentration.The solution was homogeneously mixed for 1 min in the dark and an initial (t 0 ) sample was withdrawn at this moment and analyzed for spore viability.Then hydrogen peroxide was added to the suspension and the reaction mixture was immediately irradiated.The inactivation experiment was considered as started at the precise instant that irradiation was initiated as previously reported [20,21].In all tests, 100 μL samples were obtained at every 5 min during the first 15 min of reaction time, then every 15 min during the first hour and finally every 30 min for the last hour.Once taken, all samples were diluted up to 10 7 times using 0.85% sodium chloride solution at pH 7.0.In between each dilution step, the bacterial suspensions were mixed using a vortex to ensure de homogeneity and 10 µL of each dilution was spotted on 2xYT agar.Colonies were visually identified and counted after overnight incubation at 37 o C in a microbiological incubator (Isotemp, Fisher Sci).During spore inactivation process, the DPA released was monitored by measuring the OD 270 of the supernatant fluid as reported previously [22].Briefly, heat-activated spore suspensions (OD 600 1.5) were cooled and incubated at 40 °C in 25 mM sodium phosphate buffer (pH 7.0).At different times (0, 5, 10, 15, 30, 45, 60 min), aliquots of 1 mL, were centrifuged in a microcentrifuge (13,200 rpm, 2 min), and the spore pellet was washed twice with 1 mL of distilled water (DDW) and suspended in 1 mL of DDW.Each spore suspension was boiled for 30 min, cooled on ice for 5 min, centrifuging in a microcentrifuge (13, 200 rpm, 2 min).Then, the DPA amount was determined by mixing 0.8 mL of sample plus 0.2 mL of the developing reagent [C 2 H 3 NaO 2 (0.05M pH 4.6), Cysteine (1 g/L), FeSO 4 (0.68 g/L), (NH 4 ) 2 SO 4 (0.32 g/L)] and measuring the absorbance at 270 nm (OD 270 ) as described previously [22].

Wild type spores
Similar to previous experiments [20a] was found that at acid conditions (pH 3) produce better results on mortality rate for wt spores (Figure 1) (experimental data for assays at pH 7 or 9 is found in supplementary material).This result can be explained by the improved solubility of (Figure 1).The spore inactivation using photo-assisted Fenton is in agreement with previous results reported by Bandala et al. [20] and Corona-Vasquez et al. [24] where they also mention this reaction produce high amount of ROS when pH is acid [14].

Spore mutants
Contrary to wt spores, spore mutants lacking ger E gene (4149), cot E gene (3328) and double mutant ger E/cot E -(4150) were more sensitive to lower concentrations of the photo-assisted Fenton reagents (1 mM Fe(II) and H 2 O 2 , and UV radiation) (Figure 2).The sensitivity of the mutants is due to lacking spore coat as stablish previously by Young and Setlow [23].This results means that spore coat helps to protect spores against many chemical agents as previously informed [25].Indeed, B. subtilis spores with defective coats are 10-to 100-fold times more sensitive to many oxidizing agents than intact spores [26,30].It has been suggested that the oxidizing agents used to inactivate spores cause damage to its outer layers and, perhaps, the cell membrane.However, the precise nature of the inner membrane damage caused by oxidants is not well known, although it has been demonstrated that it is not related with the oxidation of unsaturated fatty acids at least in wt spores [26].However, the lack of spore coats, permit oxidants compound is release, a compound present in high levels chelated with divalent cations in the spore core and whose loss accompanies spore inactivation by wet heat [29,32,34] or starting the spore germination.When DPA was analyzed during the photo-assisted Fenton reaction using any spore mutant or wt (Figure 3), the DPA was not detected (< 10%) from spores inactivated by any of the treatments tested except when these spore were only treated with H 2 O 2 100 mM.The absence of DPA in the most of the experiments can be due to the direct oxidation of this DPA by the generated ROS in Fenton reaction, but we have not an evidence of this, so further analysis is needed.In other hand, we observed that in the treatment with H 2 O 2 100 mM, the DPA release was constant across the time in wt without inactivation as reported previously [26].However, the question rising here is how is the spore viability and the DPA release, this analysis is showed in Figure 4    The thick proteinaceous coat of the spore protects the inner layers against many chemicals [23] although the coat apparently plays no role in spore resistance to other chemicals including acid, alkali and ethanol, and provides only a small component of spore resistance to hydrogen peroxide in wt spores [23,26].Treatment with photo-assisted Fenton reaction clearly affects the spore coat as the treatment oxidize this structure through the time, and spore resistance to photo-assisted Fenton reaction is dependent on the presence of an intact spore coat as shown by the high resistance in wt spores or the high sensitivity of cotE and gerE spores or the double mutant.The sensitivity of the mutants can be also due to a reduction on the hydrophobicity of the spores or presenting a higher pore size diameter on the coat structure increasing the ROS permeability reaching the inner membrane faster.So, the photo-assisted Fenton reaction inactivating process seems to be similar to the mechanism whereby spores are inactivated by other oxidizing agents, including chlorine dioxide, hypochlorite, Sterilox® and peroxynitrite where the inner permeability membrane of the spore is damage [23,27,28,33].

Conclusions
Spores of various Bacillus species are extremely resistant to a variety of severe treatments, including heat, radiation, and different toxic chemicals.This resistance is undesirable because can

H 2 O
2 to damage the inner membrane faster than the other strains meaning that the spore coat plays an important role in protection against oxidants.The sensitivity of these spore mutants can used for further analysis to demonstrate the oxidants effect on fatty acids and clarify the mechanisms of inner membrane damage.

Figure 3 .
Figure 3. DPA release from Bacillus subtilis wt and coat mutant spores below different treatments., Control(without treatment); , wt and UV radiation; , wt spores and Fe(II) 5mM; , wt and Fe(II) 5 mM and H 2 O 2 100 mM; , wt and H 2 O 2 ; , cot E and H 2 O 2 ; , ger E and H 2 O 2 ; , ger E/cot E and H 2 O 2 .

Pulsed electric field (kV cm -1 )
[23]wt spores are very resistant to H 2 O 2 due to spore coats as previously by [20a] in similar way reported by chlorine reagents as stablish by Young and Setlow[23]being important the integrity of the inner membrane of the spore to can inactivate the spores when it is compromised.In our assays of spore inactivation, the Zepp et al, in 1992lable to generate more hydroxyl radicals than in other pH values as seen in other previous work using the same system to inactivate helminthes eggs [20a] and this low pH no affect the hydroxyl radical production as stated byZepp et al, in 1992, where they reported that hydroxyl radicals are well produced at pH ranging from 3 to 8 using Fe(II) or Fe(III) plus H 2 O 2 , and can be used this reaction to treat contaminated water (Zepp, et al 25a).It was interesting to see that the photo-assisted Fenton reaction with the mix of the three factors (Fe(II), H 2 O 2 and UV radiation) showed better efficiency to inactivate spores than just the combination of only two components of them.It is known that Fe(II) with O 2 and UV are capable to generate hydroxyl radicals or other reactive oxygen species (ROS) [22a].
[13] this figure can be noted that cotE mutant spores showed DPA release similar to wt but the percent of surviving cells decay during the time jointly with DPA release displaying sensitivity to H 2 O 2 by the coat defect.For gerE spore mutant, the DPA release start after five minutes of treatment, together with a reduction of the viability of these spores at the same time.However, as can see, this ger E mutant is less sensitive than the cotE spores, because gerE has more coats layers than cotE[13].The double spore mutant (gerE/cotE) shows a fast DPA release just adding the H 2 O 2 This DPA amount, looks constant during the treatment time and these spores are completely inactivated at 45 minutes.This results seems to be clearly because the spore coat layers are so defective that allow the action of