Howell Matthew, Daniel Jeremy J, Brown Pamela J B
Division of Biological Sciences, University of Missouri.
Division of Biological Sciences, University of Missouri;
J Vis Exp. 2017 Nov 24(129):56497. doi: 10.3791/56497.
Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.
核心细胞过程，如DNA复制与分离、蛋白质合成、细胞壁生物合成以及细胞分裂，都依赖于对细菌生存至关重要的蛋白质的功能。一系列靶向特异性染料可用作探针，以更好地理解这些过程。用亲脂性染料染色能够观察膜结构、可视化脂质微区以及检测膜泡。使用荧光d -氨基酸（FDAAs）探测肽聚糖生物合成位点可指示细胞壁生物合成或细胞生长模式中的潜在缺陷。最后，核酸染色可指示DNA复制或染色体分离中可能存在的缺陷。花菁DNA染料可标记活细胞，适用于延时显微镜观察，能够在细胞生长过程中实时观察类核形态。细胞标记方案可应用于蛋白质缺失突变体，以识别膜结构、细胞壁生物合成或染色体分离中的缺陷。此外，延时显微镜可用于监测去除一种必需蛋白质时的形态变化，并可为蛋白质功能提供更多见解。例如，必需细胞分裂蛋白的缺失会导致细胞丝状化或分支，而细胞生长蛋白的缺失可能会使细胞变得更短或更圆。在此，为细菌植物病原体根癌农杆菌提供了细胞生长、靶向特异性标记和延时显微镜观察的方案。总之，靶向特异性染料和延时显微镜能够对根癌农杆菌中的基本过程进行表征。最后，所提供的方案可轻松修改，以探测其他细菌中的基本过程。
