About

Work in the Rudner lab focuses on fundamental questions in bacterial cell biology and development: How is information transduced across lipid bilayers? How are replicated chromosomes organized and segregated? How is the cell envelope remodeled during growth and differentiation? We address these questions in the bacterium <i>Bacillus subtilis</i>, in many cases, taking advantage of the developmental process of spore formation in this organism. Very recently, we have launched a new project in collaboration with Tom Bernhardt's lab focused on cell envelope biogenesis in two Gram-positive pathogens: <i>Staphylococcus aureus</i> and <i>Streptococcus pneumoniae</i>.

Research topics

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

Selected publications

• A potential role for acyl-phosphate in the coordination of phospholipid and lipopolysaccharide synthesis in <i>Escherichia coli</i>

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-03-14

  articleOpen accessSenior authorCorresponding

  ABSTRACT The envelope of Gram-negative bacteria like Escherichia coli is multilayered with two membranes sandwiching a peptidoglycan cell wall. The inner membrane is a typical phospholipid bilayer whereas the outer membrane is asymmetric with phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. We recently discovered that inactivation of the conserved peptidoglycan synthesis machinery responsible for cell elongation causes defects in both peptidoglycan and LPS synthesis in E. coli . This finding suggests that the isolation of suppressors that rescue the growth phenotype caused by an impaired cell elongation system is an attractive means of identifying factors involved in coordinating the biogenesis of different envelope layers. Here, we report the results of a global, transposon sequencing-based screen for such suppressors. The inactivation of a number of factors including the phospholipid synthesis enzyme PlsX was found to partially suppress the growth defects of a cell elongation mutant. Deletion of plsX also conferred increased resistance to CHIR-090, an inhibitor of the committed step of LPS synthesis catalyzed by LpxC, suggesting that loss of PlsX function stimulates LPS synthesis. Evidence is presented that increased CHIR-090 resistance is not mediated by changes in the activity of the proteolytic system (YejM-LapB-FtsH) controlling LpxC turnover. Rather, our results are consistent with a model in which the phospholipid precursor acyl-phosphate produced by PlsX serves as an inhibitor of LpxC to lower the rate of LPS synthesis when phospholipid synthesis capacity is reduced. IMPORTANCE Over the last several decades, most proteins essential for Gram-negative cell surface assembly have been characterized. However, relatively little is known about how the synthesis of different envelope layers is coordinated to promote uniform surface growth. Here, we report the results of a transposon sequencing-based genetic screen for mutants that suppress defects in the conserved peptidoglycan synthesis machinery responsible for cell elongation. Inactivation of the plsX gene encoding a phospholipid synthesis enzyme was found to both suppress the growth defect of a cell elongation mutant and to confer elevated resistance to an inhibitor of lipopolysaccharide synthesis. Our results suggest the attractive possibility that the product of PlsX, acyl-phosphate, may play a regulatory role in coordinating the phospholipid and lipopolysaccharide synthesis pathways.

  PublisherDOI

• Using fluorescently labeled wheat germ agglutinin to track lipopolysaccharide transport to the outer membrane in <i>Escherichia coli</i>

  mBio · 2025-02-24 · 10 citations

  articleOpen accessSenior author

  ABSTRACT The cell envelope of gram-negative bacteria consists of two membranes sandwiching the peptidoglycan (PG) cell wall. The outer membrane (OM) contains integrated beta-barrel proteins and has an outer leaflet composed of lipopolysaccharide (LPS). LPS is transported from the inner membrane where it is made to the OM surface by the Lpt system. In the polarly elongating alpha-proteobacterium Brucella abortus , LPS transport has been localized to the polar growth zone and division site. However, LPS transport has not been tracked in live proteobacteria like Escherichia coli that elongate by dispersed incorporation of envelope material along their cell body. Here, we report an investigation into the binding target of fluorescently labeled wheat germ agglutinin (FL-WGA) on E. coli cells that led to the development of a method for visualizing LPS transport. We show that instead of PG or enterobacterial common antigen for which FL-WGA labeling has been used to detect in the past, this probe recognizes LPS modified with a terminal N-acetylglucosamine formed by the defective O-antigen synthesis pathway of laboratory strains of E. coli . This finding enabled the construction of mutants inducible for LPS modification that were used together with FL-WGA labeling to track LPS transport to the cell surface. We show that new LPS is inserted throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously for PG synthesis and OM protein insertion in E. coli , suggesting that LPS transport to the OM is coordinated with these processes. IMPORTANCE Gram-negative bacteria like Escherichia coli are surrounded by a multilayered cell envelope that includes an outer membrane (OM) responsible for their high intrinsic resistance to antibiotics. The outer leaflet of this membrane is composed of a glycolipid called lipopolysaccharide (LPS). Here, we report the development of an imaging method to track the transport of LPS to the E. coli outer membrane. The results indicate that transport occurs throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously when cell wall synthesis and the insertion of proteins into the OM were tracked. Our results therefore suggest that LPS transport to the OM is coordinated with other essential processes that underly gram-negative cell envelope biogenesis.

  PublisherDOI

• The aPBP-type cell wall synthase PBP1b plays a specialized role in fortifying the <i>Escherichia coli</i> division site against osmotic rupture

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-04-02 · 9 citations

  preprintOpen accessSenior author

  A multi-protein system called the divisome promotes bacterial division. This apparatus synthesizes the peptidoglycan (PG) cell wall layer that forms the daughter cell poles and protects them from osmotic lysis. In the model Gram-negative bacterium Escherichia coli , PG synthases called class A penicillin-binding proteins (aPBPs) have been proposed to play crucial roles in division. However, there is limited experimental support for aPBPs playing a specialized role in division that is distinct from their general function in the expansion and fortification of the PG matrix. Here, we present in situ cryogenic electron tomography data indicating that the aPBP-type enzyme PBP1b is required to produce a wedge-like density of PG at the division site. Furthermore, atomic force and live cell microscopy showed that loss of this structure weakens the division site and renders it susceptible to lysis. Surprisingly, we found that the lipoprotein activator LpoB needed to promote the general function of PBP1b was not required for normal division site architecture or its integrity. Additionally, we show that of the two PBP1b isoforms produced in cells, it is the one with an extended cytoplasmic N-terminus that functions in division, likely via recruitment by the FtsA component of the divisome. Altogether, our results demonstrate that PBP1b plays a specialized, LpoB-independent role in E. coli cell division involving the biogenesis of a PG structure that prevents osmotic rupture. The conservation of aPBPs with extended cytoplasmic N-termini suggests that other Gram-negative bacteria may use similar mechanisms to reinforce their division site.

  PublisherDOI

• A load-bearing function for the cytoplasmic membrane of <i>Escherichia coli</i>

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-03

  preprintOpen access

  Abstract The structural integrity of bacterial cells is traditionally attributed to the peptidoglycan cell wall, and more recently to the outer membrane, with the cytoplasmic membrane assumed to be mechanically passive. Cells lacking filaments of the actin homolog MreB are more bendable, suggesting a role for the cytoskeleton in cell stiffness. Here, we show that MreB does not stiffen the envelope directly, but instead mechanically couples the cell wall to the cytoplasmic membrane through its role in peptidoglycan synthesis, increasing resistance to bending. Under hyperosmotic stress, MreB relocalized to the poles, forming linkages that prevent membrane detachment from the cell wall and attenuate cytoplasmic contraction. Disruption of MreB filament formation, nutrient starvation, or inactivation of glycan elongation factors abolished or reduced this coupling, revealing that peptidoglycan biosynthesis actively mediates stress distribution across surface layers. Our findings redefine the bacterial envelope as a mechanically integrated composite, with the cytoplasmic membrane having substantial load-bearing capacity.

  PublisherOA PDFDOI

• The hit-and-run of cell wall synthesis: LpoB transiently binds and activates PBP1b through a conserved allosteric switch

  Nature Communications · 2025-07-21 · 4 citations

  articleOpen access

  The peptidoglycan (PG) cell wall is the primary protective layer of bacteria, making the process of PG synthesis a key antibiotic target. Class A penicillin-binding proteins (aPBPs) are a family of conserved and ubiquitous PG synthases that fortify and repair the PG matrix. In gram-negative bacteria, these enzymes are regulated by outer-membrane tethered lipoproteins. However, the molecular mechanism by which lipoproteins coordinate the spatial recruitment and enzymatic activation of aPBPs remains unclear. Here we use single-molecule FRET and single-particle tracking in E. coli to show that a prototypical lipoprotein activator LpoB triggers site-specific PG synthesis by PBP1b through conformational rearrangements. Once synthesis is initiated, LpoB affinity for PBP1b dramatically decreases and it dissociates from the synthesizing enzyme. Our results suggest that transient allosteric coupling between PBP1b and LpoB directs PG synthesis to areas of low peptidoglycan density, while simultaneously facilitating efficient lipoprotein redistribution to other sites in need of fortification. Class A PBPs accomplish cell wall synthesis to enable bacterial growth and are subject to inhibition by penicillin-type antibiotics. Here, authors leverage single-molecule and bioengineering approaches to show how these essential enzymes are regulated by cognate lipoprotein cofactors.

  PublisherOA PDFDOI

• The mycomembrane proteins PorH and ProtX are inserted at polar growth zones and linked to the cell wall

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-14

  preprintOpen accessSenior authorCorresponding

  ABSTRACT The Mycobacteriales order of bacteria includes important pathogens such as Mycobacterium tuberculosis . These organisms are surrounded by a unique cell envelope architecture that includes a two-layered cell wall composed of peptidoglycan (PG) and arabinogalactan. They also build an outer membrane called the mycomembrane that is made of mycolic acids. Mycolate outer membrane proteins (MOMPs) reside within the mycomembrane and a subset are thought to form pores that allow essential nutrients to permeate the envelope. However, little is known about the structure of these proteins or the mechanism by which they are assembled. Here, we investigate MOMP assembly in the model organism Corynebacterium glutamicum ( Cglu ) using PorH as a model MOMP. PorH is encoded in an operon with the MOMP PorA, and the two small, alpha-helical proteins have been proposed to form hetero-oligomeric pores in the mycomembrane. Consistent with this proposal, AlphaFold2 predicts a high confidence structure of a hetero-oligomeric pore formed by five copies each of PorH and its partner PorA, and we show that PorA is required for the surface assembly of PorH. Using a fluorescence assay for detection of surface-exposed PorH or another MOMP called ProtX, we found that MOMP assembly occurs within zones of active PG synthesis at the cell poles. We also discovered that PorH and ProtX are linked to the cell wall. Thus, like Gram-negative bacteria, Cglu and potentially other members of Mycobacteriales order, coordinate outer membrane protein assembly with PG biogenesis and use proteins to connect the mycomembrane and the cell wall. SIGNIFICANCE Diderm bacteria in the Mycobacteriales order have a distinctive outer layer called the mycomembrane. Proteins that reside within the mycomembrane play critical roles in virulence and cell viability. However, how proteins are assembled into the mycomembrane has remained an outstanding question in the field. Here, we investigate the biogenesis of mycomembrane proteins in the model organism Corynebacterium glutamicum . We show that these proteins are inserted into the mycomembrane in a manner that correlates with polar growth and are attached to the cell wall. Many features of these mycomembrane proteins are shared between species in the Mycobacteriales, suggesting that our findings may be conserved in other species within this order.

  PublisherOA PDFDOI

• Intercepting a Mycobacterial Biosynthetic Pathway with Covalent Labeling

  Journal of the American Chemical Society · 2025-03-24 · 2 citations

  articleOpen access

  The mycobacterial cell envelope plays both infectious and protective roles. Understanding its structure is crucial for unlocking the molecular basis underlying these functions. Studying glycans, the primary components of the cell envelope, is challenging due to their limited native functional handles for chemoselective modification. New labeling methods exploit biorthogonal chemistry, using small molecule mimics that intercept cellular metabolism or late-stage glycan biosynthesis. However, these strategies can have practical limitations, including probe delivery and effectiveness. An ideal small molecule probe should be easily deployed and exploit the critical enzyme–substrate relationships of natural substrates. To this end, we developed a “probegenic” strategy to label mycobacteria. Our approach eliminates the need for explicit substrate mimicry, as the relevant functionality is revealed by a target enzyme. Specifically, we synthesized an azide-substituted trans-β-lactone probe (AzLac), which adopts a substrate-like structure upon covalent enzyme labeling. This probe is incorporated by mycolyltransferases into a core mycobacterial cell envelope glycan, including in the pathogen Mycobacterium tuberculosis. Unlike other probes of the cell envelope, AzLac facilitates selective covalent labeling of the inner leaflet of the mycomembrane. Using Corynebacterium glutamicum mycolyltransferase deletion strains, we implicated Cmt2 as the primary mycolyltransferase target. We leveraged the ability to modify the cell envelope by demonstrating that AzLac could be used to attach a DNA barcode to mycobacteria, which would help track infection dynamics. Thus, we expect AzLac will be a valuable means of monitoring and tracking the mycobacterial cell envelope. Moreover, we anticipate masking and revealing recognition motifs in probes can be applied to diverse cellular targets.

  PublisherOA PDFDOI

• Secretin-interacting plug proteins prevent antibiotic influx during type IV pilus assembly in Pseudomonas aeruginosa

  Research Square · 2025-12-01

  preprintOpen accessSenior author
  PublisherOA PDFDOI

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

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

  articleOpen access

  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

• The mycomembrane

  Current Biology · 2025-02-01 · 2 citations

  articleSenior author
  PublisherDOI

Recent grants

• Targeting cell separation systems of gram-negative bacteria.

  NIH · $462k · 2014–2016

• NIH Grant R01AI099144

  NIH · $4.3M · 2019

• Innovative Platforms for Antimicrobial Therapy and Vaccine Development

  NIH · $50.4M · 2014–2019

• Envelope biogenesis in Escherichia coli and Pseudomonas aeruginosa

  NIH · $8.3M · 2010–2029

• Targeting cell separation systems of gram-negative bacteria.

  NIH · $1.5M · 2014–2020

Frequent coauthors

• Suzanne Walker

  Harvard University

  40 shared

• David Z. Rudner

  Harvard University

  40 shared

• Elayne M. Fivenson

  Harvard University

  40 shared

• Andrew C. Kruse

  Boston VA Research Institute

  32 shared

• Patricia D. A. Rohs

  Boston VA Research Institute

  31 shared

• Irina Shlosman

  Harvard University

  25 shared

• Andrea Vettiger

  23 shared

• Tyler A. Sisley

  Boston VA Research Institute

  22 shared

Labs

• Thomas Bernhardt LabPI

  Current members of the Thomas Bernhardt Lab at Harvard Medical School and Howard Hughes Medical Institute.