The cell envelope of Thermotogae suggests a mechanism for outer membrane biogenesis.

The presence of a cell membrane is one of the major structural components defining life. Recent phylogenomic analyses have supported the hypothesis that the last universal common ancestor (LUCA) was likely a diderm. Yet, the mechanisms that guided outer membrane (OM) biogenesis remain unknown. Thermotogae is an early-branching phylum with a unique OM, the toga. Here, we use cryo-electron tomography to characterize the in situ cell envelope architecture of Thermotoga maritima and show that the toga is made of extended sheaths of ß-barrel trimers supporting small (~200 nm) membrane patches. Lipidomic analyses identified the same major lipid species in the inner membrane (IM) and toga, including the rare to bacteria membrane-spanning ether-bound diabolic acids (DAs). Proteomic analyses revealed that the toga was composed of multiple SLH-domain containing Ompα and novel ß-barrel proteins, and homology searches detected variable conservations of these proteins across the phylum. These results highlight that, in contrast to the SlpA/OmpM superfamily of proteins, Thermotoga possess a highly diverse bipartite OM-tethering system. We discuss the implications of our findings with respect to other early-branching phyla and propose that a toga-like intermediate may have facilitated monoderm-to-diderm cell envelope transitions.

Interdigitated immunoglobulin arrays form the hyperstable surface layer of the extremophilic bacterium Deinococcus radiodurans.

Deinococcus radiodurans is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, due in part to its complex cell envelope encapsulated within a hyperstable surface layer (S-layer). Despite decades of research on this cell envelope, atomic structural details of the S-layer have remained obscure. In this study, we report the electron cryomicroscopy structure of the D. radiodurans S-layer, showing how it is formed by the Hexagonally Packed Intermediate-layer (HPI) protein arranged in a planar hexagonal lattice. The HPI protein forms an array of immunoglobulin-like folds within the S-layer, with each monomer extending into the adjacent hexamer, resulting in a highly interconnected, stable, sheet-like arrangement. Using electron cryotomography and subtomogram averaging from focused ion beam-milled D. radiodurans cells, we have obtained a structure of the cellular S-layer, showing how this HPI S-layer coats native membranes on the surface of cells. Our S-layer structure from the diderm bacterium D. radiodurans shows similarities to immunoglobulin-like domain-containing S-layers from monoderm bacteria and archaea, highlighting common features in cell surface organization across different domains of life, with connotations on the evolution of immunoglobulin-based molecular recognition systems in eukaryotes.

Advanced imaging techniques: microscopy.

For decades, bacteria were thought of as "bags" of enzymes, lacking organelles and significant subcellular structures. This stood in sharp contrast with eukaryotes, where intracellular compartmentalization and the role of large-scale order had been known for a long time. However, the emerging field of Bacterial Cell Biology has established that bacteria are in fact highly organized, with most macromolecular components having specific subcellular locations that can change depending on the cell's physiological state (Barry & Gitai, 2011; Lenz & Søgaard-Andersen, 2011; Thanbichler & Shapiro, 2008). For example, we now know that many processes in bacteria are orchestrated by cytoskeletal proteins, which polymerize into surprisingly diverse superstructures, such as rings, sheets, and tread-milling rods (Pilhofer & Jensen, 2013). These superstructures connect individual proteins, macromolecular assemblies, and even two neighboring cells, to affect essential higher-order processes including cell division, DNA segregation, and motility. Understanding these processes requires resolving the in vivo dynamics and ultrastructure at different functional stages of the cell, at macromolecular resolution and in 3-dimensions (3D). Fluorescence light microscopy (fLM) of tagged proteins is highly valuable for investigating protein localization and dynamics, and the resolution power of transmission electron microscopy (TEM) is required to elucidate the structure of macromolecular complexes in vivo and in vitro. This chapter summarizes the most recent advances in LM and TEM approaches that have revolutionized our knowledge and understanding of the microbial world.

Ultrastructure of Organohalide-Respiring Dehalococcoidia Revealed by Cryo-Electron Tomography.

Dehalococcoides mccartyi (Dhc) and Dehalogenimonas spp. (Dhgm) are members of the class Dehalococcoidia, phylum Chloroflexi, characterized by streamlined genomes and a strict requirement for organohalogens as electron acceptors. Here, we used cryo-electron tomography to reveal morphological and ultrastructural features of Dhc strain BAV1 and "Candidatus Dehalogenimonas etheniformans" strain GP cells at unprecedented resolution. Dhc cells were irregularly shaped discs (890 ± 110 nm long, 630 ± 110 nm wide, and 130 ± 15 nm thick) with curved and straight sides that intersected at acute angles, whereas Dhgm cells appeared as slightly flattened cocci (760 ± 85 nm). The cell envelopes were composed of a cytoplasmic membrane (CM), a paracrystalline surface layer (S-layer) with hexagonal symmetry and ∼22-nm spacing between repeating units, and a layer of unknown composition separating the CM and the S-layer. Cell surface appendages were only detected in Dhc cells, whereas both cell types had bundled cytoskeletal filaments. Repetitive globular structures, ∼5 nm in diameter and ∼9 nm apart, were observed associated with the outer leaflet of the CM. We hypothesized that those represent organohalide respiration (OHR) complexes and estimated ∼30,000 copies per cell. In Dhgm cultures, extracellular lipid vesicles (20 to 110 nm in diameter) decorated with putative OHR complexes but lacking an S-layer were observed. The new findings expand our understanding of the unique cellular ultrastructure and biology of organohalide-respiring Dehalococcoidia. IMPORTANCEDehalococcoidia respire organohalogen compounds and play relevant roles in bioremediation of groundwater, sediments, and soils impacted with toxic chlorinated pollutants. Using advanced imaging tools, we have obtained three-dimensional images at macromolecular resolution of whole Dehalococcoidia cells, revealing their unique structural components. Our data detail the overall cellular shape, cell envelope architecture, cytoskeletal filaments, the likely localization of enzymatic complexes involved in reductive dehalogenation, and the structure of extracellular vesicles. The new findings expand our understanding of the cell structure-function relationship in Dehalococcoidia with implications for Dehalococcoidia biology and bioremediation.

Roles of LysM and LytM domains in resuscitation-promoting factor (Rpf) activity and Rpf-mediated peptidoglycan cleavage and dormant spore reactivation.

Bacterial dormancy can take many forms, including formation of Bacillus endospores, Streptomyces exospores, and metabolically latent Mycobacterium cells. In the actinobacteria, including the streptomycetes and mycobacteria, the rapid resuscitation from a dormant state requires the activities of a family of cell-wall lytic enzymes called resuscitation-promoting factors (Rpfs). Whether Rpf activity promotes resuscitation by generating peptidoglycan fragments (muropeptides) that function as signaling molecules for spore germination or by simply remodeling the dormant cell wall has been the subject of much debate. Here, to address this question, we used mutagenesis and peptidoglycan binding and cleavage assays to first gain broader insight into the biochemical function of diverse Rpf enzymes. We show that their LysM and LytM domains enhance Rpf enzyme activity; their LytM domain and, in some cases their LysM domain, also promoted peptidoglycan binding. We further demonstrate that the Rpfs function as endo-acting lytic transglycosylases, cleaving within the peptidoglycan backbone. We also found that unlike in other systems, Rpf activity in the streptomycetes is not correlated with peptidoglycan-responsive Ser/Thr kinases for cell signaling, and the germination of rpf mutant strains could not be stimulated by the addition of known germinants. Collectively, these results suggest that in Streptomyces, Rpfs have a structural rather than signaling function during spore germination, and that in the actinobacteria, any signaling function associated with spore resuscitation requires the activity of additional yet to be identified enzymes.

Roles for phthiocerol dimycocerosate lipids in Mycobacterium tuberculosis pathogenesis.

The success of Mycobacterium tuberculosis as a pathogen is well established: tuberculosis is the leading cause of death by a single infectious agent worldwide. The threat of multi- and extensively drug-resistant bacteria has renewed global concerns about this pathogen and understanding its virulence strategies will be essential in the fight against tuberculosis. The current review will focus on phthiocerol dimycocerosates (PDIMs), a long-known and well-studied group of complex lipids found in the M. tuberculosis cell envelope. Numerous studies show a role for PDIMs in several key steps of M. tuberculosis pathogenesis, with recent studies highlighting its involvement in bacterial virulence, in association with the ESX-1 secretion system. Yet, the mechanisms by which PDIMs help M. tuberculosis to control macrophage phagocytosis, inhibit phagosome acidification and modulate host innate immunity, remain to be fully elucidated.

Resuscitation-promoting factors are cell wall-lytic enzymes with important roles in the germination and growth of Streptomyces coelicolor.

Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. For Streptomyces bacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model species Streptomyces coelicolor encodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, the S. coelicolor Rpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and our in vitro biochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these five Streptomyces enzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage.

Super-resolution confocal cryo-CLEM with cryo-FIB milling for in situ imaging of Deinococcus radiodurans.

Studying bacterial cell envelope architecture with electron microscopy is challenging due to the poor preservation of microbial ultrastructure with traditional methods. Here, we established and validated a super-resolution cryo-correlative light and electron microscopy (cryo-CLEM) method, and combined it with cryo-focused ion beam (cryo-FIB) milling and scanning electron microscopy (SEM) volume imaging to structurally characterize the bacterium Deinococcus radiodurans. Subsequent cryo-electron tomography (cryo-ET) revealed an unusual diderm cell envelope architecture with a thick layer of peptidoglycan (PG) between the inner and outer membranes, an additional periplasmic layer, and a proteinaceous surface S-layer. Cells grew in tetrads, and division septa were formed by invagination of the inner membrane (IM), followed by a thick layer of PG. Cytoskeletal filaments, FtsA and FtsZ, were observed at the leading edges of constricting septa. Numerous macromolecular complexes were found associated with the cytoplasmic side of the IM. Altogether, our study revealed several unique ultrastructural features of D. radiodurans cells, opening new lines of investigation into the physiology and evolution of the bacterium.

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation.

Sporulation is a specialized developmental program employed by a diverse set of bacteria which culminates in the formation of dormant cells displaying increased resilience to stressors. This represents a major survival strategy for bacteria facing harsh environmental conditions, including nutrient limitation, heat, desiccation, and exposure to antimicrobial compounds. Through dispersal to new environments via biotic or abiotic factors, sporulation provides a means for disseminating genetic material and promotes encounters with preferable environments thus promoting environmental selection. Several types of bacterial sporulation have been characterized, each involving numerous morphological changes regulated and performed by non-homologous pathways. Despite their likely independent evolutionary origins, all known modes of sporulation are typically triggered by limited nutrients and require extensive membrane and peptidoglycan remodeling. While distinct modes of sporulation have been observed in diverse species, two major types are at the forefront of understanding the role of sporulation in human health, and microbial population dynamics and survival. Here, we outline endospore and exospore formation by members of the phyla Firmicutes and Actinobacteria, respectively. Using recent advances in molecular and structural biology, we point to the regulatory, genetic, and morphological differences unique to endo- and exospore formation, discuss shared characteristics that contribute to the enhanced environmental survival of spores and, finally, cover the evolutionary aspects of sporulation that contribute to bacterial species diversification.

Ultrastructure of Exospore Formation in Streptomyces Revealed by Cryo-Electron Tomography.

Many bacteria form spores in response to adverse environmental conditions. Several sporulation pathways have evolved independently and occur through distinctive mechanisms. Here, using cryo-electron tomography (cryo-ET), we examine all stages of growth and exospore formation in the model organism Streptomyces albus. Our data reveal the native ultrastructure of vegetative hyphae, including the likely structures of the polarisome and cytoskeletal filaments. In addition, we observed septal junctions in vegetative septa, predicted to be involved in protein and DNA translocation between neighboring cells. During sporulation, the cell envelope undergoes dramatic remodeling, including the formation of a spore wall and two protective proteinaceous layers. Mature spores reveal the presence of a continuous spore coat and an irregular rodlet sheet. Together, these results provide an unprecedented examination of the ultrastructure in Streptomyces and further our understanding of the structural complexity of exospore formation.

Liquid Chromatography-Tandem Mass Spectrometry to Define Sortase Cleavage Products.

Sortase enzymes have specific endopeptidase activity, cleaving within a defined pentapeptide sequence at the C-terminal end of their protein substrates. Here, we describe how monitoring sortase cleavage activity can be achieved using peptide substrates. Peptide cleavage can be readily analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS), which allows for the precise definition of cleavage sites. This technique could be used to analyze the peptidase activity of any enzyme, and identify sites of cleavage within any peptide.

Crystal Structure of the Streptomyces coelicolor Sortase E1 Transpeptidase Provides Insight into the Binding Mode of the Novel Class E Sorting Signal.

Many species of Gram-positive bacteria use sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of sortases (class A to F enzymes); however, class E enzymes have not been extensively studied. Class E sortases are used by soil and freshwater-dwelling Actinobacteria to display proteins that contain a non-canonical LAXTG sorting signal, which differs from 90% of known sorting signals by substitution of alanine for proline. Here we report the first crystal structure of a class E sortase, the 1.93 Å resolution structure of the SrtE1 enzyme from Streptomyces coelicolor. The active site is bound to a tripeptide, providing insight into the mechanism of substrate binding. SrtE1 possesses ß3/ß4 and ß6/ß7 active site loops that contact the LAXTG substrate and are structurally distinct from other classes. We propose that SrtE1 and other class E sortases employ a conserved tyrosine residue within their ß3/ß4 loop to recognize the amide nitrogen of alanine at position P3 of the sorting signal through a hydrogen bond, as seen here. Incapability of hydrogen-bonding with canonical proline-containing sorting signals likely contributes to class E substrate specificity. Furthermore, we demonstrate that surface anchoring of proteins involved in aerial hyphae formation requires an N-terminal segment in SrtE1 that is presumably positioned within the cytoplasm. Combined, our results reveal unique features within class E enzymes that enable them to recognize distinct sorting signals, and could facilitate the development of substrate-based inhibitors of this important enzyme family.