The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for biofilm studies. To elucidate the location of bacterial flagella throughout the biofilm life cycle, we developed a new flagella biotracking tool. Bacterial flagella were site-specifically labeled via genetic code expansion. This enabled us to track bacterial flagella during biofilm maturation. Live flagella imaging revealed the presence and synthesis of flagella throughout the biofilm life cycle. To study the possible role of flagella in a biofilm, we produced a flagella knockout strain and compared its biofilm to that of the wild-type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison with the flagella knockout strain. This suggests a possible structural role for flagella in a biofilm, conceivably as a scaffold. Our findings suggest a new model for biofilm maturation dynamic which underscores the importance of direct evidence from within the biofilm.

机会性病原体铜绿假单胞菌是一种有鞭毛的细菌，是生物膜研究的顶级模型生物之一。为了阐明细菌鞭毛在整个生物膜生命周期中的位置，我们开发了一种新的鞭毛生物追踪工具。通过遗传密码扩展对细菌鞭毛进行位点特异性标记。这使我们能够在生物膜成熟过程中追踪细菌鞭毛。活鞭毛成像揭示了整个生物膜生命周期中鞭毛的存在和合成。为了研究鞭毛在生物膜中的可能作用，我们产生了鞭毛敲除菌株，并将其生物膜与野生型菌株的生物膜进行了比较。结果显示，与鞭毛敲除菌株相比，野生型的生物膜结构强一个数量级。这表明鞭毛在生物膜中可能具有结构作用，可以想象作为支架。我们的研究结果提出了生物膜成熟动态的新模型，强调了生物膜内部直接证据的重要性。