Cosi, V. | bioRxiv Science

bioRxiv2023

A global survey of small RNA interactors identifies KhpA and KhpB as major RNA-binding proteins in Fusobacterium nucleatum

ABSTRACTThe common oral microbe Fusobacterium nucleatum has recently drawn attention after it was found to colonize tumors throughout the human body. Fusobacteria are also interesting study systems for bacterial RNA biology as these early-branching species encode many small noncoding RNAs (sRNAs) but lack homologs of the common RNA-binding proteins (RBPs) CsrA, Hfq and ProQ. To search for alternate sRNA-associated RBPs in F. nucleatum, we performed a systematic mass spectrometry analysis of proteins that co-purified with 19 different sRNAs. This approach revealed strong enrichment of the KH domain proteins KhpA and KhpB with nearly all tested sRNAs, including the {sigma}E-dependent sRNA FoxI, a regulator of several envelope proteins. KhpA/B act as a dimer to bind sRNAs with low micromolar affinity and influence the stability of several of their target transcripts. Transcriptome studies combined with biochemical and genetic analyses suggest that KhpA/B have several physiological functions, including being required for ethanolamine utilization. Our RBP search and the discovery of KhpA and KhpB as major RBPs in F. nucleatum are important first steps in identifying key players of post-transcriptional control at the root of the bacterial phylogenetic tree.

microbiology↗

bioRxiv2022

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, {sigma}E. 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 {sigma}E 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 {sigma}E 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 {sigma}E. 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 STATEMENTFusobacterium 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 {sigma}E and an associated small RNA. Our findings in an early-branching bacterium reveal surprising parallels to and differences from the {sigma}E response in well-characterized model bacteria and provide a framework that will accelerate research into the understudied phylum Fusobacteriota.

microbiology↗