Microgravity reshapes bacteriophage–host coevolution aboard the International Space Station

We ready two equivalent units of 32 sealed cryovial tubes containing experimental samples: one designated for incubation in microgravity, and the opposite designated for terrestrial incubation (Fig 1). Each set was divided into 4 prepackaged teams of eight tubes for incubation at 37 °C. Three teams have been incubated for short-term time factors (1, 2, and 4 hours), and one group for a long-term time level (23 days).

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.

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Each short-term group included three replicates of T7 and E. coli BL21 blended at a phage to host ratio (multiplicities of an infection or MOIs) of 10⁻⁶ and 10⁻⁴ and two samples with both T7 phage solely or E. coli solely. All bacterial samples contained 4 mL of log-phase (OD₆₀₀ ~0.4) E. coli with an estimated titer of ~1–2 × 10⁸ CFU/mL. The preliminary bacterial focus and MOI have been chosen to permit for measurable modifications in phage titer after incubation between timepoints, accounting for anticipated cell and phage viability loss because of freeze–thawing as a part of delivering samples to the ISS and returning them terrestrially. No cryoprotectant was added to any cultures. To isolate the results of microgravity, we used a non-motile E. coli pressure, eradicating the variable of host motility enhancing fluid mixing. The 23-day group included three replicates of T7 phage and E. coli blended at an MOI of 10⁻⁴, three replicates of T7 DMS library blended with E. coli at an MOI of 10⁻², and two samples of both T7 solely and E. coli solely. The larger MOI for the DMS group compensated for the decrease abundance of particular person variants. The DMS library contains 1,660 T7 variants, every with a single amino acid substitution within the tip area of the RBP that has been beforehand examined beneath terrestrial situations [26]. RBPs are central to phage biology, as they dictate host recognition, adsorption, and finally host vary [27–32]. Their capacity to work together with numerous bacterial floor molecules and to evolve quickly by means of genetic variation makes them key determinants of phage adaptability and therapeutic potential. Given this central position, RBPs are a very compelling goal for DMS to decode the sequence–perform guidelines underlying host specificity and an infection effectivity.

The cryovial containers handed biocompatibility, leak testing, and experimental validation (see S1 Data) to make sure pattern integrity and adjust to NASA security requirements. All samples have been ready on Earth by mixing phage and micro organism in Rhodium cryovials and instantly freezing them at −80 °C. Frozen samples have been shipped to NASA’s Wallops Flight Facility 24 days earlier than launch and transported to the ISS aboard the Northrop Grumman NG-13 Cygnus rocket. Samples have been incubated at 37 °C in microgravity in the course of the related time level, then refrozen at −80 °C, transported again 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 length of freezing and incubation aboard the ISS and evaluated the second set of samples terrestrially utilizing the identical incubation and freezing instances. Terrestrial Incubation was carried out with out shaking. An asynchronous floor management is commonplace follow for house biology flight experiments as a result of microgravity and terrestrial samples can’t be incubated in parallel precisely as precise time factors on the ISS are adjusted to accommodate astronaut scheduling and actual time monitoring of samples will not be doable.

Under regular terrestrial situations with shaking at 37 °C, the T7 phage infects and lyses E. coli BL21 inside 20–30 min and produces 100–200 progeny phages [33,34]. We hypothesized that in microgravity, decreased fluid mixing would gradual the an infection cycle by limiting productive encounters between phages and micro organism. Additionally, microgravity-induced stress would possibly disrupt host homeostasis, alter receptor expression, or intrude with intracellular processes, impeding profitable phage replication. To check this speculation, we measured phage and bacterial titers after 1-, 2-, and 4-hours, in addition to after 23 days of incubation. Because the extent of phage replication delay in microgravity was unknown, this strategy allowed us to seize a broad vary of doable delays.

Phage and micro organism have been co-cultured at pre-freeze MOIs of 10⁻⁶ and 10⁻⁴ for short-term incubation time factors (1-, 2-, and 4-hours) and 10⁻⁴ for long-term incubation (23-days). A major enhance in phage titer and reduce in bacterial host titer between incubation time factors would point out profitable phage exercise. We didn’t analyze variations in titer from the preliminary MOI beneath the idea pattern freezing would influence viability and as a substitute interpret outcomes between timepoints to find out if phage can efficiently replicate. Under terrestrial situations phage titers elevated considerably by 5–7 logs and bacterial titers decreased considerably by 4–5 logs after 4 hours, whatever the MOI (Fig 2A). Phage an infection thus occurred between two and 4 hours for the terrestrial samples, indicating the experimental situations delayed the an infection cycle by roughly two hours whereas permitting for profitable phage replication.

Fig 2. Bacteriophage T7 growth is inhibited by microgravity.

The titer of phage (green) and bacteria (orange) samples (log₁₀ 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.

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Under microgravity situations, we noticed no enhance in phage titer at any short-term incubation time factors at both MOI, however a major 4-log enhance on the 23-day time level (Fig 2B). This outcome signifies microgravity didn’t stop productive an infection and lysis however delayed it to some level previous the four-hour time level. The persistence of micro organism on the 23-day time factors (roughly 10⁷ CFU/mL for the terrestrial samples and 10² CFU/mL for microgravity samples) additionally suggests {that a} phage-resistant bacterial inhabitants emerged in each situations.

Bacterial titer with out phage remained steady at early time factors beneath terrestrial incubation however fell 6–7 logs on the 1- and 2-hour time factors in microgravity. One risk, although unproven, is that the absence of cryoprotectant contributed to decreased bacterial viability throughout freeze–thaw. We can not exclude an extra position for microgravity-related stress, however emphasize that additional experiments shall be wanted to disentangle these results. Phage with out micro organism noticed a 2-log lower in viable titer in early time factors in microgravity in comparison with terrestrial incubation, however in any other case appeared extra steady than micro organism, apart from the 23-day time level, the place we noticed a 4- and 7-log fold lower in phage titers terrestrially and in microgravity, respectively. Phages are recognized to lose stability and decay over time with no propagating host [35–37]. Although freeze–thaws complicate precisely figuring out a decay fee, this impact appeared extra pronounced in microgravity.

Next, we sought to establish mutations within the phage or bacterial genome that influenced phage-host interactions beneath microgravity. We carried out whole-genome sequencing (WGS) of T7 and E. coli BL21 earlier than and after incubation, utilizing pre-incubation genomes as references to establish de novo mutations within the 23-day samples from every situation to make sure each phage and bacterial populations had ample time to propagate. To decide whether or not de novo non-synonymous substitutions or frameshifts in T7 have been considerably enriched, we in contrast the pooled frequencies of ample non-synonymous mutations to the distribution of synonymous de novo substitutions in every situation (Mann–Whitney U check, FDR-adjusted p < 0.05; Fig 3A). To assess whether or not particular genes had considerably extra non-synonymous substitutions than different genes, we calculated this mutation density for every gene and in contrast it to the common mutation density per situation (one-tailed t check, FDR-adjusted p < 0.05, one-sided 95% CI; Figs 3B and S1). Finally, we in contrast the gene-level distribution of non-synonymous mutations between microgravity and terrestrial situations (Mann–Whitney U check, FDR-adjusted p < 0.05) to establish genes with condition-specific enrichment of those mutations (Figs 3C and S2A).

Fig 3. Enriched mutations are distributed broadly in T7 phage.

(A) Substitution frequency and abundance of phage de novo synonymous (blue) and non-synonymous substitutions or frameshifts (yellow) after microgravity (left) or terrestrial (right) incubation. (B) Average gene mutation density ±SD for de novo synonymous (blue) and non-synonymous (yellow) substitutions after microgravity (top) and terrestrial (bottom) incubation. Green bars show the number of significant mutations per gene from (A). (C) Log₁₀ abundance of mutations in selected phage genes after microgravity (left, dark green, Micro.) and terrestrial (right, light green, Terr.) incubation. Significance shown as * (adjusted p < 0.05, one-tailed t test with FDR). (D) Maximum substitution abundance in gp7.3 after microgravity (top, blue shading) or terrestrial (bottom, red shading) incubation. Wild-type (WT) sequence shown center with substitutions shaded matching bar color. Deletions shown as Δ; slashes (/) in WT sequence mark omitted unmutated regions. (E) Diagram of selected structural genes. Gp7.3 is excluded due to an uncertain structural role. (F) Maximum substitution abundance in gp11, displayed as in (D). The data underlying this Figure can be found in S1 Data.

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Significantly enriched (p < 0.05) phage substitutions have been discovered throughout each structural and non-structural proteins beneath terrestrial and microgravity situations (Fig 3B). In microgravity, gene product (gp) 7.3 and gp11 exhibited considerably extra de novo non-synonymous substitutions than different genes (Figs 3B and S1A). Mutation density was total larger terrestrially and no gene confirmed important enrichment in comparison with others terrestrially (S1B Fig). Although gp7.3 will not be absolutely characterised, it’s thought of important for T7 infectivity in E. coli BL21 beneath terrestrial situations [38]. This small 99-amino-acid protein could perform as a scaffolding protein or contribute to host adsorption, although its position within the mature virion stays unsure [39–41]. gp7.3 harbored seven considerably enriched substitutions in microgravity, the best quantity noticed in any gene beneath that situation. These substitutions have been distributed all through the protein (Figs 3D and S3), with 4 notable modifications (E48K, E61K, D68Y, D68A) involving substantial shifts away from negatively charged residues. The solely considerably enriched mutation in gp7.3 terrestrially was a six-amino-acid deletion spanning G42 to V47. The area from G39 to Q50 contained a dense cluster of substitutions and in-frame deletions, together with a 3-amino-acid deletion (G39-T41) terrestrially and a deletion from M46 to Q55 in microgravity, all occurring in a area of the protein predicted to be unstructured (S3 Fig). The excessive variety of enriched substitutions and recurring in-frame deletions on this small protein recommend that gp7.3 is each structurally versatile and important for phage exercise in each environments.

gp11 is an adaptor protein inside the T7 tail that connects the portal protein gp8, the nozzle protein gp12, and the six subunits of the tail fiber protein gp17 (Fig 3E) [38,40,42]. Enriched substitutions have been distributed all through gp11, spanning each uncovered and buried residues (Figs 3F, S4A, and S4B). One considerably enriched substitution, R2C, arose independently twice in microgravity and is situated in a versatile area able to straight interacting with gp17 tail fibers (S4C and S4D Fig). These findings recommend that the substitutions could affect phage health by altering gp11’s construction or stability moderately than by means of direct interplay with the bacterial host.

Comparison of mutation abundance revealed that de novo non-synonymous substitutions have been considerably extra prevalent within the nozzle protein gp12 after incubation in microgravity than beneath terrestrial situations, suggesting a extra outstanding position for this protein in microgravity (Fig 3C). Of the six individually enriched non-synonymous substitutions recognized throughout each situations, 5 concerned modifications towards positively charged residues (Q184R, R205H, Q242R, K404R, and W707R). These substitutions have been distributed all through the protein, with three extra seemingly contributing to host interactions (S5 Fig). Specifically, R205 is surface-exposed and positioned close to the host, Q242 lies near the terminus of the DNA supply channel, and Q184 faces straight towards the host. The cost shifts and spatial distribution of those substitutions spotlight the purposeful significance of gp12 in enhancing phage health beneath each terrestrial and microgravity situations.

Several different considerably enriched substitutions have been significantly notable. In microgravity, the V26I substitution in gp0.5 was the one mutation to brush your entire phage inhabitants—and did so independently in two replicates—indicating a robust health benefit. gp0.5 is an uncharacterized class I gene, doubtlessly related to the host membrane as a result of presence of a putative transmembrane helix [22]. Under terrestrial situations, the T115A substitution in gp4.7 was considerably enriched and extremely ample throughout all three replicates. No mutations have been detected on this gene beneath microgravity, suggesting choice stress could also be distinctive to terrestrial situations. Although the perform of gp4.7 stays unknown, BLASTP evaluation recognized homologs with ~40% similarity to putative HNH endonucleases in Klebsiella and Pectobacterium phages [43].

Lastly, quite a few considerably enriched substitutions have been discovered within the tail fiber gp17, significantly beneath terrestrial situations (Fig 3B). In each environments, substitutions have been concentrated within the C-terminal tip area, with repeated mutations at D540 and neighboring residues. This area is a recognized determinant of host vary and infectivity in terrestrial E. coli strains [26], and these outcomes suggests continued significance throughout extended incubation in each gravity situations.

De novo mutations in E. coli BL21 have been considerably extra ample in samples blended with phages than in these with out phages beneath each terrestrial and microgravity situations, indicating sturdy phage-driven selective stress in each environments (Figs 4A and S6A–S6C, Mann–Whitney U check p < 0.001, Kaplan–Meier survival and log-rank statistical check p < 0.001). Sequencing of the 4-hour pattern revealing no important deviation on this inhabitants in comparison with the pre-incubation pattern (Pearson’s R = 0.986, Jenson-Shannon divergence 0.0543, S6D and S6E Fig) and the 4-hour bacterial inhabitants was equally prone to T7 phage in comparison with the pre-incubation inhabitants (S6F Fig), indicating there was no bottlenecking impact throughout preliminary incubation that would trigger lowly ample mutations within the authentic inhabitants to seem de novo. Significantly enriched non-synonymous de novo bacterial substitutions and frameshifts have been current in each situations (Fig 4B, Mann–Whitney U check, FDR-adjusted p < 0.05, > 25% abundance), and pooling genes on Gene Ontology (GO) classes [44,45] revealed that almost all enriched genes have been related to membrane perform or the regulation of metabolic course of (Fig 4C).

Fig 4. Enriched bacterial mutations reflect phage-mediated selection.

(A) Boxplots of log₁₀ 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.

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Bacterial mutations considerably enriched (p < 0.05) solely beneath microgravity have been regularly related to the outer membrane and mobile stress response. Notable examples embody hldE (56.5%, R232C) related to the synthesis of the LPS core [46]; mrcB (51.6%, Q817 frameshift), which performs a job in cell wall synthesis and permeability [47,48]; and bipA (96.5%, H16 frameshift, also called typA) linked to LPS biosynthesis and temperature sensitivity, and beforehand related to truncated LPS phenotypes [35–37]. The important abundance of this mutation means that bipA would possibly play a job in phage sensitivity in microgravity. topA, a DNA topoisomerase related to stress response, was additionally considerably enriched in microgravity (96.1%, V73G) [49,50]. Additional mutations distinctive 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 situations, bacterial mutations considerably enriched within the presence of phage included galU (100%, L59R), concerned in UDP glucose metabolism and related to O-polysaccharide in different strains [53,54]; lptA (100%, Q43H), accountable for LPS meeting [55]; and hns (66%, I70_L75 IS1 insertion), a worldwide DNA-binding protein accountable for regulating metabolism and nutrient acquisition [56].

Several genes had considerably enriched mutations beneath each terrestrial and microgravity situations. trxA is a processivity issue for T7 DNA polymerase and is a recognized important gene for phage exercise [32,57]. Deletions in trxA have been considerably enriched in each situations (terrestrial: 44.4%, K53_V56d deletion, microgravity: 100%, I76 frameshift), indicating the gene stays important to the phage in microgravity. The identical substitution in ydcL was considerably enriched in each situations (I105K, terrestrial 100%, microgravity 33.9%). ydcL encodes a transcriptional regulator that triggers small, slow-growing persistor cell states, which may benefit micro organism throughout extended incubation situations like these on this experiment [58,59].

Finally, intriguing indels have been considerably enriched in mlaA in each situations (Fig 4D). In every situation, two samples exhibited 6-bp deletions ensuing within the lack of amino acids F44 and N45 (microgravity abundance: 95.5% and 16.5%; terrestrial abundance: 61.5% and 24.9%). In distinction, the third microgravity pattern confirmed important enrichment of a 6-bp insertion that added Asp and Phe, after N45 (30.2%, F46_N47ins), successfully inserting and repeating the identical two amino acids deleted within the different samples.

mlaA encodes an outer membrane lipoprotein believed to take away mislocalized phospholipids from the outer membrane and shuttle them again to the interior membrane [60]. This gene has not but been related to modifications in phage exercise. A mutant with the identical F44_N45 deletion has been characterised in E. coli MC4100 [61,62]. This mutation will increase outer membrane permeability, phospholipid accumulation, and vesiculation—modifications that would have an effect on phage adsorption and doubtlessly confer a aggressive benefit. A previous examine discovered that this deletion ultimately led to bacterial cell loss of life [62], however our outcomes recommend this mutation could improve bacterial survival beneath phage stress. This discrepancy may additionally mirror variations in choice context, pressure background, or the presence of suppressor mutations. Supporting this risk, we additionally recognized a considerably enriched frameshift-inducing deletion in yhdP (56.9%, L610 frameshift) in a microgravity pattern that had probably the most ample mlaA deletion. yhdP is concerned in phospholipid transport to the outer membrane, and its loss has been proven to gradual transport and cut back cell loss of life in F44_N45 mlaA mutants [61], suggesting it might equally improve survivability in microgravity.

Bacteria usually resist phage predation by mutating or downregulating floor receptors important for phage adsorption [26,63–65]. Microgravity-induced stress could amplify this response, altering the bacterial proteome, together with phage receptor profiles [15,25,66]. Such modifications can drive adaptive mutations within the phage RBP. To examine these interactions, we examined how particular person substitutions within the tip area of the T7 RBP have an effect on phage viability in microgravity.

The T7 RBP consists of six quick non-contractile tails that kind a homotrimer composed of a inflexible shaft ending with a β-sandwich tip area [67]. This area is a key determinant of host recognition and interacts with host receptor LPS to place the phage for profitable, irreversible binding [27–32,68]. We performed complete single-site saturation mutagenesis of the RBP tip area, producing a library of 1,660 variants spanning residues 472–554 (based mostly on PDB 4A0T). We then sequenced and in contrast mutational enrichment profiles following the 23-day choice beneath terrestrial and microgravity situations.

We recovered phage DNA from every pattern and scored every variant based mostly on its relative abundance earlier than and after choice (purposeful rating, F) normalized to wildtype (normalized purposeful rating, F_N). Scores have been averaged throughout replicates, and solely variants current in at the least two replicates have been retained for evaluation. Although important dropout of low-performing variants was anticipated as a result of prolonged incubation, we efficiently decided scores for 51.2% (880) of variants in microgravity and 39% (648) in terrestrial situations (Figs 5A, 5B, and S7A). Variant scores correlated nicely throughout replicates regardless of variations in phage titer and mirrored a number of rounds of replication over the 23-day incubation interval, suggesting that lower-titer samples underwent choice however subsequently misplaced viability (S7B–S7D Fig). On common phage variants have been considerably extra enriched after terrestrial incubation in comparison with microgravity (two-sample t check, Mann–Whitney U, p < 0.001) (S8A Fig). The wild-type phage was considerably depleted terrestrially in comparison with microgravity (terrestrial F = 0.58, microgravity F = 3.5, p < 0.01).

Fig 5. Deep mutational scanning reveals beneficial substitutions in microgravity.

(A, B) Heatmaps of normalized functional scores (F_N log₂) for all RBP substitutions after microgravity (A) and terrestrial (B) incubation. Scores shown on a blue to red gradient; Wildtype (WT, F_N log₂= 0) in white with a black dot; variants below the limit of detection (LOD) in gray. Line length represents standard deviation. Substitutions ordered top to bottom; residue positions (PDB 4A0T) shown left to right. (C, D) Efficiency of plating (EOP, left side) and violin plots of plaque area (right side) for (C) E. coli UTI1 and (D) E. coli UTI2 comparing wild type (WT, gray) and selected variant from the microgravity pool for that strain (right, purple). EOP Data shown as mean ± SD from three biological replicates, normalized to E. coli BL21. Significance vs. WT shown as *** (p < 0.001). The data underlying this Figure can be found in S1 Data.

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While variants that carried out worse than wildtype (F_N < 0) tended to carry out equally between microgravity and terrestrial situations (S8B Fig), enriched variants (F_N > 0) have been extremely divergent with no correlation between situations (S8C Fig). Variants enriched in microgravity regularly contained methionine and isoleucine substitutions at inside positions going through the phage (Figs 5A and S8D), in distinction to our earlier terrestrial outcomes on this host [26]. Substitutions in these areas may affect the tip area construction to facilitate adsorption with the host receptor in microgravity.

Under terrestrial situations, top-scoring variants included positively charged substitutions going through the host, in step with our earlier findings on E. coli BL21 [26]. Additional enriched variants featured negatively charged substitutions (e.g., Q488E, G521D) and glycine substitutions (e.g., G480W, G522P) which will induce structural modifications within the tip area (Figs 5B and S8B). These variants have been enriched solely after extended incubation with E. coli BL21, suggesting that such substitutions could contribute to long-term infectivity on stationary-phase hosts—an impact not noticed in shorter, nutrient-rich situations.

Because variants enriched in microgravity have been extremely distinct from these recognized beneath terrestrial situations—each on this examine and in our earlier work—we subsequent evaluated whether or not these substitutions may improve phage exercise terrestrially. If profitable, these substitution patterns might be used to enhance phage efficiency with out exhaustively sampling the total combinatorial house of the gene. We constructed two combinatorial libraries, every comprising all doable combos of 13 top-performing substitutions recognized in microgravity (L490I, N502E, F506M, F506Y, F507V, F507Y, P511M, I514M, N531Q, L533K, L533M, A539M, N546I) or beneath terrestrial situations (G521H, Q488A, Q488E, G521K, G522P, A547S, G521D, G521E, N502S, I495L, R542H, L533T, F506S). This technique decreased a possible search house of over 10²¹ variants to fewer than 5,000 per library. Variants have been synthesized in an oligo pool, assembled into an unbiased phage library utilizing ORACLE, and passaged terrestrially on two clinically remoted E. coli strains (UTI1 and UTI2) which are immune to wild-type T7 and are related to urinary tract infections [69].

We evaluated these swimming pools in effectivity of plating (EOP) experiments and in contrast their plaquing functionality versus wildtype. The combinatorial pool from microgravity confirmed important enchancment in plaquing effectivity in comparison with wildtype and had considerably bigger plaques, indicating the pool contained variants able to considerably bettering exercise on these hosts (S9A and S9B Fig). The terrestrial library carried out considerably worse or no higher than wildtype. To affirm these outcomes, we remoted particular person plaques from the microgravity pool. From UTI1, we recovered a five-substitution variant (L490I, N502E, F507V, L533K, A539M; Variant 1), and from UTI2, a six-substitution variant (L490I, N502E, P511M, L533M, A539M, N546I; Variant 2). These variants demonstrated considerably larger EOP and produced considerably bigger plaques on each UTI strains (Fig 5C and 5D). These findings assist our speculation that microgravity-enriched substitutions can enhance phage efficiency on terrestrial hosts. The prolonged incubation in microgravity revealed new mutational hotspots, enabling environment friendly navigation of sequence house to establish advanced variant combos with enhanced infectivity.

T7 bacteriophage was obtained from ATCC (ATCC BAA-1025-B2). The T7 DMS library used on this examine was the identical library inventory generated in our earlier work [26]. T7 acceptor phages used for ORACLE-based development of the combinatorial libraries have been additionally created as beforehand described [26]. Escherichia coli BL21 was sourced from laboratory shares. Uropathogenic E. coli strains UTI1 and UTI2 have been supplied by Dr. R. Welch (University of Wisconsin, Madison) and originate from a urinary tract an infection isolate assortment [69].

All PCR reactions have been carried out utilizing KAPA HiFi DNA Polymerase (Roche KK2101). The combinatorial library was generated utilizing the ORACLE methodology, as beforehand described [26]. Cloning procedures adopted producer directions except in any other case specified.

Multiplicity of an infection (MOI) was calculated by dividing the phage titer by the corresponding bacterial focus. Initial MOI is calculated based mostly on the micro organism and phage titer earlier than being frozen for transit to the ISS. The MOI for the T7 DMS library was estimated utilizing a helper plasmid, as described beforehand [26].

Deep sequencing was carried out to guage phage populations as described beforehand [26]. Phage sequencing achieved a mean depth of ~49,000× per base throughout the genome, enabling detection of low-abundance mutations. Bacterial sequencing depth averaged ~250× per base in phage-mixed samples and ~1,300× in phage-free samples, limiting mutation evaluation within the former to extra ample variants.

WGS mutations have been recognized utilizing Breseq [73]. For Fig 3, genes have been grouped based mostly on GO classifications [44,45]:

• Membrane-associated genes: GO:0016020 (Membrane), GO:0009103 (LPS biosynthesis), GO:0030288 (Outer membrane sure periplasmic house), GO:0042597 (Periplasmic house).
• Metabolism-associated genes: GO:0008152 (Metabolic course of), GO:0019222 (Regulation of metabolic course of).

To consider whether or not non-synonymous de novo substitutions and frameshift mutations have been considerably enriched in comparison with synonymous substitutions, we in contrast the frequency of every non-synonymous substitutions and frameshift (phage: >1% abundance; micro organism: >25% abundance) to the distribution of synonymous mutations utilizing a one-sided Mann–Whitney U check with Benjamini–Hochberg false discovery fee (FDR) correction (scipy.stats.mannwhitneyu, statsmodels.stats.multitest.multipletests, methodology = ‘fdr_bh’,scipy V1.10.1, statsmodel v 0.14.0) [74]. Adjusted p-values < 0.05 have been thought of important. This strategy assumes that after 23 days of choice the distribution of synonymous substitutions approximates both a impartial baseline or displays minimal selective stress, with the profit that if there may be constructive choice for synonymous substitutions there can be no enhance in false positives utilizing this methodology.

To decide whether or not non-synonymous de novo substitutions and frameshift mutations have been extra ample in bacterial samples uncovered to phage, we utilized the Mann–Whitney U check (scipy.stats.mannwhitneyu, scipy V1.10.1) to check mutation frequencies throughout teams [74,75]. Due to excessive detection limits in phage-mixed samples, we additionally carried out left-censored knowledge evaluation utilizing Kaplan–Meier survival curves (lifelines.KaplanMeierFitter, lifelines V0.27.8) and utilized a log-rank check (lifelines.statistics.log-rank_test, lifelines V0.27.8) to evaluate important variations in mutation distributions between teams [76–78].

Mutation density in phage genes was calculated by dividing the variety of non-synonymous de novo substitutions and frameshift mutations by the size (in amino acids) of every protein product. To assess whether or not any gene had considerably larger mutation density, we in contrast particular person gene densities to the condition-specific common utilizing a one-tailed t check with Benjamini-Hochberg FDR correction (scipy.stats.ttest_1samp, statsmodels.stats.multitest.multipletests, methodology = ‘fdr_bh’, different = ‘greater’, scipy V1.10.1, statsmodel v 0.14.0). Additionally, one-tailed 95% confidence intervals have been calculated utilizing scipy.stats.t.ppf (scipy V1.10.1) and visualized in volcano plots in python [74].

Structural mannequin pictures have been generated utilizing the PyMOL Molecular Graphics System, Version 3.0 (Schrödinger, LLC). Gp7.3 construction was predicted utilizing AlphaFold2 and ColabFold with MMseqs2, utilizing the anticipated construction with the best confidence [79–81]. Electron Microscopy pictures have been based mostly on PDB 7BOX (gp11) and PDB 7BOY (gp12) [41]. A composite construction of the T7 portal–tail advanced is proven from PDB 9JYZ [82]. Numbering for DMS and combinatorial library positions are based mostly on PDB 4A0T [67].