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New genetic tools enable dissection of a global stress response in the early-branching species Fusobacterium nucleatum

Fusobacterium nucleatum, long known as a common oral microbe, has recently garnered attention for its ability to colonize tissues and tumors elsewhere in the human body. Clinical and epidemiological research has now firmly established F. nucleatum as an oncomicrobe associated with several major cancer types. However, with the current research focus on host associations, little is known about gene regulation in F. nucleatum itself, including global stress response pathways that typically ensure the survival of bacteria outside their primary niche. This is due to the phylogenetic distance of Fusobacteriota to most model bacteria, their limited genetic tractability, and paucity of known gene functions. Here, we characterize a global transcriptional stress response network governed by the extracytoplasmic function sigma factor, σE. To this aim, we developed several new genetic tools for this anaerobic bacterium, including four different fluorescent marker proteins, inducible gene expression, scarless gene deletion, and transcriptional and translational reporter systems. Using these tools, we identified a σE response partly reminiscent of phylogenetically distant Proteobacteria but induced by exposure to oxygen. Although F. nucleatum lacks canonical RNA chaperones such as Hfq, we uncovered conservation of the non-coding arm of the σE response in form of the non-coding RNA FoxI. This regulatory small RNA (sRNA) acts as an mRNA repressor of several membrane proteins, thereby supporting the function of σE. In addition to the characterization of a global stress response in F. nucleatum, the genetic tools developed here will enable further discoveries and dissection of regulatory networks in this early-branching bacterium. SIGNIFICANCE STATEMENT Fusobacterium nucleatum is an abundant member of the oral microbiome that can spread throughout the body and colonize secondary sites, including cancer tissues where it promotes tumor progression. Understanding how F. nucleatum is able to adapt to this new environment might open new therapeutic opportunities, but we currently lack basic molecular knowledge of gene regulation in this phylogenetically distinct bacterium. We developed much-needed genetic tools for use in F. nucleatum and with their aid uncovered a stress response mediated by the transcriptional activator σE and an associated small RNA. Our findings in an early-branching bacterium reveal surprising parallels to and differences from the σE response in well-characterized model bacteria and provide a framework that will accelerate research into the understudied phylum Fusobacteriota.