About

Professor David Rudner is a researcher at Harvard Medical School, leading the Rudner Lab within the Department of Microbiology. His work focuses on fundamental questions in bacterial cell biology and development, specifically investigating how information is transduced across lipid bilayers, how replicated chromosomes are organized and segregated, and how the cell envelope is remodeled during growth and differentiation. His research primarily utilizes the bacterium Bacillus subtilis, often leveraging the developmental process of spore formation in this organism to address these questions. Recently, Professor Rudner has initiated a new project in collaboration with Tom Bernhardt's lab, which concentrates on cell envelope biogenesis in two Gram-positive pathogens: Staphylococcus aureus and Streptococcus pneumoniae. His research aims to deepen understanding of bacterial cell processes, contributing to the broader field of microbiology and bacterial cell biology.

Research topics

• Cell biology
• Biology
• Genetics
• Chemistry
• Organic chemistry
• Biochemistry

Selected publications

• A tetrameric SpoVA2 membrane complex is required for DPA transport into <i>Bacillus anthracis</i> spores

  mBio · 2026-02-26

  articleOpen accessSenior author

  ABSTRACT The small molecule dipicolinic acid (DPA) plays a critical role in bacterial spore resistance during dormancy and in the exit from quiescence during germination. In Bacillus subtilis , the spoVA locus is required for DPA import into the developing spore and has been implicated in its release during germination. The SpoVAC (C) and SpoVAEb (Eb) proteins form a membrane complex through which DPA transits, and the SpoVAD (D) protein binds the cytoplasmic face of the complex, resembling a plug. The human pathogen Bacillus anthracis and other members of the Bacillus cereus group have two spoVA loci. The spoVA1 operon resembles B. subtilis spoVA , while the spoVA2 locus is more distantly related and only shares the three core genes C2 , D2 , and Eb2 . Here, we show that spoVA2 is critical for DPA import and spore heat resistance, while either locus is sufficient for DPA export during germination. We report that a fourth protein (called NJ2) encoded in the spoVA2 locus is part of the C2/D2/Eb2 complex and is predicted to bind the extracytoplasmic face of the membrane complex, resembling a cap. We show that NJ2 is important for DPA import during B. anthracis sporulation and essential in B. subtilis engineered to express spoVA2 in place of its native spoVA locus. Finally, we identified an Eb2 mutant that bypasses the requirement for NJ2 in DPA import and show that the mutation impairs DPA export, providing additional support for a direct role of SpoVA complexes in the release of DPA during germination. IMPORTANCE Spore resistance and exit from dormancy during germination are central to the transmission and pathogenesis of endospore-forming pathogens like Bacillus anthracis . Our understanding of the molecular mechanisms underlying these processes has principally been informed by studies in the non-pathogenic model Bacillus subtilis . Here, we identify and characterize a membrane complex in B. anthracis that is critical for spore resistance and spore germination that is absent from B. subtilis . We show that this complex is required for the accumulation of dipicolinic acid in the spore core during sporulation and functions in its release from the core during germination. A deeper understanding of the molecular mechanisms of B. anthracis sporulation and germination will facilitate the development of strategies for more effective disease prevention and treatment.

  PublisherDOI

• Microbial GAIN domains undergo autoproteolysis and enable release of diverse cell surface associated proteins

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-05-13

  articleSenior author

  ABSTRACT Adhesion G Protein-Coupled Receptors (aGPCRs) transduce mechanical stimuli across the cytoplasmic membrane in eukaryotes. These receptors contain extracellular G PCR A utoproteolysis IN ducing (GAIN) domains that undergo autoproteolysis but maintain stable association of their cleavage products. A diverse set of adhesion domains appended to the GAIN domain bind surface ligands on neighboring cells or the extracellular matrix. Shear force is thought to disrupt the interaction between the cleavage products exposing a tethered agonist that triggers GPCR signaling. Here, we report that GAIN domains are broadly conserved among bacteria and archaea. The microbial domains lack strong sequence conservation to their eukaryotic counterparts, but are predicted to adopt a similar fold. We demonstrate that these M icrobial A utoproteolysis IN ducing (MAIN) domains undergo autoproteolysis both in vitro and in vivo, using conserved catalytic residues. Furthermore, proteolysis occurs in a conserved β-turn that allows stable non-covalent interactions between the cleavage products. MAIN domains are tethered to the cell envelope of bacteria and archaea and are fused to diverse sets of adhesion and enzymatic domains. Many of the same adhesion domains are appended to both MAIN and GAIN domains, suggesting these protein families share a common origin and function. We propose that MAIN domains allow microbes to release proteins from their cell surface in response to shear force, enabling broader nutrient scavenging, intoxication of neighboring cells, and dispersal through surface detachment.

  PublisherDOI

• Identification of sporulation genes in Bacillus anthracis highlights similarities and significant differences with Bacillus subtilis

  PLoS Biology · 2025-12-12 · 2 citations

  articleOpen accessSenior author

  The molecular basis of endospore formation in the model gram-positive bacterium Bacillus subtilis has been investigated for over half a century. Here, using high throughput and classical genetic approaches, we performed a comparative analysis of sporulation in the human pathogen Bacillus anthracis. A transposon-sequencing screen identified >150 genes required for B. anthracis sporulation. As anticipated, many of the genes that are critical for sporulation in B. subtilis were also required for B. anthracis sporulation. However, we identified >50 genes that are important for sporulation in B. anthracis but not in B. subtilis, and 22 B. anthracis sporulation genes that are absent from the B. subtilis genome. To validate the hits from our screen, we generated an ordered transposon-mutant library using Knockout Sudoku. Cytological analysis of a subset of the canonical sporulation-defective mutants revealed similar but not identical phenotypes in the pathogen compared to the model. We investigated several of the newly identified sporulation genes, with an in-depth analysis of one, ORF 04167, renamed ipdA. Sporulating cells lacking ipdA are blocked in the morphological process of engulfment, generating septal bulges. An AlphaFold-Multimer screen and a classical genetic enrichment revealed that IpdA is a secreted inhibitor of the polysaccharide deacetylase PdaN. Our data support a model in which induction of IpdA at the onset of sporulation inhibits deacetylation of the cell wall peptidoglycan (PG), enabling the sporulation-specific PG hydrolases to catalyze engulfment. Altogether, our studies reveal that B. subtilis is an excellent model for endospore formation in B. anthracis, while underscoring the importance of direct analysis in B. anthracis. The suite of tools that we have generated will catalyze the molecular dissection of sporulation and other cell biological processes in this important human pathogen.

  PublisherDOI

• Cyclic-di-AMP modulates cellular turgor in response to defects in bacterial cell wall synthesis

  Nature Microbiology · 2025-06-17 · 10 citations

  articleSenior author
  PublisherDOI

• PgpP is a broadly conserved phosphatase required for phosphatidylglycerol lipid synthesis

  Proceedings of the National Academy of Sciences · 2025-01-27 · 2 citations

  articleOpen accessSenior author

  The cytoplasmic membrane of bacteria is composed of a phospholipid bilayer made up of a diverse set of lipids. Phosphatidylglycerol (PG) is one of the principal constituents and its production is essential for growth in many bacteria. All the enzymes required for PG biogenesis in Escherichia coli have been identified and characterized decades ago. However, it has remained poorly understood how gram-positive bacteria perform the terminal step in the pathway that produces this essential lipid. In E. coli, this reaction is mediated by three functionally redundant phosphatases that convert phosphatidylglycerophosphate (PGP) into PG. Here, we show that homologs of these enzymes in Bacillus subtilis are not required for PG synthesis. Instead, we identified a previously uncharacterized B. subtilis protein, YqeG (renamed PgpP), as an essential enzyme required for the conversion of PGP into PG. Expression of B. subtilis or Staphylococcus aureus PgpP in E. coli lacking all three Pgp enzymes supported the growth of the strain. Furthermore, depletion of PgpP in B. subtilis led to growth arrest, reduced membrane lipid staining, and accumulation of PGP. PgpP is broadly conserved among Firmicutes and Cyanobacteria. Homologs are also present in yeast mitochondria and plant chloroplasts, suggesting that this widely distributed enzyme has an ancient origin. Finally, evidence suggests that PgpP homologs are essential in many gram-positive pathogens and thus the enzyme represents an attractive target for antibiotic development.

  PublisherOA PDFDOI

• Two new enzymes that liberate undecaprenyl-phosphate to replenish the carrier lipid pool during envelope stress

  mBio · 2025-01-29 · 5 citations

  articleOpen accessSenior author

  ABSTRACT The 55-carbon isoprenoid, undecaprenyl-phosphate (UndP), is a universal carrier lipid that ferries most glycans and glycopolymers across the cytoplasmic membrane in bacteria. In addition to peptidoglycan precursors, UndP transports O-antigen, capsule, wall teichoic acids, and sugar modifications. How this shared but limited lipid is distributed among competing pathways is just beginning to be elucidated. We recently reported that in the bacterium Bacillus subtilis , the stress-response sigma factor SigM and its cognate anti-sigma factor complex respond to changes in the free UndP pool. When levels are low, SigM activates genes that increase flux through the essential cell wall synthesis pathway, promote the recycling of the lipid carrier, and liberate the carrier from other polymer pathways. Here, we report that two additional enzymes under SigM control help maintain the free pool of UndP. One, UshA (YqjL), resembles alpha-beta hydrolases and liberates UndP from undecaprenyl-monophosphate-linked sugars. The other, UpsH (YpbG), resembles metallophosphoesterases and releases UndP from undecaprenyl-diphosphate-linked wall teichoic acids polymers but not lipid-linked peptidoglycan precursors. UshA becomes critical for growth when UndP-linked sugars are sequestered, and the carrier lipid pool is depleted. Similarly, UpsH becomes essential for viability when UndPP-linked intermediates accumulate. Mutations in the predicted catalytic residues of both putative hydrolases abrogate their function arguing that they act directly to release UndP. These findings define two new enzymes that liberate the carrier lipid from UndP- and UndPP-linked intermediates and bolster the model that the SigM stress-response pathway maintains the UndP pool and prioritizes its use for peptidoglycan synthesis. IMPORTANCE Motivated by the success of naturally occurring glycopeptide antibiotics like vancomycin, one arm of recent antibiotic discovery efforts has focused on compounds that bind lipid-linked precursors used to build extracytoplasmic polymers. Trapping these precursors depletes the universal carrier lipid undecaprenyl-phosphate, which is required for the synthesis of virtually all surface polymers, including peptidoglycan. Understanding how cells respond to this stress to restore the carrier lipid pool is critical to identifying effective drugs. Here, we report the identification of two new enzymes that are produced in response to the depletion of the carrier lipid pool. These enzymes recover the carrier lipid but cleave distinct lipid-linked precursors to do so.

  PublisherDOI

• Identification and characterization of the <i>Bacillus subtilis</i> spore germination protein GerY

  Journal of Bacteriology · 2024-11-12 · 3 citations

  articleOpen accessSenior author

  ABSTRACT In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth when nutrients become available. To identify uncharacterized factors involved in the exit from dormancy, we performed a transposon-sequencing screen taking advantage of the loss of spore heat resistance that accompanies germination. We reasoned that transposon insertions that impair but do not block germination will lose resistance more slowly than wild type after exposure to nutrients and will therefore survive heat treatment. Using this approach, we identified most of the known germination genes and several new ones. We report an initial characterization of 15 of these genes and a more detailed analysis of one ( ymaF ). Spores lacking ymaF (renamed gerY ) are impaired in germination in response to both L-alanine and L-asparagine, D-glucose, D-fructose, and K + . GerY is a soluble protein synthesized under σ E control in the mother cell. A YFP-GerY fusion localizes around the developing and mature spore in a manner that depends on CotE and SafA, indicating that it is a component of the spore coat. Coat proteins encoded by the gerP operon and gerT are also required for efficient germination, and we show that spores lacking two or all three of these loci have more severe defects in the exit from dormancy. Our data are consistent with a model in which GerY, GerT, and the GerP proteins are required for efficient transit of nutrients through the coat to access the germination receptors, but each acts independently in this process. IMPORTANCE Pathogens in the orders Bacillales and Clostridiales resist sterilization by differentiating into stress-resistant spores. Spores are metabolically inactive and can remain dormant for decades, yet upon exposure to nutrients, they rapidly resume growth, causing food spoilage, food-borne illness, or life-threatening disease. The exit from dormancy, called germination, is a key target in combating these important pathogens. Here, we report a high-throughput genetic screen using transposon sequencing to identify novel germination factors that ensure the efficient exit from dormancy. We identify several new factors and characterize one in greater detail. This factor, renamed GerY, is part of the proteinaceous coat that encapsulates the dormant spore. Our data suggest that GerY enables efficient transit of nutrients through the coat to trigger germination.

  PublisherOA PDFDOI

• SpoVAF and FigP assemble into oligomeric ion channels that enhance spore germination

  Genes & Development · 2024-01-01 · 16 citations

  articleOpen accessSenior author

  Bacterial spores can remain dormant for decades yet rapidly germinate and resume growth in response to nutrients. GerA family receptors that sense and respond to these signals have recently been shown to oligomerize into nutrient-gated ion channels. Ion release initiates exit from dormancy. Here, we report that a distinct ion channel, composed of SpoVAF (5AF) and its newly discovered partner protein, YqhR (FigP), amplifies the response. At high germinant concentrations, 5AF/FigP accelerate germination; at low concentrations, this complex becomes critical for exit from dormancy. 5AF is homologous to the channel-forming subunit of GerA family receptors and is predicted to oligomerize around a central pore. 5AF mutations predicted to widen the channel cause constitutive germination during spore formation and membrane depolarization in vegetative cells. Narrow-channel mutants are impaired in germination. A screen for suppressors of a constitutively germinating 5AF mutant identified FigP as an essential cofactor of 5AF activity. We demonstrate that 5AF and FigP interact and colocalize with GerA family receptors in spores. Finally, we show that 5AF/FigP accelerate germination in B. subtilis spores that have nutrient receptors from another species. Our data support a model in which nutrient-triggered ion release by GerA family receptors activates 5AF/FigP ion release, amplifying the response to germinant signals.

  PublisherOA PDFDOI

• Rapid discovery and evolution of nanosensors containing fluorogenic amino acids

  Nature Communications · 2024-09-05 · 9 citations

  articleOpen access

  Binding-activated optical sensors are powerful tools for imaging, diagnostics, and biomolecular sensing. However, biosensor discovery is slow and requires tedious steps in rational design, screening, and characterization. Here we report on a platform that streamlines biosensor discovery and unlocks directed nanosensor evolution through genetically encodable fluorogenic amino acids (FgAAs). Building on the classical knowledge-based semisynthetic approach, we engineer ~15 kDa nanosensors that recognize specific proteins, peptides, and small molecules with up to 100-fold fluorescence increases and subsecond kinetics, allowing real-time and wash-free target sensing and live-cell bioimaging. An optimized genetic code expansion chemistry with FgAAs further enables rapid (~3 h) ribosomal nanosensor discovery via the cell-free translation of hundreds of candidates in parallel and directed nanosensor evolution with improved variant-specific sensitivities (up to ~250-fold) for SARS-CoV-2 antigens. Altogether, this platform could accelerate the discovery of fluorogenic nanosensors and pave the way to modify proteins with other non-standard functionalities for diverse applications.

  PublisherOA PDFDOI

• Bacillus subtilis uses the SigM signaling pathway to prioritize the use of its lipid carrier for cell wall synthesis

  PLoS Biology · 2024-04-29 · 10 citations

  articleOpen accessSenior author

  Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These lipid-linked precursors are then flipped across the membrane and polymerized or directly transferred to surface polymers, lipids, or proteins. Despite its essential role in envelope biogenesis, UndP is maintained at low levels in the cytoplasmic membrane. The mechanisms by which bacteria distribute this limited resource among competing pathways is currently unknown. Here, we report that the Bacillus subtilis transcription factor SigM and its membrane-anchored anti-sigma factor respond to UndP levels and prioritize its use for the synthesis of the only essential surface polymer, the cell wall. Antibiotics that target virtually every step in PG synthesis activate SigM-directed gene expression, confounding identification of the signal and the logic of this stress-response pathway. Through systematic analyses, we discovered 2 distinct responses to these antibiotics. Drugs that trap UndP, UndP-linked intermediates, or precursors trigger SigM release from the membrane in <2 min, rapidly activating transcription. By contrasts, antibiotics that inhibited cell wall synthesis without directly affecting UndP induce SigM more slowly. We show that activation in the latter case can be explained by the accumulation of UndP-linked wall teichoic acid precursors that cannot be transferred to the PG due to the block in its synthesis. Furthermore, we report that reduction in UndP synthesis rapidly induces SigM, while increasing UndP production can dampen the SigM response. Finally, we show that SigM becomes essential for viability when the availability of UndP is restricted. Altogether, our data support a model in which the SigM pathway functions to homeostatically control UndP usage. When UndP levels are sufficiently high, the anti-sigma factor complex holds SigM inactive. When levels of UndP are reduced, SigM activates genes that increase flux through the PG synthesis pathway, boost UndP recycling, and liberate the lipid carrier from nonessential surface polymer pathways. Analogous homeostatic pathways that prioritize UndP usage are likely to be common in bacteria.

  PublisherDOI

Recent grants

• NIH Grant S10RR027344

  NIH · $162k · 2011

• Cell surface biogenesis in Streptococcus pneumoniae

  NIH · $2.2M · 2019–2024

• NIH Grant R01GM073831

  NIH · $3.4M · 2016

• NIH Grant RC2GM092616

  NIH · $2.0M · 2012

• Cell Envelope Homeostasis in Bacillus subtilis

  NIH · $1.4M · 2019–2023

Frequent coauthors

• Thomas G. Bernhardt

  Howard Hughes Medical Institute

  40 shared

• Erdem Karatekin

  Yale University

  29 shared

• Thierry Doan

  Institut de Microbiologie de la Méditerranée

  28 shared

• Xindan Wang

  Indiana University Bloomington

  28 shared

• Christopher D. A. Rodrigues

  University of Warwick

  26 shared

• Debora S. Marks

  Center for Systems Biology

  17 shared

• Kelly P. Brock

  Harvard University

  16 shared

• Andrew C. Kruse

  Boston VA Research Institute

  16 shared

Labs

• Rudner LabPI

  Not provided

Education

• BA, Physics

  Oberlin College

• PhD, MCB

  UC Berkeley