Biology subjects

Ramamurthi, K. S.

Publications and source records attributed to Ramamurthi, K. S..

11 recordsLinked to original sources

bioRxiv2026

A genome-wide genetic interaction platform for MRSA reveals connections between cell division and the cell envelope

The bacterial cell cycle is an ensemble of integrated pathways that coordinates cell growth and division. Although these pathways contain a suite of promising antimicrobial targets, their functional organization and interrelationships have only been sparsely mapped. Here, we established a dual-CRISPRi platform for systematic genetic interaction profiling in methicillin-resistant Staphylococcus aureus (MRSA), a major drug-resistant pathogen. Our platform achieved genome-wide coverage across a selected set of 51 cell cycle genes, surveying 115,497 gene-pairs and uncovering hundreds of genetic interactions, both positive (suppressors) and negative (synthetic sick/lethal). These interactions identified two major connections between cell division and the cell envelope. First, we identified a suppressive relationship between a defined set of cell division genes, including components of the major septal peptidoglycan biosynthesis complex PBP1-FtsWL-DivIBC, and fatty acid or phospholipid biosynthesis. Simultaneous inhibition of these lipid biosynthesis pathways suppressed the fitness and morphological defects caused by knocking down expression of cell division genes, revealing that toxic membrane accumulation contributes to the lethality of disrupting cell division. Second, we uncovered a synthetic lethal relationship between a distinct set of cell division genes and the conserved dltXABCD operon, linking cell division to the modification of teichoic acid surface polymers. Our results establish a versatile platform for interrogating selected gene sets in MRSA, providing a map of cell cycle interactions, and uncovering links between cell division and cell envelope pathways.

microbiology↗

bioRxiv2026

A lipid cue drives the subcellular localization of a self-inserting bacterial transmembrane protein

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 II 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.

microbiology↗

bioRxiv2026

16S ribosomal RNA modification drives transcript-specific translation efficiency

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 that fine-tunes ribosomal function. We identified a loss-of-function allele in mraW that corrected a late-stage sporulation defect in Bacillus subtilis by bypassing a key sporulation checkpoint via altered translational regulation. Purified ribosomes isolated from {Delta}mraW cells displayed a [~]2-fold decrease in translation efficiency; in vivo, {Delta}mraW cells produced decreased levels of the sporulation checkpoint protein CmpA. This regulation was mediated by sequences from the 5 untranslated region and the coding sequence of cmpA, which form a step-loop structure that occlude early codons of the mRNA. Proteomic analysis revealed that MraW directly or indirectly regulates the production of multiple proteins, some of which form similar structural elements as the cmpA transcript. We propose that MraW modification of 16S rRNA enhances translation efficiency in general, and that specific transcripts, whose gene products are likely required in limiting quantities, have evolved structural features that act as a regulatory mechanism to govern protein levels. This type of regulation may be most apparent in bacteria which exhibit uncoupled transcription and translation. HIGHLIGHTSO_LIA conserved 16S rRNA modification enhances translation of structured mRNAs C_LIO_LIEarly mRNA stem-loops impose translational control dependent on ribosome modification C_LIO_LImRNA structure and rRNA modifications likely co-evolved to fine-tune protein dosage C_LI

microbiology↗

bioRxiv2026

Orthogonal cell division organizes surface virulence factors to drive staphylococcal abscess community formation

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. HIGHLIGHTSO_LIOrthogonal cell division promotes staphylococcal abscess community formation C_LIO_LILoss of PcdA disrupts fibrin pseudocapsule assembly in 3D models C_LIO_LIDivision geometry ensures uniform surface deployment of adhesins C_LIO_LICell division plane selection links bacterial cell cycle control to virulence C_LI

microbiology↗

bioRxiv2025

Immunization with peptide encapsulated within synthetic spores activates T cell responses and reduces tumor growth

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", 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. Further, 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.

cancer biology↗

bioRxiv2024

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.

microbiology↗

bioRxiv2023

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.

microbiology↗

bioRxiv2023

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.

microbiology↗

bioRxiv2023

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 {sigma}F is exclusively activated in the smaller daughter cell. Compartment specific activation of {sigma}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 {sigma}F 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.

microbiology↗

bioRxiv2022

Three-dimensional structured illumination microscopy with enhanced axial resolution

We present two distinct, complementary methods for improving axial resolution in three-dimensional structured illumination microscopy (3D SIM) with minimal or no modification to the optical system. First, we show that placing a mirror directly opposite the sample enables 4-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with [~]120 nm lateral and 160 nm axial resolution. Second, we develop an improved deep learning method that can be directly applied to 3D SIM data, obviating the need for additional hardware. This procedure results in [~]120 nm isotropic resolution and can be combined with denoising to facilitate volumetric imaging spanning dozens of time points. 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 rich cytoskeletal dynamics within T-cells in the early stages of immune synapse formation.

cell biology↗

bioRxiv2020

Cell-specific cargo delivery using synthetic bacterial spores

SSHELs are synthetic bacterial spore-like particles wherein the spores cell surface is partially reconstituted around 1 {micro}m-diameter silica beads coated with a lipid bilayer. Via a unique cysteine engineered in one of the surface proteins, the surface of SSHELs may be covalently decorated with molecules of interest. Here, we modified SSHELs with an affibody directed against HER2, a cell surface protein overexpressed in some breast and ovarian cancer cells, and loaded them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduced tumor growth with lower toxicity in a mouse tumor xenograft model compared to free drug by specifically binding to HER2-positive cancer cells. We show that SSHELs bound to target cells are taken up and trafficked to acidic compartments, whereupon the cargo is released in a pH-dependent manner. Finally, we demonstrate that SHELLs can clear small tumor lesions in a complex tumor microenvironment in a zebrafish model of brain metastasis. We propose that SSHELs represent a versatile strategy for targeted drug delivery.

synthetic biology↗