Microanatomy at cellular resolution and spatial order of physiological differentiation in a bacterial biofilm.
Microanatomy at cellular resolution and spatial order of physiological differentiation in a bacterial biofilm.
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DOI：
10.1128/mbio.00103-13
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发表时间：
2013-03-19
期刊：
影响因子：
6.4
通讯作者：
Hengge R
中科院分区：
文献类型：
--
作者：
Serra DO;Richter AM;Klauck G;Mika F;Hengge R
细菌生物膜是高度结构化的多细胞群落，其形成涉及鞭毛和粘附素、淀粉样纤维和胞外多糖的细胞外基质。当营养物质变得不理想时，鞭毛是由仍在分裂的杆状大肠杆菌细胞在指数后生长期间产生的。然而，进入稳定期后，细胞停止产生鞭毛，变成卵圆形，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学的整体变化，取决于RNA聚合酶稳定期σ亚基σS (RpoS)、核苷酸第二信使环AMP (cAMP)、ppGpp和环二GMP以及生物膜控制转录因子CsgD的诱导。使用鞭毛、curli 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”标志，可以在细胞分辨率下区分大肠杆菌 K-12 大菌落生物膜中的不同生理区域。包裹在卷曲纤维网络中的小卵形细胞形成外生物膜层。内部区域的特点是异质 CsgD::GFP 和 curli 表达。大集落的底部区域具有细长的分裂细胞和紧密的缠结鞭毛网，其形成需要鞭毛运动功能。此外，外缘生长区的细胞会产生鞭毛，鞭毛将细胞包裹并束缚在一起。邻近该生长区，小链和较短的卷曲周围细胞斑块与有鞭毛的无卷曲细胞并排出现，然后卷曲覆盖最终汇合，基本上表层中的所有细胞都被包裹在“卷曲篮”中。生物膜中的异质性或细胞分化是一个普遍接受的概念，但很难获得微观尺度的直接证据。我们的研究以前所未有的细胞分辨率揭示了大肠杆菌大菌落生物膜的微观解剖学和微生理学，其生理上不同的区域和层形成为已知的全球调节网络的功能，这些网络对生物膜固有的营养供应梯度作出反应。此外，这项研究还确定了异质且潜在双稳态的 CsgD 和 curli 表达区域，显示细菌 curli 网络与阿尔茨海默病斑块惊人地相似，并表明鞭毛作为生物膜中的结构元素的新作用。
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, σS (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.