Lau Peter C Y, Dutcher John R, Beveridge Terry J, Lam Joseph S
Biophysics Interdepartmental Group, University of Guelph, Guelph, ON N1G2W1, Canada.
Biophys J. 2009 Apr 8;96(7):2935-48. doi: 10.1016/j.bpj.2008.12.3943.
Bacterial biofilms are the most prevalent mode of bacterial growth in nature. Adhesive and viscoelastic properties of bacteria play important roles at different stages of biofilm development. Following irreversible attachment of bacterial cells onto a surface, a biofilm can grow in which its matrix viscoelasticity helps to maintain structural integrity, determine stress resistance, and control ease of dispersion. In this study, a novel application of force spectroscopy was developed to characterize the surface adhesion and viscoelasticity of bacterial cells in biofilms. By performing microbead force spectroscopy with a closed-loop atomic force microscope, we accurately quantified these properties over a defined contact area. Using the model gram-negative bacterium Pseudomonas aeruginosa, we observed that the adhesive and viscoelastic properties of an isogenic lipopolysaccharide mutant wapR biofilm were significantly different from those measured for the wild-type strain PAO1 biofilm. Moreover, biofilm maturation in either strain also led to prominent changes in adhesion and viscoelasticity. To minimize variability in force measurements resulting from experimental parameter changes, we developed standardized conditions for microbead force spectroscopy to enable meaningful comparison of data obtained in different experiments. Force plots measured under standard conditions showed that the adhesive pressures of PAO1 and wapR early biofilms were 34 +/- 15 Pa and 332 +/- 47 Pa, respectively, whereas those of PAO1 and wapR mature biofilms were 19 +/- 7 Pa and 80 +/- 22 Pa, respectively. Fitting of creep data to a Voigt Standard Linear Solid viscoelasticity model revealed that the instantaneous and delayed elastic moduli in P. aeruginosa were drastically reduced by lipopolysaccharide deficiency and biofilm maturation, whereas viscosity was decreased only for biofilm maturation. In conclusion, we have introduced a direct biophysical method for simultaneously quantifying adhesion and viscoelasticity in bacterial biofilms under native conditions. This method could prove valuable for elucidating the contribution of genetic backgrounds, growth conditions, and environmental stresses to microbial community physiology.
细菌生物膜是自然界中细菌最普遍的生长方式。细菌的粘附性和粘弹性在生物膜发育的不同阶段起着重要作用。细菌细胞不可逆地附着在表面后,生物膜开始生长,其基质粘弹性有助于维持结构完整性、决定抗逆性并控制分散的难易程度。在本研究中,开发了一种力谱学的新应用,用于表征生物膜中细菌细胞的表面粘附性和粘弹性。通过使用闭环原子力显微镜进行微珠力谱分析,我们在定义的接触面积上准确地量化了这些特性。使用革兰氏阴性模式菌铜绿假单胞菌,我们观察到同基因脂多糖突变体wapR生物膜的粘附性和粘弹性特性与野生型菌株PAO1生物膜的测量结果显著不同。此外,两种菌株中的生物膜成熟也导致粘附性和粘弹性的显著变化。为了最小化实验参数变化导致的力测量中的变异性,我们开发了微珠力谱分析的标准化条件,以便能够对不同实验中获得的数据进行有意义的比较。在标准条件下测量的力曲线表明,PAO1和wapR早期生物膜的粘附压力分别为34±15 Pa和332±47 Pa,而PAO1和wapR成熟生物膜的粘附压力分别为19±7 Pa和80±22 Pa。将蠕变数据拟合到Voigt标准线性固体粘弹性模型表明,脂多糖缺乏和生物膜成熟会使铜绿假单胞菌的瞬时和延迟弹性模量大幅降低,而仅生物膜成熟会使粘度降低。总之,我们引入了一种直接的生物物理方法,用于在自然条件下同时量化细菌生物膜中的粘附性和粘弹性。该方法对于阐明遗传背景、生长条件和环境压力对微生物群落生理学的贡献可能具有重要价值。