Biology subjects

Briegel, A.

Publications and source records attributed to Briegel, A..

34 records · Page 2Linked to original sources

bioRxiv2021

UVC inactivation of pathogenic samples suitable for cryoEM analysis

Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples, from individual proteins to whole cells. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of human pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the delicate ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of two model organisms, a pathogenic bacterium (Vibrio cholerae) and a virus-like particle (the ICP1 bacteriophage). We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography (cryo-ET) followed by sub-tomogram averaging (STA), and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 [A] structure that did not reveal any discernable structural damage. Together, these results show that the UVC irradiation dose that effectively inactivates cryo-EM samples does not negatively impact their structure. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

molecular biology↗

bioRxiv2020

Microbial piggy-back: how Streptomyces spores are transported by motile soil bacteria

Streptomycetes are sessile, soil-dwelling bacteria that produce diverse metabolites that impact plant health and the behavior of microbial communities. Emerging studies have demonstrated that Streptomyces spores are distributed through a variety of mechanisms, but it remains unclear how spores are transported to their preferred micro-environments, such as plant roots. Here, we show that Streptomyces spores are capable of utilizing the motility machinery of other soil bacteria and are transported on the centimeter scale. Motility assays and microscopy studies reveal that Streptomyces spores are transported to plant tissues by interacting directly with the flagella of both gram-positive and gram-negative bacteria. Genetics experiments demonstrate that this form of motility, called piggy-backing, is facilitated by conserved structural proteins present on the surface of Streptomyces spores. These results demonstrate that non-motile bacteria are capable of utilizing the motility machinery of other microbes to complete necessary stages of their lifecycle, and that this mode of transport may be ubiquitous in nature.

microbiology↗

bioRxiv2020

Formation of wall-less cells in Kitasatospora viridifaciens requires cytoskeletal protein FilP in oxygen-limiting conditions

The 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 the presence of an intact cytoskeleton.

microbiology↗

bioRxiv2020

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Bacterial chemoreceptors, the CheA histidine kinase, and the coupling protein CheW comprise transmembrane molecular arrays with remarkable sensing properties. An unanswered question concerns how receptors turn off CheA kinase activity. Chemoreceptor cytoplasmic regions engineered to assume a trimer-of-receptor-dimers configuration form well-defined complexes with CheA and CheW and promote a kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar ESR spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The kinase-off state is especially stable, has relatively low domain mobility and associates the histidine substrate domain P1 and docking domain P2 with the kinase core. Distances measured between spin-labeled ADP molecules bound to the P4 kinase domain provide evidence for a "dipped conformation" that has been previously proposed from molecular dynamics simulations. Taken together, the data provide an experimentally restrained model for the inhibited state of the core-signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

biochemistry↗

bioRxiv2020

Spirochetes produce ordered chemoreceptor arrays of unusual composition, arrangement, and symmetry to compensate for a highly curved membrane

Abstract/SummaryThe prokaryotic chemotaxis system is arguably the best-understood signaling pathway in biology, but most insights have been obtained from only a few model organisms and many studies have relied on artificial systems that alter membrane curvature1-3. In all previously described species, chemoreceptors organize with the histidine kinase (CheA) and coupling protein (CheW) into a hexagonal (P6 symmetry) extended array that is considered universal among archaea and bacteria4,5. Here, for the first time, we report an alternative symmetry (P2) of the chemotaxis apparatus that emerges from a strict linear organization of CheA in Treponema denticola cells, which possesses arrays with the highest native curvature investigated thus far. Using cryo-ET, we reveal that the Td chemoreceptor arrays assume a truly unusual arrangement of the supra-molecular protein assembly that has likely evolved to accommodate the high membrane curvature. The arrays have several additional atypical features, such as an extended dimerization domain of CheA and a variant CheW-CheR-like fusion protein that is critical for maintaining an ordered chemosensory apparatus in an extremely curved cell. Furthermore, the previously characterized Td oxygen sensor ODP influences array integrity and its loss substantially orders CheA. These results suggest a greater diversity of the chemotaxis signaling system than previously thought and demonstrate the importance of examining transmembrane systems in vivo to retain native membrane curvature.

microbiology↗

bioRxiv2019

Teichoic acids anchor distinct cell wall lamellae in an apically growing bacterium

The bacterial cell wall is a dynamic, multicomponent structure that provides structural support for cell shape and physical protection from the environment. In monoderm species, the thick cell wall is made up predominantly of peptidoglycan, teichoic acids and a variety of capsular glycans. Filamentous monoderm Actinobacteria, such as Streptomyces coelicolor, incorporate new cell wall material at the apex of their hyphal cells during growth. In this study we use cryo-electron tomography to reveal the structural architecture of the cell wall of this bacterium. Our data shows a density difference between the apex and subapical regions of chemically isolated sacculi. Removal of the teichoic acids with hydrofluoric acid reveals a rough and patchy cell wall and distinct lamellae in a number of sacculi. Absence of the extracellular glycans poly-{beta}-1,6-[N]-acetylglucosamine and a cellulose-like polymer, produced by the MatAB and CslA proteins respectively, results in a thinner sacculus and absence of lamellae and patches. Extracellular glycans might thus form or lead to the formation of the outer cell wall lamella. Based on these findings we propose a revisited model for the complex cell wall architecture of an apically growing bacterium, in which the network of peptidoglycan together with extracellular polymers is structurally supported by teichoic acids.

microbiology↗

bioRxiv2019

Species-specific recognition of Sulfolobales mediated by UV inducible pili and S-layer glycosylation patterns

The UV-inducible pili system of Sulfolobales (Ups) mediates the formation of species-specific cellular aggregates. Within these aggregates, cells exchange DNA in order to repair DNA double strand breaks via homologous recombination. Substitution of the S. acidocaldarius pilin subunits UpsA and UpsB with their homologs from Sulfolobus tokodaii showed that these subunits facilitate species-specific aggregation. A region of low conservation within the UpsA homologs is primarily important for this specificity. Aggregation assays in the presence of different sugars showed the importance of N-glycosylation in the recognition process. In addition, the N-glycan decorating the S-layer of S. tokodaii is different from the one of S. acidocaldarius. Therefore, each Sulfolobus species seems to have developed a unique UpsA binding pocket and unique N-glycan composition to ensure aggregation and consequently also DNA exchange with cells from only the same species, which is essential for DNA repair by homologous recombination.\n\nImportanceType IV pili can be found on the cell surface of many archaea and bacteria where they play important roles in different processes. The Ups-pili from the crenarchaeal Sulfolobales species are essential in establishing species-specific mating partners, ensuring genome stability. With this work, we show that different Sulfolobus species have species-specific regions in their Ups-pilin subunits, which allow them to interact only with cells from the same species. Additionally, different Sulfolobus species all have unique S-layer N-glycosylation patterns. We propose that the unique features of each species allow the recognition of specific mating partners. This knowledge for the first time gives insights into the molecular basis of archaeal self-recognition.

microbiology↗

bioRxiv2019

Repurposing a macromolecular machine: Architecture and evolution of the F7 chemosensory system

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identified ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we then traced the evolution of the system through {gamma}-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.

microbiology↗

bioRxiv2019

Brucella EipB is periplasmic β-spiral protein required for envelope stress resistance and infection

The Gram-negative cell envelope is a remarkably diverse structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. We show that a conserved DUF1849-family protein, EipB, is secreted to the periplasmic space of Brucella, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual fourteen-stranded {beta}-spiral structure that contains a conserved disulfide bond. EipB has structural features that resemble the LolA and LolB lipoprotein delivery system, though the overall topology and fold of EipB is distinct from LolA/LolB. Deletion of eipB results in defects in both cell envelope integrity in vitro and in maintenance of spleen colonization in a mouse model of B. abortus infection. Transposon disruption of ttpA, which encodes a periplasmic tetratricopeptide repeat (TPR) protein, is synthetically lethal with eipB deletion in B. abortus. ttpA is a known virulence determinant in B. melitensis, and our studies of ttpA deletion and overexpression strains provide evidence that ttpA, like eipB, contributes to cell envelope function in Brucella. We conclude that eipB and ttpA function in the Brucella periplasmic space to maintain cell envelope integrity and to facilitate survival in a mammalian host. ImportanceBrucella species cause brucellosis, a global zoonosis. A gene encoding a conserved uncharacterized protein, EipB, is present in all sequenced Brucella and several other genera in the class Alphaproteobacteria. To our knowledge, this study presents the first functional and structural characterization of a protein from the DUF1849 family, to which EipB belongs. EipB is secreted to the periplasm where it forms a spiral-like anti-parallel {beta} structure. Deletion of Brucella eipB results in defects of the cell envelope and in reduced virulence in an animal model of disease. eipB genetically interacts with ttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in select Alphaproteobacteria.

microbiology↗

bioRxiv2018

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus

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 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 {beta}-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the integrity of the cell envelope. 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.

microbiology↗

bioRxiv2018

Structural and proteomic changes in viable but non-culturable Vibrio cholerae

Aquatic environments are reservoirs of the human pathogen Vibrio cholerae O1, which causes the acute diarrheal disease cholera. Upon low temperature or limited nutrient availability, the cells enter a viable but non-culturable (VBNC) state. Characteristic of this state are an altered morphology, low metabolic activity and lack of growth under standard laboratory conditions. Here, for the first time, the cellular ultrastructure of V. cholerae VBNC cells raised in natural waters was investigated using electron cryo-tomography complemented by comparison of the proteomes and the peptidoglycan composition of LB overnight culture and VBNC cells. The extensive remodeling of the VBNC cells was most obvious in the passive dehiscence of the cell envelope, resulting in improper embedment of flagella and pili. Only minor changes of the peptidoglycan and osmoregulated periplasmic glucans were observed. Active changes in VBNC cells included the production of cluster I chemosensory arrays and change of abundance of cluster II array proteins. Components involved in iron acquisition and storage, peptide import and arginine biosynthesis were overrepresented in VBNC cells, while enzymes of the central carbon metabolism were found at lower levels. Finally, several pathogenicity factors of V. cholerae were less abundant in the VBNC state, potentially limiting their infectious potential.

microbiology↗

bioRxiv2018

γ-proteobacteria eject their polar flagella under nutrient depletion, retaining flagellar motor relic structures

Bacteria switch only intermittently to motile planktonic lifestyles under favourable conditions. Under chronic nutrient deprivation, however, bacteria orchestrate a switch to stationary phase, conserving energy by altering metabolism and stopping motility. About two-thirds of bacteria use flagella to swim, but how bacteria deactivate this large-molecular machine remains poorly studied. Here we describe the previously unreported ejection of polar sodium-driven motors by {gamma}-proteobacteria. We show that these bacteria eject their flagella at the base of the flagellar hook when nutrients are depleted, leaving a relic of a former flagellar motor in the outer membrane. Subtomogram averages of the full motor and relic reveal that this is an active process, as a plug protein appears in the relic, likely to prevent leakage across their outer membrane. We show that this is a widespread phenomenon demonstrated by the appearance of relic structures in varied {gamma}-proteobacteria including Plesiomonas shigelloides, Vibrio cholerae, Vibrio fischeri, Shewanella putrefaciens and Pseudomonas aeruginosa.

microbiology↗

bioRxiv2017

An open-source cryo-storage solution

The field of cryo-electron microscopy is a rapidly growing method in structural biology. With this development, access to cryo-EM facilities becomes a bottleneck that results in long wait times between sample preparation and data acquisition. To improve sample storage, we developed a cryo-storage system with a more efficient and larger storage capacity that enables cryo-sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid box and storage dewar and any size cryo-EM laboratory.

molecular biology↗

bioRxiv2017

Uncharacterized bacterial structures revealed by electron cryotomography

SUMMARY STATEMENTHere we present a survey of previously uncharacterized structures we have observed in bacterial cells by electron cryotomography, in the hopes of spurring their identification and study.\n\nABSTRACTElectron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. Rather than undifferentiated bags of enzymes, we now view bacteria as structurally complex assemblies of macromolecular machines. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed several, to our knowledge, uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells.

microbiology↗

bioRxiv2017

Structure of the archaellar motor and associated cytoplasmic cone in Thermococcus kodakaraensis

Archaeal swimming motility is driven by rotary motors called archaella. The structure of these motors, and particularly how they are anchored in the absence of a peptidoglycan cell wall, is unknown. Here, we use electron cryotomography to visualize the archaellar motor in vivo in Thermococcus kodakaraensis. Compared to the homologous bacterial type IV pilus (T4P), we observe structural similarities as well as several unique features. While the position of the cytoplasmic ATPase appears conserved, it is not braced by linkages that extend upward through the cell envelope as in the T4P, but rather by cytoplasmic components that attach it to a large conical frustum up to 500 nm in diameter at its base. In addition to anchoring the lophotrichous bundle of archaella, the conical frustum associates with chemosensory arrays and ribosome-excluding material and may function as a polar organizing center for the coccoid cells.

cell biology↗

bioRxiv2016

Reversible metamorphosis in a bacterium

The cell wall is a shape-defining structure that envelopes almost all bacteria. One of its main functions is to serve as a protection barrier to environmental stresses. Bacteria can be forced in a cell wall-deficient state under highly specialized conditions, which are invariably aimed at interrupting cell wall synthesis. Therefore, the relevance of such cells has remained obscure. Here we show that many filamentous actinomycetes have a natural ability to generate a new, cell wall-deficient cell type 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 canonical mycelial mode-of-growth. Remarkably, prolonged exposure of S-cells to hyperosmotic stress yielded variants that are able to proliferate indefinitely without their cell wall. This is the first report that demonstrates the formation of wall-deficient cells as a natural adaptation strategy and their potential transition into stable wall-less forms solely caused by prolonged exposure to osmotic stress. Given that actinomycetes are potent antibiotic producers, our work also provides important insights into how biosynthetic gene clusters and resistance determinants may disseminate into the environment.

microbiology↗