{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,3,28]],"date-time":"2026-03-28T17:53:12Z","timestamp":1774720392507,"version":"3.50.1"},"posted":{"date-parts":[[2026,3,26]]},"group-title":"Microbiology","reference-count":42,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T00:00:00Z","timestamp":1774483200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,26]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>Streptococcus mutans<\/jats:italic>\n                  is a primary architect of dental caries, utilizing complex genetic networks to build resilient, acid-producing biofilms. While pooled screens (Tn-seq) have identified important fitness factors, they often fail to capture extracellular or moderate-effect determinants due to community-level masking. Therefore, to study biofilm phenotypes, we constructed a comprehensive arrayed library of 9,216 mutants and used Cartesian Pooling-Coordinate Sequencing (CP-CSeq) to establish a sequence-defined resource covering 51% of non-essential genes. By screening the entire collection in isolation, we identified several novel biofilm determinants, including the putative metal transporter SMU_635 and the glycosylation-associated protein SMU_2160. However, systematic whole-genome sequencing (WGS) of our hits revealed an interesting level of genomic instability: 25% of biofilm-defective mutants had undergone spontaneous recombination at the\n                  <jats:italic>gtfBC<\/jats:italic>\n                  locus, while 7% had lost the Tn\n                  <jats:italic>Smu1<\/jats:italic>\n                  element, an excision rate 1,000-fold higher than previously reported. While targeted mutagenesis confirmed that Tn\n                  <jats:italic>Smu1<\/jats:italic>\n                  loss does not impact biofilm integrity, the\n                  <jats:italic>gtfBC<\/jats:italic>\n                  deletions directly accounted for the most severe phenotypes, highlighting a systemic risk of misattributing gene functions to primary transposon insertions. Our findings provide a powerful new genetic resource for the\n                  <jats:italic>S. mutans<\/jats:italic>\n                  community while establishing a critical new standard: an arrayed library is only as defined as its underlying genome, making systematic genomic verification an essential prerequisite for accurate functional genomics.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Streptococcus mutans<\/jats:italic>\n                    is a major human pathogen responsible for dental caries, a global public health challenge driven in part by the organism\u2019s ability to form resilient, acidogenic biofilms. While traditional pooled genetic screens have identified many fitness factors, they often fail to capture extracellular or moderate-effect determinants because neighboring healthy bacteria can mask these defects. This work provides the scientific community with a sequence-defined arrayed mutant library, an essential resource for dissecting the individual contributions of genes to biofilm integrity in isolation. Beyond identifying well-known machinery, this study uncovers novel determinants, including the putative metal transporter SMU_635 and the putative glycosylation-associated protein SMU_2160. Crucially, the discovery of pervasive genomic instability within the library, specifically at the\n                    <jats:italic>gtfBC<\/jats:italic>\n                    and Tn\n                    <jats:italic>Smu1<\/jats:italic>\n                    loci, reveals a systemic risk in functional genomics: the potential to misattribute phenotypes to primary mutations when the underlying background has undergone large-scale rearrangements. By establishing systematic whole-genome verification as a necessary standard, this research ensures that the identification of future therapeutic targets is built upon a verified genetic foundation.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.25.714184","type":"posted-content","created":{"date-parts":[[2026,3,27]],"date-time":"2026-03-27T04:56:13Z","timestamp":1774587373000},"source":"Crossref","is-referenced-by-count":0,"title":["Genomic instability and biofilm determinants in\n                  <i>Streptococcus mutans<\/i>\n                  : insights from a sequence-defined arrayed transposon library"],"prefix":"10.64898","author":[{"given":"Ana Karen","family":"Solano Morales","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, Beck College of Sciences and Mathematics, Arkansas State University, Jonesboro, Arkansas, USA"},{"name":"Department of Oral Biology, College of Dentistry, University of Florida, 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USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrew","family":"Goode","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Beck College of Sciences and Mathematics, Arkansas State University, Jonesboro, Arkansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alejandro R.","family":"Walker","sequence":"additional","affiliation":[{"name":"Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anthony","family":"Sperduto","sequence":"additional","affiliation":[{"name":"Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bikash","family":"Dwivedi","sequence":"additional","affiliation":[{"name":"Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, 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