The extracellular matrix of bacterial biofilms consists of diverse components including polysaccharides, proteins and DNA. Extracellular RNA (eRNA) can also be present, contributing to the structural integrity of biofilms. However, technical difficulties related to the low stability of RNA make it difficult to understand the precise roles of eRNA in biofilms. Here, we show that eRNA associates with extracellular DNA (eDNA) to form matrix fibres in Pseudomonas aeruginosa biofilms, and the eRNA is enriched in certain bacterial RNA transcripts. Degradation of eRNA associated with eDNA led to a loss of eDNA fibres and biofilm viscoelasticity. Compared with planktonic and biofilm cells, the biofilm matrix was enriched in specific mRNA transcripts, including lasB (encoding elastase). The mRNA transcripts colocalised with eDNA fibres in the biofilm matrix, as shown by single molecule inexpensive FISH microscopy (smiFISH). The lasB mRNA was also observed in eDNA fibres in a clinical sputum sample positive for P. aeruginosa. Thus, our results indicate that the interaction of specific mRNAs with eDNA facilitates the formation of viscoelastic networks in the matrix of Pseudomonas aeruginosa biofilms.

细菌生物膜的细胞外基质由多种成分组成，包括多糖、蛋白质和DNA。细胞外 RNA (eRNA) 也可能存在，有助于生物膜的结构完整性。然而，与 RNA 稳定性低相关的技术困难使得人们很难理解 eRNA 在生物膜中的精确作用。在这里，我们发现，eRNA 与细胞外 DNA (eDNA) 结合，在铜绿假单胞菌生物膜中形成基质纤维，并且 eRNA 富含某些细菌 RNA 转录本。与 eDNA 相关的 eRNA 降解导致 eDNA 纤维和生物膜粘弹性的损失。与浮游细胞和生物膜细胞相比，生物膜基质富含特定的 mRNA 转录物，包括lasB（编码弹性蛋白酶）。单分子廉价 FISH 显微镜 (smiFISH) 显示，mRNA 转录物与生物膜基质中的 eDNA 纤维共定位。在铜绿假单胞菌阳性的临床痰样本的 eDNA 纤维中也观察到了 lasB mRNA 。因此，我们的结果表明，特定 mRNA 与 eDNA 的相互作用促进了铜绿假单胞菌生物膜基质中粘弹性网络的形成。