Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 03143 Kyiv, Ukraine.
Department of Biotechnology, Leather and Fur, Kyiv National University of Technologies and Design, 01011 Kyiv, Ukraine.
Int J Mol Sci. 2022 Dec 1;23(23):15096. doi: 10.3390/ijms232315096.
SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air-liquid interface biofilms in experimental microcosms and on plant surfaces. Previous investigations have shown that cellulose is the primary structural matrix component in the robust and well-attached Wrinkly Spreader biofilm, as well as in the fragile Viscous Mass biofilm. Here, we demonstrate that both biofilms include extracellular DNA (eDNA) which can be visualized using confocal laser scanning microscopy (CLSM), quantified by absorbance measurements, and degraded by DNase I treatment. This eDNA plays an important role in cell attachment and biofilm development. However, exogenous high-molecular-weight DNA appears to decrease the strength and attachment levels of mature Wrinkly Spreader biofilms, whereas low-molecular-weight DNA appears to have little effect. Further investigation with CLSM using an amyloid-specific fluorophore suggests that the Wrinkly Spreader biofilm might also include Fap fibers, which might be involved in attachment and contribute to biofilm strength. The robust nature of the Wrinkly Spreader biofilm also allowed us, using MALDI-TOF mass spectrometry, to identify matrix-associated proteins unable to diffuse out of the structure, as well as membrane vesicles which had a different protein profile compared to the matrix-associated proteins. CLSM and DNase I treatment suggest that some vesicles were also associated with eDNA. These findings add to our understanding of the matrix components in this model pseudomonad, and, as found in other biofilms, biofilm-specific products and material from lysed cells contribute to these structures through a range of complex interactions.
SBW25 是一种与土壤和植物相关的模式细菌，能够在实验微环境中和植物表面形成多种气-液界面生物膜。先前的研究表明，纤维素是坚固且紧密附着的褶皱延伸生物膜以及脆弱的粘性物质生物膜的主要结构基质成分。在这里，我们证明这两种生物膜都包含胞外 DNA（eDNA），可以使用共聚焦激光扫描显微镜（CLSM）可视化，通过吸光度测量定量，并通过 DNase I 处理降解。这种 eDNA 在细胞附着和生物膜发育中起着重要作用。然而，外源高分子量 DNA 似乎会降低成熟褶皱延伸生物膜的强度和附着水平，而低分子量 DNA 似乎影响不大。使用针对淀粉样蛋白的荧光染料的进一步 CLSM 研究表明，褶皱延伸生物膜可能还包含 Fap 纤维，它可能参与附着并有助于生物膜强度。褶皱延伸生物膜的坚固性质还使我们能够使用 MALDI-TOF 质谱法鉴定无法从结构中扩散出来的基质相关蛋白，以及与基质相关蛋白相比具有不同蛋白质谱的膜泡。CLSM 和 DNase I 处理表明，一些囊泡也与 eDNA 相关。这些发现增加了我们对这种模式假单胞菌中基质成分的理解，并且与其他生物膜一样，生物膜特异性产物和来自裂解细胞的物质通过一系列复杂的相互作用为这些结构做出贡献。
