One gene, two functions in bacterial immunity

From the Stoddard Lab, Basic Sciences Division and the Kaiser Lab, Seattle University

The pandemic has elicited an unprecedented public interest in the human immune system and its role in fighting off viral infections. While the antibodies and specialized cells that compose our immune system represent one powerful method to stave off infection, a stunning array of immunological strategies have evolved throughout the animal kingdom, and beyond. “The warfare between bacteria and the viruses that infect them probably represents one of the longest-running biological conflicts that we know of, leading to each of them building up an extraordinary arsenal of defensive and offensive weapons and strategies for neutralizing one another’s weaponry,” explains Dr. Barry Stoddard, a professor in Fred Hutch’s Basic Sciences Division and Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member. Many bacterial immune systems function by discriminating between their own DNA and that of a viral invader. Most famous among them is CRISPR-Cas, a system so powerful it has been adapted into a ubiquitous tool for genetic manipulation. A different system, termed the Bacteriophage exclusion (BREX) system, has been identified, but its function is much less well understood. In a newly published article in Nucleic Acids Research, the labs of Dr. Stoddard and Dr. Brett Kaiser, an associate professor at Seattle University, in collaboration with investigators at New England Biolabs, characterized a new BREX system and made a surprising discovery about its function.

“BREX is a largely uncharacterized defense system that restricts phage by a poorly understood mechanism,” the authors write. These systems “typically contain four to eight genes” and “utilize PglX-directed DNA methylation to distinguish self from non-self.” But very little is known of how this system is regulated, and how it actually restricts phage. While studying DNA methylation in Acinetobacter, the authors identified a new apparent BREX system that “contained the same conserved six genes present in all Type I systems, as well as a seventh gene [termed BrxR].” Recognizing an opportunity to better understand BREX function, the group cloned the entire gene cluster into E. coli and found that it was capable of phage restriction in its new host. They then set about deleting genes one by one to determine their functions and discovered that all seven are required for phage restriction and that five of them, including the enigmatic BrxR, appear to act at least in part by promoting DNA methylation to protect the host's DNA.

“We were intrigued by the observation that BrxR was required for BREX-mediated phage restriction, since it has not been reported as a core component of Type I BREX systems and is not part of the Type I systems so far described,” stated the authors. They performed X-ray crystallography to determine the structure of this protein, which revealed it to be an apparent site-specific DNA binding protein, and potentially a transcriptional regulator. DNA binding assays revealed that BrxR binds just upstream of the BREX transcription start site, suggesting it may regulate the expression of this system.

Now this is where things get weird, genetically speaking. While the authors had already examined the effects of fully deleting the BrxR gene, they decided to also generate a point mutation to introduce a premature stop codon into the gene. Since both types of mutation destroy protein function, they’d be expected to have the same phenotype. But whereas the gene deletion blocked phage restriction, the point mutation did not. Adding to the mystery, the group found that while the BrxR deletion caused upregulation of another BREX gene, BrxC, the point mutation did not. Thus, it seems, BrxR is playing multiple roles in this system. Its protein, the authors speculate, binds upstream of BREX to likely repress BREX function. And the gene itself appears to contain regulatory information that promotes BREX gene expression independently of its protein. That’s some solid multitasking.

This is one of those studies that, while illuminating, seems to leave more questions than answers. Dr. Stoddard is excited to continue unraveling the mysteries of BREX. “While we now know much about how BrxR functions as a regulator of [the BREX system]… what we do not yet know is how this protein, and/or other factors in those systems, sense the appearance and presence of such viruses, and then trigger such systems to swing into action. It’s known that these proteins, through their ‘WYL’ domains, recognize activating molecules that are generated when phage arrive, but it’s not known if those molecules originate from the viruses or are produced by the bacteria through some type of ’sound the alarm’ response. We are working hard to try to figure that out. Separately from that, there are six other factors in our BREX system, and aside from one other we really don’t know exactly what any of them do, or how they collaborate with one another, to defend against a viral attack. We have a very exciting new study of another factor (“BrxL”) ongoing in the lab that we hope to wrap up and publish before the end of the year.”

brxr structure
Structure of the BrxR protein bound to DNA Image provided by Dr. Barry Stoddard

This work was supported by the National Institutes of Health, the G. Harold and Leila Y Mathers Charitable Foundation, New England Biolabs, the Howard Hughes Medical Institute, Fred Hutchinson Cancer Center, and the US Department of Energy.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Barry Stoddard contributed to this work

Luyten YA, Hausman DE, Young JC, Doyle LA, Higashi KM, Ubilla-Rodriguez NC, Lambert AR, Arroyo CS, Forsberg KJ, Morgan RD, Stoddard BL, Kaiser BK. Identification and characterization of the WYL BrxR protein and its gene as separable regulatory elements of a BREX phage restriction system. Nucleic Acids Res. 2022 May 20;50(9):5171-5190. doi: 10.1093/nar/gkac311. PMID: 35511079; PMCID: PMC9122589.