Bacterial ribosomal RNAs (rRNAs) are decorated with conserved nucleotide modifications, but the functionality of these modifications is often underexplored. MraW (RsmH) is a 16S rRNA methyltransferase. Here, we report that deletion of mraW corrects a late-stage sporulation defect in Bacillus subtilis by bypassing a sporulation checkpoint. Ribosomes purified from ΔmraW cells display a ∼2-fold decrease in translation efficiency; in vivo, ΔmraW cells produced decreased levels of the sporulation checkpoint protein CmpA. Reduced production of CmpA is mediated by mRNA sequences that form a stem-loop which occludes early cmpA codons. Proteomic analysis revealed that MraW mediates production of multiple proteins, some of whose mRNA form similar structures as the cmpA transcript. We propose that MraW modification of 16S rRNA enhances translation efficiency in general, and that specific transcripts have evolved structural features that fine-tune protein levels. This type of control may be prevalent in bacteria which exhibit uncoupled transcription and translation.

The transition from unicellular to multicellular growth requires diversification of cellular functions within genetically identical populations. In Bacillus subtilis, biofilm formation is historically viewed as a developmental precursor to sporulation along a linear pathway. Here, we show that biofilm formation and sporulation instead diverge along a branched pathway. A subpopulation that first initiates sporulation catabolizes lipoteichoic acid through the sequential action of the enzymes ShfP (Sporulation heterogeneity factor Poison) and PhoA (alkaline phosphatase A), leading to the release of millimolar concentrations of glycerol. This glycerol impedes sporulation by disrupting cell wall synthesis and cytoplasmic pH, necessitating counteraction by another protein, ShfA (Sporulation heterogeneity factor Antidote). The extracellular glycerol, however, acts as a morphogen that directs neighboring cells to initiate biofilm formation, which we directly visualize in developing populations of cells. Thus, B. subtilis multicellularity emerges through a branched developmental program in which sporulating cells generate the cue that creates the biofilm-producing lineage via cell-cell communication through repurposing of a canonical intracellular metabolite.

The correct subcellular localization of proteins is a critical step underlying myriad biological processes, but the cues that drive the specific localization of integral membrane proteins in bacteria remain largely undeciphered. During sporulation in Bacillus subtilis, a rod-shaped outer "mother cell" constructs an internal spherical "forespore" cell that eventually matures into the spore. The integral membrane protein ShfA is made in the mother cell cytosol and localizes to the surface of the forespore. Here, we report that, despite being a multi-pass transmembrane protein, ShfA spontaneously inserts into the lipid bilayer via its N-terminal "YabQ" domain without the apparent need for a pre-localized insertase. ShfA preferentially inserts into cell division septa in multiple bacterial species, indicating that a widely conserved septal cue drives ShfA localization. Structural modeling suggested that the YabQ domain harbors a specific intramembrane groove that can bind the universal lipid carrier undecaprenyl phosphate (UndP), and UndP depletion in vivo disrupted proper ShfA localization. We propose that ShfA localizes to sites of active cell wall synthesis by binding to UndP and/or molecules like lipid I and lipid Il that contain UndP and speculate that the function of ShfA is to stabilize these precursors of cell wall biogenesis from the harsh cytosolic nanoenvironment that surrounds the forespore during sporulation.

During infection, Staphylococcus aureus forms dense multicellular structures, called staphylococcal abscess communities (SACs), that are encased in a capsule made of host fibrin to evade host immune defenses. S. aureus cells divide characteristically along successive orthogonal planes, but the contribution of this division geometry to infection is unclear. Here, we show that disrupting orthogonal cell division by deleting the cell division septum placement factor PcdA impairs SAC formation in vivo and in a three-dimensional in vitro model. Loss of PcdA leads to uneven surface distribution of adhesins containing the YSIRK signal sequence that directs their insertion into the division septum, thereby resulting in uneven interaction with fibrin fibers. Consequently, bacterial communities fail to establish a robust fibrin pseudocapsule and remain accessible to immune cells. We propose that orthogonal cell division coordinates cell cycle progression with extracellular matrix engagement, SAC architecture, and persistence within host tissues.

During infection, Staphylococcus aureus forms dense multicellular structures, called staphylococcal abscess communities (SACs) that are encased in a capsule made of host fibrin to evade host immune defenses. S. aureus cells divide characteristically along successive orthogonal planes, but the contribution of this division geometry to infection is unclear. Here, we show that disrupting orthogonal cell division by deleting the cell division septum placement factor PcdA impairs SAC formation in vivo and in a three-dimensional in vitro model. Loss of PcdA leads to uneven cell surface distribution of adhesins containing the YSIRK signal sequence that directs their insertion into the division septum, thereby resulting in uneven interaction with fibrin fibers. Consequently, bacterial communities fail to establish a robust fibrin pseudocapsule and remain accessible to immune cells. We propose that orthogonal cell division coordinates cell cycle progression with extracellular matrix engagement, SAC architecture, and persistence within host tissues.

Many bacteria divide by binary fission, producing two identical daughter cells, which requires proper placement of the division machinery at mid-cell. Spherical bacteria (cocci) face unique challenges due to their lack of natural polarity. In this review, we compile current knowledge on how cocci regulate cell division, how they select the proper division plane, and ensure accurate Z-ring positioning at mid-cell. While Streptococcus pneumoniae and Staphylococcus aureus are the most well-studied models for cell division in cocci, we also cover other less-characterized cocci across different bacterial groups and discuss the conservation of known Z-ring positioning mechanisms in these understudied bacteria.

Research on bacterial cell division has traditionally focused on rod-shaped model organisms, such as Escherichia coli and Bacillus subtilis. While these models have been important in uncovering broadly conserved factors involved in bacterial cell division, the assortment of bacterial shapes, cell wall structures, and lifestyles highlights the need to broaden the scope of study. This includes not only understanding how conserved mechanisms are adapted to diverse cellular morphologies but also discovering mechanisms that arise as specific adaptations to unique cellular shapes. In this context, alternative models such as Staphylococcus aureus are emerging to provide insight into how Gram-positive cocci overcome the challenge of lacking obvious cellular polarity to ensure accurate placement of the division septum and undergo binary fission. In this review, we highlight recent research that reveals how S. aureus performs several distinct but interrelated processes, including peptidoglycan assembly, placement of the cell division septum, and how the division septum can be used as a hub for modifying the peptidoglycan to decorate the cell surface of S. aureus.

RNA therapy, which includes delivery of mRNA or siRNA, shows promise for treating various diseases, but difficulties in targeting specific cell types and low efficiency of loading RNA into nanoparticles remain hurdles to achieving widespread use. Previously, we reported the assembly of biocompatible synthetic bacterial spore-like particles, termed “SSHELs”, which are built atop a porous silica core encased in a lipid bilayer and two bacterial proteins that form a stable proteinaceous surface that may be covalently modified with targeting proteins of interest. Here, we employ micron-scale SSHELs constructed using a fusogenic lipid and decorated with affibodies targeting cell surface HER2 to specifically deliver model mRNA and siRNA molecules specifically to HER2-positive ovarian and breast cancer cells, with high RNA loading efficiency and cargo capacity. SSHEL particles therefore represent a versatile vehicle for the delivery of not only small molecules, but also therapeutic RNA to specific cell types.

Developmental processes are carefully regulated programs that are present in multiple kingdoms of life and that generally result in cell differentiation and specialization. This regulation can be mediated in part by checkpoints that monitor the progression of development to ensure that earlier steps occur successfully before later steps are initiated. Bacterial endospore formation (i.e., sporulation) is a well-studied developmental program that transforms a progenitor cell into a dormant cell type in response to environmental stress and that serves as a model for the discussion of checkpoint mechanisms used to monitor development. This review focuses on the checkpoints monitoring bacterial sporulation, with an emphasis on the model gram-positive bacterium Bacillus subtilis , to highlight general strategies that may be broadly conserved among disparate developmental programs.

Peptide-based therapeutic immunizations represent safe approaches to elicit antigen-specific T cell responses, but their broad utility remains limited due to poor immunogenicity and short in vivo stability due to rapid degradation and clearance. Here, we employed synthetic bacterial spore-like particles, "SSHELs" (Synthetic Spore Husk-Encased Lipid), made entirely of biocompatible materials, to deliver a model peptide antigen in the absence of additional adjuvants. SSHELs carrying the peptide antigen were internalized by dendritic cells, and SSHEL-delivered peptides were then processed and cross-presented in vitro and in vivo more efficiently than free peptides. Furthermore, SSHEL-delivered peptides elicited effective antigen-specific T cell expansion in a manner that was dependent on particle size and peptide presentation mode (encased peptides were superior to surface-attached peptides). In a mouse melanoma model expressing the antigen ovalbumin, therapeutic immunization reduced tumor size and increased survival. We propose that SSHELs are a self-adjuvanting peptide delivery system that mimics a natural presentation to elicit a robust immune response.IMPORTANCEEffective delivery of antigens to the immune system is essential for activating the adaptive immune system. Synthetic Spore Husk-Encased Lipids (SSHELs) are synthetic bacterial spore-like particles, where the proteinaceous polymerized surface layer of Bacillus subtilis spores is partially reconstituted around a porous silica bead encased in a membrane. The protein surface allows easy covalent modification of the SSHEL surface, and the porous core permits high-capacity cargo loading. Here, we demonstrate that SSHELs act as a self-adjuvanting delivery system that enhances antigen uptake, processing, and MHC-I cross-presentation by dendritic cells. Importantly, we show that both particle size and antigen localization on or within the SSHEL particle profoundly influence the efficiency of T cell priming. These results establish SSHELs as a modular platform for the delivery of peptide antigens.

The bacterial pathogen, Staphylococcus aureus, grows by dividing in two alternating orthogonal planes. How these cell division planes are positioned correctly is not known. Here we used chemical genetic screening to identify PcdA as a division plane placement factor. Molecular biology and imaging approaches revealed non-orthogonal division plane selection for pcdA mutant bacteria. PcdA is a structurally and functionally altered member of the McrB AAA+ NTPase family, which are often found as restriction enzyme subunits. PcdA interacts with the tubulin-like divisome component, FtsZ, and the structural protein, DivIVA; it also localizes to future cell division sites. PcdA multimerization, localization and function are NTPase activity-dependent. We propose that the DivIVA/PcdA complex recruits unpolymerized FtsZ to assemble along the proper cell division plane. Although pcdA deletion did not affect S. aureus growth in several laboratory conditions, its clustered growth pattern was disrupted, sensitivity to cell-wall-targeting antibiotics increased and virulence in mice decreased. We propose that the characteristic clustered growth pattern of S. aureus, which emerges from dividing in alternating orthogonal division planes, might protect the bacterium from host defences. PcdA interacts with DivIVA and FtsZ, promoting Z-ring formation and division plane selection in Staphylococcus aureus, which increases virulence in mice and reduces sensitivity to cell-wall-targeting antibiotics.

Most bacteria lack membrane-enclosed organelles and rely on macromolecular scaffolds at different subcellular locations to recruit proteins for specific functions. Here, we demonstrate that the optogenetic CRY2-CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations with varying efficiencies in live Escherichia coli cells, including the nucleoid, the cell pole, the membrane, and the midcell division plane. Such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. We further show that CRY2-CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system. Finally, we test this optogenetic system in three additional bacterial species, Bacillus subtilis, Caulobacter crescentus, and Streptococcus pneumoniae, providing important considerations for this system's applicability in bacterial cell biology.

Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation nonuniformly to secure against the possibility that favorable growth conditions, which put 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 containing the proteins ShfA (YabQ) and ShfP (YvnB) that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early use a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay nonsporulating 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.

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 sporulation-specific, forespore-localized putative chaperone from a distinct higher-order clade of AAA+ ATPases that promotes the peptidoglycan glycosyltransferase activity of MurG during sporulation, even though MurG does not normally require activation during vegetative growth. MurG redeploys to the forespore surface during sporulation, where we show that the local pH is reduced and propose that this change in cytosolic nanoenvironment abrogates MurG function. Further, we show that SpoVK participates in a developmental checkpoint in which improper spore surface assembly mis-localizes SpoVK, which leads to sporulation arrest. The AAA+ ATPase clade containing SpoVK includes specialized chaperones involved in secretion, cell envelope biosynthesis, and carbohydrate metabolism, suggesting that such fine-tuning might be a widespread feature of different subcellular nanoenvironments.

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 σ F is exclusively activated in the smaller daughter cell. Compartment-specific activation of σ F 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 σ F 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.

Micro- and nanoparticles are often designed by mimicking naturally occurring structures. Bacterial spores are dormant cells elaborated by some Gram-positive bacteria during poor growth conditions to protect their genetic material from harsh environmental stresses. In Bacillus subtilis, this protection is, in part, conferred by a proteinaceous shell, the “coat”, which is composed of ~80 different proteins. The basement layer of the coat contains two unusual proteins, which we have recently reconstituted around silica beads to generate synthetic spore-like particles termed “SSHELs”. Here, we describe the protocol for generating SSHEL particles, and describe the procedure to covalently link molecules of interest (in this case an anti-HER2 affibody) to SSHEL surfaces. SSHELs therefore represent a versatile platform for the display of ligands or antigens for the site-specific delivery of cargo or vaccines.

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.

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.

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. SSHELs are synthetic spore-like particles wherein the spore’s cell surface is partially reconstituted around 1 μm-diameter silica beads. Via a unique cysteine engineered protein, the surface of SSHELs may be covalently decorated to display HER2 (SSHELs αHER2). SKOV3 and SKBR3 cells’ targeting by SSHELs αHER2was evaluated by flow cytometry and confocal microscopy. SSHELs αHER2specifically bound to target cells and were internalized proportionally to their concentration. Subsequently, SSHELs were successfully loaded with doxorubicin (Dox-SSHELs αHER2). Dox-SSHELs αHER2capabilities to kill tumor cells was evaluated by flow cytometry and caspase assay. HER2 negative cells were used as a control. Dox-SSHELs αHER2’s efficacy was tested in vivo using athymic nude mice injected with SKOV3 ovarian cancer cells and then treated for up to 40 days. Dox-SSHELs αHER2can reduce SKOV3 tumor growth up to 75% when compared to the free drug. Further, when compared to liposomal doxorubicin (Doxil TM) Dox-SSHELs αHER2showed similar efficacy in reducing tumor growth, but without any of the typical side effects correlated with Doxil TMinjections. With a facile, reproducible manufacturing process that allows for straightforward targeting towards specific cell types, and lower overall toxicity, we suggest that SSHELs may represent a versatile strategy opening new research avenues for targeted drug delivery treatment or immune system stimulation through vaccination.

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.