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However, genetic toolbox for this biotechnologically relevant thermophilic bacterium is still limited.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      We here present the establishment of a counterselection system conducive to genome modifications in\n                      <jats:italic>Bacillus methanolicus<\/jats:italic>\n                      MGA3. We first identified four candidate genes or operons feasible to become counterselection markers:\n                      <jats:italic>lacZ<\/jats:italic>\n                      from\n                      <jats:italic>Bacillus coagulans<\/jats:italic>\n                      ,\n                      <jats:italic>sacB<\/jats:italic>\n                      from\n                      <jats:italic>Bacillus subtilis<\/jats:italic>\n                      ,\n                      <jats:italic>codBA<\/jats:italic>\n                      from\n                      <jats:italic>Escherichia coli<\/jats:italic>\n                      , and\n                      <jats:italic>oroP<\/jats:italic>\n                      from\n                      <jats:italic>Lactococcus lactis<\/jats:italic>\n                      , based on their absence from the genome of\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      . We tested substrates of the encoded enzymes to confirm their lack of toxicity to wild type\n                      <jats:italic>B. methanolicus.<\/jats:italic>\n                      Experimental results confirmed that none of the tested substrates affected the growth of\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      wild type at physiologically relevant concentrations. Subsequently, the selected genes were individually cloned into a low-copy plasmid pTH1mp and used to transform\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      . We evaluated the conversion of these non-toxic substrates to toxic products upon heterologous expression of the respective marker genes in\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      . The recombinant strains were demonstrated to possess the desired counterselection activity through lack of growth in the presence of their relevant substrate. A novel transconjugation method for high-efficiency plasmid-delivery of\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      was developed and used for the establishment of genome modification via non-replicating suicide vector designed for homologous recombination. Deletion of the chromosomal\n                      <jats:italic>upp<\/jats:italic>\n                      gene, crucial for uracil metabolism, was achieved using this method.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      In this study, we confirmed the utility of the established\n                      <jats:italic>oroP<\/jats:italic>\n                      -based counterselection system for the genome modifications through deletion of\n                      <jats:italic>upp<\/jats:italic>\n                      gene in\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      . The deletion strain exhibited reduced sensitivity to 5-fluorouracil, the toxic substrate of the\n                      <jats:italic>upp<\/jats:italic>\n                      encoded enzyme, demonstrating the practical application of the counterselection markers in genome engineering of\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12934-025-02880-0","type":"journal-article","created":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T07:35:00Z","timestamp":1765352100000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development of a markerless tool for targeted chromosome modification in the thermophilic and methylotrophic bacterium Bacillus methanolicus"],"prefix":"10.1186","volume":"25","author":[{"given":"Marta","family":"Irla","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luciana Fernandes","family":"Brito","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jesper","family":"Langlo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carsten","family":"Wohlers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leonie","family":"Benninghaus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chantel","family":"Heid","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Volker F.","family":"Wendisch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jochen","family":"Schmid","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Trygve","family":"Brautaset","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,12,10]]},"reference":[{"key":"2880_CR1","doi-asserted-by":"publisher","first-page":"517","DOI":"10.1007\/s00253-014-6240-3","volume":"99","author":"AM Ochsner","year":"2015","unstructured":"Ochsner AM, Sonntag F, Buchhaupt M, Schrader J, Vorholt JA. 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