Fusobacterium nucleatum, long known as a constituent of the oral microflora, has recently garnered renewed attention for its association with several different human cancers. The growing interest in this emerging cancer-associated bacterium contrasts with a paucity of knowledge about its basic gene expression features and physiological responses. As fusobacteria lack all established small RNA-associated proteins, post-transcriptional networks in these bacteria are also unknown. In the present study, using differential RNA-sequencing, we generate high-resolution global RNA maps for five clinically relevant fusobacterial strains—F. nucleatum subspecies nucleatum, animalis, polymorphum and vincentii, as well as F. periodonticum—for early, mid-exponential growth and early stationary phase. These data are made available in an online browser, and we use these to uncover fundamental aspects of fusobacterial gene expression architecture and a suite of non-coding RNAs. Developing a vector for functional analysis of fusobacterial genes, we discover a conserved fusobacterial oxygen-induced small RNA, FoxI, which serves as a post-transcriptional repressor of the major outer membrane porin FomA. Our findings provide a crucial step towards delineating the regulatory networks enabling F. nucleatum adaptation to different environments, which may elucidate how these bacteria colonize different compartments of the human body.
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Targeting Fusobacterium nucleatum through chemical modifications of host-derived transfer RNA fragments
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RNA-seq data can be accessed at NCBI’s GEO (https://www.ncbi.nlm.nih.gov/geo) under the accession no. GSE161360. MS data can be accessed at the Proteomics Identification Database PRIDE (https://www.ebi.ac.uk/pride) under the accession no. PXD022474. The Rfam database can be accessed at http://rfam.xfam.org. The Uniprot database can be accessed at https://www.uniprot.org/uniprot/?query=taxonomy:190304. Source data are provided with this paper.
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We thank Y. el Mouali Benomar, J. Hör, D. Ryan and G. Bachrach for fruitful discussions, E. Venturini for support with the subcellular fractionation, G. Bachrach (Hebrew University of Jerusalem, Israel) for providing pORI92, E. Allen-Vercoe (University of Guelph, Canada) for providing Fusobacterium strains and L. Jenniches for help with launching the web browser. We thank S. Lamer and A. Schlosser for MS analysis. We thank the Vogel Stiftung Dr. Eckernkamp for supporting F.P. with a Dr. Eckernkamp Fellowship. This work was funded by a DFG Gottfried Wilhelm Leibniz award to J.V. (DFG Vo875‐18). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
The authors declare no competing interests.
Peer review information Nature Microbiology thanks Cari Vanderpool and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Venn diagram showing the number and class-distribution of detected TSS for F. nucleatum subsp. polymorphum. b, Length distribution and corresponding occurrences of all 800 5′-UTR yielding from a pTSS (black) or sTSS (red) are shown. The consensus Shine Dalgarno sequence is displayed with the average distance from the start codon. c, Motif search using MEME in the 50-nt region upstream of pTSS identified an extended -10 box similar to the σ70 binding site in E. coli for ~92% of pTSS. While most of the sequences harbour an AT-rich stretch preceding the -10 box, a -35 box was only detected in ~28% of promoter regions.
a, Venn diagram showing the number and class-distribution of detected TSS for F. nucleatum subsp. animalis. b, Length distribution and corresponding occurrences of all 758 5′-UTR yielding from a pTSS (black) or sTSS (red) are shown. The consensus Shine Dalgarno sequence is displayed with the average distance from the start codon. c, Motif search using MEME in the 50-nt region upstream of pTSS identified an extended −10 box similar and −35 box to the σ70 binding site in E. coli for ~92% of pTSS separated by an AT-rich stretch.
a, Venn diagram showing the number and class-distribution of detected TSS for F. nucleatum subsp. vincentii. b, Length distribution and corresponding occurrences of all 589 5′-UTR yielding from a pTSS (black) or sTSS (red) are shown. The consensus Shine Dalgarno sequence is displayed with the average distance from the start codon. c, Motif search using MEME in the 50-nt region upstream of pTSS identified an extended -10 box similar to the σ70 binding site in E. coli for ~78% of pTSS. While most of the sequences harbour an AT-rich stretch preceding the -10 box, a -35 box was only detected in ~31% of promoter regions.
a, Venn diagram showing the number and class-distribution of detected TSS for F. periodonticum. b, Length distribution and corresponding occurrences of all 638 5′-UTR yielding from a pTSS (black) or sTSS (red) are shown. The consensus Shine Dalgarno sequence is displayed with the average distance from the start codon. c, Motif search using MEME in the 50-nt region upstream of pTSS identified an extended -10 box similar and -35 box to the σ70 binding site in E. coli for ~88% of pTSS separated by an AT-rich stretch.
Overview of genomic locations for predicted riboswitches that were identified by RFAM analysis.
a, Alignment of 4.5S RNA sequences from representative strains of different Fusobacterium species and the subspecies of Fusobacterium nucleatum (FNN = F. n. subsp. nucleatum; FNA = F. n. subsp. animalis; FNP = F. n. subsp. polymorphum; FNV = F. n. subsp. vincentii; FuH = F. hwasookii; FuP = F. periodonticum; FuG = F. gonidiaiformans; FuM = F. mortiferum; FuN = F. necrophorum; FuU = F. ulcerans; FuV = F. varium). The TSS and the conserved apical tetraloop are indicated. b, Shown is prediction for the secondary structure of the 4.5S RNA in Fnn.
a, Alignment of tmRNA sequences from representative strains of Fusobacterium species and subspecies of Fusobacterium nucleatum (FNN = F. n. subsp. nucleatum; FNA = F. n. subsp. animalis; FNP = F. n. subsp. polymorphum; FNV = F. n. subsp. vincentii; FuH = F. hwasookii; FuP = F. periodonticum; FuG = F. gonidiaiformans; FuM = F. mortiferum; FuN = F. necrophorum; FuU = F. ulcerans; FuV = F. varium). The conserved tag peptide is highlighted (yellow). b, Alignment of the ORF for the tag peptide reveals sequence dichotomy between oral isolates and fusobacteria found elsewhere.
Shown is the alignment for the 6S RNA in representative strains of Fusobacterium species and subspecies of Fusobacterium nucleatum (FNN = F. n. subsp. nucleatum; FNA = F. n. subsp. animalis; FNP = F. n. subsp. polymorphum; FNV = F. n. subsp. vincentii; FuH = F. hwasookii; FuP = F. periodonticum; FuG = F. gonidiaiformans; FuM = F. mortiferum; FuN = F. necrophorum; FuU = F. ulcerans; FuV = F. varium). The region encoding for the pRNA is highlighted (blue).
a, Comparison of Fnn (ATCC 23726) growth harbouring empty vector control (ctrl.), FoxI-expressing (FoxI) or FoxI-3C-expressing (FoxI-3C) plasmid, respectively. A 24 h pre-culture was diluted 1:50 into fresh Columbia Broth and measured every 20 min with 10 s shaking prior to each measurement. Growth data are represented as the mean (± standard deviation) from three biological replicates. b, Northern blot analysis of FoxI overexpression during mid-exponential growth phase. c-f,Comparison of the excised bands for the region of interest for two replicates for either the empty vector control (ctrl.), the overexpression of FoxI (FoxI) or the mutant FoxI-3C (FoxI-3C). Shown are the estimated protein abundances by iBAQ quantification for all proteins detected in both replicates of each group. c, Comparison of total protein samples for the empty vector control. d, Comparison of total protein samples for the FoxI overexpression. e, Comparison of total protein samples for the FoxI-3C overexpression. f, Comparison of the same region for the outer membrane fraction in the control (Fig. 6b). FomA is indicated (red) and was detected as the most abundant protein in all samples with a clear reduction in the FoxI-overexpression.
Supplementary Figs. 1–4 and references.
Overview of detected TSSs.
Results of differential gene expression analysis.
Overview of predicted 5′-UTRs.
Comparison of putative virulence factors to pTSSs in Fnn.
Reannotated CDSs in Fnn.
Predicted operons in Fnn.
Overview of predicted riboregulatory elements.
Overview of predicted sRNAs.
Used oligonucleotides, plasmids and strains in the present study.
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Ponath, F., Tawk, C., Zhu, Y. et al. RNA landscape of the emerging cancer-associated microbe Fusobacterium nucleatum. Nat Microbiol 6, 1007–1020 (2021). https://doi.org/10.1038/s41564-021-00927-7