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Microgravity reshapes bacteriophage–host coevolution aboard the International Space Station [1]

['Phil Huss', 'Department Of Biochemistry', 'University Of Wisconsin-Madison', 'Madison', 'Wisconsin', 'United States Of America', 'Department Of Bacteriology', 'Microbiology Doctoral Training Program', 'Chutikarn Chitboonthavisuk', 'Anthony Meger']

Date: 2026-01 

Bacteriophage–host interactions play a fundamental role in shaping microbial ecosystems. While extensively studied on Earth, their behavior in microgravity remains largely unexplored. Here, we report the dynamics between T7 bacteriophage and Escherichia coli in microgravity aboard the International Space Station (ISS). Phage activity was initially delayed in microgravity but ultimately successful. We identified de novo mutations in both phage and bacteria that enhanced fitness in microgravity. Deep mutational scanning of the phage receptor binding domain revealed striking differences in the number, position, and mutational preferences between terrestrial and microgravity conditions, reflecting underlying differences in bacterial adaptation. Combinatorial libraries informed by microgravity selections yielded T7 variants capable of productively infecting uropathogenic E. coli resistant to wild-type T7 under terrestrial conditions. These findings help lay the foundation for future research on the impact of microgravity on phage–host interactions and microbial communities and the terrestrial benefits of this research.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: P.H. and S.R. have equity holdings are board members of Synpha Biosciences, a phage therapeutics company. Authors RPO, HM and OH are employees of Rhodium Scientific Inc. The authors declare that they have no other competing interests.

Funding: This work was supported by the Defense Threat Reduction Agency ( https://www.dtra.mil/ ) (Grant HDTRA1-16-1-0049) to S.R. C.C. was supported by a graduate training scholarship from the Anandamahidol Foundation (Thailand). The sponsors or funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2026 Huss et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we investigated how microgravity affects interactions between T7 bacteriophage and non-motile Escherichia coli BL21 aboard the International Space Station (ISS). Our evaluation of short-term (hours) and long-term (23 days) incubation of phage and host in microgravity showed significant differences in phage and bacterial viabilities and phage activity compared to terrestrial controls. Phages accumulated many de novo mutations over time that may enhance receptor binding or phage infectivity, while bacteria acquired de novo mutations in genes that may enhance fitness in microgravity and counter phage predation. Deep mutational scanning (DMS) of the phage receptor binding protein (RBP) in microgravity revealed a fitness landscape significantly different from our terrestrial experiments, suggesting substantial differences in the host receptor profile and selection pressure under microgravity. Notably, a combinatorial library of RBP variants enriched in microgravity exhibited a significant improvement in activity against terrestrial uropathogenic E. coli, while a similar library derived under terrestrial conditions showed no improvement, highlighting microgravity as a source of insights into phage–host dynamics with relevance to Earth. Overall, our findings help lay a foundation for future research into the impact of phage–host interactions on microbial communities in microgravity and in the context of space exploration.

The absence of gravity also profoundly reshapes bacterial physiology, imposing stresses that reverberate through gene regulation and metabolism [ 16 – 19 ]. Numerous studies have shown that microgravity conditions increase biofilm formation and elevate metabolic rates [ 20 – 22 ], while the reduced mixing of the surrounding medium limits the removal of metabolic waste and the replenishment of essential nutrients, thereby inducing the overexpression of starvation-associated genes, altering membrane transport processes, and driving global adjustments in cellular homeostasis [ 23 , 24 ]. Bacteria confronted with these conditions may adapt by altering their proteome, including modifying outer-membrane components that act as phage receptors, which can directly influence susceptibility to infection and the efficiency of phage adsorption [ 15 , 19 , 25 ]. Taken together, these physical and physiological perturbations underscore that microgravity constitutes a distinct and multifaceted environment capable of significantly modifying phage–host dynamics, with consequences that are likely to inform our understanding of microbial community behavior not only in extraterrestrial habitats but also in engineered and extreme terrestrial ecosystems.

Microgravity is the near-weightless condition in orbit and alters both the physical transport processes and the physiological states that shape phage predation and bacterial growth, creating an environmental niche distinct from any found terrestrially. At the physical level, phage particles typically diffuse randomly through liquid until they collide with a susceptible bacterial cell, at which point short-range forces such as van der Waals interactions and electrostatic attraction enable irreversible adsorption and subsequent injection of the phage genome [ 7 , 10 ]. Under normal gravity on Earth, this process is enhanced by natural convection. Density- and temperature-dependent buoyancy drives fluid circulation and sedimentation continually redistributes phages, nutrients, and metabolic byproducts, thereby increasing the probability of phage–host encounters. In microgravity, however, materials of differing densities fail to separate, convection currents driven by gravity no longer form, and nutrient molecules as well as motile bacteria experience restricted diffusion and disrupted motility [ 11 – 16 ].

The interaction between bacteriophages (or “phages”) and their bacterial hosts plays a fundamental role in shaping microbial ecosystems both in humans and in the environment [ 1 – 6 ]. Phages act as major drivers of bacterial diversity and evolutionary change in their bacterial prey. These interactions are determined not only by the molecular compatibility of phages and hosts but also by the larger physical context in which infections take place, with factors such as fluid mixing, nutrient gradients, and the underlying physiology of both the bacterial cell and the phage exerting a strong influence [ 7 – 9 ]. Although phage–host interactions have been extensively studied in terrestrial ecosystems, the impact of microgravity on these interactions has yet to be fully investigated. Studying phage–host interplay in microgravity may reveal new mechanisms with relevance both in space and on Earth.

Results

Design of experiments for the International Space Station We prepared two identical sets of 32 sealed cryovial tubes containing experimental samples: one designated for incubation in microgravity, and the other designated for terrestrial incubation (Fig 1). Each set was divided into four prepackaged groups of eight tubes for incubation at 37 °C. Three groups were incubated for short-term time points (1, 2, and 4 hours), and one group for a long-term time point (23 days). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Experimental design to evaluate microgravity interactions on the ISS. Samples were prepared on Earth, with quality checks to ensure cryovial integrity and prevent leakage during freeze–thaw cycles. Identical sets were frozen, then thawed and incubated either in microgravity on the ISS (left) or terrestrially (right) for defined intervals. All samples were re-frozen and later analyzed on Earth for phage and bacterial titers, whole-genome sequencing, and deep mutational scanning of the T7 receptor binding protein tip domain. https://doi.org/10.1371/journal.pbio.3003568.g001 Each short-term group included three replicates of T7 and E. coli BL21 mixed at a phage to host ratio (multiplicities of infection or MOIs) of 10−6 and 10−4 and two samples with either T7 phage only or E. coli only. All bacterial samples contained 4 mL of log-phase (OD 600 ~0.4) E. coli with an estimated titer of ~1–2 × 108 CFU/mL. The initial bacterial concentration and MOI were selected to allow for measurable changes in phage titer after incubation between timepoints, accounting for anticipated cell and phage viability loss due to freeze–thawing as part of delivering samples to the ISS and returning them terrestrially. No cryoprotectant was added to any cultures. To isolate the effects of microgravity, we used a non-motile E. coli strain, removing the variable of host motility enhancing fluid mixing. The 23-day group included three replicates of T7 phage and E. coli mixed at an MOI of 10−4, three replicates of T7 DMS library mixed with E. coli at an MOI of 10−2, and two samples of either T7 only and E. coli only. The higher MOI for the DMS group compensated for the lower abundance of individual variants. The DMS library comprises 1,660 T7 variants, each with a single amino acid substitution in the tip domain of the RBP that has been previously tested under terrestrial conditions [26]. RBPs are central to phage biology, as they dictate host recognition, adsorption, and ultimately host range [27–32]. Their ability to interact with diverse bacterial surface molecules and to evolve rapidly through genetic variation makes them key determinants of phage adaptability and therapeutic potential. Given this central role, RBPs are a particularly compelling target for DMS to decode the sequence–function rules underlying host specificity and infection efficiency. The cryovial containers passed biocompatibility, leak testing, and experimental validation (see S1 Data) to ensure sample integrity and comply with NASA safety standards. All samples were prepared on Earth by mixing phage and bacteria in Rhodium cryovials and immediately freezing them at −80 °C. Frozen samples were shipped to NASA’s Wallops Flight Facility 24 days before launch and transported to the ISS aboard the Northrop Grumman NG-13 Cygnus rocket. Samples were incubated at 37 °C in microgravity for the duration of the relevant time point, then refrozen at −80 °C, transported back to Earth, and delivered to our laboratory. We then thawed the samples, measured phage and bacterial titers, sequenced their genomes, and analyzed the DMS library (Fig 1). We recorded the duration of freezing and incubation aboard the ISS and evaluated the second set of samples terrestrially using the same incubation and freezing times. Terrestrial Incubation was performed without shaking. An asynchronous ground control is standard practice for space biology flight experiments because microgravity and terrestrial samples cannot be incubated in parallel accurately as actual time points on the ISS are adjusted to accommodate astronaut scheduling and real time tracking of samples is not possible.

Bacteriophage T7 activity is reduced in microgravity Under normal terrestrial conditions with shaking at 37 °C, the T7 phage infects and lyses E. coli BL21 within 20–30 min and produces 100–200 progeny phages [33,34]. We hypothesized that in microgravity, reduced fluid mixing would slow the infection cycle by limiting productive encounters between phages and bacteria. Additionally, microgravity-induced stress might disrupt host homeostasis, alter receptor expression, or interfere with intracellular processes, impeding successful phage replication. To test this hypothesis, we measured phage and bacterial titers after 1-, 2-, and 4-hours, as well as after 23 days of incubation. Because the extent of phage replication delay in microgravity was unknown, this approach allowed us to capture a broad range of possible delays. Phage and bacteria were co-cultured at pre-freeze MOIs of 10−6 and 10−4 for short-term incubation time points (1-, 2-, and 4-hours) and 10−4 for long-term incubation (23-days). A significant increase in phage titer and decrease in bacterial host titer between incubation time points would indicate successful phage activity. We did not analyze differences in titer from the initial MOI under the assumption sample freezing would impact viability and instead interpret results between timepoints to determine if phage can successfully replicate. Under terrestrial conditions phage titers increased significantly by 5–7 logs and bacterial titers decreased significantly by 4–5 logs after four hours, regardless of the MOI (Fig 2A). Phage infection thus occurred between two and four hours for the terrestrial samples, indicating the experimental conditions delayed the infection cycle by approximately two hours while allowing for successful phage replication. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Bacteriophage T7 growth is inhibited by microgravity. The titer of phage (green) and bacteria (orange) samples (log 10 Plaque Forming Units or Colony Forming Units, PFU/CFU/mL) after (A) terrestrial incubation or (B) incubation in microgravity mixed at a pre-freeze MOI of 10−2 (left), 10−4 (middle), or incubated separately as phage-only or bacteria-only samples (right). Bars show mean ± SD; triplicate samples shown as points, with blue indicating values at the limit of detection. The significance between adjacent time points was assessed by two-sample t test (*p < 0.05, **p < 0.01, ***p < 0.001). The data underlying this Figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003568.g002 Under microgravity conditions, we observed no increase in phage titer at any short-term incubation time points at either MOI, but a significant 4-log increase at the 23-day time point (Fig 2B). This result indicates microgravity did not prevent productive infection and lysis but delayed it to some point past the four-hour time point. The persistence of bacteria at the 23-day time points (approximately 107 CFU/mL for the terrestrial samples and 102 CFU/mL for microgravity samples) also suggests that a phage-resistant bacterial population emerged in both conditions. Bacterial titer without phage remained stable at early time points under terrestrial incubation but fell 6–7 logs at the 1- and 2-hour time points in microgravity. One possibility, though unproven, is that the absence of cryoprotectant contributed to reduced bacterial viability during freeze–thaw. We cannot exclude an additional role for microgravity-related stress, but emphasize that further experiments will be needed to disentangle these effects. Phage without bacteria saw a 2-log decrease in viable titer in early time points in microgravity compared to terrestrial incubation, but otherwise appeared more stable than bacteria, except for the 23-day time point, where we observed a 4- and 7-log fold decrease in phage titers terrestrially and in microgravity, respectively. Phages are known to lose stability and decay over time without a propagating host [35–37]. Although freeze–thaws complicate accurately determining a decay rate, this effect appeared more pronounced in microgravity. These experiments demonstrate that microgravity challenges both phage and bacterial viability. While phage infectivity is delayed compared to terrestrial conditions, phages ultimately overcome this barrier and successfully infect their hosts. Future studies targeting intermediate time points will be critical for defining the precise latent period under microgravity.

Enriched bacterial mutations reflect phage-mediated selection De novo mutations in E. coli BL21 were significantly more abundant in samples mixed with phages than in those without phages under both terrestrial and microgravity conditions, indicating strong phage-driven selective pressure in both environments (Figs 4A and S6A–S6C, Mann–Whitney U test p < 0.001, Kaplan–Meier survival and log-rank statistical test p < 0.001). Sequencing of the 4-hour sample revealing no significant deviation in this population compared to the pre-incubation sample (Pearson’s R = 0.986, Jenson-Shannon divergence 0.0543, S6D and S6E Fig) and the 4-hour bacterial population was equally susceptible to T7 phage compared to the pre-incubation population (S6F Fig), indicating there was no bottlenecking effect during initial incubation that could cause lowly abundant mutations in the original population to appear de novo. Significantly enriched non-synonymous de novo bacterial substitutions and frameshifts were present in both conditions (Fig 4B, Mann–Whitney U test, FDR-adjusted p < 0.05, > 25% abundance), and pooling genes on Gene Ontology (GO) categories [44,45] revealed that most enriched genes were associated with membrane function or the regulation of metabolic process (Fig 4C). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Enriched bacterial mutations reflect phage-mediated selection. (A) Boxplots of log 10 abundance for de novo non-synonymous substitutions and frameshifts after microgravity (left) or terrestrial (right) incubation, comparing incubation with (light shading) and without (dark shading) phage. Significance assessed by Mann–Whitney U test (***p < 0.001), with “>” indicating the more abundant population. (B) Frequency for bacterial de novo synonymous (blue) and non-synonymous substitutions or frameshifts (yellow) after microgravity (top) or terrestrial (bottom) incubation. (C) Maximum abundance of E. coli BL21 non-synonymous substitutions or frameshifts (>5%) after incubation in microgravity with phage (light purple) or without phage (dark purple), and after terrestrial incubation with phage (light green) or without phage (dark green). Grouped by membrane-associated genes (top), metabolism-associated genes (bottom-left), or other genes (bottom-right). Mutations significantly enriched in (B) marked with stars. (D) Illustration of mlaA mutation effects. Deletions and insertions result in loss or repetition of phenylalanine and asparagine residues. The data underlying this Figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003568.g004 Bacterial mutations significantly enriched (p < 0.05) only under microgravity were frequently associated with the outer membrane and cellular stress response. Notable examples include hldE (56.5%, R232C) associated with the synthesis of the LPS core [46]; mrcB (51.6%, Q817 frameshift), which plays a role in cell wall synthesis and permeability [47,48]; and bipA (96.5%, H16 frameshift, also known as typA) linked to LPS biosynthesis and temperature sensitivity, and previously associated with truncated LPS phenotypes [35–37]. The significant abundance of this mutation suggests that bipA might play a role in phage sensitivity in microgravity. topA, a DNA topoisomerase associated with stress response, was also significantly enriched in microgravity (96.1%, V73G) [49,50]. Additional mutations unique to microgravity included gltA (85%, I138 frameshift), a citrate synthase [51], and rpsF (27.7%, V18 frameshift), which encodes a 30S ribosomal protein [52]. Under terrestrial conditions, bacterial mutations significantly enriched in the presence of phage included galU (100%, L59R), involved in UDP glucose metabolism and associated with O-polysaccharide in other strains [53,54]; lptA (100%, Q43H), responsible for LPS assembly [55]; and hns (66%, I70_L75 IS1 insertion), a global DNA-binding protein responsible for regulating metabolism and nutrient acquisition [56]. Several genes had significantly enriched mutations under both terrestrial and microgravity conditions. trxA is a processivity factor for T7 DNA polymerase and is a known essential gene for phage activity [32,57]. Deletions in trxA were significantly enriched in both conditions (terrestrial: 44.4%, K53_V56d deletion, microgravity: 100%, I76 frameshift), indicating the gene remains essential to the phage in microgravity. The same substitution in ydcL was significantly enriched in both conditions (I105K, terrestrial 100%, microgravity 33.9%). ydcL encodes a transcriptional regulator that triggers small, slow-growing persistor cell states, which could benefit bacteria during prolonged incubation conditions like those in this experiment [58,59]. Finally, intriguing indels were significantly enriched in mlaA in both conditions (Fig 4D). In each condition, two samples exhibited 6-bp deletions resulting in the loss of amino acids F44 and N45 (microgravity abundance: 95.5% and 16.5%; terrestrial abundance: 61.5% and 24.9%). In contrast, the third microgravity sample showed significant enrichment of a 6-bp insertion that added Asp and Phe, after N45 (30.2%, F46_N47ins), effectively inserting and repeating the same two amino acids deleted in the other samples. mlaA encodes an outer membrane lipoprotein believed to remove mislocalized phospholipids from the outer membrane and shuttle them back to the inner membrane [60]. This gene has not yet been associated with changes in phage activity. A mutant with the same F44_N45 deletion has been characterized in E. coli MC4100 [61,62]. This mutation increases outer membrane permeability, phospholipid accumulation, and vesiculation—changes that could affect phage adsorption and potentially confer a competitive advantage. A prior study found that this deletion eventually led to bacterial cell death [62], but our results suggest this mutation may enhance bacterial survival under phage pressure. This discrepancy could also reflect differences in selection context, strain background, or the presence of suppressor mutations. Supporting this possibility, we also identified a significantly enriched frameshift-inducing deletion in yhdP (56.9%, L610 frameshift) in a microgravity sample that had the most abundant mlaA deletion. yhdP is involved in phospholipid transport to the outer membrane, and its loss has been shown to slow transport and reduce cell death in F44_N45 mlaA mutants [61], suggesting it may similarly enhance survivability in microgravity.

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