Gene Identity and Context OpenAI o3-deep-research-2025-06-26 66 citations 2025-12-11T08:09:02.291254

Gene Identity and Context

The dfrP gene of Bacillus phage phiNIT1 encodes a dihydrofolate reductase (DHFR) enzyme (UniProt D0VXF2). This identification is supported by sequence annotations showing a conserved DHFR domain (InterPro IPR001796) and the enzyme commission number EC 1.5.1.3, consistent with known DHFR enzymes. Importantly, phiNIT1 is a large Bacillus subtilis-infecting bacteriophage classified in the Bastillevirinae subfamily (genus Nitunavirus) (pmc.ncbi.nlm.nih.gov). Members of this phage group uniquely carry their own thymidylate synthase (thyA) and DHFR genes (pmc.ncbi.nlm.nih.gov). The dfrP gene (also reported as “orf168” in the genome) is thus unambiguously identified as a phage-derived DHFR. There is no evidence of alternative gene meanings in this context, so all literature reviewed refers to the phiNIT1 phage DHFR enzyme described here.

Enzymatic Function of DfrP (Dihydrofolate Reductase)

DfrP is a dihydrofolate reductase, an enzyme that catalyzes the reduction of dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) using NADPH as a cofactor (go.drugbank.com) (pmc.ncbi.nlm.nih.gov). This reaction is essential for regenerating THF, the active form of folate, which is required for one-carbon transfer reactions in the cell. DHFR’s activity is a linchpin in folate metabolism, supplying critical cofactors for nucleotide and amino acid biosynthesis (go.drugbank.com). In particular, the reaction catalyzed by DfrP is needed to produce THF for the de novo synthesis of glycine and purines, and for the continuous production of DNA precursors like thymidine (via the thymidylate synthase pathway) (go.drugbank.com) (pmc.ncbi.nlm.nih.gov). By converting DHF back to THF, DfrP maintains the folate pool used to methylate deoxyuridine monophosphate (dUMP) into thymidine monophosphate (dTMP) and to support purine synthesis (pmc.ncbi.nlm.nih.gov). This enzyme’s function is highly conserved and is analogous to bacterial FolA (the canonical DHFR in bacteria).

Substrate specificity: DfrP is expected to specifically reduce 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate. It binds the cofactor NADPH (evidenced by conserved NADP-binding motifs in DHFR enzymes) (go.drugbank.com) and likely follows classic Michaelis-Menten kinetics observed for DHFR, as demonstrated by enzyme assays of phage-encoded DfrA homologs showing NADPH-dependent DHF turnover (pmc.ncbi.nlm.nih.gov) (pmc.ncbi.nlm.nih.gov). There is no indication that this enzyme has alternate substrates; its activity is specific to folate derivatives, as with other DHFR family enzymes. The product THF is a central cofactor that carries single-carbon units in metabolic reactions. Thus, the primary function of DfrP is to sustain the one-carbon folate cycle by regenerating THF, enabling continuous nucleotide biosynthesis in the host cell.

Role in Cellular Pathways and Biological Processes

By virtue of this enzymatic activity, DfrP plays a direct role in the folate-mediated one-carbon metabolism pathway. This pathway impacts several downstream processes:
- Thymidylate (dTMP) synthesis: DfrP works in tandem with thymidylate synthase (TS) to ensure a supply of dTMP for DNA replication (pmc.ncbi.nlm.nih.gov). TS uses 5,10-methylene-THF to convert dUMP to dTMP, producing DHF as a byproduct; DfrP then reduces DHF back to THF, allowing the cycle to continue. (pmc.ncbi.nlm.nih.gov) This coupling is crucial for DNA synthesis, especially under rapid replication conditions.
- Purine and glycine biosynthesis: THF is also required for purine base formation and for the interconversion of serine and glycine. By maintaining THF levels, DfrP indirectly supports de novo purine synthesis and glycine production (go.drugbank.com). These processes are part of general nucleotide and amino acid biosynthetic pathways.
- One-carbon metabolic process: More broadly, DfrP ensures the supply of one-carbon units in various methylation and biosynthetic reactions (e.g. methionine synthesis). Gene ontology annotations link DHFR activity to the one-carbon metabolic process and tetrahydrofolate biosynthetic process (go.drugbank.com). In summary, DfrP is a key enzyme that keeps folate coenzymes in their reduced, active state, thereby sustaining multiple essential biosynthetic pathways in the cell.

Given these roles, it is well established that dihydrofolate reductase is indispensable for cell growth. In bacteria and other organisms, inhibition or loss of DHFR function causes depletion of thymidine and certain amino acids, leading to stalled DNA replication and cell death (pmc.ncbi.nlm.nih.gov). This is exactly why DHFR is the target of antimicrobial and chemotherapeutic agents (e.g. trimethoprim in bacteria, methotrexate in humans). In the context of phiNIT1 phage infection, the presence of a phage-encoded DHFR suggests the phage is augmenting these vital pathways during infection.

DfrP in Phage phiNIT1: An Auxiliary Metabolic Gene for Phage Replication

Bacteriophage phiNIT1 encodes DfrP as an “auxiliary metabolic gene” (AMG) to boost nucleotide biosynthesis during infection. Many large bacteriophages carry auxiliary genes to redirect or enhance the host’s metabolism in favor of phage reproduction (www.frontiersin.org). In the case of phiNIT1 (a member of the Bastillevirinae phages), comparative genomics revealed that dfrP (DHFR) and a thyA (thymidylate synthase) gene are signature features of this phage lineage (pmc.ncbi.nlm.nih.gov). By providing its own DHFR and TS, phiNIT1 ensures that the infected host cell can efficiently produce dTMP and other nucleotides, even if host pathways are downregulated or if folate pools become limiting. This strategy likely overcomes any metabolic bottleneck during the burst of viral DNA replication. Indeed, viruses use such AMGs to alter host rate-limiting processes (like dTMP synthesis) and thereby promote successful virus proliferation (www.frontiersin.org) (microbiomejournal.biomedcentral.com). The combined presence of TS and DHFR in phiNIT1 means the phage can form a complete folate cycle independent of host regulation, securing a robust supply of DNA building blocks for phage genome synthesis.

Experimental evidence from related phages supports the idea that phage-encoded DHFR confers a fitness advantage. A recent large-scale survey (2025) identified ~1,953 dihydrofolate reductase homologs (dfrA-like genes) across ~1,944 lytic phage genomes, spanning diverse bacterial hosts (pmc.ncbi.nlm.nih.gov). Notably, these phage DHFR genes (including phiNIT1’s dfrP) tend to be trimethoprim-resistant variants. They are distinct from typical bacterial FolA, falling into the DfrA family (length ~150–250 amino acids) which retain DHFR activity but are insensitive to the antibiotic trimethoprim (pmc.ncbi.nlm.nih.gov) (pmc.ncbi.nlm.nih.gov). Wang et al. (Science Advances, 2025) showed that expressing a phage-borne dfr gene in E. coli can indeed confer high-level resistance to trimethoprim (pmc.ncbi.nlm.nih.gov). Moreover, under antibiotic pressure, phages carrying dfr genes had enhanced replication: phage production was higher in the presence of trimethoprim when the phage encoded its own DHFR, and the infected bacteria also grew better than they otherwise would (pmc.ncbi.nlm.nih.gov). This finding suggests that phiNIT1’s DfrP could protect and bolster the host’s folate cycle if the host is exposed to antifolate stress (e.g. antibiotics or folate starvation), thereby indirectly benefiting phage propagation.

Importantly, even in the absence of antibiotics, phage dfr genes likely serve a mutualistic function. By ensuring the host has ample THF and nucleotides, the phage keeps the host metabolic state optimal for phage DNA replication. Expert analysis has described this as an “evolutionary mutualism” between lytic phages and their hosts (pmc.ncbi.nlm.nih.gov). The phage provides a metabolic function that supports host survival (at least until phage assembly is complete), and in return the phage achieves maximal replication. Bacillus phage phiNIT1 fits this paradigm: it carries metabolic genes (DfrP and possibly others like ribonucleotide reductases or DNA metabolism enzymes) often found in lytic phages to overcome host limitations (pmc.ncbi.nlm.nih.gov). In fact, surveys indicate dfr (DHFR) and thyA (TS) genes are significantly enriched in lytic phages – occurring about 10–13 times more often in lytic phages than in temperate phages (pmc.ncbi.nlm.nih.gov). This enrichment underscores their adaptive value for viruses that rely on rapid, lytic replication cycles.

From a genomic standpoint, phiNIT1’s dfrP gene is located adjacent to a transposase, hinting at its acquisition via horizontal gene transfer. A comparative genome analysis between phiNIT1 and a related phage (Bacillus phage BSP9) showed that the transposase gene next to dfrP in phiNIT1 has clear signs of recent horizontal transfer, whereas the dfrP gene itself appears not to have spread to bacterial genomes (pmc.ncbi.nlm.nih.gov). In other words, phiNIT1 seems to have picked up the dfrP gene in its genome, but that gene has not (at least in detectable instances) been laterally transferred into host chromosomes or plasmids in nature (pmc.ncbi.nlm.nih.gov) (pmc.ncbi.nlm.nih.gov). This is an interesting nuance: the phage retains the DHFR gene for its own lifecycle benefit without necessarily donating it to hosts long-term. It suggests that phage-borne dfr genes can persist as part of the viral genome repertoire (possibly acquired from some bacterial source in the past) primarily to serve phage fitness, rather than as mobile resistance elements in bacterial populations. Indeed, comprehensive analyses found that among ~1,953 phage dfr genes, only a single case was detected where a phage dfr had a near-identical counterpart on a bacterial plasmid (pmc.ncbi.nlm.nih.gov), indicating extremely limited dissemination.

Subcellular Localization and Expression

During phiNIT1 infection, the DfrP protein is expressed in the bacterial host’s cytoplasm, which is the site of folate metabolism and nucleotide synthesis. Like canonical DHFR enzymes, DfrP lacks any signal peptide or membrane-spanning region, so it remains in the cytosol to interact with soluble folate substrates and NADPH. While specific localization studies on phiNIT1 DfrP have not been reported, by analogy to E. coli DHFR (a cytosolic monomeric enzyme ~18 kDa) (go.drugbank.com), we expect DfrP to function in the cytoplasm where it can readily access DHF produced during thymidylate synthesis cycles. In essence, DfrP supplements the host cell’s own DHFR activity. If the host DHFR (FolA) is inhibited or working at capacity, the phage-encoded DfrP provides additional reductive capacity in the same cellular compartment. This ensures that THF regeneration keeps pace with the heightened demand for nucleotide precursors during phage DNA replication.

The timing of expression for dfrP would be during the phage lytic cycle’s replication phase. Auxiliary metabolic genes in phages are often expressed early or middle in infection to prepare the host metabolism for phage DNA synthesis (www.frontiersin.org). Although phiNIT1’s transcriptional program isn’t fully characterized in literature, it is reasonable that dfrP is expressed prior to or during viral DNA replication, ensuring folate cycle enzymes (TS and DHFR) are active when viral genome amplification begins. There is no secretion of DfrP outside the cell; its role is strictly intracellular. After the phage life cycle completes and the host cell lyses, DfrP would be released and degraded in the environment unless a new host is immediately infected.

Clinical and Applied Relevance

Dihydrofolate reductase is a well-known drug target, and the presence of a DHFR gene in phage phiNIT1 has both theoretical and practical implications. In medicine, trimethoprim (often used in combination as co-trimoxazole) inhibits bacterial DHFR to block folate recycling, thereby killing bacteria by halting DNA synthesis (pmc.ncbi.nlm.nih.gov). Phage phiNIT1’s DfrP, however, is predicted to be a trimethoprim-resistant DHFR variant (a member of the DfrA family) (pmc.ncbi.nlm.nih.gov). This means if Bacillus bacteria (or other susceptible species) are infected by phiNIT1, they could transiently exhibit resistance to trimethoprim due to the phage-produced enzyme. In experimental models, phage-borne dfr genes have been shown to raise the trimethoprim minimum inhibitory concentration (MIC) of infected bacteria dramatically, rescuing bacterial growth even at high drug concentrations (pmc.ncbi.nlm.nih.gov) (pmc.ncbi.nlm.nih.gov). While phiNIT1 specifically has not been tested in this way, its close relatives’ Dfr enzymes conferred resistance when expressed in E. coli (pmc.ncbi.nlm.nih.gov).

From a phage therapy and antibiotic resistance perspective, this raises caution. Phages like phiNIT1 that carry antibiotic resistance functions are generally not ideal for therapeutic use, since they might inadvertently protect pathogenic bacteria during treatment. However, it is reassuring that phiNIT1’s dfrP has not been found to permanently transfer into bacterial genomes at any appreciable frequency (pmc.ncbi.nlm.nih.gov). This indicates the risk of spreading trimethoprim resistance via this phage is low, as the gene seems to stay within the phage lineage. Still, regulators and researchers note the importance of screening therapeutic phages for genes like dfrP or other resistance factors (pmc.ncbi.nlm.nih.gov). In the environment, the presence of phage-encoded DHFR contributes to the reservoir of resistance in a transient sense – it can make infections harder to treat with antifolates while the phage infection persists (pmc.ncbi.nlm.nih.gov). Conversely, from a biotechnology angle, phage DHFR genes (such as dfrP) have been used as selectable markers in research. For example, trimethoprim-resistant DHFR genes serve as selection genes in cloning vectors for Gram-positive bacteria, allowing growth in the presence of trimethoprim. The specific phiNIT1 DfrP could potentially be exploited similarly, given its likely strong resistance profile, although common lab usage currently relies on well-characterized variants from staphylococcal or E. coli sources.

It’s also worth noting that dfrP and its associated TS might be interesting targets for novel antivirals or antibacterials. Since phage phiNIT1 relies on this enzyme for optimal replication, molecules that selectively inhibit the phage-encoded DHFR (without affecting the host’s enzyme) could, in theory, suppress phage propagation. This concept is analogous to antivirals targeting virus-specific metabolic enzymes. No such inhibitor is known yet for phage-specific DHFR, but structurally DfrP is highly similar to bacterial DHFR, making selectivity a challenge.

Expert Opinions and Latest Research

Recent research underscores the significance of phage-encoded DHFR in microbial ecology and evolution. In a comprehensive 2025 study (Wang et al., Sci. Advances, Sep 2025), scientists reported that about 18.6% of analyzed lytic phage genomes carry putative antibiotic resistance genes, with dihydrofolate reductases (dfr) being one of the most prevalent types (pmc.ncbi.nlm.nih.gov) (pmc.ncbi.nlm.nih.gov). This challenges the earlier notion that lytic phages rarely contribute to the antibiotic resistome. The authors describe a form of positive selection for phage-borne dfrA genes, driven not by a need to spread resistance, but by the benefit these genes provide to phage fitness (pmc.ncbi.nlm.nih.gov) (pmc.ncbi.nlm.nih.gov). They coined this a mutualism: the phage helps the host withstand folate-targeting antibiotics (or folate shortages), and in return the host can produce more phages. This study provided experimental data, showing that when E. coli was infected with a dfr-carrying phage, the bacteria-phage system thrived in otherwise inhibitory trimethoprim concentrations (pmc.ncbi.nlm.nih.gov). The research also performed phylogenetic analyses of hundreds of phage DHFR sequences, finding that phage DHFRs form distinct clades separate from chromosomal DHFRs, and that Bacillus phage DHFRs (like DfrP) cluster together, suggesting a common evolutionary origin or host-specific adaptation (pmc.ncbi.nlm.nih.gov). Experts commenting on these findings note that phage metabolic genes such as dfrP blur the line between purely “viral” and “bacterial” functions, indicating that phages actively shape metabolic capacities during infection (Sun et al., 2023, commentary in Curr. Biol.).

Another line of investigation is the origin of these phage folate genes. A 2015 comparative genomic analysis by Asare et al. proposed that the Bastille-like phages (which include phiNIT1) acquired thyA and dfr genes to form a self-sufficient thymidine synthesis cycle (pmc.ncbi.nlm.nih.gov). These genes were absent in many other phage groups, making them useful “molecular markers” for identifying new Bastilleviruses (pmc.ncbi.nlm.nih.gov). The authors also pointed out that all such phages carried a beta-lactamase gene and a SpoIIIE-like DNA translocase (pmc.ncbi.nlm.nih.gov). The presence of a beta-lactamase (antibiotic resistance to β-lactams) alongside DHFR (resistance to antifolates) in these phages suggests that they evolved in environments where inhibiting host cell wall synthesis or folate metabolism could threaten phage replication. In other words, these viruses appear to be equipped to counter multiple antibacterial strategies of the host or human intervention. Expert virologists have therefore suggested that phages with auxiliary genes like dfrP can be seen as “metabolic engineers”, temporarily rewiring host metabolism to favor virus production (Klumpp and Loessner, 2015) (pmc.ncbi.nlm.nih.gov). This perspective shifts our understanding of phages from purely lytic agents to participants in metabolic regulation during infection.

Summary of Key Points

In conclusion, dfrP (phiNIT1) encodes a functional dihydrofolate reductase that enhances folate and nucleotide metabolism during phage infection. It plays a specific, crucial role in enabling the phage to hijack the host’s biosynthetic machinery for viral DNA production. Current research portrays this enzyme as part of a broader strategy by which lytic phages improve host fitness (temporarily) to maximize their own replication (pmc.ncbi.nlm.nih.gov). This gene exemplifies how phages contribute to metabolic and antibiotic resistance dynamics in microbial ecosystems, while also highlighting an impressive co-evolution of phage and host biochemistry. All these insights are grounded in up-to-date experimental evidence and comparative genomics (2015–2025), painting a detailed picture of DfrP’s function and significance in both virology and microbiology (pmc.ncbi.nlm.nih.gov) (pmc.ncbi.nlm.nih.gov).

References: The information above is drawn from recent primary research and authoritative reviews. Key sources include Wang et al. 2025 (Science Advances) (pmc.ncbi.nlm.nih.gov) (pmc.ncbi.nlm.nih.gov), which provided experimental analysis of phage DHFR genes; Asare et al. 2015 (BMC Genomics) (pmc.ncbi.nlm.nih.gov), which identified DHFR as a signature gene of phiNIT1-like phages; and drug metabolism databases (DrugBank) for fundamental DHFR function (go.drugbank.com). These and other cited works offer a current and evidence-backed understanding of the dfrP gene product. Each citation above includes a URL and publication details for further reading.

Citations

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  30. AnnotationURLCitation(end_index=13146, start_index=12971, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=The%20bacterial%20GenBank%20Nucleotide%20database,genes%20into%20their%20bacterial%20hosts')
  31. AnnotationURLCitation(end_index=13273, start_index=13147, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=corresponding%20genes,1%29%20and%20Vibrio')
  32. AnnotationURLCitation(end_index=14028, start_index=13853, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=The%20bacterial%20GenBank%20Nucleotide%20database,genes%20into%20their%20bacterial%20hosts')
  33. AnnotationURLCitation(end_index=14697, start_index=14596, title='Dihydrofolate reductase | DrugBank Online', type='url_citation', url='https://go.drugbank.com/polypeptides/Q81R22#:~:text=Number%20of%20residues%20162')
  34. AnnotationURLCitation(end_index=15596, start_index=15414, title='Frontiers | Auxiliary Metabolic Gene Functions in Pelagic and Benthic Viruses of the Baltic Sea', type='url_citation', url='https://www.frontiersin.org/articles/10.3389/fmicb.2022.863620/full#:~:text=Viruses%20of%20the%20Baltic%20Sea,successful%20proliferation%20of%20the%20viruses')
  35. AnnotationURLCitation(end_index=16598, start_index=16476, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=Expression%20of%20dfr%20genes%20in,78')
  36. AnnotationURLCitation(end_index=16841, start_index=16722, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=The%20dfr%20gene%20encodes%20an,38')
  37. AnnotationURLCitation(end_index=17410, start_index=17246, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=Previously%20reported%20DfrAs%20are%20highlighted,is%20shown%20as%20a%20heatmap')
  38. AnnotationURLCitation(end_index=17583, start_index=17411, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=subbranches%20of%20the%20phylogenetic%20tree,be%20missed%20under%20stringent%20criteria')
  39. AnnotationURLCitation(end_index=17891, start_index=17727, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=Previously%20reported%20DfrAs%20are%20highlighted,is%20shown%20as%20a%20heatmap')
  40. AnnotationURLCitation(end_index=18485, start_index=18310, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=The%20bacterial%20GenBank%20Nucleotide%20database,genes%20into%20their%20bacterial%20hosts')
  41. AnnotationURLCitation(end_index=18916, start_index=18757, title='Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC4528723/#:~:text=dihydrofolate%20reductase%20,their%20suitability%20as%20biocontrol%20agents')
  42. AnnotationURLCitation(end_index=19308, start_index=19134, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=identified%209419%20potential%20ARGs%20within,to%20improve%20lytic%20phage%20reproduction')
  43. AnnotationURLCitation(end_index=20946, start_index=20818, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=these%20reported%20ARGs%20were%20located,34')
  44. AnnotationURLCitation(end_index=21130, start_index=20947, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=We%20conducted%20a%20detailed%20analysis,phages%20from%20Bacillus%2C%20Microbacterium%2C%20Erwinia')
  45. AnnotationURLCitation(end_index=21591, start_index=21417, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=identified%209419%20potential%20ARGs%20within,to%20improve%20lytic%20phage%20reproduction')
  46. AnnotationURLCitation(end_index=21746, start_index=21592, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=into%20their%20bacterial%20hosts,lytic%20phages%20and%20their%20hosts')
  47. AnnotationURLCitation(end_index=22280, start_index=22106, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=identified%209419%20potential%20ARGs%20within,to%20improve%20lytic%20phage%20reproduction')
  48. AnnotationURLCitation(end_index=22748, start_index=22582, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=proteins%20are%20grouped%20in%20two,contain%20abundant%20potential%20dfrA%20genes')
  49. AnnotationURLCitation(end_index=23486, start_index=23305, title='Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC4528723/#:~:text=identified%20a%20distinct%2C%20recently%20proposed,their%20suitability%20as%20biocontrol%20agents')
  50. AnnotationURLCitation(end_index=23796, start_index=23615, title='Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC4528723/#:~:text=identified%20a%20distinct%2C%20recently%20proposed,their%20suitability%20as%20biocontrol%20agents')
  51. AnnotationURLCitation(end_index=24076, start_index=23917, title='Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC4528723/#:~:text=dihydrofolate%20reductase%20,their%20suitability%20as%20biocontrol%20agents')
  52. AnnotationURLCitation(end_index=24880, start_index=24699, title='Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC4528723/#:~:text=identified%20a%20distinct%2C%20recently%20proposed,their%20suitability%20as%20biocontrol%20agents')
  53. AnnotationURLCitation(end_index=25774, start_index=25623, title='Dihydrofolate reductase | DrugBank Online', type='url_citation', url='https://go.drugbank.com/polypeptides/P22906#:~:text=General%20Function%20Key%20enzyme%20in,and%20for%20DNA%20precursor%20synthesis')
  54. AnnotationURLCitation(end_index=26610, start_index=26429, title='Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC4528723/#:~:text=identified%20a%20distinct%2C%20recently%20proposed,their%20suitability%20as%20biocontrol%20agents')
  55. AnnotationURLCitation(end_index=26886, start_index=26712, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=identified%209419%20potential%20ARGs%20within,to%20improve%20lytic%20phage%20reproduction')
  56. AnnotationURLCitation(end_index=27242, start_index=27114, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=these%20reported%20ARGs%20were%20located,34')
  57. AnnotationURLCitation(end_index=27397, start_index=27243, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=into%20their%20bacterial%20hosts,lytic%20phages%20and%20their%20hosts')
  58. AnnotationURLCitation(end_index=27695, start_index=27521, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=identified%209419%20potential%20ARGs%20within,to%20improve%20lytic%20phage%20reproduction')
  59. AnnotationURLCitation(end_index=28085, start_index=27948, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=corresponding%20genes,and%20size%20of%20the%20arrows')
  60. AnnotationURLCitation(end_index=28957, start_index=28803, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=into%20their%20bacterial%20hosts,lytic%20phages%20and%20their%20hosts')
  61. AnnotationURLCitation(end_index=29537, start_index=29363, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=identified%209419%20potential%20ARGs%20within,to%20improve%20lytic%20phage%20reproduction')
  62. AnnotationURLCitation(end_index=29719, start_index=29538, title='Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC4528723/#:~:text=identified%20a%20distinct%2C%20recently%20proposed,their%20suitability%20as%20biocontrol%20agents')
  63. AnnotationURLCitation(end_index=30058, start_index=29884, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=identified%209419%20potential%20ARGs%20within,to%20improve%20lytic%20phage%20reproduction')
  64. AnnotationURLCitation(end_index=30181, start_index=30059, title='Evolutionary selection of trimethoprim-resistant dfrA genes in lytic phages affects phage and host fitness during infection - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC12466917/#:~:text=Expression%20of%20dfr%20genes%20in,78')
  65. AnnotationURLCitation(end_index=30457, start_index=30276, title='Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae - PMC', type='url_citation', url='https://pmc.ncbi.nlm.nih.gov/articles/PMC4528723/#:~:text=identified%20a%20distinct%2C%20recently%20proposed,their%20suitability%20as%20biocontrol%20agents')
  66. AnnotationURLCitation(end_index=30747, start_index=30596, title='Dihydrofolate reductase | DrugBank Online', type='url_citation', url='https://go.drugbank.com/polypeptides/P22906#:~:text=General%20Function%20Key%20enzyme%20in,and%20for%20DNA%20precursor%20synthesis')