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www.nature.com/npjmgrav ARTICLE OPEN 1 1 1 1 1 1✉ Mizpha C. Fernander , Paris K. Parsons , Billal Khaled , Amina Bradley , Joseph L. Graves Jr. and Misty D. Thomas Long-term space missions have shown an increased incidence of oral disease in astronauts’ and as a result, are one of the top conditions predicted to impact future missions. Here we set out to evaluate the adaptive response of Streptococcus mutans (etiological agent of dental caries) to simulated microgravity. This organism has been well studied on earth and treatment strategies are more predictable. Despite this, we are unsure how the bacterium will respond to the environmental stressors in space. We used experimental evolution for 100-days in high aspect ratio vessels followed by whole genome resequencing to evaluate this adaptive response. Our data shows that planktonic S. mutans did evolve variants in three genes (pknB, SMU_399 and SMU_1307c) that can be uniquely attributed to simulated microgravity populations. In addition, collection of data at multiple time points showed mutations in three additional genes (SMU_399, ptsH and rex) that were detected earlier in simulated microgravity populations than in the normal gravity controls, many of which are consistent with other studies. Comparison of virulence-related phenotypes between biological replicates from simulated microgravity and control orientation cultures generally showed few changes in antibiotic susceptibility, while acid tolerance and adhesion varied signiﬁcantly between biological replicates and decreased as compared to the ancestral populations. Most importantly, our data shows the importance of a parallel normal gravity control, sequencing at multiple time points and the use of biological replicates for appropriate analysis of adaptation in simulated microgravity. npj Microgravity (2022) 8:17 ; https://doi.org/10.1038/s41526-022-00205-8 INTRODUCTION oral disease in astronauts . As a result, The Space Medicine Exploration Medical Condition List indicates that diagnostic and As NASA’s desire to explore and set up human habitation on treatment capabilities for basic dental procedures be made planets like Mars increases, preventing and/or predicting potential available . In addition, the Integrated Mathematical Medical threats to mission objectives are a main priority. Humans face 1,2 Model predicts that dental emergencies will be one of the top many challenges in space ; one example being the many conditions impacting future mission objectives . Approximately microbial challenges they encounter, potentially hindering their 20% of oral bacteria are streptococci, these organisms are own level of performance, and the integrity of their spacecraft and 3,4 responsible for both early establishment of dental plaque and habitat . Microbes are not only ubiquitous on earth, but are also 38,39 dental decay . The phenotypes of these organisms as they exist readily found on structures inhabited by humans in the space- 3,5 on earth are well studied and treatment strategies are more ﬂight environment . The crew’s normal ﬂora harbors large predictable, but these may not hold true during long-term space quantities of microbes making them the most important source 6–9 travel. of bacterial contamination . The human microbiome is essential The human mouth is a very complex community made up of for our survival as it helps break down food, protects us from over 1000 different species and is the second most complex after pathogens and even primes our immune system. Under condi- 40,41 the GI tract . These communities reach cell densities as high as tions of distress, the composition of the normal ﬂora is often 11 −142 10–14 10 CFU mL despite having to endure a constant change in altered, ultimately leading to dysbiosis and disease . This environmental stressors including food intake, temperature, pH dysbiosis could be further enhanced by the immune dysregulation 43–45 15–20 and salivary ﬂow . The oral microbiome plays a role in not only encountered during space travel . Therefore, infection result- maintaining oral health, but also in maintaining systemic health as ing from opportunistic pathogens is of concern during space all surfaces of the oral cavity (teeth, gums, tongue etc) are ﬂight . Low-shear stress and reduced gravity can promote microbial inhabited by microbes which aids in preventing colonization by 46,47 4,22–24 dysbiosis and change bacterial physiology . Experiments pathogens . Despite this, dental decay remains one of the have shown that under simulated microgravity, microbes can highest prevailing diseases in humans . Of the oral microbiome 25 26 evolve novel resistance , show enhanced growth , bioﬁlm residents, Streptococcus mutans has been actively studied for its 27,28 29 formation , extracellular polysaccharide production , increase cariogenic properties, as this organism not only causes dental production of secondary metabolites while also showing decay, but resides as a member of normal human plaque . 22,31–33 alterations in pathogenic stress response and virulence . S. mutans is a gram-positive facultative anaerobe (Firmicutes) To reduce incidence of infection in space, both the spacecraft and that normally exists as a member of the mature dental bioﬁlm the crew undergo in-depth microbial screening prior to ﬂight , community, but under certain conditions, can become the 50,51 but this mitigation strategy may not prevent novel evolutionary dominant species leading to dental caries . The formation of dental caries is reliant on two factors, 1) an ecological shift that phenotypes arising within the crew’s microﬂora that will adapt favors the growth of acid producing bacteria and 2) the presence due to their exposure to the novel environment of space. of sucrose in the environment for both fermentation and Long-term space missions and increased exposure to simulated microgravity and radiation have shown an increased incidence of production of glucans that facilitate attachment of the organism Department of Biology North Carolina Agricultural and Technical State University, Greensboro, NC, USA. email: firstname.lastname@example.org Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; M.C. Fernander et al. to the tooth, leading to formation of the plaque bioﬁlm which is growth experiments were conducted in Brain Heart Infusion (BHI) tolerant to low pH . broth (or agar) at 37 °C with 5% CO unless otherwise noted. Two short-term studies assessed the impact of simulated microgravity on S. mutans. Orsini et al. , demonstrated using a Preparation of HARV vessels High Aspect Ratio Vessel (HARV) for 48 h that S. mutans underwent High Aspect Rotating Vessels (HARVs) were purchased from both transcriptomic and metabolomic changes in carbohydrate Synthecon Inc., Houston, TX and used to culture S. mutans under metabolism and increased stress in the form of hydrogen peroxide simulated microgravity. Prior to inoculation, the HARVs were susceptibility and noted variation between biological replicates. cleaned using a mild dish soap and water and rinsed in distilled Orsini et al. also showed an increase in cellular aggregates, water twice. All the components were then soaked in a 25% indicative of an increase in cell-to-cell adhesion and/or bioﬁlm bleach solution for 15 min, rinsed extensively in tap water and formation. Subsequently, Cheng et al. found that S. mutans then rinsed in distilled water. The HARVs were then loosely displayed little changes in growth and hydrogen peroxide reassembled (as per the manufacturer), wrapped in aluminum foil tolerance while exhibiting an increase in acid tolerance in and autoclaved at 121 °C for 20 min. HARVs were then left to cool response to simulated microgravity. It is hard to determine the for 2 h. After they cooled, HARVs were loaded with 5 mL of BHI value of these short-term studies (48 h) as a typical mission can broth and placed onto the rotator backplate at 25 rpm for 24 h, at last 4–6 months at a time. It is highly likely that these short-term 37 °C and 5% CO to ensure that the HARVs were sterile. This studies only represent potential physiological acclimation, as procedure was repeated anytime that contamination was opposed to evolutionary adaptation . detected over the course of the 100-day evolution experiment. Currently, we have limited knowledge of microbial adaptation in response to long duration space ﬂight. The common human Experimental evolution (EE) of S. mutans over 100-days of resident: Streptococcus mutans, will be taken by every astronaut LSMMG exposure into space . Therefore, studies such as the present one are The physical and mechanical unloading by simulated microgravity essential for predicting which organisms have the potential to in ground-based systems have been conducted in many studies, evolve into strains with increased virulence and pose a greater risk characterizing how simulated microgravity impacts various to human health once in space. 4,6,15,22–27,29,53,54,62,66–73 organisms and biological systems . An over- Experimental evolution followed by whole genome resequen- view of the experimental methods is depicted in Fig. 1. S. mutans cing (EERseq) experiments are commonly used for evaluating the Clarke strain NCTC 10449 was used to inoculate 3 mL of fresh BHI 56–61 genomic changes associated with selection regimes . Tiruma- broth and incubated overnight with shaking at 250 rpm. This stock lai et al. adapted E. coli in simulated microgravity for 1000 was then used to streak a BHI agar plate and left to incubate generations (~50 days) using HARVs and showed it acquired overnight. A single colony was then used to make a glycerol stock genomic changes in genes involved in outer membrane protein deemed the ancestral population. Initial growth curves of the folding, ion transport and in the stress response. In a similar ancestral population in the HARVs showed saturation after ~24 h experiment, they also evaluated the consequences of periodic and the generation time was determined to be ~14 per 24 h (one exposure to antibiotic therapy . To date, these are the only long- generation per ~105 min). To begin the EE protocol the ancestral term microbial adaptation studies in simulated microgravity in the stock was used to start another overnight culture. 100 μL of this 62,63 literature . Unfortunately, likely due to the necessity of overnight culture was then used to inoculate 100 mL of fresh BHI specialty equipment, adaptation experiments to simulated micro- broth, 10 mL of the sub-culture was then loaded into a 10 mL gravity are often under-powered with few biological replicates sterile syringe and screwed into one of the two openings on the (populations evolved in parallel to capture random variation) and HARV (inlet), a second empty syringe was placed on the second often lacking normal gravity populations for comparison. The opening (outlet). 10 mL was then pushed into the inlet while the absence of a normal gravity control means that Tirumaliai syringe on the outlet collected the media from the HARV. This was 62,63 et al. could not legitimately claim that the variants that arose repeated for all 8 HARVs. Four of the HARVs were then incubated within their simulated microgravity treatment were not just on the vertical axis of rotation perpendicular to gravity and adaptations to some other aspect of their environment, such as deemed normal gravity to serve as the controls and the other four the medium. were incubated on the horizontal axis to simulate simulated In the present study, we used HARVs to evaluate the adaptive microgravity. All eight HARVs were incubated at 37 °C overnight response of four biological replicates of S. mutans to simulated with 5% CO at 25 rpm. After 24 h of growth (~14 generations, microgravity over 100-days (~1400 generations) to better Supplementary Fig. 3), the HARVs were then subcultured by understand the consequences of long-term space travel on adding 10 mL of fresh BHI into the inlet port which pushed the organisms that reside as normal residents of the host microbiome. culture from the HARV into an empty syringe attached to the The adaptive response was evaluated by performing whole- outlet port and returned to the incubator for a new 24 h cycle. This genome resequencing every three weeks and phenotypes was carried out daily for 100-days (~1400 generations). During the correlated with virulence were assessed after 100-days of EE study, the HARVs were checked daily for contamination by ﬁrst, adaptation. All adapted populations were compared to the measuring the O.D. , values greater than 1 were often indicative ancestral population as well as to their normal gravity counter- of contamination, second, we performed a simple stain with parts which differ only by the axis of rotation of the vessel crystal violet on an aliquot of the culture and observed it under a 4,65 itself . Understanding the long-term evolution of the human compound light microscope for general shape and arrangement microbiome in outer space will therefore be an important step in and anything that was uncharacteristic of S. mutans. Then twice a further understanding the effects of space travel on humans and week we made glycerol stocks and plated serial dilutions onto their resident microbes. both BHI agar and Mitis Salivarius Bacitracin agar (MSB) which is both selective and differential for S. mutans. These plates were used to validate the integrity of the glycerol stock which would be METHODS used in case of future contamination. Every milestone time point Culture strains (21-, 42-, 63- and 100-days), the remainder of the culture were Streptococcus mutans Clarke strain NCTC 10449 was purchased pelleted and stored at −80 °C for DNA sequencing. If contamina- from the ATCC [https://www.atcc.org/products/25175]. All standard tion was detected we would sterilize the HARVs as previously npj Microgravity (2022) 17 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; M.C. Fernander et al. Fig. 1 Experimental methods. Schematic representation of the experimental evolution workﬂow used to adapt Streptococcus mutans to simulated microgravity. described, inoculate with fresh media for 24 h and then inoculate were ﬁrst diluted to an O.D. of 0.05 and 5 μLwere used to with the most recently validated glycerol stock. inoculate 95 μL of fresh BHI broth supplemented with 0.1% sucrose to measure sucrose dependent adhesion or 0.1% glucose to measure sucrose-independent adhesion in a 96-well plate, in Etest® analysis for measuring antibiotic susceptibility triplicate. The plates were then incubated without shaking at All eight 100-day populations, in addition to the ancestral, were 37 °C for 24 and 48 h with 5% CO . After 24 h, the media was used to swab BHI plates in triplicate. On each of those plates, one removed, and the plate was washed with distilled water. 200 μLof Etest® strip (bioMériux) was placed in the center of the plate and a 1% crystal violet solution was then added to the washed plate left to incubate for 48 h to obtain conﬂuent lawns. Antibiotic and left to incubate for 2 h. Plates were then washed with water susceptibility was then measured by determining the minimum three times and left to dry overnight. The following day, 200 μL concentration on the strip at which growth was observed. This acetic acid was used to release the crystal violet from adhered value was then compared for each of the eight evolved cells and then was read at 595 nm. Each population was plated in populations against the values obtained for the ancestral three separate wells to acquire three independent measurements population by performing a one-way ANOVA in GraphPad Prism for each population. using the multiple comparisons function with the ancestral. In total, six antibiotics were tested including: amoxicillin, penicillin, clindamycin, erythromycin, methicillin, and vancomycin. Genome sequencing Chromosomal DNA was extracted from the pellets originating Acid tolerance assays from the 21-, 42–63- and 100-day samples for all eight evolved populations and the ancestral, using the E.Z.N.A. bacterial DNA Acid resistance is one of the main virulence traits in S. mutans,we extraction kit from Omega Biotek® as per the manufacturer’s therefore assessed our evolved populations for changes in their protocol. Eluted DNA was then quantiﬁed using the QuantiFluor® ability to survive at low pH. Acid tolerance assays were performed dsDNA system (Promega). 300 ng of puriﬁed genomic DNA was as described elsewhere . In short, after removal of the cultures used to prepare DNA libraries using the Nextera XT DNA library from the HARVs, a sample of each population was taken and prep kit (Illumina). Samples were then sequenced on an Illumina diluted to an O.D. of 0.3. These cultures were then centrifuged miSEQ sequencing platform with depth of coverage ranging from to pellet the cells and then washed using 0.2 M glycine pH 6.8. ~100X to ~200X, with most exceeding 150X coverage. Sequence Samples were again pelleted and exposed to 0.2 M glycine pH 2.8 alignment and variant calling from the samples was achieved by for 0, 20, 30 and 45 min. At the end of the incubation time, use of the breseq 0.30.0 pipeline . The breseq pipeline uses three samples were pelleted and resuspended in 0.2 M glycine pH 6.8. types of evidence to predict mutations, read alignments (RA), Serial dilutions of each time point were then plated on BHI agar missing coverage (MC), and new junctions (JC), and any reads that and CFUs were counted after 24 h of incubation and compared to indicate a difference between the sample and the reference CFU counts of the ancestral population. Each dilution was plated in triplicate to acquire accurate CFU counts. genome that cannot be resolved to describe precise genetic changes are listed as “unassigned.” These unassigned reads are not described nor interpreted here. Adhesion assays The ability to adhere to the tooth pellicle is another main Statistics virulence factor required by S. mutans to establish itself in a bioﬁlm. Therefore, at 100-days, all eight subcultured populations All phenotypic data was plotted in GraphPad Prism ® Version 9.2.0. were assessed for changes in their adhesion abilities. Cultures Statistics for pairwise comparisons between ancestral populations Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 17 M.C. Fernander et al. Table 1. Frequency of mutations detected earlier in simulated microgravity populations. Gene Day NG1 NG2 NG3 NG4 MG1 MG2 MG3 MG4 Annotation SMU_399 21 0.10 S112Y C3-GDP 0.06 0.26 C 302–313/759 0.14 C 24–36/759 0.10 E58* 42 0.29 C276–514/759 0.09 0.58 0.88 C 302–313/759 0.47 S112Y 0.09 E58* 0.10 E221* 0.58 C 538/759 63 0.30 C 276–514/759 0.08 0.85 0.63 1.00 C 302–313/759 1.00 S112Y 0.11 Q130* 0.09 C 664/759 0.23 C 29/759 100 1.00 1.00 1.00 C 7/759 1.00 0.26 0.17 0.66 1.00 C 302–313/759 0.86 S112Y 0.10 E58* 0.09 E221* ptsH 42 0.19 0.50 G54A 63 0.33 0.21 0.43 0.72 0.34 1.00 G54V 100 0.06 0.06 0.93 G54V rex 42 0.13 Q202* 0.06 T155K/G65S/D52N/ R51L/S46L 0.22 Y66C 0.83 Q193P 0.66 A33E 63 0.25 R51H 0.07 R14H 0.26 T155R 0.61 T48I 0.05 G60S 0.10 A47V 0.08 G57S 0.37 A33E 0.13 Y66C 0.53 Q193P 0.05 A115E 100 0.20 R51L 0.58 Y55D *Indicates premature stop codons. C indicates in the coding region. MG1 acquired 5 different mutations all at an f ~0.06. and each individual treatment population were calculated using was used to compare the normal gravity and simulated an unpaired T-test. Signiﬁcance is deﬁned by the two-tailed p- microgravity populations. Finally, chi-squared analysis using SPSS value using an *. * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001 and **** ≤ 0.0001. was used to compare the genomic variants to determine the Linear regression was used to compare the number of accumu- effect of environment on selection and a simple linear regression lated mutations for each normal gravity population and each was performed in GraphPad Prism with a 95% conﬁdence interval simulated microgravity population. General linear analysis in SPSS to determine the signiﬁcance in the slope for reporting of npj Microgravity (2022) 17 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M.C. Fernander et al. Table 2. Frequency of mutations detected earlier in normal gravity populations. Gene Day NG1 NG2 NG3 NG4 MG1 MG2 MG3 MG4 Annotation pknB 42 0.05 G174C 0.13 R45C 0.11 Y475* 63 0.34 Y561* 0.11 D83H 0.28 0.26 R45C 0.08 D78E 0.86 R258C 100 0.13 F58S 0.35 R45C 0.16 I2S 0.09 R258C 0.60 G19A DUF1003 domain containing protein 21 0.07 0.14 L168I 42 0.07 R181C 0.10 S231R 1 A157V 100 0.65 E195G 0.17 R199C 0.93 R181C 0.94 S231R 0.28 Q100* 0.54 R140* 0.24 0.52 D96E 0.12 K102Q 0.47 T235R 0.11 L168I 0.11 A166S 0.13 A166D 0.92 A157V DQM59_RS01880 100 0.90 E244* Cof‑type HAD‑IIB family hydrolase 0.26 0.53 A242E 0.15 G54A 0.90 E244* *Indicates a STOP codon. accumulated mutations in both normal gravity and simulated many gene functions are based on those with high sequence microgravity. homology to S. mutans UA159 genes (SMU annotated genes) . Alignment and variant calling were conducted using breseq 33.2 . Coverage distribution plots of mapped reads output from Reporting summary breseq showed a depth of coverage for unique positions ranging Further information on research design is available in the Nature from ~150x to over 200x with most showing ~200x coverage. This Research Reporting Summary linked to this article. far exceeds the minimum coverage for true variant calling of ~50x for reference guided assembly . All sequencing data with RESULTS frequencies of mutation (f) above 0.1 are summarized in DNA resequencing Supplementary Table 1. The datasets generated and analyzed in this study can be found through the NCBI BioProject database Following the 100-day EE study, we assessed the genetic changes under Bioproject number PRJNA759625, accession numbers that arose in response to both normal gravity and simulated SAMN23239391- SAMN23239423 [https://www.ncbi.nlm.nih.gov/ microgravity adaptation by whole genome DNA resequencing at sra/?term=PRJNA759625]. multiple time points (21- (~294 generations), 42- (~588 genera- We began by sequencing the ancestral population (NCTC 10449 tions), 63- (~882 generations) and 100-days (~1400 generations)). [https://www.ncbi.nlm.nih.gov/nuccore/NZ_LS483349.1]) and The S. mutans NCTC 10449 genome used in this study (Smutans_NCTC10449.gbk [https://www.ncbi.nlm.nih.gov/nuccore identiﬁed three variations when comparing it to the reference /NZ_LS483349.1]) is poorly annotated and characterized therefore, database sequence, this includes a SNP (I365S (ATT→ AGT)) in Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 17 M.C. Fernander et al. Shared-adaptation Table 3. Frequency of variants unique to normal gravity. Over the course of the 100-day experiment, variants arose that Day NG1 NG2 NG3 NG4 Annotation Gene appeared in both the normal and simulated microgravity treatments (Supplementary Table 1 - orange). On day 21, there 21 0.11 0.24 F52V DQM59_RS10195 ← (sprV) were ﬁve variants that appeared in the intergenic region between 0.27 0.18 H21N DQM59_RS10195 ← (sprV) an AAA-domain containing protein and a dihydrolipoyl dehydro- 42 0.38 0.14 Q31* DQM59_RS04330 → (ridA) genase. These were detected in at least one replicate in each 0.08 R34S DQM59_RS10195 ← (sprV) treatment, all with similar frequencies. At day 42, there were 0.05 E72K DQM59_RS10195 ← (sprV) 3 shared variants, two of them in the same intergenic region above, and a new shared variant which appeared in a C3- 0.24 K15N DQM59_RS10195 ← (sprV) glycoprotein degrading protease (C3-GDP) which, had initially 0.08 0.17 F52V DQM59_RS10195 ← (sprV) been selected for in the simulated microgravity populations at day 0.12 0.13 H21N DQM59_RS10195 ← (sprV) 21 (Table 1). Notably, the single normal gravity variant appeared at 0.10 T104A vicK very low frequency (0.089) while at high frequency in two 0.74 0.15 C 2051–2131/ spaP ← simulated microgravity populations (0.580 and 0.882); indicating that this variant could have been playing a role in simulated 0.08 S721L spaP ← microgravity adaptation. At day 63, there were again three shared 0.35 K373N DQM59_RS07770 ←(murD) variants, only one of the intergenic variants remained, the C3-GDP 63 0.28 0.46 Q31* DQM59_RS04330 → (ridA) variant had swept (f = 1.000) in one replicate of simulated 0.71 F219L DQM59_RS06715 ← microgravity while remaining at low frequency (0.087) in normal 0.22 K373N DQM59_RS07770 ←(murD) gravity. Again, supporting an adaptive role for this variant in 0.25 R34S DQM59_RS10195 ← (sprV) simulated microgravity. There was also a new shared variant in 0.08 H21Y DQM59_RS10195 ← (sprV) DQM59_RS07180, ptsH (phosphocarrier protein HPr), that was 0.54 K15N DQM59_RS10195 ← (sprV) observed at low frequency in two normal gravity replicates and at high frequency (with one sweep f = 1.000) in the simulated 0.58 F52L DQM59_RS10195 ← (sprV) microgravity (Table 1). Finally at day 100 there were six shared 0.10 F52V DQM59_RS10195 ← (sprV) variants. One in the same intergenic region between an AAA- 0.36 H21N DQM59_RS10195 ← (sprV) domain containing protein and a dihydrolipoyl dehydrogenase, E55D DQM59_RS10195 ← (sprV) one in ptsH, repeating the pattern from day 63, two in the C3-GDP 0.22 K7Q DQM59_RS02455 → with selective sweeps in both normal gravity and simulated 0.59 0.05 C 2051–2131/ spaP ← microgravity (Table 1), and two new variants DQM_RS03695 (hypothetical protein) and DQM_RSO7235 (N-acetyl-gamma-glu- 0.08 S721L spaP ← tamyl-phosphate reductase) at intermediate frequencies. 0.09 N917N spaP ← 0.21 0.05 R205C DQM59_RS03385 → Adaptation in normal gravity 100 0.55 Q31* DQM59_RS04330 → (ridA) Sequencing data also showed adaptive variants unique to the 0.33 I(+280/−96) DQM59_RS00680 → / → normal gravity populations (Supplementary Table 1 - blue). At day DQM59_RS00685 21 there were 3 unique non-synonymous (NS) SNP variants 0.53 F98V DQM59_RS03205 → ranging from f = 0.076–0.265 in all four normal gravity popula- 1.00 I(−236/−20) acnA ← / → tions in two genes. At day 42, there were 22 unique polymorph- DQM59_RS07205 isms (f = 0.051–0.744), of these, 16 were NS SNPs, 3 were 0.62 C 464–493/ DQM59_RS01355 → synonymous (S) SNPs, and the rest were in the intergenic/coding regions. At day 63 there were 58 unique variants (f = 0.051–0.711), 0.93 P169L DQM59_RS03185 →(trkA) 35 were NS SNPs, 14 S SNPs, and 9 were intergenic/coding. Finally 0.18 S191* DQM59_RS07640 →(lytS) at day 100, there were 45 unique polymorphisms, of which 32 0.15 Q229* DQM59_RS07640 →(lytS) were NS SNPs, 2 were S SNPs, and 11 were intergenic/coding. Of 0.11 K15N DQM59_RS10195 ← (sprV) these 45, three genes were shared among multiple populations 0.06 F52V DQM59_RS10195 ← (sprV) and had variants solely present in normal gravity (Tables 2 and 3). 0.60 V81F vicK This includes variants in, a DUF1003 domain-containing protein 0.38 I407F vicK (NG1-E195G and R199C, NG2-R181C and S231R, NG3-Q100* and 0.26 A237D vicK R140*), and the Cof-type HAD-IIB family hydrolase (NG3-E244* and 0.20 S721L spaP ← NG4-G54A) and sprV (NG1-K15N and NG3-F52V). In addition, many unique variants were detected in a single *Indicates premature stop codons. normal gravity population (Table 3). The only one that was C indicates in the coding region. acquired early and maintained from one sequencing time point to I indicates intergenic region. the next was the Q31* mutation in a RidA family protein in NG4. This variant ﬁrst arose in two populations (NG3; f = 0.381 and NG4; f = 0.138) by day 42 and was only maintained in NG4 (f = 0.547) at 100-days indicating these populations were likely outcompeted by DQM59_RS03765, a UDP-N-acetylglucosamine 2-epimerase, and clones encoding the wild-type gene sequence. NG2 acquired 3 an insertion in both DQM59_RS03875 a TIGR01906 high frequency mutations, this includes one sweep in the family membrane protein and in the intergenic region between intergenic region between acnA and nrdH (f = 1.000), a 30 bp trxB ← / ← DQM59_RS08210, a thioredoxin-disulﬁde reductase deletion in the coding region of the PTS sugar transporter, subunit and a DUF4059 family protein respectively. Therefore, these IIB (f = 0.619) and a P169L mutation in trkA (f = 0.925). NG3 sweeps (f = 1.000) are not the result of our adaptation studies acquired two mutations at intermediate frequency, a F98V (Supplementary Table 1, highlighted in yellow). mutation in a CBS domain-containing protein (f = 0.527) and a npj Microgravity (2022) 17 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M.C. Fernander et al. Table 4. Frequency of variants unique to simulated microgravity. Day MG1 MG2 MG3 MG4 Annotation Description 42 0.06 G88A rpoC 63 0.20 G123C ccpA → 0.12 Y636* DQM59_RS06275 ← 0.12 A78V DQM59_RS04610 →(eno) 1.00 G88A rpoC 0.06 E105G rpoC 0.12 E55D DQM59_RS10195 ← (sprV) 0.11 L22I ylqF ← 100 0.14 intergenic (+181/+120) asnS → / ← DQM59_RS04320 0.20 G77R DQM59_RS00400 → 0.84 intergenic (−381/−244) DQM59_RS00535 ← / → DQM59_RS00540 0.15 V96G DQM59_RS03190 →(trkB) 0.15 D80A DQM59_RS03980 →(lepA) 0.15 L81F ( DQM59_RS03980 →(lepA) 0.13 M267L DQM59_RS05315 ←(vex3) 0.32 pseudogene (217/304 nt) DQM59_RS08775 ← 0.88 G88A rpoC 0.65 H21N DQM59_RS10195 ← (sprV) 0.37 Y88C DQM59_RS10195 ← (sprV) 0.41 A237D vicK 0.15 W443* vicK 0.19 coding (1588–1701/4689) spaP ← 0.11 E251* gorA ← *Indicates premature stop codons. C indicates in the coding region. I indicates intergenic region. P239S mutation in a HAD family hydrolase (f = 0.331). Finally, NG4 1). At day 63, four unique mutations were detected at a frequency acquired ten mutations in nine different genes, three of the > 0.520 in the four replicates. These include: DQM59_RS01220 variants showed intermediate frequency including those detected (DUF1033 family protein), rex, pknB (Stk1 family PASTA domain- in the RidA family protein, an intergenic mutation between an containing Ser/Thr kinase) and the C3-GDP (Tables 1 and 2). Finally NAD(P)H-dependent oxidoreductase and a thiamine at day 100, 12 unique variants were detected in the four replicate pyrophosphate-dependent dehydrogenase (f = 0.333) and two populations (ten at frequencies > 0.400). Nine of these variants were separate variants in lytS (S191* f = 0.181 and Q229* f = 0.150). NS SNPs and 3 were intergenic or within a pseudogene. At 100-days there were three genes that acquired mutations in multiple Adaptation in simulated microgravity populations and carried variants which were unique to the Sequencing results showed that there were several variants simulated microgravity environment; this includes the DUF1003 unique to the simulated microgravity populations (Supplementary domain-containing protein (MG1-K102Q and T235R, MG3-A166S Table 1 - green). At day 21 there were 8 unique variants ranging and A166D and MG4-A157V), pknB (MG1-IS2, MG4-R258C and from f = 0.063–0.333 in the four simulated microgravity popula- G19A) and C3-GDP (MG1-S112Y and E58*, MG2-E221* and tions, of these, 3 were NS SNPs and 5 were intergenic/coding. At MG4–14bp and a 6 bp coding region insertion) (Tables 1 and 2). day 42 there were 17 unique polymorphisms (f = 0.053–0.575), 14 These are in addition to the single variants in each population at were NS SNPs, 1 a S SNP, and two in the intergenic/coding 100-days (Table 4). Here, MG1 adapted two high frequency regions. At day 63 there were 14 unique variants (f = 0.052–1.000), mutations, one an intergenic mutation in polymerase subunit delta 11 were NS SNPs and 3 were intergenic/coding. Finally at day 100, (f= 0.836) and a G88A mutation (f= 0.877) in rpoC in addition to there were 30 unique polymorphisms (f = 0.059–0.929), 24 were three low frequency mutations, one in the membrane protein NS SNPs and 6 were intergenic/coding. DQM59_RS00535 (G77R f= 0.199), one in the ABC transporter At 21 days, the most signiﬁcant variant was an insertion in permease vex3 (M267L f= 0.130) and one in the glutathione- pseudogene DQM59_RS099330 that occurred in MG1. In addition, disulﬁde reductase gorA (E251* f= 0.114). MG2 had a low ﬁve different variants were detected in the C3-GDP for all four frequency (f = 0.154) W443* mutation in vicK. MG3 carried four simulated microgravity populations (normal gravity populations did low frequency unique variants with f= 0.150 in trkB (V96G), two in not acquire mutations in this gene until day 42, Table 1). By day 42, lepA (D80A and L81F) and one 114 bp deletion in the coding region each of the simulated microgravity populations had a least one of spaP. In addition, MG3 had three intermediate frequency variants, variant that had arisen to a frequency > 0.470. These occurred in f= 0.315 in a pseudogene, a Y88C mutation (f= 0.370) in sprV and a four different genes: the redox-sensing transcriptional regulator rex, Y55D mutation (f= 0.582) in rex. MG4 had only one unique low the phosphocarrier protein ptsH, the C3-GDP and an intergenic mutation between an AAA domain-containing protein and frequency mutation (f= 0.141) in the intergenic region of asnS and dihydrolipoyl dehydrogenase (Table 1 and Supplementary Table a hypothetical protein. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 17 M.C. Fernander et al. Fig. 2 Adhesion phenotypes vary between biological replicates. Sucrose-dependent (SDA) was assessed after (a) 24- and (b) 48 h of static growth in BHI supplemented with 0.1% sucrose using the 100-day populations. Sucrose-independent adhesion (SIA) was assessed after (c) 24- and (d) 48 h of static growth in BHI supplemented with 0.1% glucose using the 100-day populations. Data was plotted in GraphPad Prism ® 9.2.0 and unpaired t-tests with 95% conﬁdence were used to calculate signiﬁcant differences for pairwise comparisons between the ancestral and each treatment populations. Error bars are s.e.m. and signiﬁcance is reported as a two-tailed p-value where *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. All populations show changes in adhesion sucrose-independent adhesion. The former is adequate for initial adherence, but the latter is required for virulence and pathogeni- Streptococcus pathogenicity is reliant on the ability to adhere to city . We therefore analyzed both sucrose dependent (SDA) and the tooth surface and form bioﬁlms commonly known as plaque. They do so by forming aggregates that fuse with the tooth pellicle sucrose-independent adhesion (SIA) for the four 100-day adapted via two independent mechanisms: sucrose-dependent and normal gravity populations (NG1–4) and the four 100-day npj Microgravity (2022) 17 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M.C. Fernander et al. Fig. 3 Acid-tolerance changes as a result of simulated microgravity. We assessed changes in acid-tolerance after adaptation to both normal (blue) and simulated microgravity (yellow) and compared it to that of the ancestral (black). The 100-day populations were exposed to an acidic environment (glycine pH 2.8) for 0, 10, 20, and 45 min, rescued, serial diluted and CFUs were counted after 48 h of growth on BHI agar plates. The CFU counts at the 0-time point were then normalized to 100% and the % reduction was then calculated by dividing the CFU count at the indicated time points by the CFU count at 0. The values were then plotted on a log10 scale to visualize the data. Data was plotted in GraphPad Prism ® 9.2.0 and unpaired t-tests with 95% conﬁdence were used to calculate signiﬁcant differences for pairwise comparisons between the ancestral and each treatment populations. Error bars are s.e.m. and signiﬁcance is reported as a two-tailed p-value where *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. simulated microgravity adapted populations (MG1–4) and per- (Supplementary Fig. 1A, B). For SDA, NG2 and NG3 showed a slight formed pairwise comparisons to the phenotypes exhibited by the increase over the ancestral but NG1, NG4 and MG1–4 all show no ancestral population (Fig. 2). SDA was assessed by inoculating 96- variation from the ancestral after both 24- and 48 h (Supplemen- well plates containing BHI media supplemented with 0.1% sucrose tary Fig. 1C, D). This data is important to show that the 100-day with cultures from the HARVs diluted to a starting O.D. of 0.05 phenotypic data is speciﬁc for this time point and not for any and left to incubate without shaking for both 24 and 48 h. random amount of time in the HARV. Overall, it is difﬁcult to Comparisons with the ancestral population show a decrease in predict the inﬂuence of the selection environment on this SDA in two of the normal gravity populations (NG2 and NG3) and phenotype as changes are observed in both conditions, but it is a small decrease in three of the simulated microgravity popula- important to note that phenotypes do vary greatly between tions (MG2–4) when grown for 24 h (Fig. 2a). By 48 h, all biological replicates. populations had restored similar adhesion levels (NG2, MG1–4) or higher (NG1 and NG4) than to that of the ancestral, except for Acid tolerance is speciﬁc to the adaptive environment NG3 which continued to show a signiﬁcant deﬁcit in SDA even Sustained plaque below a pH of 5.4 favors demineralization of after 48 h (Fig. 2b). tooth enamel and development of dental caries, as a result, S. The differences in SIA were more dramatic (Fig. 2c, d). Here, 96- mutans uses proton pumps to maintain intracellular pH levels and well plates were ﬁlled with BHI supplement with 0.1% glucose. BHI −1 tolerate growth in acidic environments while part of human dental broth does contain a low concentration of glucose (2 g L ) and plaque . We therefore assessed changes in acid tolerance by therefore the +0.1% is in addition to that already present in the exposing the ancestral populations, along with each of the eight medium. After 24 h in +0.1% glucose we observed a signiﬁcant evolved populations to acidic environments for 0, 20, 30 and decrease in SIA for NG1–3, MG1, MG2 and MG4. After 48 h NG1, 45 min and counted CFUs to evaluate survival and therefore their NG2, MG2 and MG4 had complete loss of SIA, whereas NG3 and ability to tolerate low pHs (Fig. 3). It is important to reiterate that MG1 had similar levels to what was observed after 24 h. our cells are planktonically grown and not in bioﬁlms as they MG3 showed a small decrease at 48 h from that of the ancestral would be in plaque. General linear analysis shows a highly and NG4 showed a signiﬁcant increase in SIA after 48 h. Apart signiﬁcant difference (p < 0.001) between the normal gravity and from NG4 and MG3, SIA is almost completely lost in both simulated microgravity populations when the 0-time point is environments. NG4 shows the lowest amount of variation, with no omitted indicating that adaptation of this phenotype is speciﬁcto signiﬁcant change for both SIA and SDA at 24 h and a slight the environment. Pairwise comparisons show that generally, acid increase in both phenotypes after 48 h. Similarly, MG3 only tolerance decreases as a result of adaptation to normal gravity exhibits a small decrease in SDA at 24 h and SIA at 48 h when when compared to the ancestral population. Most notably, all four compared to the ancestral. We also evaluated both SIA and SDA at the 21-day timepoint and for SIA, all four normal gravity and all normal gravity populations show a signiﬁcant reduction in four simulated microgravity populations showed no signiﬁcant tolerance to low pH after exposure for both 20 and 45 min. variation from that of the ancestral after both 24- and 48 h Simulated microgravity replicates on the other hand, tend to be Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 17 M.C. Fernander et al. more variable in their adaptive response and overall, quite similar scale for measuring the strength of the selective environment and to the ancestral. MG1 shows no signiﬁcant difference at both 20 it is all relative to the ﬁtness associated with the selective and 30 min, with a slight reduction after 45 min when compared environment and can only be considered weak in making a to the ancestral. MG2 shows a reduction in acid tolerance at all comparison to a second environment. Therefore, the differences in time points whereas MG3 displays an increase in acid tolerance at these slopes showed that under our conditions, simulated 20 min, then shows no change as compared to the ancestral after microgravity is accumulating mutations at a lower rate than 30 and 45 min. MG4 shows no change in acid tolerance at all time normal gravity. Mutation accumulation experiments generally points. However, this phenotype is usually required while S. show that more mutations can be observed in “weaker” selecting mutans resides as part of the bioﬁlm community (plaque), environments, this is due to the fact that most mutations are 79–82 therefore a general decrease in acid tolerance is not surprising deleterious . Thus, we can conclude that normal gravity was “weaker” than simulated microgravity. for these planktonic populations. We also collected acid tolerance Over 100-days of selection in both environments we also see data for the 21-, 42-, 63- populations (Supplementary Fig. 2a–d). reﬁnement of many of the mutations that are present in the The early data varies at each timepoint, with the earliest genomes of our sequenced populations. For example, in the timepoints showing the greatest differences from the ancestral intergenic region preceding the dihydrolipoyl dehydrogenase populations. Again, reiterating that the 100-day data is speciﬁc for (lpdA) at day 21- there were ﬁve different variants present at this time point. different frequencies across all four normal gravity populations and all four simulated microgravity populations. This was reduced Antibiotic susceptibility to only three variants across all four normal gravity populations Many others have shown changes in antibiotic susceptibility as a and three simulated microgravity populations by day 42 with an result of exposure to simulated microgravity . Here we exposed S. increase in frequency across the board. We see similar trends for mutans to six antibiotics traditionally used to treat dental 63- and 100-days and we would hypothesize that these persistent infections, this included: amoxicillin, penicillin, erythromycin, variants (+177/−52 and +49/−180), are outcompeting the minor vancomycin, methicillin, and clindamycin using Etest® strips and variants (+159/−170, +178/−51, +179/−50 and +184/−45). As found that after 100-days of adaptation, normal gravity popula- expected, we observed this exact trend for all persistent variants tions showed no change in antibiotic susceptibility when identiﬁed in our sequencing data. compared to the ancestral to any of the six antibiotics tested At 100-days, simulated microgravity treatments held unique (Supplementary Fig. 4). Generally, the simulated microgravity variants in multiple populations in the DUF1003 domain contain- populations showed no change to the tested antibiotics with the ing protein SMU_1307c (MG1, 3 and 4), pknB (MG1, 4) and in the exception of a small increase in erythromycin susceptibility for C3-GDP (MG1, 2 and 4). Normal gravity populations also held MG4 and an increase in resistance in MG1 towards clindamycin. unique variants in both SMU_1307c (NG1, 2 and 3) and in pknB (NG4). It is therefore important to determine if these unique variants could be leading to different structural and functional DISCUSSION outcomes in the resultant proteins that may be unique to the The initial goal of this study was to determine the genomic selection environment. SMU_1307c is an uncharacterized protein foundations of adaptation in Streptococcus mutans to simulated predicted to have two transmembrane (TM) segments (residues microgravity. Our data shows that after 100-days (~1400 genera- 112–136 and 148–171) (uniprot Q8DTM6 [https://www.uniprot. tions) of adaptation, planktonic S. mutans do carry speciﬁc variants org/uniprot/Q8DTM6]). Most of the unique simulated microgravity that can be uniquely attributed to simulated microgravity mutations reside within these TM regions (3/5) whereas all the populations. Our data also stresses the importance of biological unique normal gravity variants (excluding nonsense mutations) replicates, as none of the unique variants were present in all four are outside of these segments (4/4). Of the two variants in simulated microgravity populations. In addition, we show the common between normal gravity and simulated microgravity, one necessity of the normal gravity controls and reporting the lies within and the other outside of the TM segments. Overall frequency of mutation as many of the unique variants were indicating that there may be different structural outcomes detected in common genes between normal gravity and between the normal gravity and simulated microgravity variants. simulated microgravity and often at very different frequencies, PknB is a serine/threonine kinase also located in the bacterial stressing the importance of these data in assigning and validating membrane. In Staphylococcus aureus PknB has been shown to be the roles these genes may be playing in the adaptive response to important for full expression of pathogenesis and survival as it simulated microgravity. helps regulate purine biosynthesis, autolysis, its response to the When comparing the genomic variants observed in normal human immune systems and antibiotic resistance . We therefore gravity to those in simulated microgravity after 100 days of mapped both the normal gravity and simulated microgravity selection, as stated, 13 variants were shared by both normal variants on the homologous S. aureus structure (PDB 4EQM gravity and simulated microgravity; 43 were unique to normal [https://www.rcsb.org/structure/4EQM]) as there were no Strepto- gravity, and 29 were unique to simulated microgravity, for a total coccus homologs in the database. Of the eight different missense of 86 variants (chi-squared test showed that χ = 421, p < 0.0001). NS variants, six of the residues are conserved in S. aureus none of If there had been no effect of environment on selection, we would which are found in both normal gravity and simulated micro- expect the numbers of variants shared by both, normal gravity gravity. Of the four missense variants unique to normal gravity, alone, and simulated microgravity alone to be equal (~29 each). none mapped in regions that have been shown to be important Figure 4 demonstrates the number of accumulated mutations in for function (Fig. 5a – blue) . The two nonsense mutations each environment. These data show that after 100-days in either suggest that it may be beneﬁcial in our normal gravity normal gravity or simulated microgravity, both genomes are still in environment to disable the function of this protein. Of the four ﬂux. Also, in simulated microgravity, S. mutans accumulates unique simulated microgravity variants (Fig. 5a – green), two mutations at a rate signiﬁcantly slower (linear regression of the (R258C and G19A) are highly conserved residues, with G19 being slopes show p < 0.0001) than normal gravity. There is also in a glycine-rich loop which is responsible for positioning the γ- signiﬁcant variance between the populations at the different time phosphate of ATP required for signal transduction . By 100-days points indicated by the standard deviation of each environment at only the G19A variant remains at high frequency (f = 0.600) in each timepoint. We therefore suggest that simulated microgravity simulated microgravity populations along with two low frequency displays greater selection than normal gravity. There is no actual variants. In Mycobacterium marinum pknB is regulated by sigH and npj Microgravity (2022) 17 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M.C. Fernander et al. Fig. 4 Populations in simulated microgravity accumulate less mutations. The number of accumulated mutations were plotted for each normal gravity population (blue dotted lines) and each simulated microgravity population (yellow dotted lines) for each sequencing time point. The mean (solid lines) and standard deviation (error bars) were then plotted for each environment. Linear regression determined slopes were signiﬁcantly different (p < 0.0001). Data was plotted and statistical analysis were performed in GraphPad Prism ® 9.2.0. signiﬁcance is reported as a two-tailed p-value where *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. under simulated microgravity, M. marimum was shown to up- simulated microgravity, it is the ﬁrst time that it has been regulate sigH. Despite this, there was no detection of differential associated with this type of environment. expression of pknB . It is therefore possible that differential Two simulated microgravity populations also held ptsH expression is not required as the gene itself acquires novel (SMU_674) mutations by day 42, these became high frequency mutations to make it more efﬁcient under simulated microgravity. variants by 63 days when intermediate frequency variants were As previously stated, we also detected mutations in three genes ﬁrst detected in two normal gravity populations. By 100-days earlier than they were detected in the normal gravity populations, there was a single low frequency (0.063) variant remaining in the indicating that these genes may play an important role in early normal gravity populations, while a high frequency variant (0.933) adaptation to simulated microgravity. It is important to note that was maintained in MG4. The ptsH protein product, HPr, is a this information would have been lost had sequencing been phosphocarrier protein and an essential component of the performed at a single time point. Speciﬁcally, all four simulated phosphoenolpyruvate phosphotransferase system (PTS) required microgravity populations had polymorphisms in SMU_399 a C3- for sugar metabolism and virulence . G54 is highly conserved in 89–91 GDP which is believed to help in immune evasion by impairing most Hpr proteins , therefore we mapped the G54A/G54V C3b deposition and surface binding of IgGs. It has also been mutations onto the homologous HPr structure from Streptococcus shown to interact with competence genes that have been shown faecalis (PDB 1PTF [https://www.rcsb.org/structure/1PTF], Fig. 5b). to play a role in adhesion while being negatively regulated by VicK G54 is located at the center of an external surface loop suggested (which also carried mutations in both normal gravity and to play a role in protein-protein interactions. Again, the variant simulated microgravity populations) . By 100-days, mutations itself is not unique to simulated microgravity, just the time frame were maintained in three of the four simulated microgravity at which high frequency variants were ﬁrst detected. Other populations, two of which went to ﬁxation. The speciﬁc variants simulated and true space ﬂight experiments have also shown were present in both normal gravity and simulated microgravity, changes in expression of bacterial PTS systems. Staphylococcus but it is important to note the time it took to acquire these speciﬁc warneri was shown to upregulate its PTS system during long-term mutations in each of the selection environments. All four space ﬂight, which was suggested to enhance its resistance and simulated microgravity populations carried C3-GDP mutations by adaptability to its environment . In addition, changes in PTS 21-days and all four maintained variants into day 100, all at high regulation have been shown in Serratia marcescens (spaceﬂight) , frequency. One normal gravity population acquired mutations in Salmonella enteriditis (spaceﬂight) , Yersinia pestis (simulated this gene by 42-days and not until 100-days did all four normal microgravity) and Salmonella enterica serovar typhimurium gravity populations select variants. It is also worth noting that NG4 which differentially expressed ptsH speciﬁcally (simulated micro- is the only variant to not acquire a high frequency mutation, and it gravity) . Most notably, S. mutans was shown to downregulate is the only population to show a unique phenotype in both SIA PTS genes in normal gravity when compared to simulated and SDA when compared to the other populations. Others have microgravity after 48 h of exposure by Orsini et al. identiﬁed changes in the Streptococcal C3-GDP in response to Finally, mutations were detected early in all four simulated 86 87 antibiotic resistance and ﬂuoride resistance although, as this is microgravity populations (42-days) in rex, a redox sensing the ﬁrst adaptive experiment in a Streptococcal species to transcriptional regulator that plays an important role in carbon Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 17 M.C. Fernander et al. Fig. 5 Unique genomic variants map to distinct regions of adapted genes. a pknB acquired mutations that were unique to either simulated microgravity (green) or normal gravity (blue). Here we mapped all mutations detected (excluding nonsense mutations, as they lead to a premature stop codon which results in a shortened, and most often, nonfunctional protein product) at all four sequencing time points onto a homolog from Staphylococcus aureus (PDB 4EQM [https://www.rcsb.org/structure/4EQM]) as there were no solved Streptococcus homologues in the database. b ptsH acquired the same SNP in both normal gravity and simulated microgravity (G54A/V-orange). This speciﬁc residue has been shown to be important for protein-protein interactions (PDB 1PTF [https://www.rcsb.org/structure/1PTF]) . c rex acquired a total of 12 unique variants in simulated microgravity (green), 5 unique variants in normal gravity (blue) with 3 variants in common (orange). Variants mapped onto the homologous Streptococcus agalactiae structure (PDB 3KET [https://www.rcsb.org/structure/3KET]) show that they both interact directly with the DNA substrate. All structural ﬁgures were generated using The PyMOL™ Molecular Graphics System, Version 2.4.1, Schrödinger, LLC. metabolism, the oxidative stress response and bioﬁlm forma- agalactiae, most mutations (12/18) map to residues interacting 97,98 tion . Variants in all four normal gravity populations arose by directly with the DNA substrate in both normal gravity and day 63, although only two variants were maintained in MG2 and simulated microgravity populations (PDB 3KET [https://www.rcsb. NG4 at 100-days. In total, 18 variants were detected across org/structure/3KET]) . Speciﬁcally, all unique normal gravity populations, all of which display a complete change in chemical variants map to the DNA-binding region (Fig. 5c-blue) whereas composition of the resultant sidechain and when mapping those both the shared (Fig. 5c-orange) and simulated microgravity mutations on a homologous structure from Streptococcus unique (Fig. 5c-blue) variants are located both within and outside npj Microgravity (2022) 17 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M.C. Fernander et al. of the DNA-binding domain, as a result, it is more difﬁcult to mutans loses its ability to effectively adhere to surfaces via SDA. predict the effect that these will have on the structure and As SDA is required for initial attachment, this may also inﬂuence function of the protein. The two variants maintained at 100-days, these organisms’ ability to cause disease after long-term R51L (NG4 f = 0.198) and Y55D (MG2 f = 0.582) both interact adaptation. After 24 h exposure to simulated microgravity Cheng directly with the DNA, R51 with the backbone and Y55 with the et al. found that S. mutans showed an increase in acid nitrogenous base itself and the changes in chemical nature will tolerance abilities. In our study, adaptation to normal gravity likely reduce afﬁnity for DNA in both variants. Orsini et al. showed a clear reduction in acid tolerance, while in simulated detected differential expression in rex regulated genes in their microgravity we found that these results are quite variable 48 h simulated microgravity studies with S. mutans which, was between biological replicates. Salmonella also consistently and consistent with what was also found in both Staphylococcus reproducibly alters its acid tolerance response in simulated 100 101 aureus and Lactobacillus reuteri (simulated microgravity) . microgravity and this response is dependent on the presence of These studies suggest that this differential expression may be the media containing phosphate ions . BHI media also contains −1 result of the cell perceiving an alteration in its oxidation state. As 2.5 g L disodium phosphate and could potentially be inﬂuen- our study shows mutations in the DNA binding domain of Rex, this cing this change in acid tolerance under simulated microgravity is consistent with others ﬁnding differential expression of Rex in a similar fashion. regulated genes. Our phenotypic data is further supported by Fig. 4 as the It is therefore worth suggesting that different conformations of accumulation in mutations for our populations are still trending these proteins have the potential to be beneﬁcial in normal versus upwards indicating that they have yet to reach an “optimal simulated microgravity . In addition, there must be a difference genome”. This also indicates that they have not yet reached in genes that become more ﬂexible in their resultant protein optimal phenotypes. Based on the results of the Long-term structure in simulated microgravity while maintaining adequate or Evolution Experiment (LTEE), we also have reason to predict that similar growth in normal gravity vs those mutations that enhance in any environment, under which S. mutans (or any other ﬁtness in simulate microgravity and negatively affect ﬁtness in bacterium) is undergoing adaptation that there will never be an normal gravity. Computational modeling of how these mutations “optimal survival/growth point” and that the population will impact protein conformation may provide a clue to whether this continue to evolve improvements in ﬁtness indeﬁnitely . mechanism has any salience. In addition, directed mutational Finally, the 100-day normal gravity populations showed no studies to test the speciﬁc mutations in normal versus simulated change in antibiotic susceptibility when compared with the microgravity would be the deﬁnitive test of their adaptive ancestral to any of the six traditional antibiotics tested in this signiﬁcance. study. Two simulated microgravity populations showed small Finally, we detected unique variants at 100-day: one in MG1 changes towards two antibiotics (MG4 an increase in susceptibility (SMU_96 the DNA‑directed RNA polymerase subunit delta), three to erythromycin and MG1 an increase in resistance to clindamycin) in MG2 (the membrane protein, SMU_66, an ABC transporter indicating that change is possible, but generally not observed. This permease and gorA), four in MG3 (trkB, two in lepA and in data was consistent with what was found for a number of other SMU_1232c a DUF1697 domain containing protein) and one in bacterial species grown in simulated microgravity including: 104 62 105 MG4 (intergenic region between an asparagine tRNA ligase and a Yersinia pestis , Escherichia coli , Staphylococcus aureus , Vibrio 106 107 hypothetical protein). Their importance remains to be elucidated natriegens and Klebsiella pneumoniae . As our data stands, we and would require increasing the length of the experiment, this see no clear increase in assessed virulence phenotypes (acid ﬂux also continues to support that short-term experiments cannot tolerance, adhesion nor antibiotic susceptibility) when planktonic truly predict the adaptive behavior of these organisms in S. mutans is adapted to simulated microgravity. Moving forward, outer space. as bioﬁlm formation is required for pathogenesis, it will now be Along with genomic changes, we also observed changes in important to evaluate the adaptive response of S. mutans bioﬁlm phenotypes associated with our adaptation experiment. SDA as a result of long-term exposure to simulated microgravity. assays were the ﬁrsttimethatthese populationswere exposed Overall, our data makes it clear that it is imperative to perform to sucrose, as a result SDA was most notably reduced in NG2 and these adaptation experiments using multiple replicates over more NG3 as well as MG2–4 after 24 h. Ancestral phenotype was than a few generations. Furthermore, our data is confounded by restored in most populations at 48 h except for in NG3 which sequencing at multiple time points as mutation accumulation in continued to show a signiﬁcant reduction in this phenotype. SIA both the normal gravity and simulated microgravity populations is on the other hand exhibited a dramatic decrease in adhesion still in an upward ﬂux, making it hard to conclude the importance abilities in NG1–3 and MG1, 2 and 4 after 24 h. By 48 h, SIA of many mutations in adaptation speciﬁcally for simulated abilities were almost completely lost in two normal gravity and microgravity. We show that each of our four simulated two simulated microgravity populations. Changes in these traits microgravity populations do have mutations in common, but were not unique to simulated microgravity as they were also overall, appear to be on their own independent evolutionary observed in the normal gravity controls. We do hypothesize that trajectory, each with its own unique set of mutations in genes that this phenotype in the normal gravity controls may be the result span a variety of biological processes as they continue to improve of subculturing practices. In normal gravity, the cells settle to the ﬁtness. This can only be captured by having multiple biological bottom of the HARV making it possible that they can adhere to replicates and to not overstate the impact of observed mutations the base of the vessel especially with its slow rotation .During detected in the sequencing data. It is also important to note that subculturing, we primarily removed the planktonic cells thereby adaptation is occurring in response to all the culture conditions selecting those that are not adhering and potentially leaving and the evolution of side-by-side normal gravity populations is those with greater adherence abilities in the HARV. This essential in tying mutations directly to simulated microgravity. As combined with the fact that they were adapted in presence of our data concludes that for S. mutans, simulated microgravity is glucose could account for this data. As cells cannot settle in the overall a weak selection environment, albeit greater than normal simulated microgravity HARVs we believe this to be a true gravity evidenced by its reduction in accumulated mutation rate adaptive phenotype. This will be elucidated as we perform future (yet signiﬁcantly higher than a strong selection environment), experiments using S. mutans bioﬁlms. Orsini et al. also population replicates become even more important in making observed an increase in cellular aggregation, this may have connections between mutations and their role in adaptation. been the result of a short-term acclimation response to the novel Some of the identiﬁed mutations evolved in both normal gravity environment, we show here that after 100 days planktonic S. and simulated microgravity environments indicating that they Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 17 M.C. 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Rec. 1, bioﬁlm formation ability of Yersinia pestis after 40-day exposure to simulated 333–346 (2001). microgravity. Int. J. Astrobiology (2021). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 17 M.C. Fernander et al. 96. Wilson, J. W. et al. Media ion composition controls regulatory and virulence AUTHOR CONTRIBUTIONS response of Salmonella in spaceﬂight. PloS One 3, e3923 (2008). M.C.F. was responsible for overseeing and conducting all the experiments outlined in 97. Bitoun, J. P., Nguyen, A. H., Fan, Y., Burne, R. A. & Wen, Z. T. Transcriptional the manuscript. P.P., B.K. and A.B. helped in carrying out the evolution experiment, repressor Rex is involved in regulation of oxidative stress response and bioﬁlm antibiotic resistance assays and acid tolerance assays respectively. M.D.T. was formation by Streptococcus mutans. FEMS Microbiol. Lett. 320, 110–117 (2011). responsible for overseeing the work of M.C.F., P.P., B.K., and A.B., writing the 98. Bitoun, J. P., Liao, S., Yao, X., Xie, G. G. & Wen, Z. T. The redox-sensing regulator manuscript and acquiring funding. J.L.G. contributed to the acquisition of funding, Rex modulates central carbon metabolism, stress tolerance response and bio- conception and design of the study, analysis and interpretation of all data and ﬁlm formation by Streptococcus mutans. PLoS One 7, e44766 (2012). performed the statistical analysis. All authors contributed to the manuscript revision 99. Zheng, Y. et al. Distinct structural features of Rex-family repressors to sense and approved the submitted version. redox levels in anaerobes and aerobes. J. Struct. Biol. 188, 195–204 (2014). 100. Castro, S. L., Nelman-Gonzalez, M., Nickerson, C. A. & Ott, C. M. Low Fluid Shear Culture of Staphylococcus aureus Induces Attachment-Independent Bioﬁlm COMPETING INTERESTS Formation and Represses hfq Expression. Appl. Environ. Microbiol. (2011). The authors declare no competing interests 101. Senatore, G., Mastroleo, F., Leys, N. & Mauriello, G. Growth of Lactobacillus reuteri DSM17938 Under Two Simulated Microgravity Systems: Changes in Reuterin Production, Gastrointestinal Passage Resistance, and Stress Genes Expression ADDITIONAL INFORMATION Response. Astrobiology 20,1–14 (2020). Supplementary information The online version contains supplementary material 102. Hekmat, A., Hajebrahimi, Z. & Motamedzade, A. Structural changes of human available at https://doi.org/10.1038/s41526-022-00205-8. serum albumin (HSA) in simulated microgravity. Protein Pept. Lett. 24, 1030–1039 (2017). Correspondence and requests for materials should be addressed to Misty D. Thomas. 103. Wiser, M. J., Ribeck, N. & Lenski, R. E. Long-term dynamics of adaptation in asexual populations. Science 342, 1364–1367 (2013). Reprints and permission information is available at http://www.nature.com/ 104. Lawal, A. et al. The effects of modeled microgravity on growth kinetics, antibiotic reprints susceptibility, cold growth, and the virulence potential of a Yersinia pestis ymoA- deﬁcient mutant and its isogenic parental strain. Astrobiology 13, 821–832 (2013). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims 105. Rosado, H., Doyle, M., Hinds, J. & Taylor, P. W. Low-shear modeled microgravity in published maps and institutional afﬁliations. alters expression of virulence determinants of Staphylococcus aureus. Acta Astronaut 66, 408–413 (2010). 106. Garschagen, L. S., Mancinelli, R. L. & Moeller, R. Introducing Vibrio natriegens as a microbial model organism for microgravity research. Astrobiology 19, Open Access This article is licensed under a Creative Commons 1211–1220 (2019). Attribution 4.0 International License, which permits use, sharing, 107. Kalpana, D., Cha, H., Park, M. & Lee, Y. Growth, morphology, cross stress resis- adaptation, distribution and reproduction in any medium or format, as long as you give tance and antibiotic susceptibility of K. pneumoniae under simulated micro- appropriate credit to the original author(s) and the source, provide a link to the Creative gravity. J. Environ. Sci. Int. 21, 267–276 (2012). Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the ACKNOWLEDGEMENTS article’s Creative Commons license and your intended use is not permitted by statutory This work was funded via support by start-up funds provided to M.D.T. by North regulation or exceeds the permitted use, you will need to obtain permission directly Carolina Agricultural and Technical State University, by Biocomputational Evolution in from the copyright holder. To view a copy of this license, visit http://creativecommons. Action (BEACON): An NSF Center for the Study of Evolution in Action (National org/licenses/by/4.0/. Science Foundation Cooperative Agreement No. DBI-0939454). A.B. was funded by a National Institutes of Health NIGMS MARC Undergraduate NRSA Institutional Grant (Award number 5T34GM083980). © The Author(s) 2022 npj Microgravity (2022) 17 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA
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