Dynamics of the peptidoglycan biosynthetic machinery in the stalked budding bacterium <i>Hyphomonas neptunium</i>

Electron 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. We now view bacteria as structurally complex assemblies of macromolecular machines rather than as undifferentiated bags of enzymes. 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, to our knowledge, several 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.IMPORTANCE Bacteria are more structurally complex than is commonly appreciated. Here we present a survey of previously uncharacterized structures that we observed in bacterial cells by electron cryotomography, structures that will initiate new lines of research investigating their identities and roles.KEYWORDS bacteria, electron cryotomography, bacterial ultrastructure, uncharacterized structures, electron microscopy T he history of cell biology has been punctuated by advances in imaging technology. In particular, the development of electron microscopy (EM) in the 1930s produced a wealth of new information about the ultrastructure of cells (1). For the first time, the structure of cell envelopes, internal organelles, cytoskeletal filaments, and even large macromolecular complexes like ribosomes became visible. A further advance came in the 1980s and 1990s with the development of electron cryotomography (ECT) (2), which allows small cells to be imaged intact in 3D in a near-native, "frozen-hydrated" state to "macromolecular" (ϳ5 nm) resolution, without the limitations and artifacts of more traditional specimen-preparation methods (3).ECT has helped reveal the previously unappreciated complexity of "simple" bacterial cells. Our group has been using ECT to study bacteria for more than a decade, generating more than 15,000 tomograms of 88 different species. These tomograms have revealed new insights into, among other things, the bacterial cytoskeleton (4-7), cell wall architecture (8, 9), morphogenesis (10), metabolism (11), motility (12-15),

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“…Prosthecobacters were all grown according to conditions in reference 6. Hyphomonas neptunium was grown according to conditions in reference 68.…”

Section: Intracellular Structures (I) Nanospheresmentioning

confidence: 99%

Uncharacterized Bacterial Structures Revealed by Electron Cryotomography

et al. 2017

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“…Since the stalk is ~20% the width of the cell body (Wagner and Brun, ), we also considered whether geometrical constraints could contribute to the subcellular specificity of KZ144‐YFP binding. Hyphomonas neptunium forms a stalk that is morphologically similar to the C. crescentus stalk, but this organism does not form DAP‐DAP crosslinks (Cserti et al , ). KZ144‐YFP efficiently labeled DD‐crosslinked stalks in H. neptunium (Fig.…”

Section: Resultsmentioning

confidence: 99%

Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus

Stankeviciute

Miguel

Radkov

et al. 2019

Molecular Microbiology

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Summary The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod‐shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme‐resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD‐crosslinks between meso‐diaminopimelic acid and D‐alanine residues, whereas stalk peptidoglycan had more LD‐transpeptidation (meso‐diaminopimelic acid‐meso‐diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD‐transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD‐crosslinking. Despite no obvious selection pressure for LD‐crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP‐DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.

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“…In a recent study by Cserti et al (63), FDAAs were used to unravel the mechanism of PG synthesis during the division of Hyphomonas neptunium . H. neptunium replicates by stalk budding, yet the mechanistic details and enzymes involved were poorly understood.…”

Section: Development Of Fluorescent D-amino Acids and Their Applicationsmentioning

confidence: 99%

Imaging Bacterial Cell Wall Biosynthesis

Radkov

Hsu

Booher

et al. 2018

Annu. Rev. Biochem.

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Peptidoglycan is an essential component of the cell wall that protects bacteria from environmental stress. A carefully coordinated biosynthesis of peptidoglycan during cell elongation and division is required for cell viability. This biosynthesis involves sophisticated enzyme machineries that dynamically synthesize, remodel, and degrade peptidoglycan. However, when and where bacteria build peptidoglycan, and how this is coordinated with cell growth, have been long-standing questions in the field. The improvement of microscopy techniques has provided powerful approaches to study peptidoglycan biosynthesis with high spatiotemporal resolution. Recent development of molecular probes further accelerated the growth of the field, which has advanced our knowledge of peptidoglycan biosynthesis dynamics and mechanisms. Here, we review the technologies for imaging the bacterial cell wall and its biosynthesis activity. We focus on the applications of fluorescent d-amino acids, a newly developed type of probe, to visualize and study peptidoglycan synthesis and dynamics, and we provide direction for prospective research.

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Dynamics of the peptidoglycan biosynthetic machinery in the stalked budding bacterium Hyphomonas neptunium

Cited by 40 publications

References 94 publications

Uncharacterized Bacterial Structures Revealed by Electron Cryotomography

Uncharacterized Bacterial Structures Revealed by Electron Cryotomography

Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus

Imaging Bacterial Cell Wall Biosynthesis

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