Identification of genes involved in extracellular DNA release during biofilm formation in Clostridium perfringens

Clostridium perfringens forms temperature-responsive pellicle biofilms at 25 °C, characterized by a fibrous matrix composed of BsaA proteins. In addition to BsaA, extracellular DNA (eDNA) contributes to biofilm structural integrity. However, the mechanism underlying its release remains unclear. This study aimed to determine the genetic determinants of eDNA release during pellicle biofilm maturation.

We visualized the release of nucleic acids during C. perfringens proliferation using anaerobic live-cell imaging and quantified dead-cell frequency and eDNA levels. We analyzed biofilm matrix-associated RNA by RNA-seq. We deleted candidate genes to assess their roles in biofilm formation and eDNA release. Promoter activity was evaluated using fluorescence reporter assays.

Live-cell imaging demonstrated that, during growth, a subpopulation released nucleic acids via cell lysis. Dead-cell frequency and eDNA levels increased as a function of pellicle biofilm maturation, suggesting that eDNA released from dead cells contributed to biofilm integrity. We found that biofilm matrix-associated extracellular nucleic acids contained RNA, which likely reflects the transcriptome of lysed cells. RNA-seq revealed transcripts enriched in the matrix fraction, thereby identifying several genes required for pellicle biofilm maturation. Among them, deletion of cpe2430 markedly reduced eDNA levels and impaired biofilm structural stability. Promoter-reporter analysis showed bimodal expression of cpe2430, with high expression limited to ∼3 of the population.

We identified genetic factors governing eDNA release and biofilm maturation in C. perfringens. Our findings suggest a model in which regulated lysis of a subpopulation drives eDNA release, thereby reinforcing biofilm architecture.