{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T13:23:46Z","timestamp":1788269026891,"version":"build-2803163510"},"reference-count":47,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"The Royal Sociey","award":["NIF\\R1\\221190"],"award-info":[{"award-number":["NIF\\R1\\221190"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,9,1]]},"abstract":"ABSTRACT<\/jats:title>\n \n \n \n The alpha-proteobacterium\n Zymomonas mobilis<\/jats:italic>\n exhibits exceptional ethanologenic physiology, which makes it a traditional alcoholic beverage producer and a promising chassis for biofuel production. Although genetic tools for this organism have expanded in recent years, a fundamental aspect of its chromosome organization remains to be understood. In particular,\n Z. mobilis<\/jats:italic>\n has been suggested to exhibit polyploidy, but this feature is not fully confirmed because of discrepancies among studies reporting the copy number of chromosomes. Here, we tagged the chromosome-partitioning protein ParB with a fluorescent marker to visualize its cellular localization and estimate chromosome copy number in individual cells. Imaging showed that\n Z. mobilis<\/jats:italic>\n exhibits several distinctive ParB foci throughout the cytoplasm and an accumulated focus at the pole, indicating that a single\n Z. mobilis<\/jats:italic>\n cell contains >5 copies of the chromosome at the\n oriC<\/jats:italic>\n regions. We then sought to establish an efficient counter-selection system, which is crucial for engineering multiple copies of the chromosome. We assessed the efficacy of levan-sucrase (SacB) toxicity in\n Z. mobilis<\/jats:italic>\n . We found that, despite\n Z. mobilis<\/jats:italic>\n secreting a native extracellular sucrase SacB, heterologous periplasmically localized\n Bacillus subtilis<\/jats:italic>\n SacB rendered\n Z. mobilis<\/jats:italic>\n cells sensitive to sucrose. We successfully used this effect for counter-selection when deleting and inserting targeted DNA sequences into the\n Z. mobilis<\/jats:italic>\n genome. Together, this work provides important insights and tools for advancing\n Z. mobilis<\/jats:italic>\n genetics and its biotechnological applications.\n <\/jats:p>\n <\/jats:sec>\n \n IMPORTANCE<\/jats:title>\n \n Zymomonas mobilis<\/jats:italic>\n is a promising industrial bacterium with the capacity to convert sugars into ethanol at nearly maximum theoretical yield. With its expanding use in industrial applications, it is crucial to clarify if individual\n Z. mobilis<\/jats:italic>\n cells carry multiple copies of the chromosome, as this has important implications for genome engineering. Two previous studies have used quantitative PCR to address this question, but their reported chromosome copy numbers varied widely from 20 to 100. Here, we used a cell biological approach to estimate the copy number and confirmed that a single\n Z. mobilis<\/jats:italic>\n cell possesses multiple copies. In addition, we show that a SacB-based counter-selection works in\n Z. mobilis<\/jats:italic>\n , enabling efficient and complete mutation of all chromosome copies.\n <\/jats:p>\n <\/jats:sec>","DOI":"10.1128\/spectrum.00324-26","type":"journal-article","created":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T13:00:12Z","timestamp":1784638812000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Periplasmic SacB as a robust counter-selection tool for genome engineering in the polyploid bacterium\n Zymomonas mobilis<\/i>"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1825-2500","authenticated-orcid":true,"given":"Katsuya","family":"Fuchino","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01kj2bm70","id-type":"ROR","asserted-by":"publisher"}],"name":"Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle 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