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

> **NIH NIH R35** · PRINCETON UNIVERSITY · 2021 · $803,341

## Abstract

PROJECT SUMMARY
The cell envelope of Gram-negative bacteria contains two membranes, inner (IM) and outer (OM), and an
aqueous compartment termed the periplasm that is located between them. A long-term goal of my lab has always
been to understand the mechanisms of envelope biogenesis using Escherichia coli as a model system. This
proposal concerns OM biogenesis and the stress responses that maintain cell envelope physiology. All of the
components of the OM, phospholipids (PL), lipopolysaccharide (LPS), lipoproteins, and β-barrel proteins
(OMPs), are synthesized in the cytoplasm or the inner leaflet of the IM. We have identified the essential proteins
required to assemble LPS (LptABCDEFG) and OMPs (BamABCDE) in the OM and we have provided evidence
of a diffusive mechanism of phospholipid transport between the IM and the OM. In the current funding period,
we have shown that the conditional lethal phenotype of bamB bamE double mutants can be suppressed simply
by deleting a surface-exposed lipoprotein, we revealed the existence of an alternate lipoprotein trafficking
pathway, we uncovered a role for the cyclic form of Enterobacterial Common Antigen in maintaining the OM
barrier, and we identified mutations that activate or prime the σE stress response that suppress a variety of OMP
and Bam defects. In translational studies, we used our knowledge of OM biogenesis to discover a new class of
antibiotics that work to inhibit BamA at the cell surface.
We propose to use our large collection of mutations that alter the Bam components or various OMP substrates
together with our collection of suppressors as tools to probe the OMP assembly process. In particular, we will
probe the function of the non-essential BamBCE lipoproteins and test our hypothesis that BamD does not
perform a truly essential mechanistic role, but rather functions as a regulator to control the activity of BamA. We
will test the role of the chaperone Skp as a specific adaptor for the periplasmic protease DegP. We also posit
that the trimeric nature of the major OMPs functions as a global organizer of OM architecture by providing
multiple interacting faces to allow the protein-protein interactions necessary for the formation of protein islands.
Our studies on LPS assembly will utilize a mutant O-antigen ligase and the enzyme sortase to attach peptides
or proteins to LPS to challenge the capabilities of the LptDE translocon. We will also test our model that three
essential IM proteins, YejM, YciM, and FtsH comprise a novel pathway that regulates LPS synthesis in response
to the lipid status of the OM.
The mlaA* mutation destabilizes the OM by increasing LPS levels. This causes membrane loss by OM
vesiculation and IM PLs flow into the OM to replace the loss. We have identified a mutation that slows this lipid
flow and we believe that continued study of this gene may provide insights into the poorly understood process
of anterograde PL transport.

## Key facts

- **NIH application ID:** 10085086
- **Project number:** 2R35GM118024-06
- **Recipient organization:** PRINCETON UNIVERSITY
- **Principal Investigator:** Thomas J. Silhavy
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $803,341
- **Award type:** 2
- **Project period:** 2016-05-15 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10085086

## Citation

> US National Institutes of Health, RePORTER application 10085086, Biogenesis and maintenance of the outer membrane of Gram-negative bacteria (2R35GM118024-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10085086. Licensed CC0.

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