Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien 970, Taiwan.
Department of Medicine, Mackay Medical College, New Taipei 252, Taiwan.
J Microbiol Immunol Infect. 2013 Dec;46(6):405-12. doi: 10.1016/j.jmii.2012.08.004. Epub 2012 Sep 25.
Flagella contribute to the virulence of pathogenic bacteria through chemotaxis, motility, and adhesion. Understanding the various functions of flagella may provide insight into mechanisms of bacterial infection and transmission. The objectives of our study were to apply biophysical and biochemical methods to investigate the mechanisms of pH-dependent changes in flagella functions.
Atomic force microscopy (AFM) was used to analyze the flagellum morphology of Escherichia coli cultured in various pH conditions. The swarming plate method was used to identify pH-dependent changes in bacterial motility. Western blot analysis and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) were also carried out to study pH-dependent expression and structural changes of flagellin C.
E coli cultured at pH 7 produced the flagella with the greatest average length and diameter. When the bacteria were grown at pH 6 or pH 8, shorter and thinner forms of flagella were produced. The morphology of the flagella was correlated to the bacterial motility. While western blot analysis showed only a slight change in the expression of the flagellin C protein in response to changes in the pH of the culture medium, ATR-FTIR showed significant pH-dependent changes in the secondary structure of the flagellin C assembled in sheared flagella.
Our results show that both acidification and alkalization of the culture medium restricted bacterial motility, and indicate that the reduced motility may be caused by incorrect assembly of the flagellum proteins.
鞭毛通过趋化性、运动性和黏附性促进病原菌的毒力。了解鞭毛的各种功能可能有助于深入理解细菌感染和传播的机制。我们的研究目的是应用生物物理和生化方法来研究鞭毛功能随 pH 值变化的机制。
原子力显微镜（AFM）用于分析在不同 pH 条件下培养的大肠杆菌的鞭毛形态。趋化性平板法用于鉴定细菌运动性随 pH 值的变化。还进行了 Western blot 分析和衰减全反射傅里叶变换红外光谱（ATR-FTIR）分析，以研究鞭毛蛋白 C 的 pH 值依赖性表达和结构变化。
在 pH 7 下培养的大肠杆菌产生的鞭毛平均长度和直径最大。当细菌在 pH 6 或 pH 8 下生长时，会产生较短和较细的鞭毛形式。鞭毛的形态与细菌的运动性相关。虽然 Western blot 分析显示，培养基 pH 值变化对 flagellin C 蛋白的表达仅有轻微影响，但 ATR-FTIR 显示，在剪切的鞭毛中组装的 flagellin C 的二级结构存在显著的 pH 值依赖性变化。
我们的结果表明，培养基的酸化和碱化均限制了细菌的运动性，表明运动性降低可能是由于鞭毛蛋白组装不正确所致。
