Scientists Just Made Bacteria 1000x Bigger – And Discovered Something Incredible

By combining MERFISH imaging with expansion microscopy, researchers have unlocked a new way to study bacteria at the single-cell level.

This allows them to see how bacteria activate different genes in response to their environment, offering insights into microbial behavior, antibiotic resistance, and infection strategies.

How Bacteria Organize Their Activities

How do bacteria — whether beneficial ones in our bodies or harmful disease-causing strains — coordinate their activities? A recent study has provided new insights by combining advanced genomic-scale microscopy with an innovative technique to track which genes bacteria activate in different conditions and environments. Published recently in the journal Science, this breakthrough is set to advance bacterial research significantly.

Jeffrey Moffitt, PhD, and his colleagues at the Program in Cellular and Molecular Medicine (PCMM) at Boston Children’s Hospital used MERFISH, a molecular imaging technique Moffitt helped develop, to analyze messenger RNAs (mRNAs) in thousands of individual bacteria at once. This method not only mapped gene expression on a massive scale but also revealed how spatial factors influence which genes bacteria turn on — something never before achieved.

Overcoming the Challenges of Imaging Bacteria

However, the team first had to overcome a major challenge: bacterial RNAs, or the bacterial transcriptome, are densely packed inside tiny cells, making them difficult to distinguish and image. “It was a complete disaster, we couldn’t see anything,” says Moffitt.

Borrowing a technique developed in the laboratory of Ed Boyden, PhD, at MIT— expansion microscopy — they embedded the samples in a special hydrogel. They anchored the RNAs to this gel and changed the chemical buffer in the gel. This triggered it to swell, expanding the sample 50- to 1000-fold in volume. “All the bacterial RNAs become individually resolvable,” Moffitt says.

Why Measure Bacterial Gene Expression?

Until now, scientists have averaged bacterial behavior across a given bacterial population. The ability to determine what genes individual bacteria are using can give powerful new insights into bacterial interactions, virulence, stress responses, the ability to resist antibiotics, the ability to form biofilms like those in catheters and more.

“We now have the tools to answer fascinating questions about host-microbe and microbe-microbe interactions,” Moffitt says. “We can explore how bacteria might communicate and compete for spatial niches and define the structure of microbial communities. And we can ask how pathogenic bacteria adjust their gene expression as they infect mammalian cells.”

Bacterial-MERFISH can also provide insights into bacteria that are difficult to grow in a culture dish. “Now we don’t have to culture them, we can just go image them in their native environment,” Moffitt says.

Single-Cell Insights into Bacterial Survival Strategies

Several experiments the team performed illustrate the kinds of questions that bacterial-MERFISH can answer. For example, Moffitt and colleagues were able to show that individual E. coli, when starved of glucose, try utilizing alternative food sources one after another, altering their gene expression in a specific sequence. Taking a series of genomic snapshots over time enabled the team to piece together this survival strategy.

The team also got insights into how bacteria organize their RNAs within their cells, which may be important in how different aspects of gene expression are regulated. Finally, they showed that intestinal bacteria tap different genes depending on their physical location in the colon.

A New Era in Bacterial Research

“The same bacteria could be doing very different things over a space of tens of microns,” Moffit says. “They’re seeing different environments and responding differently to them. It was very difficult to address such variation before, but now we can answer the types of questions people have been dreaming about.”

Reference: “Highly multiplexed spatial transcriptomics in bacteria” by Ari Sarfatis, Yuanyou Wang, Nana Twumasi-Ankrah and Jeffrey R. Moffitt, 24 January 2025, Science.
DOI: 10.1126/science.adr0932

The paper’s coauthors were Ari Sarfatis, Yuanyou Wang, PhD, and Nana Twumasi-Ankrah in the Moffitt Lab.

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