Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a 'glue', facilitating cell-cell and cell-substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.

中文翻译：

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多个孔蛋白有助于铜绿假单胞菌生物膜中的细胞外 DNA 释放

细菌生物膜由包裹在细胞外聚合物 (EPS) 基质中的细胞聚集体组成。EPS 的一个关键成分是细胞外 DNA (eDNA)，它充当“胶水”，促进细胞-细胞和细胞-基质相互作用。我们之前已经证明 eDNA 是通过爆炸性细胞裂解在铜绿假单胞菌生物膜中产生的。由于内溶素 Lys 降解肽聚糖，这种现象涉及细菌种群的一个子集爆炸性裂解。在这里，我们证明了在铜绿假单胞菌中，AlpB、CidA 和 Hol 三个穴蛋白都参与了浸没（水合）和间质（主动扩张）生物膜中 Lys 介导的 eDNA 释放，尽管程度不同，具体取决于生物膜的类型和生物膜发展阶段。我们还证明 eDNA 释放事件决定了在淹没生物膜发展的早期阶段细胞开始聚集以启动微菌落形成的位点。此外，我们的结果表明，eDNA 的持续释放是淹没生物膜中细胞簇整合和随后的微菌落发育所必需的。总体而言，这项研究增加了我们对铜绿假单胞菌生物膜内 eDNA 释放如何在时间和空间上受到控制的理解。