Cover Page
The handle http://hdl.handle.net/1887/138247 holds various files of this Leiden
University dissertation.
Author: Ultee, E.
Title: Structural characterization of the cell envelope in Actinobacteria under changing
environments
Chapter 1
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Chapter 1
Bacterial morphology
Bacteria are successful microorganisms that are found in virtually all environments. The beginning of microbiology is marked by a discovery of Antoni van Leeuwenhoek in the 17th century. Van Leeuwenhoek (1632-1723) was the first person to use a self-made microscope to observe and document “animalcules”- small animals or microorganisms 1. In the early days of microbial research, bacteria were identified and categorized into different classes based on cellular shape or morphology. In 1884, Christian Gram developed a staining procedure to further distinguish bacteria based on the build-up of their cell envelope 2,3. The bacterial cell envelope is a complex and multilayered structure, which protects the bacterium and mediates interaction with its surroundings. Bacteria that stain positive for the Gram stain (Gram-positive or monoderm) contain a cytoplasmic membrane and a thick cell wall layer, whereas bacteria that cannot retain the stain (Gram-negative or diderm) have a cytoplasmic membrane, an outer membrane and a thin cell wall layer located in between these two membranes (in the so-called periplasmic space). Modern techniques such as DNA sequencing have allowed species identification and characterization much more accurately than the Gram-staining procedure alone 4–6. Despite a wealth of information available today, the bacterial cell envelope remains a fascinating research topic for understanding important processes such as growth, virulence and its role in immune evasion in pathogenic bacteria. To address these questions, modern microscopy techniques such as fluorescence microscopy, super-resolution microscopy and atomic-force microscopy are valuable tools for further research on all aspects of the bacterial cell envelope 7–10. As I will show in this thesis, especially cryo-electron microscopy (cryo-EM) provides a unique opportunity to study bacterial cell envelope and morphology in structural detail.
Growth of filamentous actinomycetes
The cell envelope is a dynamic structure that needs to be sufficiently rigid to maintain bacterial morphology while also being elastic enough in order to allow for bacterial growth, division or morphogenesis. During growth, the cell wall needs to expand by including new peptidoglycan (PG) monomers into the existing PG network or sacculus that surrounds the bacterium 11. In most bacteria, this process is spatially coordinated by well-conserved cytoskeletal-like proteins associating with the PG synthesis machinery. Most rod-shaped bacteria use lateral-elongation, involving the protein MreB (or related isoforms) that forms a scaffold for the cell wall biosynthetic machinery 12–14. Filamentous actinomycetes however, utilize apical elongation by which new cell wall material is synthesized and exclusively incorporated at the apex of the filament 15,16. This apical growth is enabled by the TIPOC (Tip-Organizing Center) multi-protein complex of which DivIVA is the best-studied member 17. Although more insight is being gained on the how the TIPOC functions 18–21 and on the
General introduction
chemical composition of the cell wall 22, it remains unclear how the cell envelope of apically growing bacteria is structurally organized.
Morphological development and stress adaptation in actinomycetes
Filamentous actinomycetes such as Streptomyces species, are best-known for their ability to produce antibiotics and their complex lifecycle 23. Streptomyces form spores, from which they germinate and form long filamentous vegetative hyphae that grow via apical elongation. In liquid growth medium, young vegetative hyphae aggregate via glue-like extracellular polymers to form dense pellets 24–27. On solid substrates, vegetative hyphae will develop an aerial mycelium upon nutrient depletion. These aerial hyphae produce spore chains that are better suited to resist the adverse conditions. Subsequently, the spores can be released and dispersed into the environment.
It was recently shown that in addition to spore formation upon nutrient stress, a selection of filamentous actinomycetes is capable of forming cell wall-deficient cells upon exposure to osmotic stress 28. These so-called S-cells (Stress-induced cells) are spherical due to the fact that the shape-defining cell wall is absent. When the hyperosmotic stress is relieved, the S-cells can reinitiate cell wall synthesis and reconstruct a cell wall and continue growth as a filamentous bacterium. This stress-induced formation of wall-deficient cells could potentially be a more wide-spread survival mechanism to cope with cell wall-targeting compounds or environmental stresses 29–31. The molecular mechanisms driving S-cell formation remains to be elucidated, as well as the structural changes preceding S-cell release from the hyphal tip of filamentous actinomycetes.
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Chapter 1
Outline of the thesis
This thesis focusses on the role of the cell envelope during the development of filamentous actinomycetes. More specifically, I have investigated the complexity of the cell envelope, the apical elongation of the cell wall, and stress-induced morphological changes. To address these research questions, I have used cryo-EM techniques to study the cell envelope in unprecedented detail.
Chapter 2 introduces the biological background of this thesis. It explains our current knowledge of bacterial morphology, the composition of the bacterial envelope, the synthesis of the cell wall and the organization of the genome. This review further introduces the large plethora of bacterial shapes and provides an elaborate overview on how these are altered upon exposure to different stresses. It illustrates how stress-induced morphogenesis can be a survival strategy during rapidly changing environments or inside a mammalian host.
Chapter 3 provides and overview of various electron microscopy sample preparation techniques and recent advances in cryo-EM. Here, the advantages and shortcomings of cryo-EM techniques for the study of the bacterial cell envelope of various microbes are discussed. Results presented in this chapter demonstrate how cryo-EM techniques can help in determining cell envelope thickness in Brucella abortus, in studying the Mycobacterial cell envelope, and in analyzing cell wall-deficient cells and septa of filamentous actinomycetes.
Chapter 4 is an in-depth study on the Streptomyces cell wall using cryo-electron tomography (cryo-ET). The cell wall architecture of an apically growing bacterium such as the model actinomycete Streptomyces coelicolor had not yet been described. The results presented in this chapter show that the apical regions of vegetative cells are structurally different from more distal regions. Furthermore, the study revealed that the cell wall is not merely composed of PG but forms a complex multilayered network together with teichoic acids and extracellular glycan polymers. Together the data presented in this chapter provides a new fundamental insight in the cell wall architecture of an apically growing filamentous bacterium.
In Chapter 5, the extrusion of wall-deficient S-cells from the apical regions of the filamentous actinomycete Kitasatospora viridifaciens is investigated. Exposure to hyperosmotic stress induces DNA condensation and results in the extrusion of excess membrane, empty vesicles and S-cells from the hyphal tips, a process that is further characterized in this study using cryo-ET. Interestingly, this study reveals how the cytoskeletal-like protein FilP plays a role in S-cell extrusion and dramatically affects S-cell formation in oxygen-limiting conditions.
Chapter 6 describes how stress can affect the internal membrane organization in
Streptomyces. Cryo-EM data presented here shows that both Streptomyces albus and Streptomyces venezuelae sporadically form membranous structures when cultured in
General introduction
various growth media and conditions. The formation of intracellular membranes structures is strongly induced by exposure to lysozyme and thus appears to be stress-related.
Chapter 7 describes the design of a technical tool for the storage of cryo-EM samples. Storage of samples at a cryogenic temperature can be challenging in laboratories with limited capacity, certainly when samples are stacking up for processing. A simple, yet efficient and cost-effective storage solution was designed to solve this complication.
