About

Thomas J. Silhavy is the Warner-Lambert Parke-Davis Professor of Molecular Biology at Princeton University. He is a bacterial geneticist known for his fundamental contributions to the fields of protein secretion, membrane biogenesis, and signal transduction. Using Escherichia coli as a model system, his lab was the first to isolate signal sequence mutations, identify a component of cellular protein secretion machinery, and characterize an integral membrane component of the outer membrane assembly machinery. His current research focuses on the mechanisms of outer membrane biogenesis and the regulatory systems that sense and respond to envelope stress, which trigger developmental pathways allowing cells to survive starvation. Silhavy has authored more than 275 research articles and three books, and his work has significantly advanced understanding of subcellular organization in Gram-negative bacteria, particularly the biogenesis of the outer membrane and its role as a permeability barrier. His contributions have earned him numerous honors, including election to the National Academy of Sciences, the American Academy of Arts and Sciences, and fellowships in the American Academy of Microbiology and the AAAS. He has received awards such as the NIH MERIT award, the Novitski Prize, and the ASM Lifetime Achievement Award, and is recognized for his dedication to teaching and mentoring.

Research topics

• Biology
• Cell biology
• Genetics
• Biochemistry
• Chemistry

Selected publications

• Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation

  Nature Communications · 2024-07-13 · 30 citations

  articleOpen access

  Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.

  PublisherOA PDFDOI

• OmpA controls order in the outer membrane and shares the mechanical load

  Proceedings of the National Academy of Sciences · 2024-12-04 · 28 citations

  articleOpen accessSenior authorCorresponding

  , affects virulence, adhesion, and bacterial OM integrity. However, despite more than 50 y of research, the molecular basis for the role of OmpA has remained elusive. In this study, we demonstrate that OmpA organizes the OM protein lattice and mechanically connects it to the cell wall (CW). Using gene fusions, atomic force microscopy, simulations, and microfluidics, we show that the β-barrel domain of OmpA is critical for maintaining the permeability barrier, but both the β-barrel and CW-binding domains are necessary to enhance the cell envelope's strength. OmpA integrates the compressive properties of the OM protein lattice with the tensile strength of the CW, forming a mechanically robust composite that increases overall integrity. This coupling likely underpins the ability of the entire envelope to function as a cohesive, resilient structure, critical for the survival of bacteria.

  PublisherOA PDFDOI

• Periplasmic Chaperones: Outer Membrane Biogenesis and Envelope Stress

  Annual Review of Microbiology · 2024-07-15 · 16 citations

  reviewOpen accessSenior author

  Envelope biogenesis and homeostasis in gram-negative bacteria are exceptionally intricate processes that require a multitude of periplasmic chaperones to ensure cellular survival. Remarkably, these chaperones perform diverse yet specialized functions entirely in the absence of external energy such as ATP, and as such have evolved sophisticated mechanisms by which their activities are regulated. In this article, we provide an overview of the predominant periplasmic chaperones that enable efficient outer membrane biogenesis and envelope homeostasis in Escherichia coli . We also discuss stress responses that act to combat unfolded protein stress within the cell envelope, highlighting the periplasmic chaperones involved and the mechanisms by which envelope homeostasis is restored.

  PublisherDOI

• A novel mechanism that maintains outer membrane lipid asymmetry in Pseudomonas aeruginosa

  2023-02-17 · 1 citations

  datasetOpen accessSenior author

  Newick file the PA2800 phylogenetic tree in "A novel mechanism that maintains outer membrane lipid asymmetry in Pseudomonas aeruginosa"

  PublisherDOI

• Antibiotics and hexagonal order in the bacterial outer membrane

  Nature Communications · 2023-08-09 · 11 citations

  letterOpen access
  PublisherOA PDFDOI

• Mechanism of outer membrane destabilization by global reduction of protein content

  bioRxiv (Cold Spring Harbor Laboratory) · 2023-02-20 · 1 citations

  preprintOpen accessSenior authorCorresponding

  Abstract The outer membrane (OM) of Gram-negative bacteria such as Escherichia coli is an asymmetric bilayer with the glycolipid lipopolysaccharide (LPS) in the outer leaflet and glycerophospholipids in the inner. Nearly all integral OM proteins (OMPs) have a characteristic β-barrel fold and are assembled in the OM by the BAM complex, which contains one essential β-barrel protein (BamA), one essential lipoprotein (BamD), and three non-essential lipoproteins (BamBCE). A gain-of-function mutation in bamA enables survival in the absence of BamD, showing that the essential function of this protein is regulatory. We demonstrate that the global reduction in OMPs caused by BamD loss weakens the OM, altering cell shape and causing OM rupture in spent medium. To fill the void created by OMP loss, PLs flip into the outer leaflet. Under these conditions, mechanisms that remove PLs from the outer leaflet create tension between the OM leaflets, which contributes to membrane rupture. Rupture is prevented by suppressor mutations that release the tension by halting PL removal from the outer leaflet. However, these suppressors do not restore OM stiffness or normal cell shape, revealing a possible connection between OM stiffness and cell shape. Significance Statement The outer membrane (OM) is a selective permeability barrier that contributes to the intrinsic antibiotic resistance of Gram-negative bacteria. Biophysical characterization of the roles of the component proteins, lipopolysaccharides, and phospholipids is limited by both the essentiality of the OM and its asymmetrical organization. In this study, we dramatically change OM physiology by limiting the protein content, which requires phospholipid localization to the outer leaflet and thus disrupts OM asymmetry. By characterizing the perturbed OM of various mutants, we provide novel insight into the links among OM composition, OM stiffness, and cell shape regulation. These findings deepen our understanding of bacterial cell envelope biology and provide a platform for further interrogation of OM properties.

  PublisherOA PDFDOI

• Trade-offs constrain adaptive pathways to type VI secretion system survival

  iScience · 2023-10-26 · 13 citations

  articleOpen access

  The Type VI Secretion System (T6SS) is a nano-harpoon used by many bacteria to inject toxins into neighboring cells. While much is understood about mechanisms of T6SS-mediated toxicity, less is known about the ways that competitors can defend themselves against this attack, especially in the absence of their own T6SS. Here we subjected eight replicate populations of Escherichia coli to T6SS attack by Vibrio cholerae. Over ∼500 generations of competition, isolates of the E. coli populations evolved to survive T6SS attack an average of 27-fold better, through two convergently evolved pathways: apaH was mutated in six of the eight replicate populations, while the other two populations each had mutations in both yejM and yjeP. However, the mutations we identified are pleiotropic, reducing cellular growth rates, and increasing susceptibility to antibiotics and elevated pH. These trade-offs help us understand how the T6SS shapes the evolution of bacterial interactions.

  PublisherOA PDFDOI

• A novel mechanism that maintains outer membrane lipid asymmetry in Pseudomonas aeruginosa

  Zenodo (CERN European Organization for Nuclear Research) · 2023-02-17

  datasetOpen accessSenior author

  Newick file the PA2800 phylogenetic tree in "A novel mechanism that maintains outer membrane lipid asymmetry in Pseudomonas aeruginosa"

  PublisherDOI

• Mechanism of outer membrane destabilization by global reduction of protein content

  Nature Communications · 2023-09-15 · 41 citations

  articleOpen accessSenior author

  The outer membrane (OM) of Gram-negative bacteria such as Escherichia coli is an asymmetric bilayer with the glycolipid lipopolysaccharide (LPS) in the outer leaflet and glycerophospholipids in the inner. Nearly all integral OM proteins (OMPs) have a characteristic β-barrel fold and are assembled in the OM by the BAM complex, which contains one essential β-barrel protein (BamA), one essential lipoprotein (BamD), and three non-essential lipoproteins (BamBCE). A gain-of-function mutation in bamA enables survival in the absence of BamD, showing that the essential function of this protein is regulatory. Here, we demonstrate that the global reduction in OMPs caused by BamD loss weakens the OM, altering cell shape and causing OM rupture in spent medium. To fill the void created by OMP loss, phospholipids (PLs) flip into the outer leaflet. Under these conditions, mechanisms that remove PLs from the outer leaflet create tension between the OM leaflets, which contributes to membrane rupture. Rupture is prevented by suppressor mutations that release the tension by halting PL removal from the outer leaflet. However, these suppressors do not restore OM stiffness or normal cell shape, revealing a possible connection between OM stiffness and cell shape.

  PublisherOA PDFDOI

• A periplasmic phospholipase that maintains outer membrane lipid asymmetry in <i>Pseudomonas aeruginosa</i>

  Proceedings of the National Academy of Sciences · 2023-07-18 · 24 citations

  articleOpen accessSenior author

  The outer membrane of Gram-negative bacteria is unique in both structure and function. The surface-exposed outer leaflet is composed of lipopolysaccharide, while the inner leaflet is composed of glycerophospholipids. This lipid asymmetry creates mechanical strength, lowers membrane permeability, and is necessary for virulence in many pathogens. Glycerophospholipids that mislocalize to the outer leaflet are removed by the Mla pathway, which consists of the outer membrane channel MlaA, the periplasmic lipid carrier MlaC, and the inner membrane transporter MlaBDEF. The opportunistic pathogen Pseudomonas aeruginosa has two proteins of the MlaA family: PA2800 and PA3239. Here, we show that PA2800 is part of a canonical Mla pathway, while PA3239 functions with the putative lipase PA3238. While loss of either pathway individually has little to no effect on outer membrane integrity, loss of both pathways weakens the outer membrane permeability barrier and increases production of the secondary metabolite pyocyanin. We propose that mislocalized glycerophospholipids are removed from the outer leaflet by PA3239 (renamed MlaZ), transferred to PA3238 (renamed MlaY), and degraded. This pathway streamlines recycling of glycerophospholipid degradation products by removing glycerophospholipids from the outer leaflet prior to degradation.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01GM035791

  NIH · $2.8M · 2002

• NIH Grant R01GM065216

  NIH · $3.5M · 2016

• NIH Grant R01GM034821

  NIH · $8.8M · 2017

• Predoctoral Training in Genetics and Molecular Biology

  NIH · $31.9M · 1977–2023

• Biogenesis and maintenance of the outer membrane of Gram-negative bacteria

  NIH · $8.1M · 2016–2026

Frequent coauthors

• Daniel Kahne

  Harvard University

  31 shared

• Michael N. Hall

  University of Basel

  23 shared

• Winfried Boos

  University of Konstanz

  22 shared

• Juliana C. Malinverni

  United States Military Academy

  21 shared

• Howard A. Shuman

  University of Chicago

  20 shared

• Scott D. Emr

  Cornell University

  20 shared

• Natividad Ruiz

  The Ohio State University

  20 shared

• Marcin Grabowicz

  Emory University

  18 shared

Labs

• Silhavy LabPI

Awards & honors

• Fellow of the American Academy of Microbiology (1994)
• Fellow of the American Association for the Advancement of Sc…
• Fellow of the American Academy of Arts and Sciences (2005)
• Member of the National Academy of Sciences (2005)
• Associate member of EMBO (2008)