Elitza I Tocheva

• PhD in Microbiology
• California Institute of Technology

Type VII secretion systems participate in protein export, virulence, conjugation, and metabolic regulation. Five subtypes (ESX-1 to ESX-5) exist, each with specific roles and well-characterized secretion profiles in various mycobacterial species. Mycobacterium abscessus, encodes only ESX-3 and ESX-4. Here, single and double M. abscessus mutants lac...

The bacterial cell envelope is a complex multilayered structure conserved across all bacterial phyla. It is categorized into two main types based on the number of membranes surrounding the cell. Monoderm bacteria are enclosed by a single membrane, whereas diderm cells are distinguished by the presence of a second, outer membrane (OM). An ancient di...

Introduction. Mycobacterium abscessus (MABS) is a pathogenic bacterium that can cause severe lung infections, particularly in individuals with cystic fibrosis. MABS colonies can exhibit either a smooth (S) or rough (R) morphotype, influenced by the presence or absence of glycopeptidolipids (GPLs) on their surface, respectively. Despite the clinical...

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 t...

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...

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 s...

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, exometab...

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...

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 bac...

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease by the horizontal transfer of oncogenic DNA that is integrated into the host’s genome. The conjugation is mediated by the conjugative VirB/D4 type 4 secretion system (T4SS). A. tumefaciens T4SS assembles an extracellular filament, the T-pilus, that is involved in the forma...

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 bacterial common ancestor 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...

Deinococcus radiodurans is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, partly because of its complex cell envelope encapsulated within a hyperstable surface layer (S-layer). Despite decades of research into this cell envelope, atomic structural details of the S-layer have remained obscure. In t...

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 is lost in descendant lineages classified as asporogenic. While Kurthia spp. is considered to comprise only aspor...

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 ‘...

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) millin...

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 prevente...

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 anti...

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...

A rod-shaped, motile anaerobic bacterium, designated CCRI-22567 T , 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 (optima...

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...

Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength and stiffness prevents dehydration and mechanical disruption of the biofilm. Bacteria in the Gluconacetobacter genus secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How...

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 ultra...

Endospore formation is used by members of the phylum Firmicutes to withstand extreme environmental conditions. Several recent studies have documented 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 formati...

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 ultr...

Sporulation is a complex morphophysiological process resulting in a cellular structure that is more resistant than the vegetative form. In Firmicutes, this structure is produced within the mother cell, and is called an endospore. Endospore formation is thought to have evolved in the common ancestor of Firmicutes. However, sporulation has apparently...

Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength and stiffness prevents dehydration and mechanical disruption of the biofilm. Bacteria in the Gluconacetobacter genus secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How...

Significance In order to understand the evolution of complex biological machines like the bacterial flagellar motor, it is crucial to know what each component does and when it arose. Here, we show that a subcomplex of the motor thought to act as a bushing for the spinning motor likely also serves another function—it plugs the hole in the outer memb...

The bacterial flagellar motor 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 sub-complex comprising the P- and L-rings persists in the outer membrane after flagellum disassembly. Im...

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 ver...

Mycobacteria are well known for their taxonomic diversity, their impact on global health, and for their atypical cell wall and envelope. In addition to a cytoplasmic membrane and a peptidoglycan layer, the cell envelope of members of the order Corynebacteriales, which include Mycobacterium tuberculosis, also have an arabinogalactan layer connecting...

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. Rather than undifferentiated bags of enzymes, we now view bacteria as structurally complex assemblies of macromolecular machine...

The Use of Cryotomography to Study the Complex Morphological Remodeling of Membranes in Bacteria - Volume 23 Issue S1 - Elitza I. Tocheva

Significance Quiescent bacteria are intrinsically resistant to antibiotics and the host immune response. A conserved bacterial starvation survival response is the consumption of ATP to make an inorganic polymer, polyphosphate (polyP), which then forms granule superstructures. PolyP granules occur in all three domains of life, yet how and why cells...

STATEMENT Here 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. ABSTRACT Electron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique h...

Four recent papers from our group exploiting the power of electron cryotomography to produce 3-D reconstructions of intact cells in a near-native state have led to the proposal that an ancient sporulation-like event gave rise to the second membrane in diderm bacteria. Here we review the images of sporulating monoderm and diderm cells which show how...

Four recent papers from our group exploiting the power of electron cryotomography to produce 3-D reconstructions of intact cells in a near-native state have led to the proposal that an ancient sporulation-like event gave rise to the second membrane in diderm bacteria. Here we review the images of sporulating monoderm and diderm cells which show how...

This preprint is available on arXiv.org as well: https://arxiv.org/abs/1512.09347 This is a method to find reoccuring patterns in the single cell cryo-electron tomograms. The paper is under review at Structure.

Cryo-electron tomography enables 3D visualization of cells in a near native state at molecular resolution. The produced cellular tomograms contain detailed information about all macromolecular complexes, their structures, their abundances and their specific spatial locations in the cell. However, extracting this information is very challenging and...

Cell division is essential for spore formation but not for viability in the filamentous streptomycetes bacteria. Failure to complete cell division instead blocks spore formation, a phenotype that can be visualized by the absence of gray (in Streptomyces coelicolor) and green (in Streptomyces venezuelae) spore-associated pigmentation. Despite the la...

Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction l...

Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction l...

Using electron cryotomography we show that the Gram-negative sporulating bacterium A. longum synthesizes high-density storage granules at the leading edges of engulfing membranes. The granules appear in the prespore and increase in size and number as engulfment proceeds. Typically, a cluster of 8-12 storage granules closely associates with the inne...

While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross-linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ΔponA mutant that enabled high-resolution electron...

Two hallmarks of the Firmicute phylum, which includes the Bacilli and Clostridia classes, are their ability to form endospores and their "Gram-positive" single-membraned, thick-cell-wall envelope structure. Acetonema longum is part of a lesser-known family (the Veillonellaceae) of Clostridia that form endospores but that are surprisingly "Gram nega...

The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial king...

Panel A. Negative stain of individual nanopods. Note the crystalline-like outer surface. Also note a point (indicated by arrow) where the interior structure transitions from spherical to a spiral form. Panel B. Longitudinal section of a nanopod showing a single outer layer surrounding internal vesicle-like structures (example indicated by arrow). P...

Negatively stained culture fluid samples containing nanopods from other Comamonadaceae bacteria. Nanopods are indicated by thick arrows, thin arrows point to other apparent S-layer-derived structures (indicated by crystalline surface). Samples are from:, A. avenae subsp. citrulli AAC00-1 (Panel A), A. delafieldii (Panel B), D. acidovorans SPH-1 (Pa...

Three-dimensional reconstruction of a phenanthrene-grown culture of Delftia sp. Cs1-4 imaged by electron cryotomography. The sample is supported on a lacy carbon grid (outer circle) and contains a cell (inner circular structure) and nanopods (undulating structures in the extracellular space). (MOV)

Panel A. Analysis of NpdA in Delftia sp. Cs1-4 wild type (Lanes 1 and 3) and NpdA mutant (Lanes 2 and 4) cells. Lanes 1–2 are SDS-PAGE profiles; Lanes 3 and 4 are Western blots of whole cell SDS-PAGE profiles probed with anti-NpdA. Panel B. Negatively stained culture of wild type Delftia sp. Cs1-4 showing nanopods. Panel C. Negatively stained cultu...

Bacterial outer membrane vesicles (OMV) are packets of periplasmic material that, via the proteins and other molecules they contain, project metabolic function into the environment. While OMV production is widespread in proteobacteria, they have been extensively studied only in pathogens, which inhabit fully hydrated environments. However, many (ar...

Electron cryotomography (ECT) is an emerging technology that allows thin samples such as macromolecular complexes and small bacterial cells to be imaged in 3-D in a nearly native state to "molecular" ( approximately 4 nm) resolution. As such, ECT is beginning to deliver long-awaited insight into the positions and structures of cytoskeletal fi lamen...

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bri...

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandr...

Chemoreceptors are key components of the high-performance signal transduction system that controls bacterial chemotaxis. Chemoreceptors are typically localized in a cluster at the cell pole, where interactions among the receptors in the cluster are thought to contribute to the high sensitivity, wide dynamic range, and precise adaptation of the sign...

The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate,...

Nitrite reductase (NiR) is an enzyme that uses type 1 and type 2 copper sites to reduce nitrite to nitric oxide during bacterial denitrification. A copper-nitrosyl intermediate is a proposed, yet poorly characterized feature of the NiR catalytic cycle. This intermediate is formally described as Cu(I)-NO+ and is proposed to be formed at the type 2 c...

Copper-containing nitrite reductase harbors a type-1 and a type-2 Cu site. The former acts as the electron acceptor site of the enzyme, and the latter is the site of catalytic action. The effect of the methionine ligand on the reorganization energy of the type-1 site was explored by studying the electron-transfer kinetics between NiR (wild type (wt...

Prokaryotic glutathione S-transferases are as diverse as their eukaryotic counterparts but are much less well characterized. BphK from Burkholderia xenovorans LB400 consumes two GSH molecules to reductively dehalogenate chlorinated 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), inhibitory polychlorinated biphenyl metabolites. Crystallographic str...

A lanthanide complex, named CLaNP (caged lanthanide NMR probe) has been developed for the characterisation of proteins by paramagnetic NMR spectroscopy. The probe consists of a lanthanide chelated by a derivative of DTPA (diethylenetriaminepentaacetic acid) with two thiol reactive functional groups. The CLaNP molecule is attached to a protein by tw...

A copper-nitrosyl intermediate forms during the catalytic cycle of nitrite reductase, the enzyme that mediates the committed step in bacterial denitrification. The crystal structure of a type 2 copper-nitrosyl complex of nitrite reductase reveals an unprecedented side-on binding mode in which the nitrogen and oxygen atoms are nearly equidistant fro...