Genomic instability and biofilm determinants in <i>Streptococcus mutans</i> : insights from a sequence-defined arrayed transposon library

ABSTRACT Streptococcus mutans 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 gtfBC locus, while 7% had lost the Tn Smu1 element, an excision rate 1,000-fold higher than previously reported. While targeted mutagenesis confirmed that Tn Smu1 loss does not impact biofilm integrity, the gtfBC 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 S. mutans 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. IMPORTANCE Streptococcus mutans causes dental caries through resilient, acidogenic biofilm formation. While pooled screens often overlook extracellular or moderate-effect determinants due to community masking, this study presents a sequence-defined arrayed mutant library to dissect individual gene functions in isolation. Beyond known machinery, we identified novel biofilm determinants, including metal transporter SMU_635 and glycosylation-associated protein SMU_2160. Crucially, we uncovered pervasive genomic instability at the gtfBC and Tn Smu1 loci. This reveals a systemic functional genomics risk: misattributing phenotypes to primary mutations when backgrounds undergo large-scale rearrangements. By establishing whole-genome verification as a necessary standard, this research ensures that future therapeutic target identification is built upon a verified genetic foundation.