A new tool that reveals how bacteria switch genes on and off in response to environmental changes could help researchers develop new antibiotics. The tool, called TRIP, uses single-cell sequencing and fluorescence imaging to monitor gene activity in real time.
Introduction
Bacterial infections cause millions of deaths each year, with the global threat made worse by the increasing resistance of the microbes to antibiotic treatments. This is due in part to the ability of bacteria to switch genes on and off as they sense environmental changes, including the presence of drugs. Such switching is accomplished through transcription, which converts the DNA in genes into its chemical cousin in mRNA, which guides the building of proteins that make up the microbe’s structure. For this reason, understanding how mRNA production is regulated for each bacterial gene is central to efforts to counter resistance, but approaches used to study this regulation to date have been laborious.
TRIP: A New Tool for Gene Regulation
In a new study, scientists revealed a trick that may speed such efforts. Researchers from NYU Grossman School of Medicine and the University of Illinois Urbana-Champaign showed that the way in which genes are turned on and off as bacteria grow provide clues to their regulation .
According to the study authors, organisms from bacteria to humans grow as their cells multiply by dividing, with each cell becoming two. Before cells divide, they must copy their DNA such that each of the two daughter cells has a copy. To do so, a molecular machine called DNA polymerase ticks down the DNA chain, reading and making a copy of each gene one by one.
Publishing in the journal Nature, the study adds to explanations of how gene expression throughout the genome is shaped by DNA replication during bacterial growth. Specifically, the research team found that when DNA polymerase arrives at any specific gene, it disrupts the transcription in a way that reveals the state of that gene’s regulatory status.
“Our study results show that the constant replication of genes during the cell cycle as the bacterial cells reproduce and grow can be exploited to learn about many aspects of how genes are regulated,” said study lead investigator Andrew Pountain, Ph.D., a postdoctoral research fellow at NYU Langone Health and its Institute for Systems Genetics.
“We like the analogy of the electrocardiogram in medicine,” said Itai Yanai, the senior investigator of the study and professor at NYU Langone’s Institute for Systems Genetics. By monitoring patterns of electrical activity in the heart, the ECG reveals a series of waves that provide a detailed, graphical view into a patient’s cardiac health. Similarly, waves of changes in abundance of mRNA in response to a gene’s replication produce a signature on a graph, which the authors termed the transcription-replication interaction profile, or TRIP. The researchers showed how specific waves can be linked to certain features. For example, whether a gene is under a specific form of control, known as repression, where a protein blocks that gene’s mRNA from being made.
Implications for Antibiotic Development
The new study was made possible because of technological advances in tracking gene activity in individual cells in real time through scRNA-seq, or single-cell sequencing , and smFISH, short for single-molecule fluorescence in situ hybridization. These methods allow researchers to measure how much mRNA is produced from each gene at any given moment.
The researchers applied TRIP to Escherichia coli (E. coli), one of the most studied bacteria and a common cause of infections. They found that TRIP could accurately identify genes that were regulated by different mechanisms, such as transcription factors (proteins that bind to DNA and control gene expression) or small RNAs (molecules that interfere with mRNA production or stability).
The researchers also discovered new features of gene regulation that were not previously known, such as how DNA supercoiling (the twisting of DNA strands) affects gene expression during replication.
The authors say that TRIP could be used to study other bacteria, including those that cause diseases such as tuberculosis or anthrax. By revealing how bacteria adapt to different environments and stresses, TRIP could help identify new targets for antibiotic development or ways to enhance existing drugs.
“TRIP is a powerful tool that can reveal hidden aspects of bacterial gene regulation that are otherwise difficult to measure,” said Yanai. “We hope that TRIP will enable researchers to uncover new strategies to combat antibiotic resistance and discover new ways to manipulate bacterial behavior.”