Expanding the genetic toolkit helps dissect a global stress response in the early-branching species Fusobacterium nucleatum

Abstract

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, σ^E. To this aim, we developed several 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 σ^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 noncoding arm of the σ^E response in form of the noncoding RNA FoxI. This regulatory small RNA acts as an mRNA repressor of several membrane proteins, thereby supporting the function of σ^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.

Keywords: Fusobacterium; extracytoplasmic sigma factor; noncoding RNA; posttranscriptional control; small RNA.

Fig. 1.

Phylogenetic positioning of Fusobacteriota and comparison of the ECF locus. (A) A phylogenetic tree of 265 bacterial species based on the alignments provided by Coleman et al. (14). (B) Schematic representation of the rpoE operon in E. coli and F. nucleatum. rpoE genes are in red; the anti-σ factor rseA and its putative homolog in F. nucleatum are in purple; the remaining genes in the respective rpoE operons are in gray.

Fig. 2.

A genetic toolbox for F. nucleatum. (A) Overview of the plasmid-based genetic tools developed for F. nucleatum based on the vector pVoPo-00. catP, chloramphenicol resistance cassette; ColE1, replication of origin; FP, fluorescent protein (GFP, mCherry, mNeonGreen, mScarlet-I); G.O.I, gene of interest; ORI_FN, origin of replication for F. nucleatum; tetR, tetracycline repressor. (B) Representative images of F. nucleatum carrying pVoPo-FP expressing different fluorescent proteins. The cells were PFA-fixated prior to overnight maturation at 4 °C. The emission was detected at the indicated wavelength. (Scale bars, 5 µm.) (C) Western blot analysis of lysates of F. nucleatum carrying a plasmid with mCherry introduced in the inducible system (pVoPo-03) after 30-min exposure to different ATc concentrations. mCherry runs as a duplet likely representing the full-length protein as well as a truncated version arising from an internal translational start site (53). Ponceau S (PonS) staining is shown as loading control. (D) Quantification of colony-forming units (CFU) for F. nucleatum carrying the empty vector control pVoPo-03 (p.empty) or the pVoPo-03-mazF plasmid (p.mazF). The bacteria were grown to mid-exponential phase and treated with 100 ng mL⁻¹ ATc to induce mazF expression. Serial dilutions of the samples were plated after 0 min (input), 30 min, and 3 h. Untreated samples were used as control. Data are presented as the average and SD for three biological replicates relative to the input CFUs. (E) Growth curves for F. nucleatum carrying either p.empty or p.mazF in the presence or absence of 100 ng mL⁻¹ ATc. No selection pressure for plasmid maintenance was included. Displayed is the average of three biological replicates with SD. (F) Schematic representation of allelic exchange (Left) and experimental workflow (Right) using the pVoPo-04 system to generate unmarked deletion strains. A/B, up- and downstream homology regions. (G) Northern blot detection of fadA using total RNA samples extracted from F. nucleatum WT or a ΔfadA strain generated via the deletion system pVoPo-04. 5S rRNA served as loading control.

Fig. 3.

The σ^E regulon in F. nucleatum. (A) Volcano plot of the global gene-expression changes in F. nucleatum after 30 min of σ^E induction. Gene expression of bacteria carrying pVoPo-03-σ^E is compared to cells carrying pVoPo-03 serving as empty vector control. Genes were considered significantly up-regulated with a log₂ fold-change ≥ 1 (black) and significantly down-regulated with a log₂ fold-change ≤ −1 (blue) with an FDR ≤ 0.05 (dashed horizontal line). The σ^E regulon (red) includes all transcriptional units that harbor the identified σ^E binding motif in their promoter region. (B, Upper) motif analysis via MEME (102) for all genes significantly up-regulated upon σ^E induction. TSS of all up-regulated genes were manually annotated and 50 nt upstream of the identified TSSs were used as input for MEME. The conserved −10 and −35 boxes are indicated, as well as the AT-rich spacer in between both boxes. (Lower) The previously identified promoter motif for σ⁷⁰ with an extended −10 box and a less pronounced −35 box (17). (C) Alignment of the promoter regions for selected genes identified as part of the σ^E regulon. A point mutation inserted into transcriptional reporter constructs (see D) is indicated. (D) Western blot analysis for mCherry expressed from transcriptional reporter plasmids harboring the native promoters (GTC) or a point mutation in the conserved −10 box (GTG) for selected genes shown in C. Total proteins samples were collected during mid-exponential phase for western blot analysis. PonS staining is shown as loading control. Representative images of three independent experiments are shown. Unspecific bands are marked by an asterisk. (E) qRT-PCR analysis for rpoE mRNA after exposing F. nucleatum to the indicated stress conditions for 60 min. Data are normalized to the control and plotted as the average of three biological replicates with the SD. (F) Northern blot probed for the sRNA FoxI using total RNA samples extracted from F. nucleatum treated with the indicated stress conditions for 60 min. The smaller band represents a degradation or degradation event.

Fig. 4.

Transcriptional response of the anaerobe F. nucleatum to oxygen. (A) Volcano plot of the global gene-expression changes in F. nucleatum after 20 min of oxygen exposure. Differential expression analysis was carried out by comparing the treated samples to an untreated control kept in the anaerobic chamber. Genes were considered significantly up-regulated with a log₂ fold-change ≥ 1 (black) and significantly down-regulated with a log₂ fold-change ≤ −1 (blue) with an FDR ≤ 0.05 (dashed horizontal line). The σ^E regulon (red) contains all transcriptional units that harbor the identified binding motif in their promoter region. (B) Overview of the gene-expression changes for all members of the σ^E regulon upon exposure to 400 ng mL⁻¹ polymyxin B or oxygen exposure for 20 min. The heatmap displays the log₂ fold-changes. The sRNA FoxI and rpoE are indicated.

Fig. 5.

The sRNA FoxI as a negative regulator of the σ^E response. (A) Northern blot detection of FoxI using total RNA samples extracted from F. nucleatum WT or ΔfoxI generated via the deletion system pVoPo-04. The 5S rRNA served as loading control. (B) Differential gene expression upon σ^E induction in WT F. nucleatum or in the FoxI deletion strain (ΔfoxI). The heatmap displays log₂ fold-changes of genes that are significantly down-regulated in either background (log₂ fold-change ≤ −1; FDR ≤ 0.05). mglB is marked in bold as the only gene that is not down-regulated in the ΔfoxI background upon σ^E induction. Members of the three multicistronic operons started by FadA-domain containing genes are marked in purple. (C) Schematic representation of IntaRNA (,) prediction of base-pairing between mglB mRNA and FoxI. The AUG start codon of mglB is marked in red. The mutation of the sRNA for FoxI-3C is indicated in gray. (D) Schematic representation of the translational reporter constructs used in E. F. nucleatum was either transformed with pVoPo-02 plasmids carrying mCherry fused to the 5′-region of the target gene only (control), or in combination with the expression cassette for FoxI (FoxI) or the seed region mutant FoxI-3C. (E) Representative western blots for each gene tested in the translational reporter system. C4N14_09375 served as control gene as it does not harbor any predicted FoxI-binding site. PonS staining is shown as loading control. (F) Quantification of mCherry by flow cytometry for the same constructs as shown in E. The average of three biological replicates relative to that of the control (control) is displayed together with the SD.

Fig. 6.

Extended target spectrum of the FoxI sRNA. (A) Overview of all significantly down-regulated genes (log₂ fold-change ≤ −0.5; FDR ≤ 0.05) upon pulse expression of FoxI or the seed region mutants FoxI-3C or FoxI-C4A in the ΔfoxI background. The heatmap displays the log₂ fold-changes. Genes of the identified σ^E regulon (Fig. 3) are marked in purple. (B) Schematic representation of IntaRNA target predictions between different target mRNAs and FoxI. The AUG start codons are marked in red. (C) Quantification of mCherry by flow cytometry for the translational reporter plasmids carrying the target 5′ region shown in B alone (control) or in the presence of FoxI or FoxI-3C. The average of three biological replicates relative to that of the control is displayed together with the SD. (D) Representative images of western blots of total protein samples for bacteria expressing the indicated reported constructs probed for mCherry. PonS staining is shown as loading control. (E) Model of the σ^E regulon in F. nucleatum. σ^E is released from its putative anti-σ factor (C4N14_09825) and up-regulates expression of its regulon consisting of >100 genes (marked in red). This includes bamA and skp, important for insertion of OMPs as well as lepB, ftsY, or secA, which are involved in protein-translocation across the IM. σ^E also activates the transcription of the sRNA FoxI. FoxI, in turn, down-regulates membrane-associated proteins such as the IM protein complex mglBAC and the OMP fomA as well as other OMPs.