Institut für Biologie, Mikrobiologie, Freie Universität Berlin, Berlin, Germany.
mBio. 2013 Mar 19;4(2):e00103-13. doi: 10.1128/mBio.00103-13.
Bacterial biofilms are highly structured multicellular communities whose formation involves flagella and an extracellular matrix of adhesins, amyloid fibers, and exopolysaccharides. Flagella are produced by still-dividing rod-shaped Escherichia coli cells during postexponential growth when nutrients become suboptimal. Upon entry into stationary phase, however, cells stop producing flagella, become ovoid, and generate amyloid curli fibers. These morphological changes, as well as accompanying global changes in gene expression and cellular physiology, depend on the induction of the stationary-phase sigma subunit of RNA polymerase, σ(S) (RpoS), the nucleotide second messengers cyclic AMP (cAMP), ppGpp, and cyclic-di-GMP, and a biofilm-controlling transcription factor, CsgD. Using flagella, curli fibers, a CsgD::GFP reporter, and cell morphology as "anatomical" hallmarks in fluorescence and scanning electron microscopy, different physiological zones in macrocolony biofilms of E. coli K-12 can be distinguished at cellular resolution. Small ovoid cells encased in a network of curli fibers form the outer biofilm layer. Inner regions are characterized by heterogeneous CsgD::GFP and curli expression. The bottom zone of the macrocolonies features elongated dividing cells and a tight mesh of entangled flagella, the formation of which requires flagellar motor function. Also, the cells in the outer-rim growth zone produce flagella, which wrap around and tether cells together. Adjacent to this growth zone, small chains and patches of shorter curli-surrounded cells appear side by side with flagellated curli-free cells before curli coverage finally becomes confluent, with essentially all cells in the surface layer being encased in "curli baskets."
Heterogeneity or cellular differentiation in biofilms is a commonly accepted concept, but direct evidence at the microscale has been difficult to obtain. Our study reveals the microanatomy and microphysiology of an Escherichia coli macrocolony biofilm at an unprecedented cellular resolution, with physiologically different zones and strata forming as a function of known global regulatory networks that respond to biofilm-intrinsic gradients of nutrient supply. In addition, this study identifies zones of heterogeneous and potentially bistable CsgD and curli expression, shows bacterial curli networks to strikingly resemble Alzheimer plaques, and suggests a new role of flagella as an architectural element in biofilms.
细菌生物膜是高度结构化的多细胞群落，其形成涉及鞭毛和细胞外基质的黏附素、淀粉样纤维和胞外多糖。鞭毛是在指数生长后期营养物质变得不足时由仍在分裂的杆状大肠杆菌细胞产生的。然而，当进入静止期时，细胞停止产生鞭毛，变成椭圆形，并产生淀粉样卷曲纤维。这些形态变化以及伴随的基因表达和细胞生理的全局变化，依赖于 RNA 聚合酶的静止期 σ 亚基（RpoS）、核苷酸第二信使环腺苷酸（cAMP）、ppGpp 和环二鸟苷酸（cyclic-di-GMP）的诱导，以及一个生物膜控制转录因子 CsgD。使用鞭毛、卷曲纤维、CsgD::GFP 报告基因和细胞形态作为荧光和扫描电子显微镜中的“解剖学”标志，可以在细胞分辨率下区分大肠杆菌 K-12 宏观菌落生物膜中的不同生理区带。被卷曲纤维网络包裹的小椭圆形细胞形成外层生物膜层。内部区域的特征是 CsgD::GFP 和卷曲表达的异质性。宏观菌落的底部区域以伸长分裂的细胞和纠缠的鞭毛紧密网格为特征，鞭毛的形成需要鞭毛马达功能。此外，外边缘生长区的细胞产生鞭毛，鞭毛缠绕并将细胞系在一起。在这个生长区旁边，短链和短卷曲包裹细胞的斑块与鞭毛无卷曲的细胞并排出现，然后卷曲覆盖最终变得连续，表面层中的几乎所有细胞都被“卷曲篮子”包裹。
生物膜中的异质性或细胞分化是一个被普遍接受的概念，但在微观尺度上获得直接证据一直很困难。我们的研究以空前的细胞分辨率揭示了大肠杆菌宏观菌落生物膜的微观解剖结构和微观生理学，不同的生理区带和层形成是对生物膜内在养分供应梯度做出反应的已知全局调控网络的功能。此外，本研究确定了 CsgD 和卷曲表达异质和潜在双稳态的区域，表明细菌卷曲网络与阿尔茨海默斑块惊人地相似，并提出了鞭毛作为生物膜结构元素的新作用。
