Evolution of new genes under intermittent selection

Evolution of new genes under intermittent selection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Evolution of new genes under intermittent selection Joakim Näsvall, Hind Abdalaal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4087552/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract New genes can evolve by mutations that generate a new function in an existing gene 1 . However, these mutations often have a negative impact on the original function, leading to trade-offs that constrain their further evolution 2 . Genes that exhibit a strong trade-off between the original and the new function are expected to evolve through gene duplication, which can increase the expression of a weak new activity, buffer against negative effects on the original function, and provide more targets for beneficial mutations to arise 3 . The expected outcome of evolution in conditions where both functions are beneficial is a new pair of paralogs, each specialized for one function. Despite this, there are examples in nature where bi-functional generalist enzymes have evolved from a presumed specialist ancestor 4 . This study tests the hypothesis that generalist enzymes can evolve from specialist ancestral enzymes if selection for the new function is repeatedly interrupted by periods without selection ( i.e. selection for the new function is intermittent). In evolution experiments using bacteria lacking an enzyme in the tryptophan synthesis pathway, with intermittent selection for restoring tryptophan synthesis, multiple examples were found where initially specialized enzymes in two different pathways evolved towards becoming bi-functional generalist enzymes supporting both their new and original functions. Our results highlight the importance of considering selection in nature not as a constant, but as a force that may fluctuate, and that fluctuating selection can drastically change the outcome by forcing evolution along paths that are highly constrained by conflicting selection pressures. Especially for duplications, which are mechanistically unstable and often costly, intermittent selection is expected to have a huge impact: If selection for a beneficial limiting function fluctuates, duplications may be counter selected and lost at regular intervals, forcing alternative paths of evolution that do not require duplications. Biological sciences/Evolution/Experimental evolution Biological sciences/Microbiology/Bacteria/Bacterial evolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Since Susumo Ohno’s influential book Evolution By Gene Duplication 5 , gene duplication has been recognized as major driver of evolutionary innovation. Ohno’s hypothesis proposes that gene duplications allow redundant copies to evolve new functions while freed from purifying selection on the original function. However, the hypothesis relies on several assumptions: ( i ) a gene needs to be duplicated before it is allowed to evolve a new function, ( ii ) gene duplications are selectively neutral, ( iii ) gene duplications are stable, and, ( iv ) mutations in the absence of selection can generate new, beneficial functions. The first assumption appears reasonable at a first glance. Mutations that generate a new function often disrupts the original function due to evolutionary trade-offs between the two functions 6 . The latter three of these assumptions do not align with empirical observations. Gene duplications are seldomly neutral and often confer disadvantages to their carriers 7 . Furthermore, tandem gene duplications, the most common type of duplications in bacteria 8 and eukaryotes 9 , are inherently unstable 10 , 11 . This instability limits their lifespan, making it difficult to accumulate beneficial mutations 3 . This is further confounded by the fact that deleterious mutations are far more frequent than beneficial mutations 3 . Therefore, a gene duplicate persisting for a long time without selection is much more likely to acquire loss-of-function mutations, making it a pseudogene, than to evolve a new beneficial function. Despite these shortcomings of Ohno’s model for new gene evolution through duplication – divergence, the role of gene duplication in generating new genes remains undeniable. The innovation-amplification-divergence (IAD) model offers an alternative pathway, addressing the limitations of Ohno's model 3 , 12 . Unlike Ohno's proposal that gene duplications persist due to neutrality, the IAD model suggests they are maintained because they provide a crucial benefit: boosting the expression of a weak, newly evolving activity of a gene. As such, under long-term selection favoring the improvement of this weak activity, duplications and amplifications accumulate and are further stabilized by selection. This above assumption is corroborated in our previous study, where we investigated the evolutionary trade-offs associated with mutations enabling the Salmonella enterica HisA enzyme to gain a new function in tryptophan synthesis (replacing TrpF) while maintaining its original function in histidine biosynthesis (details in Box 1 and Figure S1 ). Most mutations conferring the new function resulted in complete loss of the original activity, highlighting a strong trade-off between the two. Additionally, mutations further improving the new function rapidly eliminated any remaining ancestral function 13 . These findings suggest that the evolution of a bi-functional enzyme that could perform both functions is unlikely. However, some species within the Actinobacteria phylum challenge this notion. They lack a trpF gene and possess a single HisA ortholog, PriA, that performs both histidine and tryptophan biosynthesis functions 4 , 14 . One explanation comes from the suggestion that ancient enzymes were more promiscuous than their modern counterparts 1 . According to this explanation, PriA has retained its promiscuity for over 2 billion years, despite no selection pressure to maintain TrpF activity for much of that time, while most other HisA orthologs have become specialized 15 . While this explanation holds merit, an alternative exists: PriA could have evolved from a specialized ancestral HisA under the right conditions. This possibility suggests that modern specialized HisA orthologs might evolve TrpF activity without necessarily losing their original function. Continuous Selection Favors Divergence Through Gene Duplication Under continuous selection pressure, gene duplications and amplifications are enriched and persist due to increased expression of a weak evolving activity of a gene 3 , 12 . Duplication enables one copy to retain the original function while the other accumulates mutations that enhance the new activity. This frees the evolving copy from the constraints of maintaining the original function, allowing selection to act on the full spectrum of mutations that enhance the new activity, even if they disrupt the original one. Additionally, every mutation that further improves the new activity increases the probability of losing the original function. Consequently, even with a low frequency of loss of the original function, evolution after gene duplication is expected to be biased towards divergence into two distinct, specialized genes. Hypothesis: Intermittent Selection Favors Bi-functional Genes Gene duplications and amplifications often incur fitness costs 7 and exhibit inherent instability 10 , 11 . In environments where selection for the new activity is interrupted by periods without selection pressure, duplications may be frequently lost. Under these conditions, only mutations that maintain the original function are available for selection. We propose that intermittent selection may limit the range of selectable mutations, biasing evolution towards the creation of bi‑functional generalist genes. The relative frequencies of mutations that retain or disrupt the original function (essentially, the dominant trade-off within the system, as illustrated in Fig. 1 ) will further influence the "choice" between the two evolutionary pathways: duplication-divergence or the evolution of a bi-functional gene. Here, we have tested this hypothesis in evolution experiments with intermittent selection for a new function (Fig. 1 b, c). Briefly, populations of bacteria lacking the trpF gene, and therefore unable to grow without an external source of tryptophan, were passaged in a minimal medium with a limiting amount of tryptophan. Under these conditions the ancestral strain can grow for a few generations using the added tryptophan but once tryptophan is depleted growth ceases and only mutants able to synthesize tryptophan are able to continue growth. Thus, each growth cycle can be divided into two stages; an initial phase without selection for the new function and a second phase with selection. Box 1. Relevant enzymes in the histidine and tryptophan biosynthetic pathways. HisA – ProFAR isomerase ; catalyzes the conversion of N'-[(5'-phosphoribosyl)-formimino-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N'-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR), the fourth step in histidine biosynthesis. Encoded by the hisA gene in the his operon. TrpA – Tryptophan synthase subunit A ; together with tryptophan synthase subunit B (TrpB) catalyzes the two-step conversion of (3-indolyl)-glycerol-phosphate (IGP) to L-tryptophan, the final step in tryptophan biosynthesis. Encoded by the trpA gene in the trp operon. TrpF – Phosphoribosyl anthranilate isomerase ; catalyzes the conversion of N-(5’-phosphoribosyl)-anthranilate (PRA) to 1-(2-carboxyphenylamino)-1’-deoxyribulose-5’-phosphate (CdRP), the third step in tryptophan biosynthesis. In Enterobacteriaceae fused to the C-terminal of the TrpC protein (catalyzing the fourth step in the pathway). Here, I refer to the 202 amino acid C-terminal domain of the fused TrpCF peptide as TrpF and the corresponding 606 bp DNA sequence as the trpF gene. Mutants lacking any of these activities are unable to synthesize the respective amino acids and are therefore unable to grow in medium without added histidine ( hisA -mutants) or tryptophan ( trpA and trpF mutants) and are said to be auxotrophic for histidine or tryptophan, respectively. Strains that can synthesize both amino acids are said to be prototrophic for histidine and tryptophan. For simplicity, the proteins and their enzymatic activities are referred to in the text by the corresponding gene product names (HisA – HisA activity etc. for the enzyme encoded by the hisA gene and its native activity). When HisA or TrpA catalyzes the reaction that is normally catalyzed by TrpF, the activity is referred to as TrpF activity. Results To test the hypothesis that intermittent selection for a new evolving function leads to evolution of bi-functional generalist genes rather than duplication and divergence into two specialized genes, we conducted evolution experiments with Salmonella enterica . We grew populations of strains lacking the trpF gene through serial passages in batch cultures in medium with a limiting amount of tryptophan (Fig. 1 ). This design created intermittent selection for TrpF activity. Initially, sufficient tryptophan was available in each growth cycle, allowing all cells to grow. However, after a few generations, tryptophan became depleted, making TrpF activity essential for continued growth. Evolution of TrpF activity in HisA under intermittent selection Eight populations were evolved for approximately 430 generations. By the end of the experiment, three populations exhibited growth after tryptophan depletion (Fig. 2 b, Table S1 ). Whole genome sequencing (WGS) of these populations revealed that two populations harbored multiple amino acid substitutions within the hisA gene (Table S1 ). Additionally, these populations contained mutations known to elevate mutation rates (mutators; frameshift mutations in mutH and mutS , respectively; Table S2) and many additional mutations scattered across their genomes. The third population lacked mutations in any readily identifiable target gene and its mechanism of adaptation remains unclear (Supplementary text, Table S2). Mutators cause a shift in mutation target and type To investigate the role of mutator mutations in acquiring TrpF activity and explore alternative evolutionary paths, a new experiment was designed. It compared 24 populations with mutator mutations ( mutS ) to 24 populations without them ( mutS +; non-mutators). These populations were subjected to the same intermittent selection conditions for approximately 78 population doublings. Notably, after this period, 10 of the non-mutator populations and 21 of the mutator populations exhibited detectable growth within 7 days after tryptophan depletion (Fig. 2 c and d). Whole genome sequencing (WGS) of a subset of populations, followed by targeted Sanger sequencing in the remaining ones, identified candidate mutations responsible for TrpF activity. These mutations were found in either hisA (in 9 mutator populations) or trpA (in 11 mutator and 10 non-mutator populations). Interestingly, all sequenced non-mutator populations and 6 of the mutator populations shared the same trpA mutation: a 7-bp duplication causing a frameshift (details in Supplementary Text and Figure S6). One non-mutator population also acquired an additional amino acid substitution (A67T) in trpA within a subpopulation lacking the frameshift mutation. Notably, one mutator population lacked mutations in either hisA or trpA and remains unexplained (Table S2; Supplementary text). These results demonstrate that while mutator mutations were not essential for the evolution of TrpF activity under intermittent selection, they influenced the types of mutations found. Thus, S. enterica can evolve TrpF activity through mutations in two distinct genes ( hisA and trpA ). Additionally, at least one other pathway exists for which we have not identified the mechanism. In the minimal medium used for the evolution experiment, both HisA and TrpA activities are essential for growth. HisA is required throughout each growth cycle, while TrpA becomes necessary only after tryptophan depletion. Notably, only one whole-genome sequenced population harbored a duplication/amplification event within the target gene ( hisA ). This single instance suggests the possibility of further evolution by duplication-divergence. However, analysis of the sequence reads for this population did not suggest the presence of diverging mutations in any hisA copy that might have lost one of the original or evolving activities. These observations support our hypothesis that intermittent selection can limit the chance of evolution by duplication-divergence by restricting the window of opportunity for duplications to become enriched and persist before selection pressure for the new function is relaxed. All HisA paths start with the same mutation To determine the order of appearance for mutations in populations harboring multiple hisA or trpA mutations, we sequenced the target genes in clones isolated from earlier time points (Table S3). Consistent with our previous findings that single amino acid substitutions in HisA rarely confer TrpF activity without compromising HisA function 13 , all evolutionary paths involving hisA mutations began with the same mutation (Q18R). Conversely, trpA displayed a greater diversity of starting mutations, suggesting a potentially weaker trade-off between the two enzymatic activities in this gene compared to hisA . To validate the identified hisA and trpA mutations as responsible for TrpF-independent growth, and to isolate the effects of these variants from the numerous background mutations accumulated during the evolution experiments, we reconstructed each identified hisA and trpA variant from the final populations, along with most intermediate variants from earlier time points, for further analysis. Successive mutations improve overall fitness at the conditions of the evolution experiments To assess the fitness benefit conferred by the reconstructed hisA and trpA variants, we measured the growth of strains harboring these variants in the same minimal medium used in the evolution experiments (Fig. 3 ). Following tryptophan depletion, growth becomes dependent on all three activities (the native HisA and TrpA activities, and the evolved TrpF activity). Notably, all identified hisA and trpA variants enabled growth after tryptophan depletion (Fig. 3 ). Furthermore, in most cases where the complete mutational pathways were reconstructed, each additional mutation within a pathway resulted in improved growth after tryptophan depletion. HisA, but not TrpA, tends to lose its native function when TrpF activity improves Prior to tryptophan depletion, all reconstructed trpA mutants exhibited growth rates indistinguishable from the ancestral strain, which itself grew similarly to a prototrophic (TrpF+) strain (Figures S2 and S5). This is unsurprising, as TrpA activity is not essential when tryptophan is present in the medium. In contrast, all hisA mutants displayed slower growth compared to the ancestral strain, and each additional hisA mutation further reduced the growth rate (Figs. 4 a, S3, and S4). This growth reduction in hisA mutants could be partially restored by supplementing the medium with guanosine. This rescue effect likely reflects a metabolic link between histidine, tryptophan, and purine nucleotide biosynthesis (Figures S3 and S4; Supplementary text). However, even with guanosine supplementation, all hisA mutants grew significantly slower than a wild-type hisA control strain (Fig. 4 a), indicating a measurable decline in the native HisA function. Since TrpF and TrpA act within the same pathway, growth curves of trpA mutants cannot reveal the extent of activity for either function. Hypothetically, a trpA mutant with very weak TrpA activity but strong TrpF activity could exhibit a growth curve identical to one with the opposite functional relationship. To evaluate the impact on native TrpA activity, we introduced a wild-type trpF gene under a strong constitutive promoter at an unrelated locus in ten reconstructed trpA mutants. Following complementation with wild-type trpF , six trpA mutants displayed growth rates indistinguishable from their isogenic wild-type counterparts, suggesting minimal loss of TrpA activity (Fig. 4 b). However, four mutants grew significantly slower, indicating a decline in TrpA function. One of these activity-compromised variants harbored the P62fs frameshift mutation, which is expected to be poorly expressed due to the requirement for frameshift suppression (Supplementary text, Figure S6). Interestingly, three variants originated from the same evolutionary pathway, where the initial mutation (G61S) resulted in TrpA activity loss, and the subsequent mutation (P62S) restored TrpA function. In contrast to the hisA mutants, these findings suggest that some trpA mutants acquired TrpF activity without a substantial trade-off in their native function. This observation may point towards a weaker functional trade-off between the two activities within TrpA compared to HisA. Discussion Several models for the evolution of new genes by modification of existing genes build on the assumptions that the new function either appears after the duplication of the ancestral gene 5 , or that it appears as a selectively neutral promiscuous activity that at some point happens to become beneficial due to a change in the environment 3 . Therefore, in a previous evolution experiment, we started with a HisA double mutant. One mutation generated weak TrpF activity but led to the complete loss of the original HisA activity, and an additional mutation restored some of the HisA activity 12 . Furthermore, the mutant gene was placed under a strong constitutive promoter and in a location known to be prone to amplification. The mutant bacteria were subjected to continuous selection for both HisA and TrpF activities for roughly 3,000 generations. During this experiment, the mutant hisA gene amplified to a high copy number and accumulated divergent mutations (up to three per gene copy). These mutations improved one or the other activity in separate gene copies, ultimately leading to the evolution of two specialized enzymes in most cases. Here, we instead started with strains with the wild-type hisA and trpA genes in their native loci and with their native regulation intact. In addition to the interruptions in selection pressure for the new function, which is likely to reduce the chance of selection of gene duplications, the native location of the his - and trp -operons near the replication terminus region leads to low rates of duplication formation 8 . Still, in these experiments quite rapid evolution with several mutations in the hisA or trpA genes were seen in relatively short time-span. Curiously, evolution through the generation of TrpF activity in HisA was only seen in the presence of mutators, while in the absence of mutators a frameshift mutation in trpA was found in multiple independent populations and only one population had a SNP (in trpA ) that conferred TrpF activity. The absence of other mutations in trpA or hisA in the non-mutator populations can be explained by three factors: ( i ) Relatively short time span: The experiment only lasted for approximately 78 population doublings, limiting the opportunity for mutations to accumulate. ( ii ) Small population size: The total population size at the end of each cycle was small (around 10⁹ cells in 5 ml), reducing the chance of beneficial mutations arising. ( iii ) Very small mutational target size: There were likely less than ten possible single nucleotide substitutions that could generate TrpF activity in hisA or trpA . The cumulative number of cells per population in the experiment corresponds to less than 10¹¹ cells. With mutation rates for single nucleotide substitutions ranging between 10⁻¹⁰ – 10⁻¹² per cell/nucleotide/generation in non-mutator strains 16 , the specific nucleotide substitutions in hisA or trpA would likely occur at least once per population during our experiments. In contrast, the observed frameshift mutation is a duplication of 7 bp in a 12 bp sequence containing two short tandem repeats (5 bp each; Figure S6). Mutations of this type are known to occur at rates that are orders of magnitude above the rates of single nucleotide substitutions in E. coli 17 . Furthermore, the frameshift mutation outperforms all of the reconstructed single amino acid substitutions in both hisA and trpA (Fig. 3 ). Thus, in the absence of mutators, the frameshift mutation is expected to happen more often (many times in each population) and once it does, it outcompetes any single nucleotide substitutions that may have been generated. Our findings support the notion that interrupting selection for the new activity with periods lacking selection pressure disfavors gene duplication as a solution for evolving the new function while retaining the original one. While gene duplication offers the advantages of both increased beneficial activity and buffering against loss of the original function, periods without selection are expected to lead to the loss of unstable duplications and counter-selection against mutants with reduced original function. Although we lack direct evidence that gene duplications or amplifications were not present at some point in some of the populations, the absence of evolved clones harboring duplicated hisA genes with the expected loss of HisA activity in one copy serves as indirect evidence against stabilization of duplications by selection. Mutations that improve TrpF activity in HisA, at the expense of complete HisA loss, are demonstrably more common than those that retain both functions 12 , 13 . Therefore, if duplications were present, they would almost invariably lead to the divergence into two separate enzymes. Analysis of reconstructed mutant growth curves under the same conditions as the evolution experiment (Figures S3a and S4a) reveals that these conditions present a more complex evolutionary landscape than what can be solely predicted by growth rates before and after tryptophan depletion. Notably, even non-growing or slow-growing cells consume resources from the medium. Consequently, after tryptophan depletion, the ancestral strain continues to utilize other essential resources (carbon, nitrogen, phosphorus, sulfur, minerals, trace elements) until the medium becomes exhausted and no further growth is possible. This introduces a time-dependent selection pressure: a beneficial mutation must enable significant growth between tryptophan depletion and depletion of the next essential resource. Additionally, post-depletion growth needs to compensate for any growth deficit incurred before tryptophan depletion. This complex interplay between maintaining the original function and acquiring the new activity imposes a stringent selective filter, potentially limiting the spectrum of viable mutations. The three enzymes HisA, TrpA and TrpF are structurally related proteins with an (αβ) 8 -barrel fold that are proposed to have evolved from a common ancestor 18 . Additionally, HisA catalyzes the same isomerization reaction as TrpF, albeit on a bulkier substrate. Previous evolution experiments have succeeded in regenerating TrpF activity in HisA, but have often resulted in complete loss of the original function 12 , 19 . Similarly, our research investigating the functional trade-offs between HisA and TrpF activities in Salmonella enterica revealed that mutations generating or enhancing TrpF activity rarely preserve HisA activity 13 . Intriguingly, in Actinobacteria lacking a native TrpF ortholog, PriA (a HisA ortholog) is active in both the histidine and tryptophan pathways 4 . This suggests the existence of evolutionary paths towards bifunctional enzymes, even in the presence of a seemingly strong functional trade-off. Our findings indicate that during intermittent selection for the new function, evolution can proceed through rare mutations with weak trade-offs. However, when selection for the original activity is relaxed (by gene duplication), these mutations are likely outnumbered by more frequent mutations with strong trade-offs. Evran et al. 20 employed rational design to introduce putative catalytic residues from TrpF into TrpA, but only achieved TrpF activity after extensive mutagenesis and gene shuffling. Notably, their proposed essential mutations (F22C and L177D) were not sufficient for detectable TrpF activity. In contrast, our evolutionary approach identified several single amino acid substitutions (G98C, D27Y, G61S, Y102H) that directly conferred TrpF activity, without introducing the proposed catalytic residues. Interestingly, some mutations identified by Evran et al. 20 as "permissive" (G61S, T24S) were sufficient for TrpF activity in our experiments, suggesting these mutations may play a more significant role than previously thought. These findings highlight the potential limitations of structure-guided design compared to experimental evolution for introducing new activities in enzymes. However, structural analysis of evolved mutants remains valuable for elucidating the mechanistic basis of the newly acquired function. Conclusion The evolution of a novel function in an enzyme, while preserving its essential original function, is usually seen as an adaptive conflict that require gene duplication. However, this study reveals an intriguing alternative: even in the face of a seemingly strong trade-off, rare evolutionary paths may exist that allow an enzyme to acquire a new function without compromising the original one. These findings support our hypothesis that fluctuating selection pressures, by preventing the selection of gene duplications and disfavoring mutations with severe effects on the original function, can guide evolution towards the emergence of generalist enzymes. Methods Bacterial strains and growth conditions All bacterial strains are derivatives of Salmonella enterica ssp. enterica serovar Typhimurium strain LT2 (except for three Escherichia coli strains that were used as PCR templates for amplification of selection cassettes used in λ Red recombineering). For transferring chromosomal markers between strains, generalized transduction with phage P22 HT 105/1 int-201 21 or a defective prophage derivative of it (see below; “Construction and use of an artificial Gene Transfer Agent”) was used. Lysogeny broth (LB; 10 g/L Tryptone [Sigma], 5 g/L yeast extract [Sigma], 10 g/L NaCl) was used as rich medium, and was supplemented with 15 g/L Bacto agar to make LB agar (LA) plates. Salt-free LB (LB prepared without NaCl) was used for preparing cells for transformation by electroporation. SOC medium 22 was used for recovery after transformations. As minimal medium, M9 medium 23 was prepared with twice the amounts of M9 salts, glucose, CaCl 2 and MgCl 2 in order to allow growth to a higher cell density. Minimal medium was supplemented with tryptophan (W; 0.1 mM [1x] or 5 µM [0.05x, for tryptophan-limited medium]), histidine (H; 0.1 mM), and guanosine (Guo; 0.3 mM) when needed. For simplicity, the base medium is referred to as 2× M9, with any additional additions indicated as + H (histidine) + W (tryptophan) and + Guo (guanosine). To make solid M9 plates, 15 g/L of Bacto agar was added. All growth of bacteria was done at 37°C (except when preparing cells for λ Red recombineering). Antibiotics (trimethoprim [tmp; 10 mg/L; Sigma], tetracyclin [tet; 7.5 mg/L; Sigma], chloramphenicol [cam; 12.5 mg/L; Sigma]) were added to the medium only when needed for selecting recombinants after transformations and transductions. PCR and Sanger sequencing DNA for λ Red recombineering and Sanger sequencing was prepared by PCR using Phusion DNA polymerase (Thermo Fisher). All PCR reactions were made with bacterial cell suspensions as templates; either part of a single fresh colony from an agar plate or a ~ 1 µl sample from a frozen (-80°C) glycerol- or DMSO stock culture was re‑suspended in 100 µl ultrapure water (Sigma). Of this, 1 µl was used per 20 µl of PCR reaction. PCR cycling parameters have been described elsewhere 24 – 26 . For de-salting, removal of oligonucleotides and primer-dimers, and concentration of the DNA, PCR products were precipitated with polyethylene glycol (PEG). Briefly, PCR reactions were mixed 1:1 with a 2× PEG precipitation solution (24% [w/v] PEG 8000 [Sigma], 20 mM MgCl 2 in ultra-pure water [Sigma]) and incubated for 10 min at room temperature. DNA was pelleted by centrifugation at ≥ 14,000 × g for 10 min, washed once in ≥ 1 volume of 70% ethanol and re-centrifuged at ≥ 14,000 × g for 10 min, after which it was air-dried. For local sequencing, DNA was dissolved in ultra-pure water, mixed with a sequencing primer (Table S4), and sent to Eurofins Genomics (Ebersberg, Germany) for Sanger sequencing. DNA for recombineering was dissolved in 2–10 µl of ultra-pure water. λ Red Recombineering Mutations, genetic markers, and reporter genes were constructed and inserted using different λ Red recombineering strategies as described elsewhere 25 – 27 . Cultures of bacterial strains carrying the λ Red helper plasmid pSIM5-Tet 28 were grown in salt-free LB plus 0.2% (w/v) glucose at 30°C overnight, after which the cultures were diluted 100-fold into 25 ml of the same medium and allowed to grow at 30°C for one hour. To induce expression of the λ recombination genes, the cultures were transferred to a 42°C shaking water bath and incubated for another 15 min. After cooling in an ice-water bath for several minutes the cultures were pelleted by centrifugation and washed once in 1 ml ice-cold de-ionized water or 10% glycerol. The cells were resuspended in 200 µl of ice-cold de-ionized water or 10% glycerol. For each transformation, DNA (0.2–2 µl of de-salted and concentrated PCR product) was mixed with 20 µl cell-suspension in an electroporation cuvette (Bio-Rad, 1 mm gap) and electroporated (2.5 kV, 200 Ω, 25 µF in a Gene Pulser Xcell; BioRad). Immediately after electroporation the cell suspensions were re-suspended in 200 µl pre-warmed (42°C) SOC, transferred to a 10- or 50 ml plastic tube and incubated at 42°C for 15 minutes before plating on selective medium. All DNA oligonucleotides are listed in Table S4. For re-construction of mutations in hisA and trpA , the target genes containing the mutations were PCR amplified from evolved populations or isolated clones from evolved populations. In some cases, the gene was amplified as two overlapping fragments using a mutant and a wildtype as template in order to avoid one mutation present in a mutant containing several mutations. The PCR products were transformed into strains carrying the selectable and counter-selectable cassette Atox1 26 (GenBank accession: MN207489; containing dhfr [trimethoprim resistance], P rhaB - orph11 [DNAse toxin orphan-11 from E. coli EC869 under the control of the rhamnose inducible rhaB promoter], and amilCP [blue chromoprotein from Acropora millepora ]) in hisA or trpA , selecting for loss of the cassette on M9 + 0.3% (w/v) rhamnose + histidine + tryptophan plates. Deletions, some single nucleotide substitutions, and small insertions were constructed using DIRex 25 , 26 , using “half-cassettes” that were generated by amplifying overlapping parts of Atox1 and Atox2 (GenBank accession: MN207490) as described previously 25 , 26 . Evolution experiments Experiment 1 (populations 1–1 to 1–8) was started from separate cultures of DA52864 (Δ trpF Δ P gal Δ galE :: T lux ). In addition to the deletion of the trpF coding sequence (corresponding to the C-terminal domain of the fused TrpCF protein), it carries a deletion of the gal operon promoter and has the galE gene replaced by the lux transcriptional terminator 24 . These mutations make the strain conditionally resistant to phage P22 infection (resistant in the absence of galactose but sensitive in the presence of galactose), and were used in order to avoid contamination by phage P22 and avoid selection of P22 resistant mutants, which we have occasionally seen in previous experiments. After a first overnight growth in medium containing both histidine and tryptophan, samples from each population were passaged 1:100 into 5 mL tryptophan-limited medium (2× M9 + 0.05× W) in 50 mL conical bottom screwcap tubes, and were continually passaged into the same medium once every seven days for 20 cycles, after which they were passaged 1:1000 into the same medium every 3–4 days (twice a week) for an additional 30 cycles, totaling approximately 430 (20*6.64 + 30*10) generations. As each cycle started with fresh tryptophan-containing medium, growth for the first few generations occurred without any selection for TrpF activity. Once tryptophan was depleted, only mutants able to synthesize tryptophan (due to mutations that generated TrpF activity in another enzyme) could grow, making selection for TrpF activity intermittent. Samples from the populations were frozen at -80°C in 20% DMSO after cycle 10, 15, 20, 25, 40 and 50. Experiment 2 (lineages 2 mut + 1–2 mut + 24 and 2 mutS 1–2 mutS 24) was done similarly to experiment 1, but was started from separate cultures of DA62207 and DA64168 (Δ trpF and Δ trpF mutS ; see supplementary Table S5 for the detailed genotypes of these strains). In addition to the trpF deletion and the mutS mutation, these strains contain the arabinose-inducible defective P22 prophage GTA22 (below), which makes them resistant to lytic growth of phage P22 and makes them convenient both as donors and recipients in transductions. These 48 populations were passaged with 100× dilutions into 5 ml of fresh tryptophan-limited medium (2× M9 + 0.05× W) once every seven days for 13 cycles, at which point all but three mutS populations grew visibly denser. Samples from the populations were frozen at -80°C in 20% glycerol after cycle 4, 5, 6, 7, 8, 10, 12 and 13. Growth curves and growth rate determinations Overnight cultures (in 1 mL 2× M9 + 0.4% glucose + 0.1 mM histidine + 0.1 mM tryptophan) were started either from isolated colonies of pure re-constructed clones or from thawed 50 µL samples from freezer stocks of evolved populations. Pure clones were grown as five biological replicates while populations were grown as a single sample per population. After approximately 24 hours of growth at 37°C the cultures were diluted 1:1000 into tryptophan-free (2× M9 + 0.4% glucose) and tryptophan-limited (same but with 5 µM tryptophan) medium, after which 300 µL of each diluted culture was transferred to a well in a Bioscreen honeycomb plate. To avoid evaporation of media, all empty wells were filled with 300 µL medium or water, and the plates were sealed by taping around the edges. The plates were incubated in a Bioscreen C reader (Oy Growth Curves, Turku, Finland) at 37°C, with continuous moderate shaking, and reading of optical density at 600 nm (OD 600 ) every 4 minutes for 7 days. Despite taping the edges, the wells around the edges of the plate reached a higher final OD than other replicates of the same strain and upon inspection these cultures showed a significant loss of volume. For growth rate determinations, the OD 600 data was plotted to find the exponential phase (which ended at about OD 600 = 0.18 for the cultures in tryptophan-limited media) and the data for only the exponential phase was transferred to Prism 9 or 10 (GraphPad Software). The data was fitted (without subtracting any blank value) to the exponential growth equation Y(t) = b + Y 0 * e kt , where Y(t) is the OD 600 at time t , b is the contribution of the medium and the plastic of the individual well of the Bioscreen plate to the OD 600 (“blank”), Y 0 is the contribution of cells to the OD at time t = 0, k is the growth rate (min − 1 ), and t is the time (min). Differences of < 5% in k between two strains were considered insignificant due to limitations such as the precision of OD measurements and well-to-well variation in the Bioscreen plates. The average growth rate up to 90% of the maximum OD for each culture (Fig. 3 ) was determined as follows: First the OD 600 data was blank-subtracted using the b value from the curve fitting (above) as blank. For each culture the first data point above Y 0 ( Y > Y0 ) and the first point where OD reached above 90% of the maximum OD ( Y 90%ODmax ) within the first three days were determined. The average growth rates between those two points were determined as k avg = [ln( Y 90%ODmax )-ln( Y > Y0 )]/[ t 90%ODmax -t > Y0 ] and are expressed in day − 1 . Whole Genome Re-sequencing Genomic DNA was prepared from 1–2 ml samples from evolving populations using MasterPure (Epicentre). Libraries for MiSeq sequencing (in-house) were made using Nextera DNA library prep and indexing kits (Illumina). Some samples were sequenced by BGI (Beijing, China) using their proprietary technology. Analysis of sequence reads were done with CLC Genomics Workbench (Qiagen). After quality-based trimming to remove any ambiguities and low-quality reads, the reads were mapped against a reference genome containing all modifications present in the ancestral strains. Indels and SNPs were detected using the low frequency variant detector, and structural rearrangements (duplications or deletions) were found using visual scanning of mapped read depth in combination with the structural rearrangement tool in CLC. Amplification copy number in the single population (DA65458; 2mutS8) that contained an amplification of a relevant target gene ( hisA ) was estimated by dividing the mean read depth of the structural genes in the his -operon (7136 bp at 156.49× read depth) with the mean of the mean read depths of four identically-sized regions (4× 7136 bp at 19.95× read depth) just outside of the amplified region. One relevant mutation ( trpA [Pro62Fs]) was reported by CLC genomics workbench to be detected in 30–60% of the reads covering that position. However, when the reads were re-mapped to a reference sequence containing the mutation, 82–100% of the reads from the relevant populations mapped perfectly to the mutation. Isolation and sequencing of clones from evolved populations In order to determine the order of appearance of mutations in populations with more than one mutation in the target gene ( hisA or trpA ) during the evolution experiments, clones were isolated from frozen stocks of earlier time-points, and the target genes were PCR amplified to generate templates for Sanger sequencing. For each frozen population, a streak was made to isolate single colonies on LA plates. Eight colonies from each streak were picked and streaked once more on LA plates, and from each of these eight clones a single colony was used as template for PCR. Some clones with known hisA or trpA alleles were frozen in the strain collection and used as templates in PCR reactions for re-construction of mutations in fresh (unevolved) genetic background. Construction and use of an artificial Gene Transfer Agent After transductions with phage P22, it is of critical importance to isolate phage-free bacterial clones to avoid the lytic spread of phage in cultures as well as to avoid the selection of phage-resistant mutants or lysogens that are impossible to use in later transductions. Additionally, in long-term evolution experiments, we have occasionally found evolving populations to be infected by P22, or to carry mutations that provide resistance to P22, which could be problematic for later genetic experiments. Therefore, an inducible artificial Gene Transfer Agent (GTA; here referred to as GTA22) was constructed (using λ Red recombineering) by three modifications to a P22 HT 105/1 sieA44 lysogen. The mutation HT 105/1 21 in makes DNA packaging into new P22 virions less specific, allowing a high frequency of generalized transduction. The sieA44 mutation prevents the lysogen from blocking entry of DNA from P22 (and related phages) in the periplasm, making the lysogen work as a recipient in transductions. However, P22 also encodes a phase-variable O-antigen conversion locus, gtrABC 29 , which when it is expressed prevents adsorption of P22 to the cell (P22 uses the O-antigen as receptor), making the cells resistant to superinfection (and prevents transduction). To convert this prophage into an inducible gene transfer agent, both ends, including the attachment site, of the prophage were deleted (using DIRex 25 , 26 ). One deletion (Δ thrW - kil ) removes 6.9 kb of DNA from the “left” end of the prophage, including the left copy of the P22 attachment site (in the thrW tRNA gene), the integrase and excisionase genes ( int and xis ), the recombination genes ( abc1 , abc2 and erf ) and the septation inhibitor ( kil ). The other deletion removes 3 kb of DNA including the right copy of the attachment site (a P22 encoded partial duplicate of the thrW gene) and the phage-encoded O-antigen conversion locus ( gtrABC ). Secondly, in order to make strains containing GTA22 work as donor in transductions the prophage was made inducible by placing (using λ Red recombineering) a copy of the P22 antirepressor ( ant P22 ) after the L-arabinose inducible P araBAD promoter in the host genome. Without the attachment site and the integrase/excisionase the prophage cannot excise and circularize its genome when it is induced, and any replication forks starting at the phage origin of replication extends into the surrounding bacterial host genome 30 . Without the O-antigen conversion genes and a functional sieA gene the lysogen cannot prevent adsorption of P22, and will therefore accept incoming DNA from P22 virions. But, as GTA22 still contains a functional P22 repressor ( c2 ) and other regulatory elements, the lysogen is resistant to lytic growth of any superinfecting P22 genome. As packing of the P22 genome into new virions is unidirectional and initiates at a single pac site in the phage genome 30 , GTA22 (being unable to excise and circularize) is unable to pack a complete copy of its genome into new virions, making it essentially avirulent (but it could theoretically form rare virulent virions if a copy of its genome is circularized through illegitimate recombination). Thus, a strain containing GTA22 is resistant to lytic infection by P22 but works efficiently as recipient in transductions. Furthermore, it never (or very rarely) forms virulent virions, which removes the need for screening to avoid phage infected clones. In order to induce GTA22, overnight cultures were diluted 1:20 into LB containing 0.1% (w/v) L-arabinose. After incubation for at least 6 hours to allow lysis, any surviving bacteria were killed by vigorous shaking with chloroform. Lysates were cleared by centrifugation (≥ 14,000 ×g for 1–3 min) before being used in transductions. For transductions, 0.1–10 µl of cleared lysate was mixed with 100 µl LB supplemented with 0.4% glucose (to prevent expression of P araBAD -ant P22 in the recipient due to remaining arabinose in the lysate), after which 100 µl overnight culture of the recipient strain was added. After 30 min incubation at 37°C the transductions were plated on appropriate selective media, and the plates were incubated until colonies appeared (overnight on LA, 24–48 h on M9). Generation of a mutator allele In order to generate a reversible knock-out of mutS , an insertion of the selectable and counter selectable Acatsac1 cassette (GenBank accession: MF124798; containing the genes amilCP , cat , and sacB , conferring blue color, chloramphenicol resistance, and sucrose sensitivity, respectively) was designed as a duplication-insertion (DUP-In) 24 so that it would generate a duplication with a single copy of the Acatsac1 cassette inserted between two truncated mutS copies, one containing the first ~ 1.5 kb of the 2.5 kb mutS gene, and the other containing the last ~ 1.5 kb. As none of the mutS copies are complete they are unlikely to be functional (as was verified from the large amounts of genomic mutations in the evolved mutator populations compared to the isogenic non-mutator populations; Table S2, parts 2 and 3), and due to the presence of the cassette the colonies are blue, chloramphenicol resistant and sucrose sensitive. The reason for making it as a DUP-In was to be able to revert the mutator allele (by plating on sucrose to find cells that lost the cassette by recombination between the 500 bp repeats) in clones isolated from the evolved populations, but this was not tested. Declarations Acknowledgements We thank Anna Knöppel, Omar Warsi, Ramith Nair, and Dan Andersson for critical reading of the manuscript. This work was founded by the Swedish Research Council (VR-NT 2020-03512) and the Carl Trygger Foundation (CTS 22:2094). Author Contributions JN conceived the study, constructed bacterial strains, made bacterial growth rate assays and analyzed data. HA and JN performed evolution experiments, and isolated and sequenced clones from evolved populations. HA wrote an early draft of the manuscript as part of her PhD thesis, JN re-worked and extended the manuscript. Competing interests None. References Jensen, R. A. Enzyme recruitment in evolution of new function. Annu Rev Microbiol 30 , 409–425 (1976). Khersonsky, O. & Tawfik, D. S. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu Rev Biochem 79 , 471–505 (2010). Bergthorsson, U., Andersson, D. I. & Roth, J. R. Ohno’s dilemma: Evolution of new genes under continuous selection. Proc. Natl. Acad. Sci. 104 , 17004–17009 (2007). Barona-Gomez, F. & Hodgson, D. A. Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis. EMBO Rep 4 , 296–300 (2003). Ohno, S. Evolution by Gene Duplication . (Springer-Verlag, 1970). doi:10.1007/978-3-642-86659-3. Soskine, M. & Tawfik, D. S. Mutational effects and the evolution of new protein functions. Nat. Rev. Genet. 2015 168 11 , 572–582 (2010). Reams, A. B., Kofoid, E., Savageau, M. & Roth, J. R. Duplication Frequency in a Population of Salmonella enterica Rapidly Approaches Steady State With or Without Recombination. Genetics 184 , 1077–1094 (2010). Anderson, P. & Roth, J. Spontaneous tandem genetic duplications in Salmonella typhimurium arise by unequal recombination between rRNA (rrn) cistrons. Proc Natl Acad Sci U A 78 , 3113–3117 (1981). Fan, C., Chen, Y. & Long, M. Recurrent Tandem Gene Duplication Gave Rise to Functionally Divergent Genes in Drosophila. Mol. Biol. Evol. 25 , 1451–1458 (2008). Haack, K. R. & Roth, J. R. Recombination between chromosomal IS200 elements supports frequent duplication formation in Salmonella typhimurium. Genetics 141 , 1245–1252 (1995). Hill, C. W., Foulds, J., Soll, L. & Berg, P. Instability of a missense suppressor resulting from a duplication of genetic material. J. Mol. Biol. 39 , 563–581 (1969). Näsvall, J., Sun, L., Roth, J. R. & Andersson, D. I. Real-time evolution of new genes by innovation, amplification, and divergence. Science 338 , 384–387 (2012). Lundin, E., Näsvall, J. & Andersson, D. I. Mutational Pathways and Trade-Offs Between HisA and TrpF Functions: Implications for Evolution via Gene Duplication and Divergence. Front. Microbiol. 11 , 588235 (2020). Due, A. V., Kuper, J., Geerlof, A., von Kries, J. P. & Wilmanns, M. Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis. Proc. Natl. Acad. Sci. U. S. A. 108 , 3554–3559 (2011). Plach, M. G., Reisinger, B., Sterner, R. & Merkl, R. Long-Term Persistence of Bi-functionality Contributes to the Robustness of Microbial Life through Exaptation. PLOS Genet. 12 , e1005836 (2016). Ellis Hudson, R., Bergthorsson, U. & Ochman, H. Transcription increases multiple spontaneous point mutations in Salmonella enterica. Nucleic Acids Res. 31 , 4517–4522 (2003). Torres-Cruz, J. & van der Woude, M. W. Slipped-Strand Mispairing Can Function as a Phase Variation Mechanism in Escherichia coli. J. Bacteriol. 185 , 6990–6994 (2003). Lang, D., Thoma, R., Henn-Sax, M., Sterner, R. & Wilmanns, M. Structural Evidence for Evolution of the β/α Barrel Scaffold by Gene Duplication and Fusion. Science 289 , 1546–1550 (2000). Jürgens, C. et al. Directed evolution of a (βα)8-barrel enzyme to catalyze related reactions in two different metabolic pathways. Proc. Natl. Acad. Sci. 97 , 9925–9930 (2000). Evran, S., Telefoncu, A. & Sterner, R. Directed evolution of (βα)8-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase α-subunit. Protein Eng. Des. Sel. 25 , 285–293 (2012). Schmieger, H. Phage P22-mutants with increased or decreased transduction abilities. Mol. Gen. Genet. MGG 119 , 75–88 (1972). Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166 , 557–580 (1983). Miller, J. H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia Coli and Related Bacteria . (Cold Spring Harbor Laboratory Press, Plainview, N.Y, 1992). Näsvall, J., Knöppel, A. & Andersson, D. I. Duplication-Insertion Recombineering: a fast and scar-free method for efficient transfer of multiple mutations in bacteria. Nucleic Acids Res. 45 , e33 (2017). Näsvall, J. Direct and Inverted Repeat stimulated excision (DIRex): Simple, single-step, and scar-free mutagenesis of bacterial genes. PloS One 12 , e0184126 (2017). Näsvall, J. Dup-In and DIRex: Techniques for Single-Step, Scar-Free Mutagenesis with Marker Recycling. in Recombineering: Methods and Protocols (ed. Reisch, C. R.) 85–104 (Springer US, New York, NY, 2022). doi:10.1007/978-1-0716-2233-9_7. Yu, D. et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 97 , 5978–5983 (2000). Koskiniemi, S., Pränting, M., Gullberg, E., Näsvall, J. & Andersson, D. I. Activation of cryptic aminoglycoside resistance in Salmonella enterica. Mol. Microbiol. 80 , 1464–1478 (2011). Leavitt, J. C. et al. Bacteriophage P22 SieA-mediated superinfection exclusion. mBio 15 , e0216923 (2024). Susskind, M. M. & Botstein, D. Molecular genetics of bacteriophage P22. Microbiol. Rev. 42 , 385–413 (1978). Additional Declarations There is NO Competing Interest. Supplementary Files TableS1S5.xlsx Supplementary Table S1 - S5 Supplementarytext.pdf Supplementary Text and Figures Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4087552","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":281489151,"identity":"26630d97-658c-44fe-910a-53f5ef3665bd","order_by":0,"name":"Joakim Näsvall","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYBACxgYGZoYEGO9BgQ0PgwRQjHgtCQZphLUAATOCmWBwmIFBgpD69ubHBg9zGPL4+88YfkgwOC+jO7u5dQNj22EG+XbstjH2HDNOSNzGUCxxI8dYIsHgNo/ZnYNtN0BaDM4cwK5lRoLxAaCWxIYbbAkQLTcSoVokEnBoSf8M1jL//LHkHwkG5xBa5Oc/wKElB+ywxA0Hko8BbTmA0MJwA7v3GXvOFBskbpNI3Hgj+ZhFgkEyxC8J59J5DM5gd5hhe/tmyZ/bbBLnnT/YfONDhZ292e32Zzc+lFnLybdj975hA5hCjwug+TzYncXAII9LYhSMglEwCkYBHAAAJmVmJajcoEcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6831-3105","institution":"Uppsala University","correspondingAuthor":true,"prefix":"","firstName":"Joakim","middleName":"","lastName":"Näsvall","suffix":""},{"id":281489152,"identity":"b66ba1b2-4076-4cd3-b2e9-30d38ed311a0","order_by":1,"name":"Hind Abdalaal","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Hind","middleName":"","lastName":"Abdalaal","suffix":""}],"badges":[],"createdAt":"2024-03-12 21:00:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4087552/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4087552/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53268653,"identity":"311d2914-5fda-4fe7-b1ca-c8f09180d150","added_by":"auto","created_at":"2024-03-22 16:02:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":511651,"visible":true,"origin":"","legend":"\u003cp\u003eHypothesis and experimental outline. \u003cstrong\u003e(a)\u003c/strong\u003e Two main paths are depicted for the evolution of a new function while maintaining the original function in an ancestral gene (yellow rectangle). Both paths require the ancestral gene to possess a weak activity for the new function, either as a fortuitous side-activity or due to a mutation. Selection for increased expression of the new function (right) can lead to gene duplication/amplification events. Subsequent mutations can improve the new activity in some gene copies while preserving the original function in others. Conversely, in the absence of duplication (left), only mutations that improve the new function with minimal loss of the original function can be selected. The strength of the trade-off between the two functions (risk of losing the original function as the new function improves) determines the favored path. A strong trade-off or continuous selection favors duplication, while a weak trade-off or intermittent selection allows evolution towards a bifunctional enzyme. \u003cstrong\u003e(b - c)\u003c/strong\u003e Serial passage experiments with \u003cem\u003eSalmonella enterica\u003c/em\u003e lacking the \u003cem\u003etrpF\u003c/em\u003egene, grown in media with limiting tryptophan. Each 7-day cycle consisted of a short period with unlimited growth (tryptophan present) followed by a longer period without tryptophan (tryptophan-limited selection). Only prototrophic mutants (that had gained TrpF activity) could grow during this phase. \u003cstrong\u003e(c)\u003c/strong\u003e The expected dynamics during the takeover of a population by a TrpF+ mutant competing with its TrpF- ancestor in tryptophan-limited medium. Each cycle includes a brief window between tryptophan depletion and depletion of other essential nutrients, where the TrpF+ mutant can grow and outcompete the TrpF- ancestor.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4087552/v1/24b9f5432a077266b5e5f98c.jpg"},{"id":53267634,"identity":"cb2bf51d-15e7-4129-8881-187aae76393f","added_by":"auto","created_at":"2024-03-22 15:54:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":744826,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth Dynamics of Prototrophic and Auxotrophic Strains under Limiting Resources.\u003cstrong\u003e (a)\u003c/strong\u003e Hypothetical growth curves: the expected growth behavior of a prototrophic strain (black), an auxotrophic strain (red), and an evolving prototroph (blue) under limiting resource conditions. The auxotroph (red) can only grow until the limiting resource is depleted (stationary phase at low density). The prototroph (black) can continue growing until depletion of other essential nutrients or changes in medium composition (e.g., pH or metabolite accumulation) halt growth. The evolved prototroph (blue) exhibits growth after the limiting resource depletion, with a rate dependent on the rate of its newly acquired activity. If the original enzyme function is crucial for growth, reduced activity due to a mutation might lead to a slower growth rate before the limiting resource is depleted. \u003cstrong\u003e(b – d)\u003c/strong\u003e Batch growth curves of a tryptophan prototroph (\u003cem\u003etrpF\u003c/em\u003e+, black), the ancestral tryptophan auxotroph (Δ\u003cem\u003etrpF\u003c/em\u003e, red), the evolving populations from the final time-point that gained TrpF activity (blue), and those that did not gain TrpF activity (grey). \u003cstrong\u003e(b)\u003c/strong\u003e The eight lineages from the first experiment. \u003cstrong\u003e(c)\u003c/strong\u003e The 24 non-mutator lineages from the second experiment. \u003cstrong\u003e(d)\u003c/strong\u003e The 24 mutator lineages from the second experiment.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4087552/v1/5403628f24b73aa88ed2cde0.jpg"},{"id":53267636,"identity":"2b31f9ee-9586-4b2e-991b-6a18671b9ade","added_by":"auto","created_at":"2024-03-22 15:54:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":570117,"visible":true,"origin":"","legend":"\u003cp\u003eOverall growth rates of re-constructed mutants in tryptophan-limited medium. \u003cstrong\u003e(a)\u003c/strong\u003e \u003cem\u003ehisA \u003c/em\u003emutants. \u003cstrong\u003e(b)\u003c/strong\u003e \u003cem\u003etrpA\u003c/em\u003e mutants. Reconstructed mutants were grown in M9 glucose minimal medium supplemented with limiting tryptophan (5 μM) and guanosine (3 mM). As growth under these conditions deviates from exponential growth (except for the TrpF+ control strain), growth rates are reported as the average rates until cultures reached 90% of their maximum OD₆₀₀ (see Methods for details). Black bars represent average growth rates. NS; non-significant (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). *; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005, ***; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0005, based on a two-tailed Student’s t-test with equal variance. Each mutant was compared to its immediate ancestor, indicated by the grey lines. All strains were grown in at least four independent biological replicates. The complete growth curves can be found in Figures S3, S4, and S5.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4087552/v1/ad0f35f2020bbec9e13a7b3d.jpg"},{"id":53267638,"identity":"5ed82513-2665-4175-bf50-c1b943af5467","added_by":"auto","created_at":"2024-03-22 15:54:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":501380,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of Mutations on Original Activity During TrpF Evolution. \u003cstrong\u003e(a)\u003c/strong\u003e HisA Activity: Exponential growth rates were measured in the early, rapid growth phase (0.095 \u0026lt; OD₆₀₀ \u0026lt; 0.184) before tryptophan depletion in M9 glucose medium supplemented with limiting tryptophan (5 μM) and guanosine (3 mM). Rates were normalized to the growth rate of the \u003cem\u003etrpF\u003c/em\u003e+ wild-type strain grown in the same experiment. Under these conditions, reductions in HisA activity lead to decreased growth rates. Strains were grown in four or five biological replicates. Grey lines connect each mutant to its immediate ancestor. The question mark indicates an unknown mutation order in the evolution experiment, preventing complete reconstruction of the path. \u003cstrong\u003e(b)\u003c/strong\u003e TrpA Activity: Exponential growth rates were measured in M9 glucose medium without supplements for strains expressing wild-type trpF from a constitutive promoter at a different locus. Here, reductions in TrpA activity lead to decreased growth rates. For growth curves of \u003cem\u003etrpA\u003c/em\u003e mutants under the same conditions as (a), see Figure S2. NS denotes non-significant (difference in growth rate \u0026lt; 5% or p \u0026gt; 0.05). Asterisks indicate significance levels (*; p \u0026lt; 0.05, **; p \u0026lt; 0.005, ***; p \u0026lt; 0.0005) based on two-tailed Student's t-tests with equal variance, comparing each mutant to its immediate ancestor (grey lines).\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4087552/v1/b40c0c45106602312b7801dd.jpg"},{"id":53269306,"identity":"f58aa159-cc1c-4b0d-83d0-abf1164b5ee4","added_by":"auto","created_at":"2024-03-22 16:10:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":797283,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4087552/v1/a0dba004-e69d-4bae-9233-46b0537ad166.pdf"},{"id":53267632,"identity":"f07ce3c5-4009-401d-b221-a3f4761ee3ec","added_by":"auto","created_at":"2024-03-22 15:54:11","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":137659,"visible":true,"origin":"","legend":"Supplementary Table S1 - S5","description":"","filename":"TableS1S5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4087552/v1/b6289fdaf37cea700b5e6db5.xlsx"},{"id":53267635,"identity":"8c37a31b-4c3c-4421-a1b9-bf0fddfd960b","added_by":"auto","created_at":"2024-03-22 15:54:12","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1300816,"visible":true,"origin":"","legend":"Supplementary Text and Figures","description":"","filename":"Supplementarytext.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4087552/v1/842a1eaa169780313529bef8.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Evolution of new genes under intermittent selection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSince Susumo Ohno\u0026rsquo;s influential book \u003cem\u003eEvolution By Gene Duplication\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, gene duplication has been recognized as major driver of evolutionary innovation. Ohno\u0026rsquo;s hypothesis proposes that gene duplications allow redundant copies to evolve new functions while freed from purifying selection on the original function. However, the hypothesis relies on several assumptions: (\u003cem\u003ei\u003c/em\u003e) a gene needs to be duplicated before it is allowed to evolve a new function, (\u003cem\u003eii\u003c/em\u003e) gene duplications are selectively neutral, (\u003cem\u003eiii\u003c/em\u003e) gene duplications are stable, and, (\u003cem\u003eiv\u003c/em\u003e) mutations in the absence of selection can generate new, beneficial functions. The first assumption appears reasonable at a first glance. Mutations that generate a new function often disrupts the original function due to evolutionary trade-offs between the two functions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The latter three of these assumptions do not align with empirical observations. Gene duplications are seldomly neutral and often confer disadvantages to their carriers\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Furthermore, tandem gene duplications, the most common type of duplications in bacteria\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and eukaryotes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, are inherently unstable\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This instability limits their lifespan, making it difficult to accumulate beneficial mutations\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This is further confounded by the fact that deleterious mutations are far more frequent than beneficial mutations\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, a gene duplicate persisting for a long time without selection is much more likely to acquire loss-of-function mutations, making it a pseudogene, than to evolve a new beneficial function.\u003c/p\u003e\n\u003cp\u003eDespite these shortcomings of Ohno\u0026rsquo;s model for new gene evolution through duplication \u0026ndash; divergence, the role of gene duplication in generating new genes remains undeniable. The innovation-amplification-divergence (IAD) model offers an alternative pathway, addressing the limitations of Ohno's model\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Unlike Ohno's proposal that gene duplications persist due to neutrality, the IAD model suggests they are maintained because they provide a crucial benefit: boosting the expression of a weak, newly evolving activity of a gene. As such, under long-term selection favoring the improvement of this weak activity, duplications and amplifications accumulate and are further stabilized by selection.\u003c/p\u003e\n\u003cp\u003eThis above assumption is corroborated in our previous study, where we investigated the\u0026nbsp;evolutionary trade-offs associated with mutations enabling the \u003cem\u003eSalmonella enterica\u003c/em\u003e HisA enzyme to gain a new function in tryptophan synthesis (replacing TrpF) while maintaining its original function in histidine biosynthesis (details in Box 1 and Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Most mutations conferring the new function resulted in complete loss of the original activity, highlighting a strong trade-off between the two. Additionally, mutations further improving the new function rapidly eliminated any remaining ancestral function\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. These findings suggest that the evolution of a bi-functional enzyme that could perform both functions is unlikely.\u003c/p\u003e\n\u003cp\u003eHowever, some species within the \u003cem\u003eActinobacteria\u003c/em\u003e phylum challenge this notion. They lack a \u003cem\u003etrpF\u003c/em\u003e gene and possess a single HisA ortholog, PriA, that performs both histidine and tryptophan biosynthesis functions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. One explanation comes from the suggestion that ancient enzymes were more promiscuous than their modern counterparts\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. According to this explanation, PriA has retained its promiscuity for over 2\u0026nbsp;billion years, despite no selection pressure to maintain TrpF activity for much of that time, while most other HisA orthologs have become specialized\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. While this explanation holds merit, an alternative exists: PriA could have evolved from a specialized ancestral HisA under the right conditions. This possibility suggests that modern specialized HisA orthologs might evolve TrpF activity without necessarily losing their original function.\u003c/p\u003e\n\u003ch3\u003eContinuous Selection Favors Divergence Through Gene Duplication\u003c/h3\u003e\n\u003cp\u003eUnder continuous selection pressure, gene duplications and amplifications are enriched and persist due to increased expression of a weak evolving activity of a gene\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Duplication enables one copy to retain the original function while the other accumulates mutations that enhance the new activity. This frees the evolving copy from the constraints of maintaining the original function, allowing selection to act on the full spectrum of mutations that enhance the new activity, even if they disrupt the original one. Additionally, every mutation that further improves the new activity increases the probability of losing the original function. Consequently, even with a low frequency of loss of the original function, evolution after gene duplication is expected to be biased towards divergence into two distinct, specialized genes.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch3\u003eHypothesis: Intermittent Selection Favors Bi-functional Genes\u003c/h3\u003e\n\u003cp\u003eGene duplications and amplifications often incur fitness costs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and exhibit inherent instability\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In environments where selection for the new activity is interrupted by periods without selection pressure, duplications may be frequently lost. Under these conditions, only mutations that maintain the original function are available for selection. We propose that intermittent selection may limit the range of selectable mutations, biasing evolution towards the creation of bi‑functional generalist genes. The relative frequencies of mutations that retain or disrupt the original function (essentially, the dominant trade-off within the system, as illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) will further influence the \"choice\" between the two evolutionary pathways: duplication-divergence or the evolution of a bi-functional gene. Here, we have tested this hypothesis in evolution experiments with intermittent selection for a new function (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb, c).\u003c/p\u003e\n\u003cp\u003eBriefly, populations of bacteria lacking the \u003cem\u003etrpF\u003c/em\u003e gene, and therefore unable to grow without an external source of tryptophan, were passaged in a minimal medium with a limiting amount of tryptophan. Under these conditions the ancestral strain can grow for a few generations using the added tryptophan but once tryptophan is depleted growth ceases and only mutants able to synthesize tryptophan are able to continue growth. Thus, each growth cycle can be divided into two stages; an initial phase without selection for the new function and a second phase with selection.\u003c/p\u003e\n\u003ctable style=\"height: 88px; width: 629px;\" border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 619px;\"\u003e\n\u003cp\u003e\u003cstrong\u003eBox 1. Relevant enzymes in the histidine and tryptophan biosynthetic pathways.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHisA \u0026ndash; ProFAR isomerase\u003c/strong\u003e; catalyzes the conversion of N'-[(5'-phosphoribosyl)-formimino-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N'-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR), the fourth step in histidine biosynthesis. Encoded by the \u003cem\u003ehisA\u003c/em\u003e gene in the \u003cem\u003ehis\u003c/em\u003e operon.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrpA \u0026ndash; Tryptophan synthase subunit A\u003c/strong\u003e; together with tryptophan synthase subunit B (TrpB) catalyzes the two-step conversion of (3-indolyl)-glycerol-phosphate (IGP) to L-tryptophan, the final step in tryptophan biosynthesis. Encoded by the \u003cem\u003etrpA\u003c/em\u003e gene in the \u003cem\u003etrp\u003c/em\u003e operon.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrpF \u0026ndash; Phosphoribosyl anthranilate isomerase\u003c/strong\u003e; catalyzes the conversion of N-(5\u0026rsquo;-phosphoribosyl)-anthranilate (PRA) to 1-(2-carboxyphenylamino)-1\u0026rsquo;-deoxyribulose-5\u0026rsquo;-phosphate (CdRP), the third step in tryptophan biosynthesis. In \u003cem\u003eEnterobacteriaceae\u003c/em\u003e fused to the C-terminal of the TrpC protein (catalyzing the fourth step in the pathway). Here, I refer to the 202 amino acid C-terminal domain of the fused TrpCF peptide as TrpF and the corresponding 606 bp DNA sequence as the \u003cem\u003etrpF\u003c/em\u003e gene.\u003c/p\u003e\n\u003cp\u003eMutants lacking any of these activities are unable to synthesize the respective amino acids and are therefore unable to grow in medium without added histidine (\u003cem\u003ehisA\u003c/em\u003e-mutants) or tryptophan (\u003cem\u003etrpA\u003c/em\u003e and \u003cem\u003etrpF\u003c/em\u003e mutants) and are said to be \u003cstrong\u003eauxotrophic\u003c/strong\u003e for histidine or tryptophan, respectively. Strains that can synthesize both amino acids are said to be \u003cstrong\u003eprototrophic\u003c/strong\u003e for histidine and tryptophan.\u003c/p\u003e\n\u003cp\u003eFor simplicity, the proteins and their enzymatic activities are referred to in the text by the corresponding gene product names (HisA \u0026ndash; HisA activity \u003cem\u003eetc.\u003c/em\u003e for the enzyme encoded by the \u003cem\u003ehisA\u003c/em\u003e gene and its native activity). When HisA or TrpA catalyzes the reaction that is normally catalyzed by TrpF, the activity is referred to as TrpF activity.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eTo test the hypothesis that intermittent selection for a new evolving function leads to evolution of bi-functional generalist genes rather than duplication and divergence into two specialized genes, we conducted evolution experiments with \u003cem\u003eSalmonella enterica\u003c/em\u003e. We grew populations of strains lacking the \u003cem\u003etrpF\u003c/em\u003e gene through serial passages in batch cultures in medium with a limiting amount of tryptophan (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). This design created intermittent selection for TrpF activity. Initially, sufficient tryptophan was available in each growth cycle, allowing all cells to grow. However, after a few generations, tryptophan became depleted, making TrpF activity essential for continued growth.\u003c/p\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eEvolution of TrpF activity in HisA under intermittent selection\u003c/h2\u003e\n\u003cp\u003eEight populations were evolved for approximately 430 generations. By the end of the experiment, three populations exhibited growth after tryptophan depletion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Whole genome sequencing (WGS) of these populations revealed that two populations harbored multiple amino acid substitutions within the \u003cem\u003ehisA\u003c/em\u003e gene (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Additionally, these populations contained mutations known to elevate mutation rates (mutators; frameshift mutations in \u003cem\u003emutH\u003c/em\u003e and \u003cem\u003emutS\u003c/em\u003e, respectively; Table S2) and many additional mutations scattered across their genomes. The third population lacked mutations in any readily identifiable target gene and its mechanism of adaptation remains unclear (Supplementary text, Table S2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eMutators cause a shift in mutation target and type\u003c/h2\u003e\n\u003cp\u003eTo investigate the role of mutator mutations in acquiring TrpF activity and explore alternative evolutionary paths, a new experiment was designed. It compared 24 populations with mutator mutations (\u003cem\u003emutS\u003c/em\u003e) to 24 populations without them (\u003cem\u003emutS\u003c/em\u003e+; non-mutators). These populations were subjected to the same intermittent selection conditions for approximately 78 population doublings. Notably, after this period, 10 of the non-mutator populations and 21 of the mutator populations exhibited detectable growth within 7 days after tryptophan depletion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec and d).\u003c/p\u003e\n\u003cp\u003eWhole genome sequencing (WGS) of a subset of populations, followed by targeted Sanger sequencing in the remaining ones, identified candidate mutations responsible for TrpF activity. These mutations were found in either \u003cem\u003ehisA\u003c/em\u003e (in 9 mutator populations) or \u003cem\u003etrpA\u003c/em\u003e (in 11 mutator and 10 non-mutator populations). Interestingly, all sequenced non-mutator populations and 6 of the mutator populations shared the same \u003cem\u003etrpA\u003c/em\u003e mutation: a 7-bp duplication causing a frameshift (details in Supplementary Text and Figure S6). One non-mutator population also acquired an additional amino acid substitution (A67T) in \u003cem\u003etrpA\u003c/em\u003e within a subpopulation lacking the frameshift mutation. Notably, one mutator population lacked mutations in either \u003cem\u003ehisA\u003c/em\u003e or \u003cem\u003etrpA\u003c/em\u003e and remains unexplained (Table S2; Supplementary text). These results demonstrate that while mutator mutations were not essential for the evolution of TrpF activity under intermittent selection, they influenced the types of mutations found.\u003c/p\u003e\n\u003cp\u003eThus, \u003cem\u003eS. enterica\u003c/em\u003e can evolve TrpF activity through mutations in two distinct genes (\u003cem\u003ehisA\u003c/em\u003e and \u003cem\u003etrpA\u003c/em\u003e). Additionally, at least one other pathway exists for which we have not identified the mechanism. In the minimal medium used for the evolution experiment, both HisA and TrpA activities are essential for growth. HisA is required throughout each growth cycle, while TrpA becomes necessary only after tryptophan depletion. Notably, only one whole-genome sequenced population harbored a duplication/amplification event within the target gene (\u003cem\u003ehisA\u003c/em\u003e). This single instance suggests the possibility of further evolution by duplication-divergence. However, analysis of the sequence reads for this population did not suggest the presence of diverging mutations in any \u003cem\u003ehisA\u003c/em\u003e copy that might have lost one of the original or evolving activities. These observations support our hypothesis that intermittent selection can limit the chance of evolution by duplication-divergence by restricting the window of opportunity for duplications to become enriched and persist before selection pressure for the new function is relaxed.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eAll HisA paths start with the same mutation\u003c/h2\u003e\n\u003cp\u003eTo determine the order of appearance for mutations in populations harboring multiple \u003cem\u003ehisA\u003c/em\u003e or \u003cem\u003etrpA\u003c/em\u003e mutations, we sequenced the target genes in clones isolated from earlier time points (Table S3). Consistent with our previous findings that single amino acid substitutions in HisA rarely confer TrpF activity without compromising HisA function\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, all evolutionary paths involving \u003cem\u003ehisA\u003c/em\u003e mutations began with the same mutation (Q18R). Conversely, \u003cem\u003etrpA\u003c/em\u003e displayed a greater diversity of starting mutations, suggesting a potentially weaker trade-off between the two enzymatic activities in this gene compared to \u003cem\u003ehisA\u003c/em\u003e. To validate the identified \u003cem\u003ehisA\u003c/em\u003e and \u003cem\u003etrpA\u003c/em\u003e mutations as responsible for TrpF-independent growth, and to isolate the effects of these variants from the numerous background mutations accumulated during the evolution experiments, we reconstructed each identified \u003cem\u003ehisA\u003c/em\u003e and \u003cem\u003etrpA\u003c/em\u003e variant from the final populations, along with most intermediate variants from earlier time points, for further analysis.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eSuccessive mutations improve overall fitness at the conditions of the evolution experiments\u003c/h2\u003e\n\u003cp\u003eTo assess the fitness benefit conferred by the reconstructed \u003cem\u003ehisA\u003c/em\u003e and \u003cem\u003etrpA\u003c/em\u003e variants, we measured the growth of strains harboring these variants in the same minimal medium used in the evolution experiments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Following tryptophan depletion, growth becomes dependent on all three activities (the native HisA and TrpA activities, and the evolved TrpF activity). Notably, all identified \u003cem\u003ehisA\u003c/em\u003e and \u003cem\u003etrpA\u003c/em\u003e variants enabled growth after tryptophan depletion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, in most cases where the complete mutational pathways were reconstructed, each additional mutation within a pathway resulted in improved growth after tryptophan depletion.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eHisA, but not TrpA, tends to lose its native function when TrpF activity improves\u003c/h2\u003e\n\u003cp\u003ePrior to tryptophan depletion, all reconstructed \u003cem\u003etrpA\u003c/em\u003e mutants exhibited growth rates indistinguishable from the ancestral strain, which itself grew similarly to a prototrophic (TrpF+) strain (Figures S2 and S5). This is unsurprising, as TrpA activity is not essential when tryptophan is present in the medium.\u003c/p\u003e\n\u003cp\u003eIn contrast, all \u003cem\u003ehisA\u003c/em\u003e mutants displayed slower growth compared to the ancestral strain, and each additional \u003cem\u003ehisA\u003c/em\u003e mutation further reduced the growth rate (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, S3, and S4). This growth reduction in \u003cem\u003ehisA\u003c/em\u003e mutants could be partially restored by supplementing the medium with guanosine. This rescue effect likely reflects a metabolic link between histidine, tryptophan, and purine nucleotide biosynthesis (Figures S3 and S4; Supplementary text). However, even with guanosine supplementation, all \u003cem\u003ehisA\u003c/em\u003e mutants grew significantly slower than a wild-type \u003cem\u003ehisA\u003c/em\u003e control strain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea), indicating a measurable decline in the native HisA function.\u003c/p\u003e\n\u003cp\u003eSince TrpF and TrpA act within the same pathway, growth curves of \u003cem\u003etrpA\u003c/em\u003e mutants cannot reveal the extent of activity for either function. Hypothetically, a \u003cem\u003etrpA\u003c/em\u003e mutant with very weak TrpA activity but strong TrpF activity could exhibit a growth curve identical to one with the opposite functional relationship. To evaluate the impact on native TrpA activity, we introduced a wild-type \u003cem\u003etrpF\u003c/em\u003e gene under a strong constitutive promoter at an unrelated locus in ten reconstructed \u003cem\u003etrpA\u003c/em\u003e mutants.\u003c/p\u003e\n\u003cp\u003eFollowing complementation with wild-type \u003cem\u003etrpF\u003c/em\u003e, six \u003cem\u003etrpA\u003c/em\u003e mutants displayed growth rates indistinguishable from their isogenic wild-type counterparts, suggesting minimal loss of TrpA activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). However, four mutants grew significantly slower, indicating a decline in TrpA function. One of these activity-compromised variants harbored the P62fs frameshift mutation, which is expected to be poorly expressed due to the requirement for frameshift suppression (Supplementary text, Figure S6). Interestingly, three variants originated from the same evolutionary pathway, where the initial mutation (G61S) resulted in TrpA activity loss, and the subsequent mutation (P62S) restored TrpA function.\u003c/p\u003e\n\u003cp\u003eIn contrast to the \u003cem\u003ehisA\u003c/em\u003e mutants, these findings suggest that some \u003cem\u003etrpA\u003c/em\u003e mutants acquired TrpF activity without a substantial trade-off in their native function. This observation may point towards a weaker functional trade-off between the two activities within TrpA compared to HisA.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSeveral models for the evolution of new genes by modification of existing genes build on the assumptions that the new function either appears after the duplication of the ancestral gene\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, or that it appears as a selectively neutral promiscuous activity that at some point happens to become beneficial due to a change in the environment\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, in a previous evolution experiment, we started with a HisA double mutant. One mutation generated weak TrpF activity but led to the complete loss of the original HisA activity, and an additional mutation restored some of the HisA activity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Furthermore, the mutant gene was placed under a strong constitutive promoter and in a location known to be prone to amplification. The mutant bacteria were subjected to continuous selection for both HisA and TrpF activities for roughly 3,000 generations. During this experiment, the mutant hisA gene amplified to a high copy number and accumulated divergent mutations (up to three per gene copy). These mutations improved one or the other activity in separate gene copies, ultimately leading to the evolution of two specialized enzymes in most cases.\u003c/p\u003e\n\u003cp\u003eHere, we instead started with strains with the wild-type \u003cem\u003ehisA\u003c/em\u003e and \u003cem\u003etrpA\u003c/em\u003e genes in their native loci and with their native regulation intact. In addition to the interruptions in selection pressure for the new function, which is likely to reduce the chance of selection of gene duplications, the native location of the \u003cem\u003ehis\u003c/em\u003e- and \u003cem\u003etrp\u003c/em\u003e-operons near the replication terminus region leads to low rates of duplication formation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Still, in these experiments quite rapid evolution with several mutations in the \u003cem\u003ehisA\u003c/em\u003e or \u003cem\u003etrpA\u003c/em\u003e genes were seen in relatively short time-span. Curiously, evolution through the generation of TrpF activity in HisA was only seen in the presence of mutators, while in the absence of mutators a frameshift mutation in \u003cem\u003etrpA\u003c/em\u003e was found in multiple independent populations and only one population had a SNP (in \u003cem\u003etrpA\u003c/em\u003e) that conferred TrpF activity.\u003c/p\u003e\n\u003cp\u003eThe absence of other mutations in \u003cem\u003etrpA\u003c/em\u003e or \u003cem\u003ehisA\u003c/em\u003e in the non-mutator populations can be explained by three factors: (\u003cem\u003ei\u003c/em\u003e) Relatively short time span: The experiment only lasted for approximately 78 population doublings, limiting the opportunity for mutations to accumulate. (\u003cem\u003eii\u003c/em\u003e) Small population size: The total population size at the end of each cycle was small (around 10⁹ cells in 5 ml), reducing the chance of beneficial mutations arising. (\u003cem\u003eiii\u003c/em\u003e) Very small mutational target size: There were likely less than ten possible single nucleotide substitutions that could generate TrpF activity in \u003cem\u003ehisA\u003c/em\u003e or \u003cem\u003etrpA\u003c/em\u003e. The cumulative number of cells per population in the experiment corresponds to less than 10\u0026sup1;\u0026sup1; cells. With mutation rates for single nucleotide substitutions ranging between 10⁻\u0026sup1;⁰ \u0026ndash; 10⁻\u0026sup1;\u0026sup2; per cell/nucleotide/generation in non-mutator strains\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, the specific nucleotide substitutions in hisA or trpA would likely occur at least once per population during our experiments. In contrast, the observed frameshift mutation is a duplication of 7 bp in a 12 bp sequence containing two short tandem repeats (5 bp each; Figure S6). Mutations of this type are known to occur at rates that are orders of magnitude above the rates of single nucleotide substitutions in \u003cem\u003eE. coli\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Furthermore, the frameshift mutation outperforms all of the reconstructed single amino acid substitutions in both \u003cem\u003ehisA\u003c/em\u003e and \u003cem\u003etrpA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Thus, in the absence of mutators, the frameshift mutation is expected to happen more often (many times in each population) and once it does, it outcompetes any single nucleotide substitutions that may have been generated.\u003c/p\u003e\n\u003cp\u003eOur findings support the notion that interrupting selection for the new activity with periods lacking selection pressure disfavors gene duplication as a solution for evolving the new function while retaining the original one. While gene duplication offers the advantages of both increased beneficial activity and buffering against loss of the original function, periods without selection are expected to lead to the loss of unstable duplications and counter-selection against mutants with reduced original function. Although we lack direct evidence that gene duplications or amplifications were not present at some point in some of the populations, the absence of evolved clones harboring duplicated \u003cem\u003ehisA\u003c/em\u003e genes with the expected loss of HisA activity in one copy serves as indirect evidence against stabilization of duplications by selection. Mutations that improve TrpF activity in HisA, at the expense of complete HisA loss, are demonstrably more common than those that retain both functions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Therefore, if duplications were present, they would almost invariably lead to the divergence into two separate enzymes.\u003c/p\u003e\n\u003cp\u003eAnalysis of reconstructed mutant growth curves under the same conditions as the evolution experiment (Figures S3a and S4a) reveals that these conditions present a more complex evolutionary landscape than what can be solely predicted by growth rates before and after tryptophan depletion. Notably, even non-growing or slow-growing cells consume resources from the medium. Consequently, after tryptophan depletion, the ancestral strain continues to utilize other essential resources (carbon, nitrogen, phosphorus, sulfur, minerals, trace elements) until the medium becomes exhausted and no further growth is possible. This introduces a time-dependent selection pressure: a beneficial mutation must enable significant growth between tryptophan depletion and depletion of the next essential resource. Additionally, post-depletion growth needs to compensate for any growth deficit incurred before tryptophan depletion. This complex interplay between maintaining the original function and acquiring the new activity imposes a stringent selective filter, potentially limiting the spectrum of viable mutations.\u003c/p\u003e\n\u003cp\u003eThe three enzymes HisA, TrpA and TrpF are structurally related proteins with an (\u0026alpha;\u0026beta;)\u003csub\u003e8\u003c/sub\u003e-barrel fold that are proposed to have evolved from a common ancestor\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Additionally, HisA catalyzes the same isomerization reaction as TrpF, albeit on a bulkier substrate. Previous evolution experiments have succeeded in regenerating TrpF activity in HisA, but have often resulted in complete loss of the original function\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Similarly, our research investigating the functional trade-offs between HisA and TrpF activities in \u003cem\u003eSalmonella enterica\u003c/em\u003e revealed that mutations generating or enhancing TrpF activity rarely preserve HisA activity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Intriguingly, in \u003cem\u003eActinobacteria\u003c/em\u003e lacking a native TrpF ortholog, PriA (a HisA ortholog) is active in both the histidine and tryptophan pathways\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This suggests the existence of evolutionary paths towards bifunctional enzymes, even in the presence of a seemingly strong functional trade-off. Our findings indicate that during intermittent selection for the new function, evolution can proceed through rare mutations with weak trade-offs. However, when selection for the original activity is relaxed (by gene duplication), these mutations are likely outnumbered by more frequent mutations with strong trade-offs.\u003c/p\u003e\n\u003cp\u003eEvran \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e employed rational design to introduce putative catalytic residues from TrpF into TrpA, but only achieved TrpF activity after extensive mutagenesis and gene shuffling. Notably, their proposed essential mutations (F22C and L177D) were not sufficient for detectable TrpF activity. In contrast, our evolutionary approach identified several single amino acid substitutions (G98C, D27Y, G61S, Y102H) that directly conferred TrpF activity, without introducing the proposed catalytic residues. Interestingly, some mutations identified by Evran \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e as \"permissive\" (G61S, T24S) were sufficient for TrpF activity in our experiments, suggesting these mutations may play a more significant role than previously thought. These findings highlight the potential limitations of structure-guided design compared to experimental evolution for introducing new activities in enzymes. However, structural analysis of evolved mutants remains valuable for elucidating the mechanistic basis of the newly acquired function.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe evolution of a novel function in an enzyme, while preserving its essential original function, is usually seen as an adaptive conflict that require gene duplication. However, this study reveals an intriguing alternative: even in the face of a seemingly strong trade-off, rare evolutionary paths may exist that allow an enzyme to acquire a new function without compromising the original one. These findings support our hypothesis that fluctuating selection pressures, by preventing the selection of gene duplications and disfavoring mutations with severe effects on the original function, can guide evolution towards the emergence of generalist enzymes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n\u003ch2\u003eBacterial strains and growth conditions\u003c/h2\u003e\n\u003cp\u003eAll bacterial strains are derivatives of \u003cem\u003eSalmonella enterica\u003c/em\u003e ssp. \u003cem\u003eenterica\u003c/em\u003e serovar Typhimurium strain LT2 (except for three \u003cem\u003eEscherichia coli\u003c/em\u003e strains that were used as PCR templates for amplification of selection cassettes used in \u0026lambda; Red recombineering). For transferring chromosomal markers between strains, generalized transduction with phage P22 HT 105/1 int-201\u003csup\u003e21\u003c/sup\u003e or a defective prophage derivative of it (see below; \u0026ldquo;Construction and use of an artificial Gene Transfer Agent\u0026rdquo;) was used. Lysogeny broth (LB; 10 g/L Tryptone [Sigma], 5 g/L yeast extract [Sigma], 10 g/L NaCl) was used as rich medium, and was supplemented with 15 g/L Bacto agar to make LB agar (LA) plates. Salt-free LB (LB prepared without NaCl) was used for preparing cells for transformation by electroporation. SOC medium\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e was used for recovery after transformations. As minimal medium, M9 medium\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e was prepared with twice the amounts of M9 salts, glucose, CaCl\u003csub\u003e2\u003c/sub\u003e and MgCl\u003csub\u003e2\u003c/sub\u003e in order to allow growth to a higher cell density. Minimal medium was supplemented with tryptophan (W; 0.1 mM [1x] or 5 \u0026micro;M [0.05x, for tryptophan-limited medium]), histidine (H; 0.1 mM), and guanosine (Guo; 0.3 mM) when needed. For simplicity, the base medium is referred to as 2\u0026times; M9, with any additional additions indicated as +\u0026thinsp;H (histidine)\u0026thinsp;+\u0026thinsp;W (tryptophan) and +\u0026thinsp;Guo (guanosine). To make solid M9 plates, 15 g/L of Bacto agar was added. All growth of bacteria was done at 37\u0026deg;C (except when preparing cells for \u0026lambda; Red recombineering). Antibiotics (trimethoprim [tmp; 10 mg/L; Sigma], tetracyclin [tet; 7.5 mg/L; Sigma], chloramphenicol [cam; 12.5 mg/L; Sigma]) were added to the medium only when needed for selecting recombinants after transformations and transductions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003ePCR and Sanger sequencing\u003c/h2\u003e\n\u003cp\u003eDNA for \u0026lambda; Red recombineering and Sanger sequencing was prepared by PCR using Phusion DNA polymerase (Thermo Fisher). All PCR reactions were made with bacterial cell suspensions as templates; either part of a single fresh colony from an agar plate or a\u0026thinsp;~\u0026thinsp;1 \u0026micro;l sample from a frozen (-80\u0026deg;C) glycerol- or DMSO stock culture was re‑suspended in 100 \u0026micro;l ultrapure water (Sigma). Of this, 1 \u0026micro;l was used per 20 \u0026micro;l of PCR reaction. PCR cycling parameters have been described elsewhere\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. For de-salting, removal of oligonucleotides and primer-dimers, and concentration of the DNA, PCR products were precipitated with polyethylene glycol (PEG). Briefly, PCR reactions were mixed 1:1 with a 2\u0026times; PEG precipitation solution (24% [w/v] PEG 8000 [Sigma], 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e in ultra-pure water [Sigma]) and incubated for 10 min at room temperature. DNA was pelleted by centrifugation at \u0026ge;\u0026thinsp;14,000 \u0026times; g for 10 min, washed once in \u0026ge;\u0026thinsp;1 volume of 70% ethanol and re-centrifuged at \u0026ge;\u0026thinsp;14,000 \u0026times; g for 10 min, after which it was air-dried. For local sequencing, DNA was dissolved in ultra-pure water, mixed with a sequencing primer (Table S4), and sent to Eurofins Genomics (Ebersberg, Germany) for Sanger sequencing. DNA for recombineering was dissolved in 2\u0026ndash;10 \u0026micro;l of ultra-pure water.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e\u0026lambda; Red Recombineering\u003c/h2\u003e\n\u003cp\u003eMutations, genetic markers, and reporter genes were constructed and inserted using different \u0026lambda; Red recombineering strategies as described elsewhere\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Cultures of bacterial strains carrying the \u0026lambda; Red helper plasmid pSIM5-Tet\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e were grown in salt-free LB plus 0.2% (w/v) glucose at 30\u0026deg;C overnight, after which the cultures were diluted 100-fold into 25 ml of the same medium and allowed to grow at 30\u0026deg;C for one hour. To induce expression of the \u0026lambda; recombination genes, the cultures were transferred to a 42\u0026deg;C shaking water bath and incubated for another 15 min. After cooling in an ice-water bath for several minutes the cultures were pelleted by centrifugation and washed once in 1 ml ice-cold de-ionized water or 10% glycerol. The cells were resuspended in 200 \u0026micro;l of ice-cold de-ionized water or 10% glycerol. For each transformation, DNA (0.2\u0026ndash;2 \u0026micro;l of de-salted and concentrated PCR product) was mixed with 20 \u0026micro;l cell-suspension in an electroporation cuvette (Bio-Rad, 1 mm gap) and electroporated (2.5 kV, 200 Ω, 25 \u0026micro;F in a Gene Pulser Xcell; BioRad). Immediately after electroporation the cell suspensions were re-suspended in 200 \u0026micro;l pre-warmed (42\u0026deg;C) SOC, transferred to a 10- or 50 ml plastic tube and incubated at 42\u0026deg;C for 15 minutes before plating on selective medium.\u003c/p\u003e\n\u003cp\u003eAll DNA oligonucleotides are listed in Table S4. For re-construction of mutations in \u003cem\u003ehisA\u003c/em\u003e and \u003cem\u003etrpA\u003c/em\u003e, the target genes containing the mutations were PCR amplified from evolved populations or isolated clones from evolved populations. In some cases, the gene was amplified as two overlapping fragments using a mutant and a wildtype as template in order to avoid one mutation present in a mutant containing several mutations. The PCR products were transformed into strains carrying the selectable and counter-selectable cassette \u003cem\u003eAtox1\u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e (GenBank accession: MN207489; containing \u003cem\u003edhfr\u003c/em\u003e [trimethoprim resistance], \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003erhaB\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003eorph11\u003c/em\u003e [DNAse toxin orphan-11 from \u003cem\u003eE. coli\u003c/em\u003e EC869 under the control of the rhamnose inducible \u003cem\u003erhaB\u003c/em\u003e promoter], and \u003cem\u003eamilCP\u003c/em\u003e [blue chromoprotein from \u003cem\u003eAcropora millepora\u003c/em\u003e]) in \u003cem\u003ehisA\u003c/em\u003e or \u003cem\u003etrpA\u003c/em\u003e, selecting for loss of the cassette on M9\u0026thinsp;+\u0026thinsp;0.3% (w/v) rhamnose\u0026thinsp;+\u0026thinsp;histidine\u0026thinsp;+\u0026thinsp;tryptophan plates. Deletions, some single nucleotide substitutions, and small insertions were constructed using DIRex\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, using \u0026ldquo;half-cassettes\u0026rdquo; that were generated by amplifying overlapping parts of \u003cem\u003eAtox1\u003c/em\u003e and \u003cem\u003eAtox2\u003c/em\u003e (GenBank accession: MN207490) as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eEvolution experiments\u003c/h2\u003e\n\u003cp\u003eExperiment 1 (populations 1\u0026ndash;1 to 1\u0026ndash;8) was started from separate cultures of DA52864 (\u0026Delta;\u003cem\u003etrpF\u003c/em\u003e \u0026Delta;\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003egal\u003c/em\u003e\u003c/sub\u003e \u0026Delta;\u003cem\u003egalE\u003c/em\u003e::\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003elux\u003c/em\u003e\u003c/sub\u003e). In addition to the deletion of the \u003cem\u003etrpF\u003c/em\u003e coding sequence (corresponding to the C-terminal domain of the fused TrpCF protein), it carries a deletion of the \u003cem\u003egal\u003c/em\u003e operon promoter and has the \u003cem\u003egalE\u003c/em\u003e gene replaced by the \u003cem\u003elux\u003c/em\u003e transcriptional terminator\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These mutations make the strain conditionally resistant to phage P22 infection (resistant in the absence of galactose but sensitive in the presence of galactose), and were used in order to avoid contamination by phage P22 and avoid selection of P22 resistant mutants, which we have occasionally seen in previous experiments. After a first overnight growth in medium containing both histidine and tryptophan, samples from each population were passaged 1:100 into 5 mL tryptophan-limited medium (2\u0026times; M9\u0026thinsp;+\u0026thinsp;0.05\u0026times; W) in 50 mL conical bottom screwcap tubes, and were continually passaged into the same medium once every seven days for 20 cycles, after which they were passaged 1:1000 into the same medium every 3\u0026ndash;4 days (twice a week) for an additional 30 cycles, totaling approximately 430 (20*6.64\u0026thinsp;+\u0026thinsp;30*10) generations. As each cycle started with fresh tryptophan-containing medium, growth for the first few generations occurred without any selection for TrpF activity. Once tryptophan was depleted, only mutants able to synthesize tryptophan (due to mutations that generated TrpF activity in another enzyme) could grow, making selection for TrpF activity intermittent. Samples from the populations were frozen at -80\u0026deg;C in 20% DMSO after cycle 10, 15, 20, 25, 40 and 50.\u003c/p\u003e\n\u003cp\u003eExperiment 2 (lineages 2\u003cem\u003emut\u003c/em\u003e\u0026thinsp;+\u0026thinsp;1\u0026ndash;2\u003cem\u003emut\u003c/em\u003e\u0026thinsp;+\u0026thinsp;24 and 2\u003cem\u003emutS\u003c/em\u003e1\u0026ndash;2\u003cem\u003emutS\u003c/em\u003e24) was done similarly to experiment 1, but was started from separate cultures of DA62207 and DA64168 (\u0026Delta;\u003cem\u003etrpF\u003c/em\u003e and \u0026Delta;\u003cem\u003etrpF mutS\u003c/em\u003e; see supplementary Table S5 for the detailed genotypes of these strains). In addition to the \u003cem\u003etrpF\u003c/em\u003e deletion and the \u003cem\u003emutS\u003c/em\u003e mutation, these strains contain the arabinose-inducible defective P22 prophage GTA22 (below), which makes them resistant to lytic growth of phage P22 and makes them convenient both as donors and recipients in transductions. These 48 populations were passaged with 100\u0026times; dilutions into 5 ml of fresh tryptophan-limited medium (2\u0026times; M9\u0026thinsp;+\u0026thinsp;0.05\u0026times; W) once every seven days for 13 cycles, at which point all but three \u003cem\u003emutS\u003c/em\u003e populations grew visibly denser. Samples from the populations were frozen at -80\u0026deg;C in 20% glycerol after cycle 4, 5, 6, 7, 8, 10, 12 and 13.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eGrowth curves and growth rate determinations\u003c/h2\u003e\n\u003cp\u003eOvernight cultures (in 1 mL 2\u0026times; M9\u0026thinsp;+\u0026thinsp;0.4% glucose\u0026thinsp;+\u0026thinsp;0.1 mM histidine\u0026thinsp;+\u0026thinsp;0.1 mM tryptophan) were started either from isolated colonies of pure re-constructed clones or from thawed 50 \u0026micro;L samples from freezer stocks of evolved populations. Pure clones were grown as five biological replicates while populations were grown as a single sample per population. After approximately 24 hours of growth at 37\u0026deg;C the cultures were diluted 1:1000 into tryptophan-free (2\u0026times; M9\u0026thinsp;+\u0026thinsp;0.4% glucose) and tryptophan-limited (same but with 5 \u0026micro;M tryptophan) medium, after which 300 \u0026micro;L of each diluted culture was transferred to a well in a Bioscreen honeycomb plate. To avoid evaporation of media, all empty wells were filled with 300 \u0026micro;L medium or water, and the plates were sealed by taping around the edges. The plates were incubated in a Bioscreen C reader (Oy Growth Curves, Turku, Finland) at 37\u0026deg;C, with continuous moderate shaking, and reading of optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) every 4 minutes for 7 days. Despite taping the edges, the wells around the edges of the plate reached a higher final OD than other replicates of the same strain and upon inspection these cultures showed a significant loss of volume. For growth rate determinations, the OD\u003csub\u003e600\u003c/sub\u003e data was plotted to find the exponential phase (which ended at about OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.18 for the cultures in tryptophan-limited media) and the data for only the exponential phase was transferred to Prism 9 or 10 (GraphPad Software). The data was fitted (without subtracting any blank value) to the exponential growth equation \u003cem\u003eY(t)\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eb\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e*\u003cem\u003ee\u003c/em\u003e\u003csup\u003e\u003cem\u003ekt\u003c/em\u003e\u003c/sup\u003e, where \u003cem\u003eY(t)\u003c/em\u003e is the OD\u003csub\u003e600\u003c/sub\u003e at time \u003cem\u003et\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e is the contribution of the medium and the plastic of the individual well of the Bioscreen plate to the OD\u003csub\u003e600\u003c/sub\u003e (\u0026ldquo;blank\u0026rdquo;), \u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e is the contribution of cells to the OD at time \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0, \u003cem\u003ek\u003c/em\u003e is the growth rate (min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and \u003cem\u003et\u003c/em\u003e is the time (min). Differences of \u0026lt;\u0026thinsp;5% in \u003cem\u003ek\u003c/em\u003e between two strains were considered insignificant due to limitations such as the precision of OD measurements and well-to-well variation in the Bioscreen plates. The average growth rate up to 90% of the maximum OD for each culture (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) was determined as follows: First the OD\u003csub\u003e600\u003c/sub\u003e data was blank-subtracted using the \u003cem\u003eb\u003c/em\u003e value from the curve fitting (above) as blank. For each culture the first data point above \u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e (\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u0026gt;\u0026thinsp;Y0\u003c/em\u003e\u003c/sub\u003e) and the first point where OD reached above 90% of the maximum OD (\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003e90%ODmax\u003c/em\u003e\u003c/sub\u003e) within the first three days were determined. The average growth rates between those two points were determined as \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003eavg\u003c/em\u003e\u003c/sub\u003e = [ln(\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003e90%ODmax\u003c/em\u003e\u003c/sub\u003e)-ln(\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u0026gt;\u0026thinsp;Y0\u003c/em\u003e\u003c/sub\u003e)]/[\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e90%ODmax\u003c/em\u003e\u003c/sub\u003e-t\u003csub\u003e\u003cem\u003e\u0026gt;\u0026thinsp;Y0\u003c/em\u003e\u003c/sub\u003e] and are expressed in day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eWhole Genome Re-sequencing\u003c/h2\u003e\n\u003cp\u003eGenomic DNA was prepared from 1\u0026ndash;2 ml samples from evolving populations using MasterPure (Epicentre). Libraries for MiSeq sequencing (in-house) were made using Nextera DNA library prep and indexing kits (Illumina). Some samples were sequenced by BGI (Beijing, China) using their proprietary technology. Analysis of sequence reads were done with CLC Genomics Workbench (Qiagen). After quality-based trimming to remove any ambiguities and low-quality reads, the reads were mapped against a reference genome containing all modifications present in the ancestral strains. Indels and SNPs were detected using the low frequency variant detector, and structural rearrangements (duplications or deletions) were found using visual scanning of mapped read depth in combination with the structural rearrangement tool in CLC. Amplification copy number in the single population (DA65458; 2mutS8) that contained an amplification of a relevant target gene (\u003cem\u003ehisA\u003c/em\u003e) was estimated by dividing the mean read depth of the structural genes in the \u003cem\u003ehis\u003c/em\u003e-operon (7136 bp at 156.49\u0026times; read depth) with the mean of the mean read depths of four identically-sized regions (4\u0026times; 7136 bp at 19.95\u0026times; read depth) just outside of the amplified region. One relevant mutation (\u003cem\u003etrpA\u003c/em\u003e[Pro62Fs]) was reported by CLC genomics workbench to be detected in 30\u0026ndash;60% of the reads covering that position. However, when the reads were re-mapped to a reference sequence containing the mutation, 82\u0026ndash;100% of the reads from the relevant populations mapped perfectly to the mutation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eIsolation and sequencing of clones from evolved populations\u003c/h2\u003e\n\u003cp\u003eIn order to determine the order of appearance of mutations in populations with more than one mutation in the target gene (\u003cem\u003ehisA\u003c/em\u003e or \u003cem\u003etrpA\u003c/em\u003e) during the evolution experiments, clones were isolated from frozen stocks of earlier time-points, and the target genes were PCR amplified to generate templates for Sanger sequencing. For each frozen population, a streak was made to isolate single colonies on LA plates. Eight colonies from each streak were picked and streaked once more on LA plates, and from each of these eight clones a single colony was used as template for PCR. Some clones with known \u003cem\u003ehisA\u003c/em\u003e or \u003cem\u003etrpA\u003c/em\u003e alleles were frozen in the strain collection and used as templates in PCR reactions for re-construction of mutations in fresh (unevolved) genetic background.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eConstruction and use of an artificial Gene Transfer Agent\u003c/h2\u003e\n\u003cp\u003eAfter transductions with phage P22, it is of critical importance to isolate phage-free bacterial clones to avoid the lytic spread of phage in cultures as well as to avoid the selection of phage-resistant mutants or lysogens that are impossible to use in later transductions. Additionally, in long-term evolution experiments, we have occasionally found evolving populations to be infected by P22, or to carry mutations that provide resistance to P22, which could be problematic for later genetic experiments. Therefore, an inducible artificial Gene Transfer Agent (GTA; here referred to as GTA22) was constructed (using \u0026lambda; Red recombineering) by three modifications to a P22 HT 105/1 \u003cem\u003esieA44\u003c/em\u003e lysogen. The mutation HT 105/1\u003csup\u003e21\u003c/sup\u003e in makes DNA packaging into new P22 virions less specific, allowing a high frequency of generalized transduction. The \u003cem\u003esieA44\u003c/em\u003e mutation prevents the lysogen from blocking entry of DNA from P22 (and related phages) in the periplasm, making the lysogen work as a recipient in transductions. However, P22 also encodes a phase-variable O-antigen conversion locus, \u003cem\u003egtrABC\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, which when it is expressed prevents adsorption of P22 to the cell (P22 uses the O-antigen as receptor), making the cells resistant to superinfection (and prevents transduction).\u003c/p\u003e\n\u003cp\u003eTo convert this prophage into an inducible gene transfer agent, both ends, including the attachment site, of the prophage were deleted (using DIRex\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e). One deletion (\u0026Delta;\u003cem\u003ethrW\u003c/em\u003e-\u003cem\u003ekil\u003c/em\u003e) removes 6.9 kb of DNA from the \u0026ldquo;left\u0026rdquo; end of the prophage, including the left copy of the P22 attachment site (in the \u003cem\u003ethrW\u003c/em\u003e tRNA gene), the integrase and excisionase genes (\u003cem\u003eint\u003c/em\u003e and \u003cem\u003exis\u003c/em\u003e), the recombination genes (\u003cem\u003eabc1\u003c/em\u003e, \u003cem\u003eabc2\u003c/em\u003e and \u003cem\u003eerf\u003c/em\u003e) and the septation inhibitor (\u003cem\u003ekil\u003c/em\u003e). The other deletion removes 3 kb of DNA including the right copy of the attachment site (a P22 encoded partial duplicate of the \u003cem\u003ethrW\u003c/em\u003e gene) and the phage-encoded O-antigen conversion locus (\u003cem\u003egtrABC\u003c/em\u003e). Secondly, in order to make strains containing GTA22 work as donor in transductions the prophage was made inducible by placing (using \u0026lambda; Red recombineering) a copy of the P22 antirepressor (\u003cem\u003eant\u003c/em\u003e\u003csub\u003e\u003cem\u003eP22\u003c/em\u003e\u003c/sub\u003e) after the L-arabinose inducible \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003earaBAD\u003c/em\u003e\u003c/sub\u003e promoter in the host genome. Without the attachment site and the integrase/excisionase the prophage cannot excise and circularize its genome when it is induced, and any replication forks starting at the phage origin of replication extends into the surrounding bacterial host genome\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Without the O-antigen conversion genes and a functional \u003cem\u003esieA\u003c/em\u003e gene the lysogen cannot prevent adsorption of P22, and will therefore accept incoming DNA from P22 virions. But, as GTA22 still contains a functional P22 repressor (\u003cem\u003ec2\u003c/em\u003e) and other regulatory elements, the lysogen is resistant to lytic growth of any superinfecting P22 genome. As packing of the P22 genome into new virions is unidirectional and initiates at a single \u003cem\u003epac\u003c/em\u003e site in the phage genome\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, GTA22 (being unable to excise and circularize) is unable to pack a complete copy of its genome into new virions, making it essentially avirulent (but it could theoretically form rare virulent virions if a copy of its genome is circularized through illegitimate recombination). Thus, a strain containing GTA22 is resistant to lytic infection by P22 but works efficiently as recipient in transductions. Furthermore, it never (or very rarely) forms virulent virions, which removes the need for screening to avoid phage infected clones.\u003c/p\u003e\n\u003cp\u003eIn order to induce GTA22, overnight cultures were diluted 1:20 into LB containing 0.1% (w/v) L-arabinose. After incubation for at least 6 hours to allow lysis, any surviving bacteria were killed by vigorous shaking with chloroform. Lysates were cleared by centrifugation (\u0026ge;\u0026thinsp;14,000 \u0026times;g for 1\u0026ndash;3 min) before being used in transductions. For transductions, 0.1\u0026ndash;10 \u0026micro;l of cleared lysate was mixed with 100 \u0026micro;l LB supplemented with 0.4% glucose (to prevent expression of \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003earaBAD\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-ant\u003c/em\u003e\u003csub\u003e\u003cem\u003eP22\u003c/em\u003e\u003c/sub\u003e in the recipient due to remaining arabinose in the lysate), after which 100 \u0026micro;l overnight culture of the recipient strain was added. After 30 min incubation at 37\u0026deg;C the transductions were plated on appropriate selective media, and the plates were incubated until colonies appeared (overnight on LA, 24\u0026ndash;48 h on M9).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eGeneration of a mutator allele\u003c/h2\u003e\n\u003cp\u003eIn order to generate a reversible knock-out of \u003cem\u003emutS\u003c/em\u003e, an insertion of the selectable and counter selectable \u003cem\u003eAcatsac1\u003c/em\u003e cassette (GenBank accession: MF124798; containing the genes \u003cem\u003eamilCP\u003c/em\u003e, \u003cem\u003ecat\u003c/em\u003e, and \u003cem\u003esacB\u003c/em\u003e, conferring blue color, chloramphenicol resistance, and sucrose sensitivity, respectively) was designed as a duplication-insertion (DUP-In)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e so that it would generate a duplication with a single copy of the \u003cem\u003eAcatsac1\u003c/em\u003e cassette inserted between two truncated \u003cem\u003emutS\u003c/em\u003e copies, one containing the first\u0026thinsp;~\u0026thinsp;1.5 kb of the 2.5 kb \u003cem\u003emutS\u003c/em\u003e gene, and the other containing the last\u0026thinsp;~\u0026thinsp;1.5 kb. As none of the \u003cem\u003emutS\u003c/em\u003e copies are complete they are unlikely to be functional (as was verified from the large amounts of genomic mutations in the evolved mutator populations compared to the isogenic non-mutator populations; Table S2, parts 2 and 3), and due to the presence of the cassette the colonies are blue, chloramphenicol resistant and sucrose sensitive. The reason for making it as a DUP-In was to be able to revert the mutator allele (by plating on sucrose to find cells that lost the cassette by recombination between the 500 bp repeats) in clones isolated from the evolved populations, but this was not tested.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Anna Kn\u0026ouml;ppel, Omar Warsi, Ramith Nair, and Dan Andersson for critical reading of the manuscript. This work was founded by the Swedish Research Council (VR-NT 2020-03512) and the Carl Trygger Foundation (CTS 22:2094).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJN conceived the study, constructed bacterial strains, made bacterial growth rate assays and analyzed data. HA and JN performed evolution experiments, and isolated and sequenced clones from evolved populations. HA wrote an early draft of the manuscript as part of her PhD thesis, JN re-worked and extended the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJensen, R. A. Enzyme recruitment in evolution of new function. \u003cem\u003eAnnu Rev Microbiol\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 409\u0026ndash;425 (1976).\u003c/li\u003e\n\u003cli\u003eKhersonsky, O. \u0026amp; Tawfik, D. S. Enzyme promiscuity: a mechanistic and evolutionary perspective. \u003cem\u003eAnnu Rev Biochem\u003c/em\u003e \u003cstrong\u003e79\u003c/strong\u003e, 471\u0026ndash;505 (2010).\u003c/li\u003e\n\u003cli\u003eBergthorsson, U., Andersson, D. I. \u0026amp; Roth, J. R. Ohno\u0026rsquo;s dilemma: Evolution of new genes under continuous selection. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 17004\u0026ndash;17009 (2007).\u003c/li\u003e\n\u003cli\u003eBarona-Gomez, F. \u0026amp; Hodgson, D. A. Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis. \u003cem\u003eEMBO Rep\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 296\u0026ndash;300 (2003).\u003c/li\u003e\n\u003cli\u003eOhno, S. \u003cem\u003eEvolution by Gene Duplication\u003c/em\u003e. (Springer-Verlag, 1970). doi:10.1007/978-3-642-86659-3.\u003c/li\u003e\n\u003cli\u003eSoskine, M. \u0026amp; Tawfik, D. S. Mutational effects and the evolution of new protein functions. \u003cem\u003eNat. Rev. Genet. 2015 168\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 572\u0026ndash;582 (2010).\u003c/li\u003e\n\u003cli\u003eReams, A. B., Kofoid, E., Savageau, M. \u0026amp; Roth, J. R. Duplication Frequency in a Population of Salmonella enterica Rapidly Approaches Steady State With or Without Recombination. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e184\u003c/strong\u003e, 1077\u0026ndash;1094 (2010).\u003c/li\u003e\n\u003cli\u003eAnderson, P. \u0026amp; Roth, J. Spontaneous tandem genetic duplications in Salmonella typhimurium arise by unequal recombination between rRNA (rrn) cistrons. \u003cem\u003eProc Natl Acad Sci U A\u003c/em\u003e \u003cstrong\u003e78\u003c/strong\u003e, 3113\u0026ndash;3117 (1981).\u003c/li\u003e\n\u003cli\u003eFan, C., Chen, Y. \u0026amp; Long, M. Recurrent Tandem Gene Duplication Gave Rise to Functionally Divergent Genes in Drosophila. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1451\u0026ndash;1458 (2008).\u003c/li\u003e\n\u003cli\u003eHaack, K. R. \u0026amp; Roth, J. R. Recombination between chromosomal IS200 elements supports frequent duplication formation in Salmonella typhimurium. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e141\u003c/strong\u003e, 1245\u0026ndash;1252 (1995).\u003c/li\u003e\n\u003cli\u003eHill, C. W., Foulds, J., Soll, L. \u0026amp; Berg, P. Instability of a missense suppressor resulting from a duplication of genetic material. \u003cem\u003eJ. Mol. Biol.\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 563\u0026ndash;581 (1969).\u003c/li\u003e\n\u003cli\u003eN\u0026auml;svall, J., Sun, L., Roth, J. R. \u0026amp; Andersson, D. I. Real-time evolution of new genes by innovation, amplification, and divergence. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e338\u003c/strong\u003e, 384\u0026ndash;387 (2012).\u003c/li\u003e\n\u003cli\u003eLundin, E., N\u0026auml;svall, J. \u0026amp; Andersson, D. I. Mutational Pathways and Trade-Offs Between HisA and TrpF Functions: Implications for Evolution via Gene Duplication and Divergence. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 588235 (2020).\u003c/li\u003e\n\u003cli\u003eDue, A. V., Kuper, J., Geerlof, A., von Kries, J. P. \u0026amp; Wilmanns, M. Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 3554\u0026ndash;3559 (2011).\u003c/li\u003e\n\u003cli\u003ePlach, M. G., Reisinger, B., Sterner, R. \u0026amp; Merkl, R. Long-Term Persistence of Bi-functionality Contributes to the Robustness of Microbial Life through Exaptation. \u003cem\u003ePLOS Genet.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e1005836 (2016).\u003c/li\u003e\n\u003cli\u003eEllis Hudson, R., Bergthorsson, U. \u0026amp; Ochman, H. Transcription increases multiple spontaneous point mutations in Salmonella enterica. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 4517\u0026ndash;4522 (2003).\u003c/li\u003e\n\u003cli\u003eTorres-Cruz, J. \u0026amp; van der Woude, M. W. Slipped-Strand Mispairing Can Function as a Phase Variation Mechanism in Escherichia coli. \u003cem\u003eJ. Bacteriol.\u003c/em\u003e \u003cstrong\u003e185\u003c/strong\u003e, 6990\u0026ndash;6994 (2003).\u003c/li\u003e\n\u003cli\u003eLang, D., Thoma, R., Henn-Sax, M., Sterner, R. \u0026amp; Wilmanns, M. Structural Evidence for Evolution of the \u0026beta;/\u0026alpha; Barrel Scaffold by Gene Duplication and Fusion. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e289\u003c/strong\u003e, 1546\u0026ndash;1550 (2000).\u003c/li\u003e\n\u003cli\u003eJ\u0026uuml;rgens, C. \u003cem\u003eet al.\u003c/em\u003e Directed evolution of a (\u0026beta;\u0026alpha;)8-barrel enzyme to catalyze related reactions in two different metabolic pathways. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 9925\u0026ndash;9930 (2000).\u003c/li\u003e\n\u003cli\u003eEvran, S., Telefoncu, A. \u0026amp; Sterner, R. Directed evolution of (\u0026beta;\u0026alpha;)8-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase \u0026alpha;-subunit. \u003cem\u003eProtein Eng. Des. Sel.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 285\u0026ndash;293 (2012).\u003c/li\u003e\n\u003cli\u003eSchmieger, H. Phage P22-mutants with increased or decreased transduction abilities. \u003cem\u003eMol. Gen. Genet. MGG\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 75\u0026ndash;88 (1972).\u003c/li\u003e\n\u003cli\u003eHanahan, D. Studies on transformation of Escherichia coli with plasmids. \u003cem\u003eJ. Mol. Biol.\u003c/em\u003e \u003cstrong\u003e166\u003c/strong\u003e, 557\u0026ndash;580 (1983).\u003c/li\u003e\n\u003cli\u003eMiller, J. H. \u003cem\u003eA Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia Coli and Related Bacteria\u003c/em\u003e. (Cold Spring Harbor Laboratory Press, Plainview, N.Y, 1992).\u003c/li\u003e\n\u003cli\u003eN\u0026auml;svall, J., Kn\u0026ouml;ppel, A. \u0026amp; Andersson, D. I. Duplication-Insertion Recombineering: a fast and scar-free method for efficient transfer of multiple mutations in bacteria. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, e33 (2017).\u003c/li\u003e\n\u003cli\u003eN\u0026auml;svall, J. Direct and Inverted Repeat stimulated excision (DIRex): Simple, single-step, and scar-free mutagenesis of bacterial genes. \u003cem\u003ePloS One\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e0184126 (2017).\u003c/li\u003e\n\u003cli\u003eN\u0026auml;svall, J. Dup-In and DIRex: Techniques for Single-Step, Scar-Free Mutagenesis with Marker Recycling. in \u003cem\u003eRecombineering: Methods and Protocols\u003c/em\u003e (ed. Reisch, C. R.) 85\u0026ndash;104 (Springer US, New York, NY, 2022). doi:10.1007/978-1-0716-2233-9_7.\u003c/li\u003e\n\u003cli\u003eYu, D. \u003cem\u003eet al.\u003c/em\u003e An efficient recombination system for chromosome engineering in Escherichia coli. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 5978\u0026ndash;5983 (2000).\u003c/li\u003e\n\u003cli\u003eKoskiniemi, S., Pr\u0026auml;nting, M., Gullberg, E., N\u0026auml;svall, J. \u0026amp; Andersson, D. I. Activation of cryptic aminoglycoside resistance in Salmonella enterica. \u003cem\u003eMol. Microbiol.\u003c/em\u003e \u003cstrong\u003e80\u003c/strong\u003e, 1464\u0026ndash;1478 (2011).\u003c/li\u003e\n\u003cli\u003eLeavitt, J. C. \u003cem\u003eet al.\u003c/em\u003e Bacteriophage P22 SieA-mediated superinfection exclusion. \u003cem\u003emBio\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, e0216923 (2024).\u003c/li\u003e\n\u003cli\u003eSusskind, M. M. \u0026amp; Botstein, D. Molecular genetics of bacteriophage P22. \u003cem\u003eMicrobiol. Rev.\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 385\u0026ndash;413 (1978).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4087552/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4087552/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNew genes can evolve by mutations that generate a new function in an existing gene\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, these mutations often have a negative impact on the original function, leading to trade-offs that constrain their further evolution\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Genes that exhibit a strong trade-off between the original and the new function are expected to evolve through gene duplication, which can increase the expression of a weak new activity, buffer against negative effects on the original function, and provide more targets for beneficial mutations to arise\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The expected outcome of evolution in conditions where both functions are beneficial is a new pair of paralogs, each specialized for one function. Despite this, there are examples in nature where bi-functional generalist enzymes have evolved from a presumed specialist ancestor\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This study tests the hypothesis that generalist enzymes can evolve from specialist ancestral enzymes if selection for the new function is repeatedly interrupted by periods without selection (\u003cem\u003ei.e.\u003c/em\u003e selection for the new function is intermittent). In evolution experiments using bacteria lacking an enzyme in the tryptophan synthesis pathway, with intermittent selection for restoring tryptophan synthesis, multiple examples were found where initially specialized enzymes in two different pathways evolved towards becoming bi-functional generalist enzymes supporting both their new and original functions.\u003c/p\u003e \u003cp\u003eOur results highlight the importance of considering selection in nature not as a constant, but as a force that may fluctuate, and that fluctuating selection can drastically change the outcome by forcing evolution along paths that are highly constrained by conflicting selection pressures. Especially for duplications, which are mechanistically unstable and often costly, intermittent selection is expected to have a huge impact: If selection for a beneficial limiting function fluctuates, duplications may be counter selected and lost at regular intervals, forcing alternative paths of evolution that do not require duplications.\u003c/p\u003e","manuscriptTitle":"Evolution of new genes under intermittent selection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 15:54:07","doi":"10.21203/rs.3.rs-4087552/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"28f3e5e6-8f46-4e01-a124-3107b5de8242","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":29642044,"name":"Biological sciences/Evolution/Experimental evolution"},{"id":29642045,"name":"Biological sciences/Microbiology/Bacteria/Bacterial evolution"}],"tags":[],"updatedAt":"2024-04-16T16:00:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-22 15:54:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4087552","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4087552","identity":"rs-4087552","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}