Yuting Huang, Liyang Wang, Xue Zhang, Nan Su, Heping Li, Yoshimitsu Oda, Xinhui Xing,6,7,8,*

1 Key Laboratory for Industrial Biocatalysis, Ministry of Education of China, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China

2 Biobreeding Research Center, Wuxi Research Institute of Applied Technologies, Tsinghua University, Wuxi 214072, China

3 Department of Entomology, College of Plant Protection, China Agricultural University, Beijing 100193, China

4 Department of Engineering Physics, Tsinghua University, Beijing 100084, China

5 Institute of Life and Environmental Sciences, Osaka Shin-Ai College, Tsurumi, Tsurumi-ku, Osaka 5380052, Japan

6 Centre for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China

7 Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Shenzhen 518055, China

8 Institute of Biomedical Health Technology and Engineering, Shenzhen Bay Laboratory, Shenzhen 518055, China

Keywords:ARTP mutagenesis umu-Microplate test Biological engineering Cell engineering Biotechnology

A B S T R A C T Mutagenesis is an important technique for microbial mutation breeding.As the source of mutations,DNA damage extent is a key indicator for the effectiveness of mutagenesis. Therefore, a rapid and easy DNA damage quantification method is required for the comparison of mutagenesis effects and development of mutagenesis tools. Here, we used the umu-microplate test system to quantitatively compare the DNA damage strength caused by atmospheric and room-temperature plasma(ARTP)and other traditional mutagenesis methods including: ultraviolet radiation (UV), diethyl sulfate (DES) and 4-nitroquinoline-1-oxide (4-NQO). The test strain of Salmonella typhimurium TA1535/pSK1002 was used to monitor the time-course profile of β-galactosidase activity induced by DNA damage caused by different mutagenesis methods using a microplate reader. The umu-microplate test results showed that ARTP caused higher extent of DNA damage than UV and chemical mutagens, which agrees well with the result obtained by SOS-FACS-based quantification method as reported previously. This umu-microplate test is accessible for broad researchers who are lack of the expensive FACS instruments and allows the quick quantitative evaluation of DNA damage among living cells for different mutagenesis methods in the study of the microbial mutation breeding.

1. Introduction

Microbial mutation breeding is an important technique which has been broadly employed for microbial improvement,especially in the fields of biotechnology, biomanufacturing, food fermentation and environmental protection [1]. Mutagenesis tools, including chemical mutagens, physical or biological approaches are used to increase mutation rate and accelerate the subsequent evolutionary process to obtain mutant strains with desirable phenotypes. Mutagenesis is also an alternative choice in the fields where only non-GMOs (genetically modified organisms) are allowed to be used [2,3]. Given the ability or potential to generate random mutant libraries, mutagenesis methods have also been proved to be an effective approach in exploring of unknown molecular functions of microbes by combining omics analysis[4].Therefore, the development of new mutagenesis tools is still of importance.

DNA damages are indispensable for mutation breeding [5], the extent of which is a crucial indicator of the effectiveness of mutagenesis. Therefore, a feasible quantification method of DNA damage is required for a comparative evaluation of different mutation methods,since the DNA damage strength will be responsible for the mutation rate for selecting the interested phenotypes.A number of bioassay methods have been developed to detect the genotoxicity caused by various environment mutagens and carcinogens for environmental protection [6-9]. Theumutest is widely used so far among all the methods due to its simple, rapid and economic features in practical applications [10-14]. In this method, the reporter genelacZwas fused to theumuCoperon,which is regulated by the SOS response induced by DNA damages.The plasmid pSK1002 carrying the SOS inducedlacZcircuit was transformed into theSalmonella typhimuriumstrainTA1535 and was used in theumutest system [15-18]. The activity of βgalactosidase is quantitatively measured to evaluate the genotoxicity of mutagens using colorimetric microplate test or fluorescence-activated cell sorting (FACS) method [8,19].

As a mutagenesis tool, Atmospheric and Room-Temperature Plasma (ARTP) mutagenesis developed by our group has attracted a great of attentions in the recent years[20,21].To date,hundreds of microorganisms were successfully mutated by ARTP mutagenesis [22-25]. It featured not only in wide application and high efficiency of mutation diversity, but also in its safety and ease-of-use[20,21]. In a previous study, we developed aumu-FACS DNA damage quantification method, in which by using a fluorescent substrate of β-galactosidase and PI reagent, the activity of β-galactosidase induced in the model strain and cell viability after mutagenesis treatment was determined, respectively [19].However, theumu-FACS analysis requires the expensive FACS instrument which is not broadly accessible in many institutions and companies, and its operation needs an user with technical skills, such that limits its wide utilization in microbial breeding research and industry [26-28]. Therefore, a rapid and feasible DNA damage quantification method is also desired.

Here we used the rapid and simpleumu-microplate test to develop a quantitative evaluation method for the DNA damage extent upon the treatments by different mutagenesis methods and correlate with that reported by theumu-FACS evaluation method established previously for mutation breeding. The DNA damages caused by ARTP, UV, and chemical mutagens (DES and 4-NQO) were analyzed and compared.

2. Materials and Methods

2.1. Strains and culture

The strainS. typhimuriumTA1535/pSK1002 (hisG46, rfa, uvrB)was used forumu-microplate test in this study [8]. The βgalactosidase was encoded by theumuC-lacZfused gene carried on plasmid pSK1002 and its expression was induced by SOS response caused by DNA damages.S. typhimuriumTA1535/pSK1002 was kindly supplied by Dr. Y. Oda of Osaka Shin-Ai College, which is the model strain forumutest.

S.typhimuriumTA1535/pSK1002 was grown in 10 ml TGA medium (tryptone 10 g·L-1, NaCl 5 g·L-1, HEPES 11.9 g·L-1, glucose 2 g·L-1; final pH adjusted to 7.0 ± 0.2) containing ampicillin(50 μg·ml-1) at 37 °C overnight with vigorous shaking. 1 ml of the overnight cultures (optical density at 600 nm (OD600) reached 2) were added into 9 ml TGA medium and was incubated at 37 °C for 1.5 h before mutagenesis treatment.

2.2. Chemical, ARTP and UV mutagenesis

For chemical mutagenesis, 4-nitroquinoline-1-oxide (4-NQO)and diethyl sulfate(DES)were obtained from Sinopharm Chemical Reagent Beijing,China.4-NQO and DES were dissolved in dimethyl sulfoxide(DMSO)and diluted in a 1:1 series respectively to appropriate concentrations.180 μl of each concentration and 70 μl of the cultures were mixed in a 96-well microplate.70 μl of TGA medium mixed with 180 μl H2O as blank control and 70 μl of the cultures mixed with 180 μl H2O as negative control were also added to the same microplate. The microplate was then cultured at 37 °C for 2 h.

DNA damage caused by ARTP and UV irradiation is a transient effect,without the need of long period reaction as for the chemical mutagenesis. Instead of the microplate process as mentioned above, 7 ml of the cultures were transferred in the 100 ml flask with 20 ml TGA medium,then cultured at 37°C for 2 h.An aliquot of growing cells was exposed to UV or ARTP irradiation at different dosages as described below.

UV irradiation was generated by a germicidal lamp (30 W,253.7 nm). A distance of 30 cm was set up between the UV lamp and the bacterial sample during mutagenesis,and the UV radiation energy was set up at 90 μW·m-2. ARTP mutagenesis was performed with the ARTP Mutation Breeding System [21], jointly developed by our group and Wuxi TMAXTREE Biotechnology Co.Ltd (Wuxi, Jiangsu, China). Helium was used as the working gas at a flow rate of 10 slpm (standard liters per minute). The power input of the ARTP mutation system at a driving frequency of 13.56 MHz was 100 W. The distance between the plasma torch nozzle and the sample was 2 mm and the plasma jet temperature was controlled at 25-35 °C.

2.3. SOS/umu assay

During the mutagenesis treatment, the second microplate was preincubated at 37°C with 270 μl TGA medium in each well corresponding to the mutagenesis samples. 30 μl of the treated culture from either the first microplate with chemical mutagens or samples of ARTP/UV irradiation was added to the second microplate.After incubation at 37°C for 2 h with 200 r·min-1shaking,the bacterial growth was measured using microplate reader for the OD600.Subsequently, 30 μl of the culture was transformed to the third microplate, which was preincubated at 37 °C and containing 120 μl B-buffer (3.87 mol·L-1Na2HPO4·12H2O, 0.017 mol·L-1NaH2PO4·12H2O, 0.01 mol·L-1KCl, 0.002 mol·L-1MgSO4·7H2O,0.003 mol·L-1NaC12H25SO4and 0.03 mol·L-1β-mercaptoethanol)in each well.30 μl ofO-nitrophenyl-β-D-galactopyranoside(ONPG)(4.5 mg·ml-1) was added immediately into each well and incubated with shaking at 28 °C for 30 min. Subsequently, 120 μl of the stopping solution (1 mol·L-1Na2CO3) was added and mixed with the cultures before measuring the β-galactosidase activity at 420 nm using the microplate reader. The schematic illustration of theumu-microplate test was shown in Fig. 1. The SOS induction factor was used to quantify the DNA damage extent caused by different mutagenesis treatments as described previously with some modifications [29] and was calculated using the following equations:

Fig. 1. Schematic of umu-microplate test protocol.

A600,T: The optical absorbance of treatment sample at 600 nm

A420,T: The optical absorbance of treatment sample at 420 nm

A600,N: The optical absorbance of negative control at 600 nm

A420,N: The optical absorbance of negative control at 420 nm

A600,B: The optical absorbance of blank control at 600 nm

A420,B: The optical absorbance of blank control at 420 nm

G: The growth factor

IR: The relative SOS induction rate

E/Biomass: The β-galactosidase activity of unit cell density

The results were calculated as the mean values of triplicate wells from two or three independent experiments and the error rate of each data was lower than 3.5%. A significant higherIRover the untreated bacteria sample represents as positive SOS induction.

3. Results and Discussion

3.1. Comparison of DNA damage by different mutagenesis methods

The growth factor (G) indicates the ability of testing bacterial strain to survive and maintain the metabolic activity for cell growth after mutagenesis treatment, and the relative SOS induction rate (IR) reflects the ability of the living bacterial strain to be induced to expressumuCdue to the DNA damage or genotoxicity by the mutagenesis treatment. The values ofGandIRfor each mutagenesis methods were calculated as listed in Table 1. TheIRvalues of 4-NQO treatment increased with the increase of the 4-NQO concentration,while theGvalues showed opposite trend.This result implied that the 4-NQO mutagen inhibited the bacterial growth,and damaged DNA which led to the induction of the microbial SOS response. The similar trends in the values ofGandIRfor other mutation sources could also be seen in the other groups. It was worth noting that the highestIRvalue of UV group reached 8.4, while the highestIRfor ARTP mutagenesis treatment was as high as 11.42. The higher theIRvalue indicated the higher DNA damage extent of the living bacterial cells caused by the mutation treatment. According to Table 1, it was found that ARTP mutagenesis method caused more severe DNA damage to the testing microbes comparing to other conventional mutagenesis, which agreed well with the trend detected by theumu-FACS method reported previously.

Table 1 Comparison of S. typhimurium TA1535/pSK1002 genotoxicity treated with different mutagenesis methods

3.2. The time course changes of the SOS response by mutagenesis treatments

In order to analyze the time-course changes of the SOS response induction by the exposure to different mutagenesis treatments,we determined the β-galactosidase activity per unit of cell biomass concentration (E/Biomass) at the time interval of 15 minutes within 2 hours of incubation after the mutagenesis treatments(Fig.2).The β-galactosidase activity increased initially and reached the plateau at 1 h incubation for ARTP treatment and 1.25 h for UV radiation, respectively. TheE/Biomass reached a higher value for ARTP treatment for 20 and 30 seconds comparing to ARTP treatment for 10 and 40 seconds (Fig. 2(a)). This result was suspected to because that at 40 seconds ARTP treatment, the cells have programmed to death,such that less DNA damage repair was induced.The highest β-galactosidase activity induction caused by ARTP was higher than that by UV radiation.This result also implied that ARTP caused higher extent of DNA damages comparing to UV mutagenesis.

We then analyzed the net changes in β-galactosidase activity per unit of cell biomass concentration at 15 minutes time interval(Δ(E/Biomass)) during the incubation, which represents the SOS response change rate by a mutation treatment to determine the dynamics of the SOS response for different mutagenesis methods.The Δ(E/Biomass) dynamic changes during the incubation were shown in Fig. 3.

The Δ(E/Biomass) varied with the subsequent incubation time for ARTP and UV treatments. The highest Δ(E/Biomass) showed up between 0.5 h to 1.5 h incubation by the physical mutagenesis treatment methods.Interestingly,the Δ(E/Biomass)was higher for ARTP at intermediate treatment dosages.The highest Δ(E/Biomass)value of ARTP treatment was much higher than that of the UV mutagenesis treatments, implying that ARTP treatment caused more severer DNA damages as indicated by the SOS responses than UV mutagenesis. In contrast, the Δ(E/Biomass) of chemical mutagenesis showed a monotonic decrease trend with the incubation time after the chemical mutagen addition (Fig. 3(c)). The varied changing trends of Δ(E/Biomass) for physical and chemical mutagenesis might be caused by their different mutagenic treatment operations. For chemical mutagenesis, the testing cells were incubated with chemical mutagens for a relative long period time(2 h)for letting the mutagen get into the cell,and the DNA damages and repair were processed simultaneously. In contrast, the physical mutagenesis was performed by a short time separately and the treated cells were transferred to incubation condition for the evaluation of DNA damage and cell growth.The different chemical and physical mutagenesis treatment fashions were presumed to be the reason for the varied Δ(E/Biomass)changing trends among the different mutagenesis methods.

Fig. 2. Kinetic of the β-galactosidase activity induced by (a) ARTP and (b) UV radiation.

Fig. 3. Dynamic change of the β-galactosidase activity induced by ARTP (a), UV (b), chemical mutagens (c, 4-NQO and DES).

The highest Δ(E/Biomass)implying the maximum SOS response rate detected in this work was compared to the maximum SOS induction factor (Fi) for the living cells after mutation treatment measured byumu-FACS method in our previous study [19]. The value ofFireflected the DNA damage extent per living cells and was positively related to the subsequent mutation rates per generation for evaluating the mutation efficiency [19]. As shown in Fig. 4, the mutagenesis method with higherFialso showed higher value of Δ(E/Biomass), indicating that the dynamic SOS response changes upon the mutation treatment evaluated in this study was positively correlated with the SOS induction factor determined by our previousumu-FACS method [19]. Therefore, by detecting the Δ(E/Biomass)using the feasibleumu-microplate test, we were able to easily quantify DNA damage extent which is positively correlated with mutation rates [19].

Fig. 4. Relationship between DNA damage extents evaluated by umu-microplate test and that by umu-based FACS method. Fi is the SOS induction factor per living cells after mutation treatment determined using the umu-based FACS method[20].

4. Conclusions

The study aimed to develop a rapid and relative cheapumumicroplate test to quantitatively evaluate the DNA damage for the comparison of different mutagenesis methods. The βgalactosidase gene was engineered into theS. typhimuriumTA1535/pSK1002 [29,30], and its expression was induced by DNA damages. This strain was originally developed to detect environment mutagens and carcinogens with the feature of high sensitivity and operation feasibility [17,18,31].

In this study,the DNA damage of the test strain after treated by ARTP,UV and chemical mutagens(DES and 4-NQO)was compared using theumu-microplate test. Value ofIRreflects the relative SOS response after mutagenesis treatment, which is corresponding to the degree of DNA damage. The value ofIRwas higher as the dose of mutagens increased and the highestIRwas observed in the ARTP group among all the mutagens used in the study, implying ARTP could damage the bacterial DNA more severely than the other conventional mutagenesis methods. The net increment of β-galactosidase activity per cell biomass (Δ(E/Biomass)) after mutation treatment by different mutagenesis methods were determined, which was an indicator for the DNA damage level and the repair ability caused by different mutagenesis methods. The Δ(E/Biomass)changed in a fluctuated way for physical mutagenesis treatment comparing to chemical mutagenesis treatment, whose value monotonicallydecreased with time. The ARTP treatment caused the highest Δ(E/Biomass)which was much higher than that of UV and chemical treatments. This result suggested that ARTP can cause more stronger DNA damages than the other mutagenesis methods. The highest Δ(E/Biomass) for the model strain upon the mutation treatment was well correlated to the maximum SOS factor (Fi) per living cell concentration evaluated by the FACS-umuestablished previously[19]for each mutagenesis method.The positive correlation between these two evaluation parameters for DNA damage extent implied theumu-microplate test can be feasibly applied in quantitative evaluation of DNA damage extent which determines the subsequent mutation rate for mutation breeding,and can be used for the further study on development of mutagenesis tools.

The capability of ARTP causing stronger DNA damage extent might be accounted for effective ARTP mutation breeding reported so far [22-25]. ARTP can generate various reactive radical species including ROS and RNS by the reaction of activated chemical particles of plasma with air, water contained in the treated samples and/or the medium components as well as the biomolecules[21,32,33]. The activated chemical particles of the plasma have short life time and penetration depth, while the various reactive oxygen and nitrogen species (RONS) after the complicated reactions are presumably the main players for DNA damage in the cells,leading to mutations [34,35]. Although the detailed molecular mechanism of ARTP mutation breeding still remains unclear, our previous studies have demonstrated the breakage effects on artificially synthesized oligonucleotides [36] and plasmid DNA [35].Besides,the mutation rate of viable bacterial cells treated with different treatment (ARTP, UV irradiation and chemical mutagens)was analyzed in our previous study [19]. We found that ARTP caused the highest mutation rate among all the mutagenesis methods studied. Taken together, DNA damage extent caused by a mutation method is an important indicator for studies on the mutation breeding and evolution process.

Although the FACS-umutest for quantification of DNA damage extent has been established in our previous study [19], the needs of expensive FACS instrument and the complicated operating protocol limit its broad application in mutation study. In this study,we demonstrated the feasibility of the rapid microplate test based on SOS/umusystem to estimate the DNA damage extent caused by ARTP and other traditional mutagenesis methods.The highest Δ(E/Biomass) to DNA damage by the different mutagen treatment in thisumu-microplate test was highly correlated to the maximum DNA damage extent (Fi) per living cell established in our previous FACS-umutest, in which we had shown the positive correlation ship between DNA damage and the subsequent mutation rate. In brief summary,umu-microplate test is a rapid and relatively cheap method to quantitatively evaluate the DNA damage extent for comparing different mutagenesis methods and the development of mutation breeding technology.

Acknowledgements

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by National Key Research and Development Project of China (2016YFD0102106) and National Key Scientific Instrument and Equipment Project of National Natural Science Foundation of China (21627812).