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Understanding the growth of Corynebacterium glutamicum
Understanding the growth of Corynebacterium glutamicum
From a cell biological standpoint, there is a great similarity between the harmless soil bacterium Corynebacterium glutamicum and Mycobacterium tuberculosis, the causative agent of the lung disease tuberculosis. Longitudinal growth of individual cells occurs exclusively from the poles, differing from the majority of known bacteria. Another characteristic of the so-called CMN (Corynebacteria, Mycobacteria, Nocardia) group is the structure of the cell wall. It is characterized by an outer hydrophobic mycomembrane (MM) and a layer of arabinogalactan (AG) covalently bound to the peptidoglycan (PG) of the cell wall. In the present work, various aspects of C. glutamicum growth and division were characterized. The spatio-temporal organization of individual cells and their components were the focus. Analyses were performed using biochemical, microscopic, and data processing methods. A developed image analysis environment found application in several publications. By using a microfluidic chamber, microscopic time-lapse images from growing C. glutamicum microcolonies were obtained. Individual cells from each timeframe were measured and placed in a genealogical context using custom software developed during the thesis. The resulting data were analyzed by the co-author of the study by using a newly developed inference method. This resulted in a characterization of the growth dynamics of individual cells. The mode called 'asymptotic-linear' describes an initial acceleration of the elongation velocity, which then changes into plateau. The explanation for this lies in organization of growth zones and maturation of the new cell pole after division. Further, the data showed that strict division symmetry in apically growing bacteria is unnecessary for a normal length distribution of cells within a population. In another sub-project, the cellular effects of two antibiotics were compared. These are the well-established anti-tuberculosis drug ethambutol (EMB) and the experimental compound benzothiazinone 043 (BTZ). Both substances act at different points in the metabolic pathway building up the AG layer. By using special staining techniques, fluorescence microscopy and image analysis, it was shown that sublethal concentrations of both substances cause apical growth in individual cells to cease. This is accompanied by a change in morphology. Treated cells appear shorter and broader. In addition to this commonality, the antibiotics also showed a different effect. In the case of EMB, discontinuity of MM at the cell poles was observed after appropriate staining. Upon the addition of BTZ, no change occurred here compared to the control. The corresponding finding was confirmed by electron microscopy. This also showed that the organization of the individual layers of the cell wall is strongly influenced by both substances. The integrity of the MM plays a protective role for the cell. This was shown by combined dilution series. Together with EMB, β-lactam antibiotics showed a synergistic effect. In contrast, when combined with BTZ, only an additive effect was observed. Furthermore, the analysis of static fluorescence microscopic images, described in this thesis contributed to the discovery of the diploid chromosome organization of C. glutamicum. By extracting fluorescence profiles of single cells and subsequent sorting, the dynamic localization of ParB over the cell cycle could be visualized. This shows an opposing pattern of foci, migrating from the cell poles to the center, depending on the length of the cell. The known mode of action of the ParABS system and the apical growth mode support the discovery of diploidy. Finally, the cell cycle-dependent localization of the DNA pump FtsK was demonstrated by using image analysis. With the help of a transposon mutagenesis experiment it was first determined that ftsK gets essential in the context of the absence of the DNA topology organizing protein SMC. The longer dwell-time of the labeled FtsZ derivative in mutant cells was found to be a consequence of increased entropy on cell cycle progression and was further confirmed by single particle tracking (SPT). In summary, several new insights into the growth and division of C. glutamicum were gained. Newly developed methods were extensively used and allow for further application. Important aspects of the regulation of cell length were presented for the first time for apical growth. The cell biological characterization of EMB and BTZ contributes to the understanding of the mode of action of both antibiotics. Finally, the contributions to the discovery of the diploid chromosome organization of C. glutamicum and the description of the influence of chromatid structure on the progress of the cell cycle, a deeper insight into the dynamic development of this cell structure was gained.
Mycobacteriales, Corynebacterium, Image analysis, Microscopy, Elongasome, Benzothiazione, Ethambutol
Meyer, Fabian Mark
2024
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Meyer, Fabian Mark (2024): Understanding the growth of Corynebacterium glutamicum. Dissertation, LMU München: Faculty of Biology
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Abstract

From a cell biological standpoint, there is a great similarity between the harmless soil bacterium Corynebacterium glutamicum and Mycobacterium tuberculosis, the causative agent of the lung disease tuberculosis. Longitudinal growth of individual cells occurs exclusively from the poles, differing from the majority of known bacteria. Another characteristic of the so-called CMN (Corynebacteria, Mycobacteria, Nocardia) group is the structure of the cell wall. It is characterized by an outer hydrophobic mycomembrane (MM) and a layer of arabinogalactan (AG) covalently bound to the peptidoglycan (PG) of the cell wall. In the present work, various aspects of C. glutamicum growth and division were characterized. The spatio-temporal organization of individual cells and their components were the focus. Analyses were performed using biochemical, microscopic, and data processing methods. A developed image analysis environment found application in several publications. By using a microfluidic chamber, microscopic time-lapse images from growing C. glutamicum microcolonies were obtained. Individual cells from each timeframe were measured and placed in a genealogical context using custom software developed during the thesis. The resulting data were analyzed by the co-author of the study by using a newly developed inference method. This resulted in a characterization of the growth dynamics of individual cells. The mode called 'asymptotic-linear' describes an initial acceleration of the elongation velocity, which then changes into plateau. The explanation for this lies in organization of growth zones and maturation of the new cell pole after division. Further, the data showed that strict division symmetry in apically growing bacteria is unnecessary for a normal length distribution of cells within a population. In another sub-project, the cellular effects of two antibiotics were compared. These are the well-established anti-tuberculosis drug ethambutol (EMB) and the experimental compound benzothiazinone 043 (BTZ). Both substances act at different points in the metabolic pathway building up the AG layer. By using special staining techniques, fluorescence microscopy and image analysis, it was shown that sublethal concentrations of both substances cause apical growth in individual cells to cease. This is accompanied by a change in morphology. Treated cells appear shorter and broader. In addition to this commonality, the antibiotics also showed a different effect. In the case of EMB, discontinuity of MM at the cell poles was observed after appropriate staining. Upon the addition of BTZ, no change occurred here compared to the control. The corresponding finding was confirmed by electron microscopy. This also showed that the organization of the individual layers of the cell wall is strongly influenced by both substances. The integrity of the MM plays a protective role for the cell. This was shown by combined dilution series. Together with EMB, β-lactam antibiotics showed a synergistic effect. In contrast, when combined with BTZ, only an additive effect was observed. Furthermore, the analysis of static fluorescence microscopic images, described in this thesis contributed to the discovery of the diploid chromosome organization of C. glutamicum. By extracting fluorescence profiles of single cells and subsequent sorting, the dynamic localization of ParB over the cell cycle could be visualized. This shows an opposing pattern of foci, migrating from the cell poles to the center, depending on the length of the cell. The known mode of action of the ParABS system and the apical growth mode support the discovery of diploidy. Finally, the cell cycle-dependent localization of the DNA pump FtsK was demonstrated by using image analysis. With the help of a transposon mutagenesis experiment it was first determined that ftsK gets essential in the context of the absence of the DNA topology organizing protein SMC. The longer dwell-time of the labeled FtsZ derivative in mutant cells was found to be a consequence of increased entropy on cell cycle progression and was further confirmed by single particle tracking (SPT). In summary, several new insights into the growth and division of C. glutamicum were gained. Newly developed methods were extensively used and allow for further application. Important aspects of the regulation of cell length were presented for the first time for apical growth. The cell biological characterization of EMB and BTZ contributes to the understanding of the mode of action of both antibiotics. Finally, the contributions to the discovery of the diploid chromosome organization of C. glutamicum and the description of the influence of chromatid structure on the progress of the cell cycle, a deeper insight into the dynamic development of this cell structure was gained.