The cell wall is considered an essential component for bacterial survival, providing structural support, and protection from environmental insults. Under normal growth conditions, filamentous... Show moreThe cell wall is considered an essential component for bacterial survival, providing structural support, and protection from environmental insults. Under normal growth conditions, filamentous actinobacteria insert new cell wall material at the hyphal tips regulated by the coordinated activity of cytoskeletal proteins and cell wall biosynthetic enzymes. Despite the importance of the cell wall, some filamentous actinobacteria can produce wall-deficient S-cells upon prolonged exposure to hyperosmotic stress. Here, we performed cryo-electron tomography and live cell imaging to further characterize S-cell extrusion in Kitasatospora viridifaciens. We show that exposure to hyperosmotic stress leads to DNA compaction, membrane and S-cell extrusion, and thinning of the cell wall at hyphal tips. Additionally, we find that the extrusion of S-cells is abolished in a cytoskeletal mutant strain that lacks the intermediate filament-like protein FilP. Furthermore, micro-aerobic culturing promotes the formation of S-cells in the wild type, but the limited oxygen still impedes S-cell formation in the ΔfilP mutant. These results demonstrate that S-cell formation is stimulated by oxygen-limiting conditions and dependent on functional cytoskeleton remodeling. Show less
The cell wall is considered an essential component for bacterial survival, providing structural support, and protection from environmental insults. Under normal growth conditions, filamentous... Show moreThe cell wall is considered an essential component for bacterial survival, providing structural support, and protection from environmental insults. Under normal growth conditions, filamentous actinobacteria insert new cell wall material at the hyphal tips regulated by the coordinated activity of cytoskeletal proteins and cell wall biosynthetic enzymes. Despite the importance of the cell wall, some filamentous actinobacteria can produce wall-deficient S-cells upon prolonged exposure to hyperosmotic stress. Here, we performed cryo-electron tomography and live cell imaging to further characterize S-cell extrusion in Kitasatospora viridifaciens. We show that exposure to hyperosmotic stress leads to DNA compaction, membrane and S-cell extrusion, and thinning of the cell wall at hyphal tips. Additionally, we find that the extrusion of S-cells is abolished in a cytoskeletal mutant strain that lacks the intermediate filament-like protein FilP. Furthermore, micro-aerobic culturing promotes the formation of S-cells in the wild type, but the limited oxygen still impedes S-cell formation in the Delta filP mutant. These results demonstrate that S-cell formation is stimulated by oxygen-limiting conditions and dependent on functional cytoskeleton remodeling. Show less
Bacteria have the ability to alter their morphology in order to adapt to changing environments. We have investigated the role of the cell envelope in the development and stress-adaptation... Show moreBacteria have the ability to alter their morphology in order to adapt to changing environments. We have investigated the role of the cell envelope in the development and stress-adaptation strategies of Actinobacteria. Here, we demonstrate how cryo-electron microscopy techniques can be used as efficient tools to study cell envelope of various bacteria. An in-depth study on the Streptomyces cell wall with cryo-electron tomography reveals the structural complexity of the Gram-positive cell wall in apically growing bacteria. Additionally, we report the formation of intracellular membrane accumulations in Streptomyces as a result of exposure to a stress-inducing agent. Furthermore, we studied the ability of the filamentous actinomycete Kitasatospora viridifaciens to extrude wall-deficient cells or S-cells upon exposure to hyperosmotic stress. We characterized the structural alterations associated with S-cell formation using cryo-electron microscopy and reveal that S-cell formation requires cytoskeletal protein FilP in K. viridifaciens. To summarize, this thesis provides new insights in the structural complexity and stress-induced alterations of the bacterial cell envelope. Show less
The bacterial cell wall is a multicomponent structure that provides structural support and protection. In monoderm species, the cell wall is made up predominantly of peptidoglycan, teichoic acids... Show moreThe bacterial cell wall is a multicomponent structure that provides structural support and protection. In monoderm species, the cell wall is made up predominantly of peptidoglycan, teichoic acids and capsular glycans. Filamentous monoderm Actinobacteria incorporate new cell-wall material at their tips. Here we use cryo-electron tomography to reveal the architecture of the actinobacterial cell wall ofStreptomyces coelicolor. Our data shows a density difference between the apex and subapical regions. Removal of teichoic acids results in a patchy cell wall and distinct lamellae. Knock-down oftagOexpression using CRISPR-dCas9 interference leads to growth retardation, presumably because build-in of teichoic acids had become rate-limiting. Absence of extracellular glycans produced by MatAB and CslA proteins results in a thinner wall lacking lamellae and patches. We propose that theStreptomycescell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.Eveline Ultee et al. reveal the architecture of the actinobacterial cell wall of a polar growing bacteriumStreptomyces coelicolor, using cryo-electron tomography. This study suggests that theStreptomycescell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids. Show less
Bacteria thrive in virtually all environments. Like all other living organisms, bacteria may encounter various types of stresses, to which cells need to adapt. In this chapter, we describe how... Show moreBacteria thrive in virtually all environments. Like all other living organisms, bacteria may encounter various types of stresses, to which cells need to adapt. In this chapter, we describe how cells cope with stressful conditions and how this may lead to dramatic morphological changes. These changes may not only allow harmless cells to withstand environmental insults but can also benefit pathogenic bacteria by enabling them to escape from the immune system and the activity of antibiotics. A better understanding of stress-induced morphogenesis will help us to develop new approaches to combat such harmful pathogens. Show less
Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a... Show moreMolecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight‐stranded β‐barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O‐polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O‐polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O‐polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division. Show less
Ramijan Carmiol, A.K.; Ultee, E.; Willemse, J.J.; Zhang, Z.; Wondergem, A.J.; Meij, A. van der; ... ; Claessen, D. 2018
The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain... Show moreThe cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress. Show less
Cryo-electron microscopy (cryo-EM) enables the study of biological structures in situ in great detail and to solve protein structures at Ångstrom level resolution. Due to recent advances in... Show moreCryo-electron microscopy (cryo-EM) enables the study of biological structures in situ in great detail and to solve protein structures at Ångstrom level resolution. Due to recent advances in instrumentation and data processing, the field of cryo-EM is a rapidly growing. Access to facilities and national centers that house the state-of-the-art microscopes is limited due to the ever-rising demand, resulting in long wait times between sample preparation and data acquisition. To improve sample storage, we have developed a cryo-storage system with an efficient, high storage capacity that enables sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid storage box and dewar and any size cryo-EM laboratory. Show less
