The cell envelope of Mycobacterium abscessus and its role in pathogenesis.

Mycobacterium abscessus is a nontuberculosis mycobacterium (NTM) that has shown an exponential rise in its ability to cause disease. Due to its ubiquitous presence in the environment, M. abscessus is widely implicated in secondary exacerbations of many nosocomial infections and genetic respiratory disorders, such as cystic fibrosis (CF). Contrary to other rapidly growing NTMs, the cell envelope of M. abscessus harbors several prominent features and undergoes modifications that are responsible for its pathogenesis. Compositional changes of the mycobacterial outer membrane (MOM) significantly decrease the presence of glycopeptidolipids (GPLs) and enable the transition from a colonizing, smooth morphotype into a virulent, rough morphotype. The GPLs are transported to the MOM by the Mycobacterial membrane proteins Large (MmpL), which further act as drug efflux pumps and confer antibiotic resistance. Lastly, M. abscessus possesses 2 type VII secretion systems (T7SS): ESX-3 and ESX-4, both of which have recently been implicated in host-pathogen interactions and virulence. This review summarizes the current knowledge of M. abscessus pathogenesis and highlights the clinically relevant association between the structure and functions of its cell envelope.

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.

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.

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.

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past.

Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

Cryo-EM structure of the Agrobacterium tumefaciens T4SS-associated T-pilus reveals stoichiometric protein-phospholipid assembly.

Agrobacterium tumefaciens causes crown gall disease in plants by the horizontal transfer of oncogenic DNA. The conjugation is mediated by the VirB/D4 type 4 secretion system (T4SS) that assembles an extracellular filament, the T-pilus, and is involved in mating pair formation between A. tumefaciens and the recipient plant cell. Here, we present a 3 Å cryoelectron microscopy (cryo-EM) structure of the T-pilus solved by helical reconstruction. Our structure reveals that the T-pilus is a stoichiometric assembly of the VirB2 major pilin and phosphatidylglycerol (PG) phospholipid with 5-start helical symmetry. We show that PG head groups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 abolished pilus formation. While our T-pilus structure is architecturally similar to previously published conjugative pili structures, the T-pilus lumen is narrower and positively charged, raising questions of whether the T-pilus is a conduit for ssDNA transfer.

Cryptosporulation in Kurthia spp. forces a rethinking of asporogenesis in Firmicutes.

Endosporulation is a complex morphophysiological process resulting in a more resistant cellular structure that is produced within the mother cell and is called endospore. Endosporulation evolved in the common ancestor of Firmicutes, but it is lost in descendant lineages classified as asporogenic. While Kurthia spp. is considered to comprise only asporogenic species, we show here that strain 11kri321, which was isolated from an oligotrophic geothermal reservoir, produces phase-bright spore-like structures. Phylogenomics of strain 11kri321 and other Kurthia strains reveals little similarity to genetic determinants of sporulation known from endosporulating Bacilli. However, morphological hallmarks of endosporulation were observed in two of the four Kurthia strains tested, resulting in spore-like structures (cryptospores). In contrast to classic endospores, these cryptospores did not protect against heat or UV damage and successive sub-culturing led to the loss of the cryptosporulating phenotype. Our findings imply that a cryptosporulation phenotype may have been prevalent and subsequently lost by laboratory culturing in other Firmicutes currently considered as asporogenic. Cryptosporulation might thus represent an ancestral but unstable and adaptive developmental state in Firmicutes that is under selection under harsh environmental conditions.

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.

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.

Loss of the Bacterial Flagellar Motor Switch Complex upon Cell Lysis.

The bacterial flagellar motor is a complex macromolecular machine whose function and self-assembly present a fascinating puzzle for structural biologists. Here, we report that in diverse bacterial species, cell lysis leads to loss of the cytoplasmic switch complex and associated ATPase before other components of the motor. This loss may be prevented by the formation of a cytoplasmic vesicle around the complex. These observations suggest a relatively loose association of the switch complex with the rest of the flagellar machinery. IMPORTANCE We show in eight different bacterial species (belonging to different phyla) that the flagellar motor loses its cytoplasmic switch complex upon cell lysis, while the rest of the flagellum remains attached to the cell body. This suggests an evolutionary conserved weak interaction between the switch complex and the rest of the flagellum which is important to understand how the motor evolved. In addition, this information is crucial for mimicking such nanomachines in the laboratory.

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.

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.

No endospore formation confirmed in members of the phylum Proteobacteria.

Endospore formation is used by members of the phylum Firmicutes to withstand extreme environmental conditions. Several recent studies have proposed endospore formation in species outside of Firmicutes, particularly in Rhodobacter johrii and Serratia marcescens, members of the phylum Proteobacteria. Here, we aimed to investigate endospore formation in these two species by using advanced imaging and analytical approaches. Examination of the phase-bright structures observed in R. johrii and S. marcescens using cryo-electron tomography failed to identify endospores or stages of endospore formation. We determined that the phase-bright objects in R. johrii cells were triacylglycerol storage granules and those in S. marcescens were aggregates of cellular debris. In addition, R. johrii and S. marcescens containing phase-bright objects do not possess phenotypic and genetic features of endospores, including enhanced resistance to heat, presence of dipicolinic acid, or the presence of many of the genes associated with endospore formation. Our results support the hypothesis that endospore formation is restricted to the phylum Firmicutes.Importance: Bacterial endospore formation is an important process that allows the formation of dormant life forms called spores. As such, organisms able to sporulate can survive harsh environmental conditions for hundreds of years. Here, we follow up on previous claims that two members of Proteobacteria, Serratia marcescens and Rhodobacter johrii, are able to form spores. We conclude that those claims were incorrect and show that the putative spores in R. johrii and S. marcescens are storage granules and cellular debris, respectively. This study concludes that endospore formation is still unique to the phylum Firmicutes.

Structure of the Bacterial Cellulose Ribbon and Its Assembly-Guiding Cytoskeleton by Electron Cryotomography.

Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength, and stiffness prevent dehydration and mechanical disruption of the biofilm. Bacteria in the genus Gluconacetobacter secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How this higher-order structure is produced is poorly understood. We used cryo-electron tomography and focused-ion-beam milling of native bacterial biofilms to image cellulose-synthesizing Gluconacetobacter hansenii and Gluconacetobacter xylinus bacteria in a frozen-hydrated, near-native state. We confirm previous results suggesting that cellulose crystallization occurs serially following its secretion along one side of the cell, leading to a cellulose ribbon that can reach several micrometers in length and combine with ribbons from other cells to form a robust biofilm matrix. We were able to take direct measurements in a near-native state of the cellulose sheets. Our results also reveal a novel cytoskeletal structure, which we have named the cortical belt, adjacent to the inner membrane and underlying the sites where cellulose is seen emerging from the cell. We found that this structure is not present in other cellulose-synthesizing bacterial species, Agrobacterium tumefaciens and Escherichia coli 1094, which do not produce organized cellulose ribbons. We therefore propose that the cortical belt holds the cellulose synthase complexes in a line to form higher-order cellulose structures, such as sheets and ribbons.IMPORTANCE This work's relevance for the microbiology community is twofold. It delivers for the first time high-resolution near-native snapshots of Gluconacetobacter spp. (previously Komagataeibacter spp.) in the process of cellulose ribbon synthesis, in their native biofilm environment. It puts forward a noncharacterized cytoskeleton element associated with the side of the cell where the cellulose synthesis occurs. This represents a step forward in the understanding of the cell-guided process of crystalline cellulose synthesis, studied specifically in the Gluconacetobacter genus and still not fully understood. Additionally, our successful attempt to use cryo-focused-ion-beam milling through biofilms to image the cells in their native environment will drive the community to use this tool for the morphological characterization of other studied biofilms.

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.

Bacterial flagellar motor PL-ring disassembly subcomplexes are widespread and ancient.

The bacterial flagellum is an amazing nanomachine. Understanding how such complex structures arose is crucial to our understanding of cellular evolution. We and others recently reported that in several Gammaproteobacterial species, a relic subcomplex comprising the decorated P and L rings persists in the outer membrane after flagellum disassembly. Imaging nine additional species with cryo-electron tomography, here, we show that this subcomplex persists after flagellum disassembly in other phyla as well. Bioinformatic analyses fail to show evidence of any recent horizontal transfers of the P- and L-ring genes, suggesting that this subcomplex and its persistence is an ancient and conserved feature of the flagellar motor. We hypothesize that one function of the P and L rings is to seal the outer membrane after motor disassembly.

De Novo Structural Pattern Mining in Cellular Electron Cryotomograms.

Electron cryotomography enables 3D visualization of cells in a near-native state at molecular resolution. The produced cellular tomograms contain detailed information about a plethora of macromolecular complexes, their structures, abundances, and specific spatial locations in the cell. However, extracting this information in a systematic way is very challenging, and current methods usually rely on individual templates of known structures. Here, we propose a framework called "Multi-Pattern Pursuit" for de novo discovery of different complexes from highly heterogeneous sets of particles extracted from entire cellular tomograms without using information of known structures. These initially detected structures can then serve as input for more targeted refinement efforts. Our tests on simulated and experimental tomograms show that our automated method is a promising tool for supporting large-scale template-free visual proteomics analysis.

Criibacterium bergeronii gen. nov., sp. nov., a new member of the family Peptostreptococcaceae, isolated from human clinical samples.

A rod-shaped, motile anaerobic bacterium, designated CCRI-22567T, was isolated from a vaginal sample of a woman diagnosed with bacterial vaginosis and subjected to a polyphasic taxonomic study. The novel strain was capable of growth at 30-42 °C (optimum, 42 °C), at pH 5.5-8.5 (optimum, pH 7.0-7.5) and in the presence of 0-1.5 % (w/v) NaCl (optimally at 0.5 % NaCl). The phylogenetic trees based on 16S rRNA gene sequences showed that strain CCRI-22567T forms a distinct evolutionary lineage independent of other taxa in the family Peptostreptococcaceae. Strain CCRI-22567T exhibited 90.1 % 16S rRNA gene sequence similarity to Peptoanaerobacter stomatis ACC19aT and 89.7 % to Eubacterium yurii subsp. schtitka ATCC 43716. The three closest organisms with an available whole genome were compared to strain CCRI-22567T for genomic relatedness assessment. The genomic average nucleotide identities (OrthoANIu) obtained with Peptoanaerobacter stomatis ACC19aT, Eubacterium yurii subsp. margaretiae ATCC 43715 and Filifactor alocis ATCC 35896T were 71.8, 70.3 and 69.6 %, respectively. Strain CCRI-22567T contained C18 : 1 ω9c and C18 : 1 ω9c DMA as the major fatty acids. The DNA G+C content of strain CCRI-22567T based on its genome sequence was 33.8 mol%. On the basis of the phylogenetic, chemotaxonomic and other phenotypic properties, strain CCRI-22567T is considered to represent a new genus and species within the family Peptostreptococcaceae, for which the name Criibacterium bergeronii gen. nov., sp. nov., is proposed. The type strain of Criibacterium bergeronii is CCRI-22567T (=LMG 31278T=DSM 107614T=CCUG 72594T).