New Imaging Technique Illuminates Bacterial Gene Activity

The studies also captured spatial data, revealing how spatial factors influence the genes bacteria turn on. This had never been done before, the authors said.

A tale of two technologies

Moffitt helped develop MERFISH (short for multiplexed error-robust fluorescence in situ hybridization) about 10 years ago to directly image genome-wide properties of individual cells within intact tissues and use that information to study a range of important biological problems.

To use MERFISH to study bacterial RNA, also known as the bacterial transcriptome, the team had to overcome a major barrier. Because bacterial cells are tiny, their RNAs are crammed tightly inside and mingle with one another, making it hard to image and identify them.

“It was a complete disaster. We couldn’t see anything,” said Moffitt.

So the team borrowed a technique known as expansion microscopy that had been developed in the lab of Edward Boyden at MIT.

Moffitt and colleagues began by embedding the samples in a special hydrogel. They then anchored the RNAs to this gel and changed the chemical buffer in the gel. This triggered the samples to swell, expanding 50- to 1,000-fold in volume.

“All the bacterial RNAs become individually resolvable,” Moffitt said.

The team dubbed the combination of techniques bacterial-MERFISH.

What bacterial-MERFISH enables

The ability to determine the genes that individual bacteria are using can provide powerful new insights into questions related to human health, including bacteria’s:

• Interactions with one another and with other cells
• Ability to inflict harm on an infected host
• Stress responses
• Ability to resist antibiotics
• Ability to form hard-to-treat biofilms, such as in catheters

“We now have the tools to answer fascinating questions about host-microbe and microbe-microbe interactions,” Moffitt said. “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 on 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 said.

Early insights into bacterial gene expression

Moffitt and colleagues showed that individual E. coli, when starved of glucose, try using 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 gained insight into how bacteria organize their RNAs, which may be important for regulating different aspects of gene expression.

Finally, they showed that intestinal bacteria tap different genes depending on their physical location in the colon.

“The same bacteria could be doing very different things over a space of tens of microns,” Moffit said. “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.”

Adapted from a Boston Children’s blog post.