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Decades-old mystery solved: How Gram-negative bacteria keep their outer membrane intact

Researchers from the National University of Singapore (NUS) have solved a 60-year-old mystery in bacterial cell envelope biology, defining the primary function of an important protein complex responsible for maintaining the stability of the outer membrane (OM).

The work has been published in .

Bacterial infections impose a significant health care burden globally, worsened by the rise in antimicrobial resistance (AMR) among pathogenic species. Gram-negative bacteria, in particular, possess an additional lipid bilayer, the OM, which is impermeable to many antibiotics, thereby limiting treatment options.

Extensive efforts have been devoted in the past 30 years to study and understand how the bacterial OM is built and maintained, with the vision that such knowledge will fuel the future development of new OM-targeting antibiotics. Indeed, several promising compounds have recently been discovered as novel classes of antibiotics that target some well-studied systems involved in OM assembly.

The Gram-negative bacterial Tol-Pal complex was discovered in the 1960s and has since been known to be important for OM integrity, and later also ascribed a role in cell division.

In 2017, scientists from the research group of Associate Professor Chng Shu Sin at the Department of Chemistry, NUS, and the Singapore Center for Environmental Life Sciences Engineering (SCELSE-NUS), demonstrated using intricate lipid profiling and transport assays that the Tol-Pal complex plays a role in maintaining OM lipid homeostasis—the act of balancing lipid content—by moving excess lipids back to the cytoplasmic membrane.

This work provided the first logical explanation for how the lack of the Tol-Pal complex gave rise to OM defects. However, confident assignment of a definitive biological function was still elusive, as Tol-Pal mutants exhibited broad-ranging phenotypes that could also be attributed to cell division defects.

Fast-forward to 2025, a team led by Associate Professor Chng has now made a significant breakthrough, and conclusively defined the primary function of the Tol-Pal complex in OM lipid homeostasis.

Using an elegant genetic approach, the team engineered a variant of the Tol-Pal complex that was re-directed away from, and thus could not perform associated roles at the cell division site (where the bacterial cell constricts and eventually separates into daughter cells). Strikingly, this Tol-Pal variant was fully capable of maintaining OM lipid homeostasis, and thus OM stability and barrier function.

Dr. Tan Wee Boon, a senior research fellow in the Chng group and first author of the study said, "We have unambiguously established the true physiological function of the Tol-Pal complex, more than 60 years after its first discovery."

This new knowledge of the Tol-Pal complex sets the foundation for the development of future drugs that target OM lipid homeostasis to disrupt the OM barrier and/or kill the bacteria.

"This breakthrough marks an important conceptual advance in our overall understanding of bacterial outer membrane assembly and lipid transport, and is a testament to our consistent efforts towards elucidating the fundamental mechanics of the living cell," added Prof Chng.