“Silent” genes in bacteria may hold the key to new antibiotics

Silences are potentially golden in the search for antibiotics to slow the ongoing crisis of drug resistance.

Rice University bioscientists have engineered new on-off switches to control “silent” genes in a strain of bacteria. Their strategy could fuel the ongoing search for new antibiotics.

The researchers customized CRISPR tools to control the expression of genes in Streptomyces bacteria that, in nature, are only expressed when needed. Until now, these genes have been challenging for synthetic biologists to access.

“As laboratories began to sequence the genomes of these organisms known to produce one or more antibiotics, we realized that the pathways responsible for producing antibiotics and other molecules of interest are much more abundant than previously thought,” said James Chappell. , an assistant professor of biosciences whose lab studies bacteria and ways to engineer them.

“Each Streptomyces strain is now predicted to be able to produce on average up to 40 different molecules of interest, including antibiotics,” he said.

Work led by Chappell and graduate student Andrea Ameruoso could allow labs to quickly develop libraries of potential antibiotics to test on pathogens. Significantly, they said that while CRISPR-Cas9 has been used to create a platform to activate genes in organisms such as Escherichia coli, this is the first time it has been applied to Streptomyces.

Their study appears in Nucleic Acids Research.

“Bacteria like Streptomyces have evolved to produce antibiotics only when they need to, in natural environments like soil,” Chappell explained. “When we grow them in the lab, it’s an artificial environment and very different from how they grow naturally, so the gene pools are silenced.

“They are a kind of genetic dark matter,” he said. “We can’t isolate the chemicals they express to perform a functional screen.”

The lab’s new strategy eliminates the time-consuming task of exposing their proof-of-concept bacteria, S. venezuelae, the source of the common antibiotic chloramphenicol, to potential inducers of gene expression. “Andrea’s technology adds synthetic regulators to the cell to artificially stimulate or suppress the expression of these pathways,” Chappell said.

“Now we only need a protein and a small piece of RNA, and we can go anywhere we want to directly repress or activate a particular target,” Ameruoso added.

The emergence of CRISPR technology, which adapts the mechanisms of the bacterial immune system to locate specific genes along a DNA strand, has simplified access to previously hidden clusters of genes, he said.

“Streptomyces is a genus of bacteria that includes up to 500 species, and each species may have between 20 and 40 of these gene clusters capable of producing antibiotics or other molecules of interest,” Ameruoso said. “So once we find a way to scale up our technology, it could be incredibly powerful.”

Chappell said it’s a simple matter of designing CRISPR to bind to different DNA sequences. “We use it to control gene expression,” he said. “If we want to do this in a set of different species in a set of different ways, theoretically it should be possible. So this paper lays the foundation for a new kind of approach.”

Ameruoso said he is working on a fluorescent technique to observe cluster activation in real time. “The main challenge is that observing the activation depths of an array relies on purifying the molecule from the extracts we generate,” he said. “This is a low-yield, labor-intensive process. We want to develop a reporter to observe a fluorescent signal when a pathway is being activated.”

The researchers noted that the process could be used to produce molecules for antifungal and anticancer agents or for agriculture. “We focus on antibiotics because at some point in history, we’ve noticed that they kill microbes,” Chappell said. “But that’s not necessarily what they evolved for, because they’re also often used as communication signals between cells. So there’s a lot of potential uses.”

He said the study demonstrates an important new approach to activating silent pathways. “The vision for the next generation of work is to go straight,” he said. “We showed that it works in a silent single pathway. Now let’s do it in 40 pathways in this species, and then let’s do it in thousands of microbes.

“The power of CRISPR-Cas9 is that it’s really scalable for that,” Chappell said.

Reference: Ameruoso A, Villegas Kcam MC, Cohen KP, Chappell J. Activation of natural product synthesis using CRISPR interference and activation systems in Streptomyces. Nucleic Acids Res. 2022:gkac556. doi: 10.1093/nar/gkac556

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