Lifecycle of a predatory bacterium vampirizing its prey through the cell envelope and S-layer.

Predatory bacteria feed upon other bacteria in various environments. Bdellovibrio exovorus is an obligate epibiotic predator that attaches on the prey cell surface, where it grows and proliferates. Although the mechanisms allowing feeding through the prey cell envelope are unknown, it has been proposed that the prey's proteinaceous S-layer may act as a defensive structure against predation. Here, we use time-lapse and cryo-electron microscopy to image the lifecycle of B. exovorus feeding on Caulobacter crescentus. We show that B. exovorus proliferates by non-binary division, primarily generating three daughter cells. Moreover, the predator feeds on C. crescentus regardless of the presence of an S-layer, challenging its assumed protective role against predators. Finally, we show that apparently secure junctions are established between prey and predator outer membranes.

Programmed Flagellar Ejection in Caulobacter crescentus Leaves PL-subcomplexes.

The bacterial flagellum consists of a long extracellular filament that is rotated by a motor embedded in the cell envelope. While flagellar assembly has been extensively studied,1 the disassembly process remains less well understood. In addition to the programmed flagellar ejection that occurs during the life cycle of Caulobacter crescentus, we and others have recently shown that many bacterial species lose their flagella under starvation conditions, leaving relic structures in the outer membrane.2-7 However, it remains unknown whether the programmed flagellar ejection of C. crescentus leaves similar relics or not. Here, we imaged the various stages of the C. crescentus life cycle using electron cryo-tomography (cryo-ET) and found that flagellar relic subcomplexes, akin to those produced in the starvation-induced process, remain as a result of flagellar ejection during cell development. This similarity suggests that the programmed flagellar ejection of C. crescentus might share a common evolutionary path with the more general, and likely more ancient,3 starvation-related flagellar loss.

Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale.

We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.

Flagellar Structures from the Bacterium Caulobacter crescentus and Implications for Phage ϕ CbK Predation of Multiflagellin Bacteria.

Caulobacter crescentus is a Gram-negative alphaproteobacterium that commonly lives in oligotrophic fresh- and saltwater environments. C. crescentus is a host to many bacteriophages, including ϕCbK and ϕCbK-like bacteriophages, which require interaction with the bacterial flagellum and pilus complexes during adsorption. It is commonly thought that the six paralogs of the flagellin gene present in C. crescentus are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in C. crescentus can indeed attenuate ϕCbK adsorption efficiency, although no single deletion completely ablates ϕCbK adsorption. Thus, the bacteriophage ϕCbK likely recognizes a common motif among the six known flagellins in C. crescentus with various degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by ϕCbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe posttranslational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and ϕCbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 Å. Altogether, this work builds a platform for future investigations of how phage ϕCbK infects C. crescentus at the molecular level.IMPORTANCE Bacterial flagellar filaments serve as an initial attachment point for many bacteriophages to bacteria. Some bacteria harbor numerous flagellin genes and are therefore able to generate flagellar filaments with complex compositions, which is thought to be important for evasion from bacteriophages. This study characterizes the importance of the six flagellin genes in C. crescentus for infection by bacteriophage ϕCbK. We find that filaments containing the FljK flagellin are the preferred substrate for bacteriophage ϕCbK. We also present a high-resolution structure of a flagellar filament containing only the FljK flagellin, which provides a platform for future studies on determining how bacteriophage ϕCbK attaches to flagellar filaments at the molecular level.

Self-propelling and rolling of a sessile-motile aggregate of the bacterium Caulobacter crescentus.

Active dispersal of microorganisms is often attributed to the cells' motile organelles. However, much less is known about whether sessile cells can access such motility through aggregation with motile counterparts. Here, we show that the rosette aggregates of the bacterium Caulobacter crescentus, although predominantly sessile, can actively disperse through the flagellar motors of motile members. Comparisons in kinematics between the motile rosettes and solitary swimming cells indicate that the rosettes can be powered by as few as a single motor. We further reconstructed the 3D movements of the rosettes to reveal that their proximity to a solid-liquid interface promotes a wheel-like rolling, as powered by the flagellar torque. This rolling movement also features a sequence of sharp turns, a reorientation mechanism distinct from that of swimming cells. Overall, our study elucidates an unexplored regime of aggregation-based motility that can be widely applied to sessile-motile composites.

GapR binds DNA through dynamic opening of its tetrameric interface.

GapR is a nucleoid-associated protein that is an essential regulator of chromosome replication in the cell cycle model Caulobacter crescentus. Here, we demonstrate that free GapR is a homotetramer, but not a dimer as previously reported (Guo et al., Cell 175: 583-597, 2018). We have determined the crystal structure of GapR in complex with a 10-bp A-tract DNA, which has an open tetrameric conformation, different from the closed clamp conformation in the previously reported crystal structure of GapR/DNA complex. The free GapR adopts multiple conformations in dynamic exchange equilibrium, with the major conformation resembling the closed tetrameric conformation, while the open tetrameric conformation is a representative of minor conformers. As it is impossible for the circular genomic DNA to get into the central DNA binding tunnel of the major conformation, we propose that GapR initially binds DNA through the open conformation, and then undergoes structural rearrangement to form the closed conformation which fully encircles the DNA. GapR prefers to bind DNA with 10-bp consecutive A/T base pairs nonselectively (Kd ∼12 nM), while it can also bind GC-rich DNA sequence with a reasonable affinity of about 120 nM. Besides, our results suggest that GapR binding results in widening the minor groove of AT-rich DNA, instead of overtwisting DNA.

Formation and function of bacterial organelles.

Advances in imaging technologies have revealed that many bacteria possess organelles with a proteomically defined lumen and a macromolecular boundary. Some are bound by a lipid bilayer (such as thylakoids, magnetosomes and anammoxosomes), whereas others are defined by a lipid monolayer (such as lipid bodies), a proteinaceous coat (such as carboxysomes) or have a phase-defined boundary (such as nucleolus-like compartments). These diverse organelles have various metabolic and physiological functions, facilitating adaptation to different environments and driving the evolution of cellular complexity. This Review highlights that, despite the diversity of reported organelles, some unifying concepts underlie their formation, structure and function. Bacteria have fundamental mechanisms of organelle formation, through which conserved processes can form distinct organelles in different species depending on the proteins recruited to the luminal space and the boundary of the organelle. These complex subcellular compartments provide evolutionary advantages as well as enabling metabolic specialization, biogeochemical processes and biotechnological advances. Growing evidence suggests that the presence of organelles is the rule, rather than the exception, in bacterial cells.

Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter.

Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ∼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.

In situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog.

Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity.

A bacterial surface layer protein exploits multistep crystallization for rapid self-assembly.

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. This pathway involves 2 domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using a time course of electron cryo-microscopy (Cryo-EM) imaging reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.

In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer.

Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus.

We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in Escherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E's function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.

High-resolution 3D models of Caulobacter crescentus chromosome reveal genome structural variability and organization.

High-resolution three-dimensional models of Caulobacter crescentus nucleoid structures were generated via a multi-scale modeling protocol. Models were built as a plectonemically supercoiled circular DNA and by incorporating chromosome conformation capture based data to generate an ensemble of base pair resolution models consistent with the experimental data. Significant structural variability was found with different degrees of bending and twisting but with overall similar topologies and shapes that are consistent with C. crescentus cell dimensions. The models allowed a direct mapping of the genomic sequence onto the three-dimensional nucleoid structures. Distinct spatial distributions were found for several genomic elements such as AT-rich sequence elements where nucleoid associated proteins (NAPs) are likely to bind, promoter sites, and some genes with common cellular functions. These findings shed light on the correlation between the spatial organization of the genome and biological functions.

The intrinsically disordered C-terminal linker of FtsZ regulates protofilament dynamics and superstructure in vitro.

The bacterial tubulin FtsZ polymerizes to form a discontinuous ring that drives bacterial cell division by directing local cell wall synthesis. FtsZ comprises a polymerizing GTPase domain, an intrinsically disordered C-terminal linker (CTL), and a C-terminal conserved peptide (CTC). FtsZ protofilaments align circumferentially in the cell, with the CTC mediating attachment to membrane-associated division proteins. The assembly of FtsZ protofilaments into dynamic clusters is critical for cell division, but the interactions between protofilaments and regulatory mechanisms that mediate cluster assembly and dynamics are unknown. Here, we describe a role for the CTL of Caulobacter crescentus FtsZ as an intrinsic regulator of lateral interactions between protofilaments in vitro FtsZ lacking its CTL (ΔCTL) shows a dramatically increased propensity to form long multifilament bundles compared with wild type (WT). ΔCTL also displays a reduced GTP hydrolysis rate compared with WT, but this altered activity does not account for bundle formation, as reducing protofilament turnover in WT is not sufficient to induce bundling. Surprisingly, binding of the membrane-anchoring protein FzlC disrupts ΔCTL bundling in a CTC-dependent manner. Moreover, the CTL affects the ability of the FtsZ curving protein FzlA to promote formation of helical bundles. We conclude that the CTL of FtsZ influences polymer structure and dynamics both through intrinsic effects on lateral interactions and turnover and by influencing extrinsic regulation of FtsZ by binding partners. Our characterization of CTL function provides a biochemical handle for understanding the relationship between FtsZ-ring structure and function in bacterial cytokinesis.

FtsEX-mediated regulation of the final stages of cell division reveals morphogenetic plasticity in Caulobacter crescentus.

During its life cycle, Caulobacter crescentus undergoes a series of coordinated shape changes, including generation of a polar stalk and reshaping of the cell envelope to produce new daughter cells through the process of cytokinesis. The mechanisms by which these morphogenetic processes are coordinated in time and space remain largely unknown. Here we demonstrate that the conserved division complex FtsEX controls both the early and late stages of cytokinesis in C. crescentus, namely initiation of constriction and final cell separation. ΔftsE cells display a striking phenotype: cells are chained, with skinny connections between cell bodies resulting from defects in inner membrane fusion and cell separation. Surprisingly, the thin connections in ΔftsE cells share morphological and molecular features with C. crescentus stalks. Our data uncover unanticipated morphogenetic plasticity in C. crescentus, with loss of FtsE causing a stalk-like program to take over at failed division sites.

Stalk formation of Brevundimonas and how it compares to Caulobacter crescentus.

The Caulobacter crescentus cell extension known as a stalk represents an unusual bacterial morphology. C. crescentus produces stalks under multiple nutrient conditions, but the length of the stalk is increased in response to phosphate starvation. However, the exact function of the stalk is not known, nor is it known how much stalk biogenesis or function is conserved with other stalked bacteria. Work presented here shows that many organisms in the Caulobacter genus and the next closest genus (Brevundimonas) generally do not synthesize stalks in the relatively-rich PYE growth medium, suggesting that the synthesis of a stalk under nutrient-rich conditions by C. crescentus may be the exception instead of the norm among its phylogenetic group. Brevundimonas subvibrioides can be induced to synthesize stalks by genetically mimicking phosphate starvation conditions, indicating stalk synthesis in this organism may be performed on an as-need basis. This mutation, however, does not appear to increase the incidence of holdfast synthesis. While B. subvibrioides stalks appear to be synthesized with the same polarity with respect to holdfast as C. crescentus stalks, evidence is presented that suggests B. subvibrioides may disassemble stalks when they are no longer needed. Many homologs of C. crescentus genes encoding stalk-associated proteins are absent in the B. subvibrioides genome, and B. subvibrioides PstA-GFP as well as C. crescentus StpX-GFP are able to enter the B. subvibrioides stalk compartment, calling into question the level of compartmentalization of the B. subvibrioides stalk. In summary, this work begins to address how much the C. crescentus model for this unusual morphological adaptation can be extended to related organisms.

Super-Resolution Microscopy and Single-Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule.

Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically fuses the protein of interest to a photostable fluorescent tag. Several labeling schemes are available to accomplish this. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types. However, their use in bacteria has been limited due to challenges imposed by a complex bacterial cell wall. Here, we describe the use of a genetically encoded photostable fluoromodule that can be targeted to cytosolic and membrane proteins in the Gram negative bacterium Caulobacter crescentus. Additionally, we summarize methods to use this fluoromodule for single protein imaging and super-resolution microscopy using stimulated emission depletion. © 2017 by John Wiley & Sons, Inc.

Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer.

Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Often the outermost cell envelope component, S-layers serve diverse functions including aiding pathogenicity and protecting against predators. We report that the S-layer of Caulobacter crescentus exhibits calcium-mediated structural plasticity, switching irreversibly between an amorphous aggregate state and the crystalline state. This finding invalidates the common assumption that S-layers serve only as static wall-like structures. In vitro, the Caulobacter S-layer protein, RsaA, enters the aggregate state at physiological temperatures and low divalent calcium ion concentrations. At higher concentrations, calcium ions stabilize monomeric RsaA, which can then transition to the two-dimensional crystalline state. Caulobacter requires micromolar concentrations of calcium for normal growth and development. Without an S-layer, Caulobacter is even more sensitive to changes in environmental calcium concentration. Therefore, this structurally dynamic S-layer responds to environmental conditions as an ion sensor and protects Caulobacter from calcium deficiency stress, a unique mechanism of bacterial adaptation. These findings provide a biochemical and physiological basis for RsaA's calcium-binding behavior, which extends far beyond calcium's commonly accepted role in aiding S-layer biogenesis or oligomerization and demonstrates a connection to cellular fitness.

Relative Rates of Surface and Volume Synthesis Set Bacterial Cell Size.

Many studies have focused on the mechanisms underlying length and width determination in rod-shaped bacteria. Here, we focus instead on cell surface area to volume ratio (SA/V) and demonstrate that SA/V homeostasis underlies size determination. We propose a model whereby the instantaneous rates of surface and volume synthesis both scale with volume. This model predicts that these relative rates dictate SA/V and that cells approach a new steady-state SA/V exponentially, with a decay constant equal to the volume growth rate. To test this, we exposed diverse bacterial species to sublethal concentrations of a cell wall biosynthesis inhibitor and observed dose-dependent decreases in SA/V. Furthermore, this decrease was exponential and had the expected decay constant. The model also quantitatively describes SA/V alterations induced by other chemical, nutritional, and genetic perturbations. We additionally present evidence for a surface material accumulation threshold underlying division, sensitizing cell length to changes in SA/V requirements.