From antibiotics to antibacterial wipes, humans have developed ways to battle the threats microbes pose to our health. But bacteria are also contending with their own biological threats. These threats include viruses called the bacteriophage (often called “phage” for short) that infect bacteria and other single-celled organisms. Like humans, bacteria have developed innovations to fight phage and the threat they pose to bacterial health.
The conflict between single-celled organisms, which include bacteria and archaea, and phage is ancient and ongoing. Throughout this evolutionary battle, bacteria have evolved a variety of mechanisms to outsmart phage. These include the well-known restriction endonucleases and CRISPR system, as well as the bacteriophage exclusion, or BREX, system. Despite BREX systems being ubiquitous among most bacteria and archaea species, scientists still have a poor understanding of how these species utilize the multi-protein BREX systems to fight phage.
In a new study published in Nucleic Acids Research, researchers from the Fred Hutchinson Cancer Center described how bacteria utilize BrxL, a key protein in the BREX system, to prevent phage from replicating inside host cells.
“This study provides considerable new insight into a poorly understood ‘backup' mechanism by which bacteria defend themselves against viral challenges,” said Dr. Barry Stoddard, a professor in the Basic Sciences Division and a co-senior author on the study. “More specifically, investigators have recently uncovered evidence that in addition to well-known innate and adaptive anti-viral defense systems (known as Restriction-Modification (‘RM’) and CRISPR systems, respectively), they appear to possess and exchange a large number of additional factors and pathways that are also employed for self-defense against foreign invaders.”
The BREX system is one such “backup” bacterial self-defense mechanism, but it has been a challenging one to study. “The BREX system has been particularly enigmatic, as it consists of a rather bewildering number of proteins (as many as 7) that are all required for the system to work properly,” explained Dr. Stoddard. The team decided to focus on one of those enigmatic proteins called BrxL, which is present in some, but not all, of BREX systems.
The researchers used cryogenic electron microscopy, or cryo-EM, to obtain high-resolution images of the BrxL protein, both on its own and bound to DNA. They followed up on the structural finding with biochemical assays to determine how BrxL functions in the BREX system to fight phage.
The results suggested that “the BREX system appears to target foreign DNA in a manner that prevents it from replicating once inside a bacterial cell, in a manner that depends upon DNA binding and subsequent ATP hydrolysis by the BrxL factor,” said Dr. Stoddard.
This was a surprising result. “The biochemical function (DNA binding) that we elucidated for BrxL was a completely unexpected finding from this study,” said Dr. Stoddard. “Prior to this work, it was believed (based on sequence homology) that BrxL was likely a protease (the ‘L’ in its name actually stands for ‘Lon Protease homologue), which was an incorrect assumption.”
The results from this study provide valuable insights to motivate future research. “The determination of the structure of BrxL bound to DNA, along with the prediction of additional DNA-binding folds for several other BREX factors, leads us to now hypothesize that the BREX system acts by searching for and recognizing phage-specific DNA replication origins and then imposing a block on the ability of that DNA to undergo replication during the natural infective cycle of the virus,” explained Dr. Stoddard. “We are now designing experiments to prove or disprove this idea, by examining 1) whether BrxL specifically recognizes DNA sequences found within or near the viral (bacteriophage) DNA replication origin, and 2) if such a recognition event triggers the subsequent assembly of additional BREX factors at that site, leading to inhibition of viral DNA replication.”
This work highlights the importance of curiosity-driven basic science research in making discoveries that advance fundamental scientific knowledge. “The Center itself has provided significant seed and bridge funding, partially through the Basic Sciences endowment, that has allowed us to pursue this line of research,” said Dr. Stoddard. “This study is one of the first to report on structures that were determined using the Center’s new CryoEM microscopes and data collection facility.”
Dr. Stoddard also emphasized the collaborative nature of this work, which was conducted with investigators at New England Biolabs and Dr. Brett Kaiser’s research group at Seattle University. “Brett is the co-senior author of this study, along with myself,” said Dr. Stoddard. “His group is comprised entirely of talented young undergraduates, most of whom experienced their first true research laboratory experience while working on this project.” He also credits Dr. Betty Shen, a staff scientist in his lab and the first author of this paper. “These findings and observations were all the result of Betty’s extraordinary efforts to visualize multiple forms of the BrxL protein.”
This work was supported by New England Biolabs, Fred Hutchinson Cancer Center, the National Institutes of Health, the National Institute of General Medical Sciences, Howard Hughes Medical Institute, and the U.S Department of Energy.
The Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Barry Stoddard contributed to this work.
Shen BW, Doyle LA, Werther R, Westburg AA, Bies DP, Walter SI, Luyten YA, Morgan RD, Stoddard BL, Kaiser BK. 2023. Structure, substrate binding and activity of a unique AAA+ protein: the BrxL phage restriction factor. Nucleic Acids Research. Epub ahead of print.