WT replicate1
Fusobacterium nucleatum is a gram-negative, spindle-shaped bacterium that plays a central role in oral biofilms, particularly in periodontal disease-associated subgingival plaque, a polymicrobial community of over 400 bacterial species. It acts as a bridging organism, facilitating the integration of early and late colonizers, a process critical for dental plaque formation. Beyond its ecological role, F. nucleatum is associated with systemic diseases, including preterm birth and colorectal cancer, underscoring its clinical significance.Fusobacterium nucleatum is taxonomically diverse, comprising at least four well-defined subspecies: nucleatum (FNN), polymorphum (FNP), animalis (FNA), and vincentii (FNV). Among these, we found that only FNA strains produce autoinducer-2 (AI-2), a conserved signaling molecule that mediates interspecies communication in both Gram-positive and Gram-negative bacteria. AI-2 biosynthesis depends on the luxS gene, which encodes the enzyme S-ribosylhomocysteinase (LuxS), responsible for generating the AI-2 precursor. Given the role of FNA in colorectal cancer and periodontitis, we investigated how AI-2 production influences the physiology of this subspecies.FNA cells were cultured anaerobically in tryptic soy broth supplemented with 1% Bacto peptone and 0.25% freshly prepared cysteine (TSPC medium) or in artificial gingival crevicular fluid (GCF) medium. Two RNA-seq experiments were performed: one using cultures grown to OD  0.8 under anaerobic conditions (slow-growing strain), and the other using cultures grown to OD  1.3 from a starting OD of 0.3. Cells from the wild-type strain 7_1 and its luxS mutant were harvested by centrifugation, resuspended in 1 mL of TRIzol reagent (Ambion), and lysed via mechanical disruption using 0.1 mm silicon beads (MP Biomedicals).Total RNA was extracted using the Direct-zol RNA MiniPrep Kit (ZYMO Research, R2051) following the manufacturers protocol. RNA samples were submitted to the Cancer Genomics Center at The University of Texas Health Science Center at Houston (CPRITRP240610) for quality control and sequencing. RNA integrity was assessed using the Agilent RNA 6000 Pico Kit (#5067-1513) on the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), and samples with RNA integrity numbers (RIN) greater than 7 were used for library construction.RNA-seq libraries were prepared from rRNA-depleted RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L, New England Biolabs) in combination with NEBNext Multiplex Oligos for Illumina (E6609S, New England Biolabs), following the manufacturers instructions. Library quality was evaluated using the Agilent High Sensitivity DNA Kit (#5067-4626) on the Bioanalyzer 2100, and concentrations were quantified via qPCR using the Collibri Library Quantification Kit (#A38524500, Thermo Fisher Scientific). ). The libraries were pooled evenly and went for the paired-end 150-cycle sequencing on an Illumina NovaSeq X plus System (Illumina, Inc., USA).