{"doi":"10.1113/jp288355","title":"Evolutionary poker lacks a full deck when modelling the LTEE Cit<sup>+</sup> phenotype","abstract":"The recent paper by Root-Bernstein & Bernstein (2024) in The Journal of Physiology on the E-poker modelling of Lenski's Long-Term Evolution Experiment (LTEE) cites our work (van Hofwegen et al., 2016). We appreciate the insightful exploration by the authors, especially regarding the role of interactomes and evolutionary processes. We would like to clarify several points that refer to our findings and add context to the general discussion about evolutionary contingency and interpreting the LTEE Escherichia coli Cit+ phenotype. Root-Bernstein and Bernstein (p. 2523) cite that we explicitly conclude that the LTEE negates Gould's theory of contingency. To clarify, we are referencing Lenski's early interpretation (Pennesi, 2013). Lenski initially thought all LTEE E. coli populations followed a uniform evolutionary path but changed his view after a Cit+ variant emerged in one of 12 flasks after 15 years. Indeed, after another 20 years, additional Cit+ variants have not arisen in the LTEE giving the impression that the route to this phenotype is contingent on rare antecedent mutations and not repeatable. Using basic genetic analysis, our results show Cit+ mutations are not due to contingency. Gould's theory may be true, just not in this circumstance. The key points of our results are the reversible mechanism by which aerobic citrate transport arises and the genetic differences in succinate metabolism between E. coli K12 and E. coli B. Root-Bernstein and Bernstein (p. 2523) state, ‘….no previous strain of E. coli had ever been observed to be able to catabolize citrate in aerobic conditions’. To clarify, Lenski states in his original LTEE Cit+ paper (Blount et al., 2008) that aerobic citrate use by E. coli had only been observed once before (Hall, 1982) but gives no details. Hall's paper describes the isolation of E. coli K12 Cit+ variants in 14 days compared to the LTEE 15 years. We simply asked, ‘what could account for this significant time difference between these two experiments?’ Hall's work was done when genomic sequencing was not available to genotype his mutation. He mapped his mutation to the region of the chromosome responsible for anaerobic citrate metabolism and speculated he had activated the anaerobic citrate transporter. To pick up on his work, we first repeated his experiments. We isolated multiple independent Cit+ mutants that arose between 12 and 100 generations using a direct selection with citrate as the sole carbon source (a direct selection in contrast to the LTEE as Root-Bernstein and Bernstein rightly identify). Importantly, genomic sequencing shows the type/class of our Cit+ mutants are analogous to Lenski's lone Cit+ LTEE isolate. They all arise from an amplification in the citrate transporter gene, citT, followed by a promoter capture by genome restructuring/fusion with an aerobically expressed nearby gene. Gene amplifications are the most common mutations in E. coli and importantly they are reversible and subject to intermittent selection, for and against, during the 24 h cycle of the LTEE protocol. We verified this point by repeating Lenski's protocol but transferred cultures after 1 week instead of 24 h. After one or two transfers (1–2 weeks), Cit+ variants arise in multiple independent experiments. This is because extended time in stationary phase with citrate allows cells with citT amplifications to increase in number and thus increase the probability of a promoter capture. Roth and Maisnier-Patin (2016) agree with our interpretation that intermittent selection was at play vs. contingency. Finally, using phage transduction, we show that the Cit+ mutation can be transferred into wild-type E. coli K12 without prior citrate exposure, genetic proof that the aerobic Cit+ phenotypes do not require contingent single nucleotide polymorphism accumulation. Root-Bernstein and Bernstein rightly point out (their fig. 9) that two transporters are required for aerobic citrate metabolism, CitT (citrate antiporter) and D","journal":"The Journal of Physiology","year":2025,"id":561533,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9496,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":432570,"name":"Carolyn J. Hovde","orcid":"0000-0002-7383-3033","position":1,"is_corresponding":false},{"id":432569,"name":"Scott A. Minnich","orcid":"0000-0002-3202-9049","position":0,"is_corresponding":true}],"reference_count":9,"raw_metadata":null,"created_at":"2026-07-19T02:55:58.259104Z","pmid":"39907441","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}