Altruistic feeding and cell-cell signaling during bacterial differentiation actively enhance phenotypic heterogeneity.

Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation non-uniformly to secure against the possibility that favorable growth conditions, which puts sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early utilize a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay non-sporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness.

Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis.

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

Bacterial cell surface nanoenvironment requires a specialized chaperone to activate a peptidoglycan biosynthetic enzyme.

Bacillus subtilis spores are produced inside the cytosol of a mother cell. Spore surface assembly requires the SpoVK protein in the mother cell, but its function is unknown. Here, we report that SpoVK is a dedicated chaperone from a distinct higher-order clade of AAA+ ATPases that activates the peptidoglycan glycosyltransferase MurG during sporulation, even though MurG does not normally require activation by a chaperone during vegetative growth. MurG redeploys to the spore surface during sporulation, where we show that the local pH is reduced and propose that this change in cytosolic nanoenvironment necessitates a specific chaperone for proper MurG function. Further, we show that SpoVK participates in a developmental checkpoint in which improper spore surface assembly inactivates SpoVK, which leads to sporulation arrest. The AAA+ ATPase clade containing SpoVK includes other dedicated chaperones involved in secretion, cell-envelope biosynthesis, and carbohydrate metabolism, suggesting that such fine-tuning might be a widespread feature of different subcellular nanoenvironments.

Cell division machinery drives cell-specific gene activation during bacterial differentiation.

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that a unique feature of the sporulation septum, defined by the cell division machinery, drives the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

Protein coopted from a phage restriction system dictates orthogonal cell division plane selection in Staphylococcus aureus.

The spherical bacterium Staphylococcus aureus, a leading cause of nosocomial infections, undergoes binary fission by dividing in two alternating orthogonal planes, but the mechanism by which S. aureus correctly selects the next cell division plane is not known. To identify cell division placement factors, we performed a chemical genetic screen that revealed a gene which we termed pcdA. We show that PcdA is a member of the McrB family of AAA+ NTPases that has undergone structural changes and a concomitant functional shift from a restriction enzyme subunit to an early cell division protein. PcdA directly interacts with the tubulin-like central divisome component FtsZ and localizes to future cell division sites before membrane invagination initiates. This parallels the action of another McrB family protein, CTTNBP2, which stabilizes microtubules in animals. We show that PcdA also interacts with the structural protein DivIVA and propose that the DivIVA/PcdA complex recruits unpolymerized FtsZ to assemble along the proper cell division plane. Deletion of pcdA conferred abnormal, non-orthogonal division plane selection, increased sensitivity to cell wall-targeting antibiotics, and reduced virulence in a murine infection model. Targeting PcdA could therefore highlight a treatment strategy for combatting antibiotic-resistant strains of S. aureus.

Cell-specific cargo delivery using synthetic bacterial spores.

Delivery of cancer therapeutics to non-specific sites decreases treatment efficacy while increasing toxicity. In ovarian cancer, overexpression of the cell surface marker HER2, which several therapeutics target, relates to poor prognosis. We recently reported the assembly of biocompatible bacterial spore-like particles, termed "SSHELs." Here, we modify SSHELs with an affibody directed against HER2 and load them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduce tumor growth and increase survival with lower toxicity in a mouse tumor xenograft model compared with free drug and with liposomal doxorubicin by preferentially accumulating in the tumor mass. Target cells actively internalize and then traffic bound SSHELs to acidic compartments, whereupon the cargo is released to the cytosol in a pH-dependent manner. We propose that SSHELs represent a versatile strategy for targeted drug delivery, especially in cancer settings.

Three-dimensional structured illumination microscopy with enhanced axial resolution.

The axial resolution of three-dimensional structured illumination microscopy (3D SIM) is limited to ∼300 nm. Here we present two distinct, complementary methods to improve axial resolution in 3D SIM with minimal or no modification to the optical system. We show that placing a mirror directly opposite the sample enables four-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with ∼120-nm lateral and 160-nm axial resolution. We also developed a deep learning method achieving ∼120-nm isotropic resolution. This method can be combined with denoising to facilitate volumetric imaging spanning dozens of timepoints. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers and visualizing cytoskeletal dynamics within T cells in the early stages of immune synapse formation.

Bacterial developmental checkpoint that directly monitors cell surface morphogenesis.

Bacillus subtilis spores are encased in two concentric shells: an outer proteinaceous "coat" and an inner peptidoglycan "cortex," separated by a membrane. Cortex assembly depends on coat assembly initiation, but how cells achieve this coordination across the membrane is unclear. Here, we report that the protein SpoVID monitors the polymerization state of the coat basement layer via an extension to a functional intracellular LysM domain that arrests sporulation when coat assembly is initiated improperly. Whereas extracellular LysM domains bind mature peptidoglycan, SpoVID LysM binds to the membrane-bound lipid II peptidoglycan precursor. We propose that improper coat assembly exposes the SpoVID LysM domain, which then sequesters lipid II and prevents cortex assembly. SpoVID defines a widespread group of firmicute proteins with a characteristic N-terminal domain and C-terminal peptidoglycan-binding domains that might combine coat and cortex assembly roles to mediate a developmental checkpoint linking the morphogenesis of two spatially separated supramolecular structures.

Cytoskeletal proteins: lessons learned from bacteria.

Cytoskeletal proteins are classified as a group that is defined functionally, whose members are capable of polymerizing into higher order structures, either dynamically or statically, to perform structural roles during a variety of cellular processes. In eukaryotes, the most well-studied cytoskeletal proteins are actin, tubulin, and intermediate filaments, and are essential for cell shape and movement, chromosome segregation, and intracellular cargo transport. Prokaryotes often harbor homologs of these proteins, but in bacterial cells, these homologs are usually not employed in roles that can be strictly defined as 'cytoskeletal'. However, several bacteria encode other proteins capable of polymerizing which, although they do not appear to have a eukaryotic counterpart, nonetheless appear to perform a more traditional 'cytoskeletal' function. In this review, we discuss recent reports that cover the structures and functions of prokaryotic proteins that are broadly termed as cytoskeletal, either by sequence homology or by function, to highlight how the enzymatic properties of traditionally studied cytoskeletal proteins may be used for other types of cellular functions; and to demonstrate how truly 'cytoskeletal' functions may be performed by uniquely bacterial proteins that do not display homology to eukaryotic proteins.

How bacteria block their own biofilms.

Bacterial biofilms are surface-associated multicellular communities that are highly resistant to removal. Scheffler et al. discovered that Pseudomonas aeruginosa secretes a small molecule that inhibits other P. aeruginosa cells from adsorbing to surfaces by interfering with type IV pili dynamics. The inhibition of cell adsorption could present a method to prevent biofilm formation on sensitive surfaces in hospitals and industry.

Reformulation of an extant ATPase active site to mimic ancestral GTPase activity reveals a nucleotide base requirement for function.

Hydrolysis of nucleoside triphosphates releases similar amounts of energy. However, ATP hydrolysis is typically used for energy-intensive reactions, whereas GTP hydrolysis typically functions as a switch. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during Bacillus subtilis sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. We therefore reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVAGTPase functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that we report is required for polymerization. Further, incubation of SpoIVAGTPase with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. We also present data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA.

Living organisms need energy to stay alive; in cells, this energy is supplied in the form of a small molecule called adenosine triphosphate, or ATP, a nucleotide that stores energy in the bonds between its three phosphate groups. ATP is present in all living cells and is often referred to as the energy currency of the cell, because it can be easily stored and transported to where it is needed. However, it is unknown why cells rely so heavily on ATP when a highly similar nucleotide called guanosine triphosphate, or GTP, could also act as an energy currency. There are several examples of proteins that originally used GTP and have since evolved to use ATP, but it is not clear why this switch occurred. One suggestion is that ATP is the more readily available nucleotide in the cell. To test this hypothesis, Updegrove, Harke et al. studied a protein that helps bacteria transition into spores, which are hardier and can survive in extreme environments until conditions become favorable for bacteria to grow again. In modern bacteria, this protein uses ATP to provide energy, but it evolved from an ancestral protein that used GTP instead. First, Updegrove, Harke et al. engineered the protein so that it became more similar to the ancestral protein and used GTP instead of ATP. When this was done, the protein gained the ability to break down GTP and release energy from it, but it no longer performed its enzymatic function. This suggests that both the energy released and the source of that energy are important for a protein's activity. Further analysis showed that the modern version of the protein has evolved to briefly hold on to ATP after releasing its energy, which did not happen with GTP in the modified protein. Updegrove, Harke et al. also discovered that the levels of GTP in a bacterial cell fall as it transforms into a spore, while ATP levels remain relatively high. This suggests that ATP may indeed have become the source of energy of choice because it was more available. These findings provide insights into how ATP became the energy currency in cells, and suggest that how ATP is bound by proteins can impact a protein's activity. Additionally, these experiments could help inform the development of drugs targeting proteins that bind nucleotides: it may be essential to consider the entirety of the binding event, and not just the release of energy.

Lipid flip-flop and desorption from supported lipid bilayers is independent of curvature.

Flip-flop of lipids of the lipid bilayer (LBL) constituting the plasma membrane (PM) plays a crucial role in a myriad of events ranging from cellular signaling and regulation of cell shapes to cell homeostasis, membrane asymmetry, phagocytosis, and cell apoptosis. While extensive research has been conducted to probe the lipid flip flop of planar lipid bilayers (LBLs), less is known regarding lipid flip-flop for highly curved, nanoscopic LBL systems despite the vast importance of membrane curvature in defining the morphology of cells and organelles and in maintaining a variety of cellular functions, enabling trafficking, and recruiting and localizing shape-responsive proteins. In this paper, we conduct molecular dynamics (MD) simulations to study the energetics, structure, and configuration of a lipid molecule undergoing flip-flop and desorption in a highly curved LBL, represented as a nanoparticle-supported lipid bilayer (NPSLBL) system. We compare our findings against those of a planar substrate supported lipid bilayer (PSSLBL). Our MD simulation results reveal that despite the vast differences in the curvature and other curvature-dictated properties (e.g., lipid packing fraction, difference in the number of lipids between inner and outer leaflets, etc.) between the NPSLBL and the PSSLBL, the energetics of lipid flip-flop and lipid desorption as well as the configuration of the lipid molecule undergoing lipid flip-flop are very similar for the NPSLBL and the PSSLBL. In other words, our results establish that the curvature of the LBL plays an insignificant role in lipid flip-flop and desorption.

Formation and Properties of a Self-Assembled Nanoparticle-Supported Lipid Bilayer Probed through Molecular Dynamics Simulations.

We have carried out coarse-grained molecular dynamics (MD) simulations to study the self-assembly procedure of a system of randomly placed lipid molecules, water beads, and a nanoparticle (NP). The self-assembly results in the formation of the nanoparticle-supported lipid bilayer (NPSLBL), with the self-assembly mechanism being driven by events such as the formation of small lipid clusters, merging of the lipid clusters in the vicinity of the NP to form NP-embedded vesicle with a pore, and collapsing of that pore to eventually form the equilibrated NPSLBL system overcoming a large free-energy barrier. Subsequently, we quantify the properties and the configurations of this NPSLBL system. We reveal that unlike our proposition of an equal number of lipid molecules occupying the inner and outer leaflets in a recent report studying the properties of a preassembled lipid bilayer, the equilibrated self-assembled NPSLBL system demonstrates a much larger number of lipid molecules occupying the outer leaflet as compared to the inner leaflet. Second, the thickness of the water layer entrapped between the NP and the inner leaflet shows similar values as predicted by experiments and our previous study. Finally, we reveal that, similar to our previous study, the diffusivity of the lipid molecules in the outer leaflet is larger than that in the inner leaflet but, due to higher temperature employed during our simulations, are even larger than that predicted by our previous study.

A 2-dimensional ratchet model describes assembly initiation of a specialized bacterial cell surface.

Bacterial spores are dormant cells that are encased in a thick protein shell, the "coat," which participates in protecting the organism's DNA from environmental insults. The coat is composed of dozens of proteins that assemble in an orchestrated fashion during sporulation. In Bacillus subtilis, 2 proteins initiate coat assembly: SpoVM, which preferentially binds to micron-scale convex membranes and marks the surface of the developing spore as the site for coat assembly; and SpoIVA, a structural protein recruited by SpoVM that uses ATP hydrolysis to drive its irreversible polymerization around the developing spore. Here, we describe the initiation of coat assembly by SpoVM and SpoIVA. Using single-molecule fluorescence microscopy in vivo in sporulating cells and in vitro on synthetic spores, we report that SpoVM's localization is primarily driven by a lower off-rate on membranes of preferred curvature in the absence of other coat proteins. Recruitment and polymerization of SpoIVA results in the entrapment of SpoVM on the forespore surface. Using experimentally derived reaction parameters, we show that a 2-dimensional ratchet model can describe the interdependent localization dynamics of SpoVM and SpoIVA, wherein SpoVM displays a longer residence time on the forespore surface, which favors recruitment of SpoIVA to that location. Localized SpoIVA polymerization in turn prevents further sampling of other membranes by prelocalized SpoVM molecules. Our model therefore describes the dynamics of structural proteins as they localize and assemble at the correct place and time within a cell to form a supramolecular complex.

Nanovesicles Versus Nanoparticle-Supported Lipid Bilayers: Massive Differences in Bilayer Structures and in Diffusivities of Lipid Molecules and Nanoconfined Water.

We carry out molecular dynamics (MD) simulations to compare the equilibrium architecture and properties of nanoparticle-supported lipid bilayers (NPSLBLs) with the free vesicles of similar dimensions. Three key differences emerge. First, we witness that for a free vesicle, a much larger number of lipid molecules occupy the outer layer as compared to the inner layer; on the other hand, for the NPSLBL the number of lipid molecules occupying the inner and outer layers is identical. Second, we witness that the diffusivities of the lipid molecules occupying both the inner and the outer layers of the free vesicles are identical, whereas for the NPSLBLs the diffusivity of the lipid molecules in the outer layer is more than twice the diffusivity of the lipid molecules in the inner layer. Finally, the NPSLBLs entrap nanoscopic thin water film between the inner lipid layer and the NP and the diffusivity of this water film is nearly 1 order of magnitude smaller than the diffusivity of the bulk water; on the other hand, the water inside the free vesicles has a diffusivity that is only slightly lower than that of the bulk water. Our findings, possibly the first probing the atomistic details of the NPSLBLs, are anticipated to shed light on the properties of this important nanomaterial with applications in a large number of disciplines ranging from drug and gene delivery to characterizing curvature-sensitive molecules.

An essential Staphylococcus aureus cell division protein directly regulates FtsZ dynamics.

Binary fission has been well studied in rod-shaped bacteria, but the mechanisms underlying cell division in spherical bacteria are poorly understood. Rod-shaped bacteria harbor regulatory proteins that place and remodel the division machinery during cytokinesis. In the spherical human pathogen Staphylococcus aureus, we found that the essential protein GpsB localizes to mid-cell during cell division and co-constricts with the division machinery. Depletion of GpsB arrested cell division and led to cell lysis, whereas overproduction of GpsB inhibited cell division and led to the formation of enlarged cells. We report that S. aureus GpsB, unlike other Firmicutes GpsB orthologs, directly interacts with the core divisome component FtsZ. GpsB bundles and organizes FtsZ filaments and also stimulates the GTPase activity of FtsZ. We propose that GpsB orchestrates the initial stabilization of the Z-ring at the onset of cell division and participates in the subsequent remodeling of the divisome during cytokinesis.

Vaccine display on artificial bacterial spores enhances protective efficacy against Staphylococcus aureus infection.

Spores of Bacillus subtilis are encased in a protein coat composed of ∼80 different proteins. Recently, we reconstituted the basement layer of the coat, composed of two structural proteins (SpoVM and SpoIVA) around spore-sized silica beads encased in a lipid bilayer, to create synthetic spore-like particles termed 'SSHELs'. We demonstrated that SSHELs could display thousands of copies of proteins and small molecules of interest covalently linked to SpoIVA. In this study, we investigated the efficacy of SSHELs in delivering vaccines. We show that intramuscular vaccination of mice with undecorated one micron-diameter SSHELs elicited an antibody response against SpoIVA. We further demonstrate that SSHELs covalently modified with a catalytically inactivated staphylococcal alpha toxin variant (HlaH35L), without an adjuvant, resulted in improved protection against Staphylococcus aureus infection in a bacteremia model as compared to vaccination with the antigen alone. Although vaccination with either HlaH35L or HlaH35L conjugated to SSHELs similarly elicited the production of neutralizing antibodies to Hla, we found that a subset of memory T cells was differentially activated when the antigen was delivered on SSHELs. We propose that the particulate nature of SSHELs elicits a more robust immune response to the vaccine that results in superior protection against subsequent S. aureus infection.

Dash-and-Recruit Mechanism Drives Membrane Curvature Recognition by the Small Bacterial Protein SpoVM.

In Bacillus subtilis, sporulation requires that the 26-amino acid protein SpoVM embeds specifically into the forespore membrane, a structure with convex curvature. How this nanometer-sized protein can detect curves on a micrometer scale is not well understood. Here, we report that SpoVM exploits a "dash-and-recruit" mechanism to preferentially accumulate on the forespore. Using time-resolved imaging and flow cytometry, we observe that SpoVM exhibits a faster adsorption rate onto membranes of higher convex curvature. This preferential adsorption is accurately modeled as a two-step process: first, an initial binding event occurs with a faster on rate, then cooperative recruitment of additional SpoVM molecules follows. We demonstrate that both this biochemical process and effective sporulation in vivo require an unstructured and flexible SpoVM N terminus. We propose that this two-pronged strategy of fast adsorption followed by recruitment of subsequent molecules is a general mechanism that allows small proteins to detect subtle curves with a radius 1,000-fold their size.

Bacterial Cell Division: Nonmodels Poised to Take the Spotlight.

The last three decades have witnessed an explosion of discoveries about the mechanistic details of binary fission in model bacteria such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus. This was made possible not only by advances in microscopy that helped answer questions about cell biology but also by clever genetic manipulations that directly and easily tested specific hypotheses. More recently, research using understudied organisms, or nonmodel systems, has revealed several alternate mechanistic strategies that bacteria use to divide and propagate. In this review, we highlight new findings and compare these strategies to cell division mechanisms elucidated in model organisms.

Cell Death Pathway That Monitors Spore Morphogenesis.

The use of quality control mechanisms to stall developmental pathways or completely remove defective cells from a population is a widespread strategy to ensure the integrity of morphogenetic programs. Endospore formation (sporulation) is a well conserved microbial developmental strategy in the Firmicutes phylum wherein a progenitor cell that faces starvation differentiates to form a dormant spore. Despite the conservation of this strategy, it has been unclear what selective pressure maintains the fitness of this developmental program, composed of hundreds of unique genes, during multiple rounds of vegetative growth when sporulation is not required. Recently, a quality control pathway was discovered in Bacillus subtilis which monitors the assembly of the spore envelope and specifically eliminates, through cell lysis, sporulating cells that assemble the envelope incorrectly. Here, we review the use of checkpoints that govern the entry into sporulation in B. subtilis and discuss how the use of regulated cell death pathways during bacterial development may help maintain the fidelity of the sporulation program in the species.