Kai-Chih Chang1, Shun-Jia Cheng1, Yi-Cheng Chen2, Hsin-Ru Huang3, Je-Wen Liou4. 1. Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien 970, Taiwan. 2. Department of Medicine, Mackay Medical College, New Taipei 252, Taiwan. 3. Department of Biochemistry, School of Medicine, Tzu Chi University, Hualien 970, Taiwan. 4. Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien 970, Taiwan; Department of Biochemistry, School of Medicine, Tzu Chi University, Hualien 970, Taiwan. Electronic address: jwliou@mail.tcu.edu.tw.
Abstract
BACKGROUND AND PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
BACKGROUND AND PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Authors: Dhruba Acharya; Matthew J Sullivan; Benjamin L Duell; Tanguy Eveno; Mark A Schembri; Glen C Ulett Journal: Front Cell Infect Microbiol Date: 2019-04-24 Impact factor: 5.293