Mutation Analysis of the rpoB Gene in the Radiation-Resistant Bacterium Deinococcus radiodurans R1 Exposed to Space during the Tanpopo Experiment at the International Space Station

Abstract

To investigate microbial viability and DNA damage, dried cell pellets of the radiation-resistant bacterium Deinococcus radiodurans were exposed to various space environmental conditions at the Exposure Facility of the International Space Station (ISS) as part of the Tanpopo mission. Mutation analysis was done by sequencing the rpoB gene encoding RNA polymerase β-subunit of the rifampicin-resistant mutants. Samples included bacteria exposed to the space environment with and without exposure to UV radiation as well as control samples held in the ISS cabin and at ground. The mutation sites of the rpoB gene obtained from the space-exposed and ISS/ground control samples were similar to the rpoB mutation sites previously reported in D. radiodurans. Most mutations were found at or near the rifampicin binding site in the RNA polymerase β-subunit. Mutation sites found in UV-exposed samples were mostly shared with non-exposed and ISS/ground control samples. These results suggest that most mutations found in our experiments were induced during procedures that were applied across all treatments: preparation, transfer from our laboratory to the ISS, return from the ISS, and storage before analysis. Some mutations may be enhanced by specific factors in the space experiments, but the mutations were also found in the spontaneous and control samples. Our experiment suggests that the dried cells of the microorganism D. radiodurans can travel without space-specific deterioration that may induce excess mutations relative to travel at Earth's surface. However, upon arrival at a recipient location, they must still be able to survive and repair the general damage induced during travel.

1. Introduction

The transfer of life between planets has been one of the major scientific research objectives of astrobiology. The transfer process includes ejection from the doner planet (Burchell et al., 2004; Fajardo-Cavazos et al., 2009; Yang et al., 2009; Smith, 2013; Worth et al., 2013), transfer (Onofri et al., 2012; Kawaguchi et al., 2013; Yamagishi et al., 2018), and landing on the target planet (Cockell et al., 2007), and in general is often referred to as panspermia. Arrhenius and Borns (1908), among others, proposed the term radio-panspermia to label the process when propagation occurred by solar radiation (Arrhenius and Borns, 1908). Panspermia facilitated by transport on rock or via aggregated life-forms is called lithopanspermia (Horneck et al., 2002) or massapanspermia (Kawaguchi et al., 2013, 2020), respectively.

The possible transfer process has been evaluated by exposing spores of Bacillus subtilis (Horneck et al., 1994, 2002) and vegetative cells of extremophiles (Yamagishi et al., 2018; Kawaguchi et al., 2020) to space environments. These organisms are resistant to harsh environments such as exposure to UV and ionizing radiation, and space vacuum.

Deinococcus radiodurans is known to be resistant to UV and ionizing radiation and desiccation (Anderson et al., 1956; Battista 1997; Daly, 2009; Slade and Radman, 2011; Ishino and Narumi, 2015). To investigate microbial viability and DNA damage, the radiation-resistant bacteria Deinococcus spp. were exposed to the space environment at the Exposure Facility of the International Space Station (ISS) as part of the Tanpopo mission (Yamagishi et al., 2008, 2009, 2021) since May 2015 (Kawaguchi et al., 2016). Three Exposure Panels (EPs), each harboring dried deinococcal cells, were exposed to the space environment, which included space vacuum, UV and ionizing radiation, and fluctuating temperatures. Each year, one of the three EPs was detached and returned to the ground. We analyzed the survival frequency and DNA damage of dried deinococcal cells using pulsed-field gel electrophoresis and quantitative polymerase chain reaction (Kawaguchi et al., 2020).

In this report, we analyzed the mutant frequency and the mutation spectra of the samples returned from the Tanpopo mission. Rifampicin is an antibiotic that binds to RNA polymerase β-subunit encoded by rpoB gene, thus inhibiting the initial step of transcription. Certain rpoB mutations confer rifampicin resistance to bacteria (Floss and Yu, 2005; Campbell et al., 2001); we therefore assessed the mutant frequency and mutation spectrum in the rpoB gene of D. radiodurans that was exposed to space by counting and analyzing rifampicin-resistant clones. From these data, we estimated the extent of major DNA damage induced by the space environment and storage in the ISS.

2. Materials and Methods

2.1. Bacterial strain, culture, and sample preparation

Bacterial strain, culture, and sample preparation have been described previously (Kawaguchi et al., 2020). Deinococcus radiodurans strain R1 ATCC 13939 was cultured for 15 h in mTGE medium (1% [w/v] tryptone, 0.6% [w/v] beef extract, 0.2% [w/v] glucose) at 30°C. Late logarithmic phase cells of D. radiodurans R1 were harvested and washed three times with 10 mM potassium phosphate buffer (PB; pH 7.0). Sterilized aluminum plates with cylindrical wells (2.0 mm diameter and 2 mm or 100 μm depth) with a flat floor were used as sample holders. The wells were filled with different amounts of deinococcal cells corresponding approximately to 100, 500, and 1000 μm thick cell layers, in the upper plate. The wells were filled with dried cells at 1000 μm thickness in the lower plates and in the ISS cabin and ground control plates.

The details of the thickness and number of deinococcal cells in the cell layers have been reported previously (Kawaguchi et al., 2020). Upper and lower sample plates were stacked in the exposure unit. The upper sample plates in the exposure units would be UV-irradiated, whereas the lower plates would be non-UV-irradiated and act as a dark control. For the ISS cabin control, EPs were packed in zippered plastic bags with two desiccant blocks each and kept in the dark in the pressurized storeroom of the Japanese Experiment Module (JEM) of the ISS. Ground control samples were stored in an incubator with desiccant blocks at 20°C in our laboratory at Tokyo University of Pharmacy and Life Sciences, Japan.

2.2. Experimental conditions of the Tanpopo mission

Three EPs, each harboring dried deinococcal cells, were exposed to the space environment. The Tanpopo EPs were attached to the ExHAM (Exposure Handrail Attachment Mechanism) and placed on the Exposure Facility of the JEM-ISS for about 1, 2, and 3 years, as described in the references (Yamagishi et al., 2018, 2021; Kawaguchi et al., 2020). The exact exposure was 384, 769, and 1126 days, respectively. Each year, one of the three EPs was detached and returned to the ground. The space dark controls (non-UV-exposed) were exposed to the space environment in the dark in the lower aluminum sample plate. The ISS cabin control samples were stored in a pressurized area of the ISS.

We used an alanine dosimeter to estimate the UV flux (Yamagishi et al., 2018). Silver-activated phosphate glass based radiophotoluminescence dosimeters were used to measure radiation dose outside and inside the ISS (Yamagishi et al., 2018). Temperature was monitored by a mechanical thermometer (Yamagishi et al., 2018). Environmental conditions for the duration of the 3 years were reported (Kawaguchi et al., 2020; Yamagishi et al., 2021) and are shown in Table 1.

Table 1.

Three-Year Environmental Measurement Results in the Tanpopo Experiment (Kawaguchi et al., 2020)

Experimental place	Wavelength range	UV fluence^a	Ionizing radiation	Temperature range	Pressure range	Humidity
Experimental place	(nm) / window type	(MJ/m²/year)	(mGy/year)^b	(°C)^c	(Pa)^d	(%)
Space
Upper plate^e	110–400 / MgF₂	124–177	232 ± 5	29 ± 5 ∼ −42 ± 5	10^–4 ∼ 10^–7	—
Upper plate^e	170–400 / SiO₂	114–163	232 ± 5	29 ± 5 ∼ −42 ± 5	10^–4 ∼ 10^–7	—
Dark control in space
Lower plate^f		—	232 ± 5	29 ± 5 ∼ −42 ± 5	10^–4 ∼ 10^–7	—
ISS pressurized area		—	83 ± 1	19 ∼ 25	10⁵	45 ∼ 55
Ground control^g		—	1	20	10⁵	5 ∼ 15

UV fluence was estimated in the reference (Kawaguchi et al., 2020).

Ionizing radiation was measured with the dosimeters in the reference (Kawaguchi et al., 2020).

Maximum and minimum temperatures were measured as in the references (Yamagishi et al. 2018; Hashimoto et al. 2019).

Outside pressure estimated in the reference (Rabbow et al., 2015).

Sample plates were set on the upper side of the exposure unit; the plates were irradiated with UV.

Sample plates were set under the upper plates; UV was completely blocked by the upper plates.

The ground control was stored in a desiccator in an incubator at Tokyo University of Pharmacy and Life Sciences.

2.3. Survival assay

Survival of D. radiodurans dried cell was assayed as reported previously (Kawaguchi et al., 2020). After exposure, the dehydrated cells were recovered from the sample plate wells by resuspending the cell pellet in 0.5 mL sterile PB (phosphate buffer) for each well, and used for analyses. Aliquots of deinococcal cell suspension were serially diluted in sterile PB and dropped onto mTGE medium plates. Colonies were counted after incubation at 30°C for 36 h. Surviving cell fractions were determined from the quotient N _c/N _c0, where N _c was the number of colony-forming units of the sample kept in the space, ground, or ISS cabin, and N _c0 was that at the time of sample preparation.

2.4. Isolation of rifampicin-resistant clones

After the exposure experiment, three wells were analyzed independently for all samples. The cells recovered from each well were suspended in 0.5 mL sterile 10 mM PB. Based on survival estimated in a previous experiment (Kawaguchi et al., 2020), the samples corresponding to cell survival of between 10⁷ and 10⁸ were used to inoculate 10 mL of mTGE medium. Each culture was cultivated until OD_590nm reached between 1.1 and 3.0, which took between 15 and 22 h. Because each D. radiodurans cell has multiple copies of genomic DNA (Hansen, 1978), each culture was cultivated to segregate the multiple copies of genomic DNA before the colony isolation to estimate the number of mutated genomes. The cultures were plated after serial dilution onto mTGE agar supplemented with 50 μg/mL rifampicin to determine the number of rifampicin-resistant cells (Rif^R), and on mTGE agar without rifampicin to determine the total number of viable cells. Plates were incubated at 30°C for 3 or 4 days before counting the colonies.

Mutant frequency f was calculated by dividing the number of Rif^R by the number of total viable cells based upon the mutant accumulation method (Foster, 2006).

2.5. Mutation spectra analysis of rifampicin-resistant mutant clones

We also determined DNA sequences of the rpoB gene extracted from Rif^R. Rif^R clones were isolated from 500 μm thick samples that included space-exposed with UV (>110 nm), ground and ISS cabin controls, and from 1000 μm thick samples of space dark controls. Samples with 500 μm thickness were used for mutant frequency analysis, expecting the larger UV effect than that on the thicker samples, while all the space dark (–UV) samples were 1000 μm thick, which were used for the mutant frequency analysis. Each colony on mTGE-rifampicin agar plates was suspended in 100 μL sterilized pure water in a well of 96-well plates. Cells were disrupted by freezing the sample in liquid nitrogen and thawing them in a 56°C water bath and repeating the treatment five times. The 2 μL cell lysate was used for polymerase chain reaction (PCR) amplification (94°C 1 min – [98°C 10 s – 50°C 5 s – 74°C 2 min] 35 times – 74°C 7 min – 4°C storage) in a 30 μL reaction mixture containing 1.5 U LA Taq Hot Start (Takara Bio Inc., Shiga, Japan), LA PCR buffer, 0.8 mM each dNTP, 10 pmol rpoB 1058 forward (5'-AAACTGTGCCGATGGTGGAC-3') and 10 pmol rpoB 1945 reverse (5'-TAGCTCACGCGGCCATTCAC-3') primers (Kim et al., 2004).

Polymerase chain reaction product was purified with a NucleoSpin 96 PCR Clean up kit (Takara Bio Inc., Shiga, Japan). DNA samples, each equivalent to 40 ng DNA, were used for sequence analysis with rpoB 1058 forward primer, in Eurofins Genomics Inc. (Tokyo, Japan). Sequence data were evaluated with SEQUENCHER (Gene Codes Corp., MI, USA), and only the sequence regions with sufficient S/N were used for sequence alignment analysis with Clustal X (Larkin et al., 2007).

The relative mutation frequency of each type R _t and its standard deviation SD_Rt, and mutation frequency of each type f _t and its standard deviation SD_t, were estimated from the number of the clones of each type n, the number of the clones with sequence analysis for each sample N and mutant frequency f. $R_{t} = \frac{n}{N}$

S D_{R t} = \frac{\sqrt{n}}{N}

f_{t} = f \frac{n}{N}

S D_{t} = f \frac{\sqrt{n}}{N}

3. Results

3.1. Survival of the D. radiodurans cells

Survival of the D. radiodurans cells was estimated and reported in a previous paper (Kawaguchi et al., 2020). The survival of the cell pellets at 500 μm thickness over 3 years is shown in Fig. 1. Survival of the ground control samples decreased from about 20% to less than 10% from year 1 to year 3. The survival of space-exposed samples was a few times lower than the ground control after 1 year of exposure and decreased over the 3 years. UV less than 170 nm had little effect on survival, as indicated by the similar decreasing slope of the survival curves compared to quartz (>170 nm) and MgF₂ (>110 nm) windows. Survival in the ISS cabin control was 10 times less after 1 year and decreased much faster than the ground control and space-exposed samples. A similar trend was reported for the 1000 μm thick samples (Kawaguchi et al., 2020). This may be attributed to differences in humidity between the two environments, among other factors (Kawaguchi et al., 2020). Humidity in the ISS cabin and on ground controls was around 45–50% and 5–15%, respectively (Table 1). Cells inside ISS cabin samples could not be kept dry during the experimental period, and this moisture may have caused oxidative stress. Oxygen partial pressure in the ISS cabin did not differ from ground control.

FIG. 1.

Survival over time of D. radiodurans R1 500 μm thick cell pellets. Brown squares: ground control. Blue circles: ISS cabin control. Black circles: space-exposed samples in the dark. Pale blue circles: space-exposed sample under MgF₂ window (>110 nm). Pink circles: space-exposed samples under quartz window (>170 nm). Standard error of the mean (SEM) of the data obtained from three sample wells are shown. Most SEMs are small and covered by the data marks.

3.2. Mutant frequency analysis

The mutant frequencies of Rif^R are summarized in Fig. 2. Naturally occurring spontaneous mutant frequency of wild type was performed in the same way after inoculating the wild type colony. The result of the frequency analysis of spontaneous mutation confirmed that the culture was started from the cell with the wild type rpoB gene, because the majority of the colonies (1–7.0 × 10^–7) showed the wild type phenotype, that is, rifampicin sensitive. The naturally occurring mutant frequency was slightly higher but similar to the median mutation frequency 1.5 (95% confidence limits: 0.34–3.3) × 10^–8 reported previously by Kim et al. (2004) which was calculated by the method of Drake (1991). Mutant frequencies of ground control samples, as well as space-exposed samples, were about 10 times higher than the spontaneous mutant frequency. The ground control samples showed slightly higher mutant frequency in the second and third year compared to the first year. The tendency was not obvious in the space samples, although a slightly higher frequency was noted for space-exposed 1000 μm thick samples with UV exposure. The mutant frequencies of the space-exposed samples were comparable to the ground controls and space dark controls (Fig. 2). The results suggest that the effect of UV on mutation induction was marginal in dried deinococcal cells exposed to space for about 3 years. The results also suggest that any mutations observed were induced by procedures shared among the different treatments, such as preparation, storage before and after the experiments, and/or recovery of the dried samples for analysis.

FIG. 2.

Mutant frequency of rpoB gene estimated by the appearance of rifampicin-resistant clones of D. radiodurans R1 after 1-, 2-, and 3-year exposure to the space environment or stored at a ground laboratory are shown in blue, magenta, and green bars, respectively. Spontaneous mutant frequency of the samples stored in ISS cabin for the first year are also shown. Standard errors of the mean (SEMs) of the data obtained from three sample wells are shown.

3.3. Mutation spectra analysis

The mutation spectra of the rpoB gene in Rif^R obtained from samples exposed to space or stored in the ISS cabin or in the ground laboratory are shown in Fig. 3. Spontaneous mutation data reported by Kim et al. (2004) are also shown in Fig. 3. All the mutations are listed in Supplementary Table S1. From 90 to 94 sequences were read without ambiguity among 94 amplicons applied to the sequencer for each sample. The numbers are shown in Supplementary Table S1. Though UV-irradiated space samples of 1- to 3-year exposure were analyzed, only 1-year samples of space dark control (–UV) and cabin control were analyzed, to reconcile with the limited human resource. Compared with spontaneous mutations, mutation frequency was higher in space-exposed samples with and without UV as well as ground controls in most mutation directions except a transversion from AT to CG, which was not enhanced in the sample exposed to space with UV nor detected in the ISS cabin control. Enhanced frequency of a 9 bp deletion was noted in the space dark sample, as well as the ISS cabin and ground control samples.

FIG. 3.

Mutation spectra of transition, transversion, deletion, and insertion. Panel (A) shows the mutation frequency, and (B) shows relative mutation frequency among mutation types. Each error bar shows SD_t and SD_Rt of each mutation data. Spontaneous mutations were those reported by Kim et al. (2004). Spontaneous mutation: green. Ground control samples average of the samples stored for 1–3 years: brown. ISS cabin control sample stored for 1 year: blue. Space-exposed under MgF₂ window (+UV > 110 nm) samples averaged for 1- to 3-year exposure samples: pale blue. Space dark control sample exposed for 1 year: black.

Mutation frequency in most directions was not enhanced in the space exposed sample with UV compared with the space dark control (Fig. 3B). These data suggest that the mutation is, in general, caused by procedures consistent with all treatments, such as preparation, storage before and after the experiments, and/or recovery of the dried samples for analysis, although some specific mutation types may be induced by some of the conditions specific to the space, ISS, or ground environment.

3.4. Site specificity of mutation frequency

Mutation frequencies at all the sites where mutations were detected are shown in Fig. 4A. Mutation frequency was higher at most sites in the space and control samples compared with the spontaneous mutation frequency. Mutation was enhanced at D425 and G443, and a 9 bp deletion from nucleotide positions 1258 to 1266 in the ISS cabin controls compared with other space samples and the ground controls. Mutation was enhanced in space dark and ground control samples at H435 compared with other space samples.

FIG. 4.

Site-specific mutation frequency. Panel (A) shows the mutation frequency, and (B) shows relative mutation frequency among mutation sites. Four deletion mutations and two insertions are also shown. Color codes are the same as in Fig. 3. Each error bar shows SD_t and SD_Rt of each mutation point.

It is easier to see the difference in site specificity of mutations between samples in Fig. 4B, where the relative percent of mutations at each site is indicated. Most mutation sites found in the space and control samples were found where spontaneous mutations were found. It would be adequate to compare the mutations between space-exposed samples with UV and without UV to see the effect of UV radiation. However, there is no specific site showing enhanced mutation frequency with UV radiation. Though ground controls and space dark controls showed similar relative mutation frequency, space-exposed samples with UV showed the highest relative mutation freqency at H435 compared with other sites. The ISS cabin controls showed relatively enhanced mutation frequency at D425 and G443.

Mutation spectra at the hot spots, which were the residues with high mutation frequency, are indicated in Fig. 5. At D425, GC to AT transition occurred most frequently in the ground controls, ISS controls, and space-exposed samples with UV, resulting in a mutated residue D425N. H435 showed a wide spectrum of possible mutations. Among these, GC to AT transitions were frequent in space dark controls resulting in H435Y, and GC to TA transversions in space-exposed sample with UV resulting in H435N, compared with other sites and directions. At G443, GC to TA transversions were frequent in ISS cabin control samples, resulting in G443W.

FIG. 5.

Relative mutation frequency of each mutation type at each hot spot. Color codes are the same as in Fig. 3. Each error bar shows SD_Rt of each mutation point.

4. Discussion

4.1. Mutant frequency

In our space-exposure experiments with D. radiodurans cell pellets, survival ranged from only a few percent to over 50 percent after 1 to 3 years of exposure to the space environment. The mutant frequency of Rif^R was increased about 1 order of magnitude compared with spontaneous mutation in the cells that survived after the space exposure; the same trend was observed for the ground and ISS control samples. However, no obvious increase in mutant frequency was detected from the space environment including the UV radiation. The results suggest that a space environment including space vacuum, ionizing and UV radiation, and temperature fluctuations did not induce significant mutations in the surviving cells recovered from the samples. These results suggest that most mutations were induced by procedures common to all treatments: sample preparation, transfer from our laboratory to the ISS, return from the ISS, and storage before analysis. It will be interesting to test the effect of the desiccation, moisture, and oxygen on the mutation of D. radiodurans and to analyze the physical mechanism, in the future. These factors are common in the sample preparation step of our experiment.

We have exposed the cell pellets to the space environment. The surface layer of the cell pellets might shield the UV light protecting the cells below the surface layer. However, we are able to estimate the effect of UV light by comparing the survival of wild type and a mutant strain. We have exposed the UV-sensitive mutant (UVS78) as well as the wild type to the space environment (Kawaguchi et al., 2020). The survival frequency of the wild type and UVS78 in 1000 μm samples exposed in space with UV irradiation for 3 years was 3.3 ± 0.7 × 10^–2 and 1.2 ± 0.3 × 10^–7, respectively. The result suggests that most of the cells in 1000 μm thick pellet have suffered damage from UV resulting in the low surviving frequency in the mutant UVS78, while the damage induced by UV was repaired by the wild type genes that are deficient in UVS78. The low survival frequency of the mutant UVS78 suggests that the UV is affecting the cells close to the bottom of the cell pellets.

Previously, as part of the PROTECT experiment of the EXPOSE-E mission on board the ISS, the mutagenic efficiency of space was studied in spores of Bacillus subtilis 168 by Moeller et al., (2012). After 1.5 years of exposure to selected parameters of outer space or simulated martian conditions, they analyzed the rates of induced mutations to rifampicin resistance. In all their flight samples, mutations in Rif^R were induced and their rates increased by several orders of magnitude. Extraterrestrial solar UV radiation (>110 nm) led to the most pronounced increase (up to nearly 4 orders of magnitude); however, mutations were also induced in flight samples shielded from insolation (about 2 orders of magnitude), which were exposed to the same conditions except solar irradiation. In our experiments on space exposure of D. radiodurans, Rif^R mutant frequency was much lower (about 1 order of magnitude) than the PROTECT experiment, and only slight enhancement of mutant frequency was detected due to UV exposure compared with space dark (non-UV exposed) controls.

There are many factors that differ between these two space experiments, including species, cell type (vegetative cells versus spores), and thickness of the cell layers (a layer of 5–10 B. subtilis spores versus 100–1000 μm thick D. radiodurans cell layers). The 100 μm thick D. radiodurans samples were dead after 1 year of exposure to space with UV. One of the factors responsible for the different observations between these two experiments may be related to the difference in spontaneous mutation frequency in these two species. The spontaneous mutation frequencies of B. subtilis vegetative cells and spores were calculated as ∼1.1 × 10^–8 and ∼5.8 × 10^–9 by the fluctuation assay, respectively (Nicholson and Maughan 2002). The former is similar to that of D. radiodurans (1.5 × 10^–8) determined by Kim et al., (2004), but the latter is more than 2.5-fold higher than that of B. subtilis spores. Another factor may be the state of the samples: UV may be shielded by the dead-cell layer at the surface of the 500 or 1000 μm thick samples in our experiments. Accordingly, species differences combined with differences in sample morphology may explain the inconsistencies between these two experiments.

4.2. Mutation spectra

Long et al. (2015) conducted a genome-level mutation accumulation experiment using D. radiodurans strain BAA-816 by transferring the culture for 250 times without selecting the phenotype. They have reported the mutation spectrum of the wild type, with the A/T to G/C mutation rate (based on a total count of 88 AT to GC transitions and 82 AT to CG transversions) per site per generation higher than that in the other direction (based on a total count of 157 GC to AT transitions and 33 GC to TA transversions). We could not find the same tendency in the rifampicin resistance–dependent screening experiments in the data reported by Kim et al. (2004) and by ourselves (Supplementary Fig. S1). The difference may be related to the difference of the experimental system and/or strain used for the experiment. Nevertheless, in our experiment G/C to A/T mutation rate higher than the other direction was noted in all the experiments including ground control (Supplementary Fig. S1). The tendency may be related to the sample preparation including the drying step in atmospheric conditions.

4.3. Mutation site specificity

The rifampicin-resistant mechanism has been summarized previously (Campbell et al., 2001). The antibiotic rifampicin inhibits RNA polymerase upon binding to the β-subunit. A mutation at the antibiotic binding site and a site far from the binding site in RNA polymerase β-subunit may inhibit binding of the antibiotic, thus conferring rifampicin resistance to the mutant RNA polymerase. The mutation site is denoted as cluster N, I, II, and III (Fig. 6). In our experiments, most mutation sites were found in the residue reported previously for the Rif^R mutants. In our space exposure experiments and control experiments, mutations were found mostly in clusters I and II, and most of them were found at the residues at or close to the sites found in E. coli (Fig. 7).

FIG. 6.

Schematic illustration of E. coli RNA polymerase β subunit and the rifampicin binding site (Campbell et al., 2001) and the mutations reported in rpoB genes of E. coli and D. radiodurans (Kim et al., 2004).

FIG. 7.

Summary of the mutations found in E. coli (a: Kim et al., 2004), D. radiodurans (b: Kim et al., 2004, c: this work), and B. subtilis (d: Moeller et al., 2012, e: Fajardo-Cavazos et al., 2018, f: Nicholson and Park, 2005). Mutations found in the species are shown under the wild type amino acid residues, with D. radiodurans numbering at the top line. Figure in each color box is the number of Rif^R clones isolated in respective experiment. Color boxes without figure are the mutations summarized in the references for E. coli and D. radiodurans (Kim et al., 2004).

In the PROTECT experiment, nucleotide sequences at the Rif^R mutations in the rpoB gene of B. subtilis 168 were analyzed after the spore layers were exposed to space with and without UV for 1.5 years (Moeller et al., 2012). The 21 Rif^R mutations isolated from the flight experiment showed all GC to AT transitions were localized to one hotspot: H482 corresponding to the H435 in D. radiodurans (Fig. 7). In mutants isolated from the parallel mission ground reference, the spectrum was wider with predicted amino acid changes at residues Q469K/L/R (Q422 in D. radiodurans numbering), H482D/P/R(H435), and S487L(S440) (Fig. 7). The mutation sites found in the ground reference samples of PROTECT were similar to those reported for B. subtilis spores and vegetative cells previously (Fig. 7). The hot spot H482Y found in the space experiment was reported in previous ground and ISS cabin experiments (Fig. 7; Nicholson and Park 2005; Moeller et al., 2012; Fajardo-Cavazos et al., 2018).

In our space experiments with D. radiodurans, the mutation spectrum was much wider than the spectrum detected in B. subtilis, including the PROTECT experiment (Fig. 7). Mutation sites and type are similar among samples of space with or without UV and ISS and ground controls, as well as those reported previously for D. radiodurans, which are much wider than those reported for B. subtilis (Fig. 7). Accordingly, the wider spectrum of our space experiment is mostly related to differences between species. The point discussed above, the effect of the species, cell type, and the thickness of the cell layer can be further tested by additional space experiments: B. subtilis vegetative cell layers with different thicknesses, for example.

However, if we focus on the detail, space-exposed samples with UV showed higher mutation frequency than the dark and ground controls at H435N induced by GC to TA transversion (Fig. 5). ISS cabin control samples showed higher mutation frequency than other samples in D425N induced by GC to AT transition and G443W induced by GC to TA transversion. These mutations may be enhanced by the specific factors in space or ISS cabin environments.

4.4. Conclusions

There were similarities between the two space exposure experiments, PROTECT and Tanpopo, in terms of rpoB mutation. The most frequent mutation in B. subtilis (H482) was found at the corresponding site, H435, in D. radiodurans (Figs. 4 and 7), although the most frequent mutation direction was different (Fig. 7).

The results of the rpoB gene mutation analysis of Rif^R obtained from the space-exposed and ISS/ground control D. radiodurans samples are in good agreement with the previous mutation analysis results obtained from D. radiodurans vegetative cells. Most mutations were found at or near the rifampicin-binding sites of RNA polymerase β-subunit in Rif^R clones. Mutation sites found in UV-exposed samples were always shared with samples of dark (non-UV exposed), ISS, and ground controls with the exception of only one Rif^R R417P mutation. These results suggest that most mutations found in our experiments were induced by procedures common to all treatments: sample preparation, transfer from our laboratory to the ISS, return from the ISS and storage before analysis. However, some mutations may have been caused by specific factors in the experiment: H435N induced by GC to TA transversion was only found in the space-exposed sample with UV, and D425N induced by GC to AT transition and G443W induced by GC to TA transversion were only found in the ISS cabin control samples. Our experiments suggest that the dried cells of the microorganism D. radiodurans can travel without space-specific deterioration that may induce excess mutations relative to travel at Earth's surface. The common mutation observed may provide an advantage for adaptation to new habitats either on Earth or on other planets.

Footnotes

Acknowledgments

We are grateful to JAXA and NASA for the excellent support during the space exposure experiment. We also thank Ms. Yuka Togashi for the excellent technical assistance.

Authorship Confirmation Statement

Y.K., S.Y., I.N., and A.Y. designed the research. H.H. contributed to the design and manufacture of EPs and contributed as an operator representing the Tanpopo team. D.F., Y.K., I.K., and J.Y. analyzed the survival fractions and mutations. Y.K., I.N., H.H., S.Y., and A.Y. wrote the paper.

Funding Statement

The work was financially supported by JSPS KAKENHI Grant-in-Aid for Scientific Research (B) 16H04823 and for Young Scientists (B) 16K17840. This work was also supported by the Astrobiology Center of National Institutes of Natural Sciences (AB282002, AB292002, AB302005, AB312006, and AB022002).

Authors' Disclosure Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary Material

Supplementary Table S1

Supplementary Figure S1

Abbreviations Used

Associate Editor: Petra Rettberg

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