O2, replicate1
Fusobacterium nucleatum (Fn) is a Gram-negative anaerobic bacterium that plays a crucial role in oral biofilm formation and is associated with systemic diseases, including colorectal cancer and preterm birth. Despite its clinical significance, mechanistic studies of Fn have been hindered by its perceived requirement for strict anaerobic conditions. Yet paradoxically, Fn mainly resides in the oxygen-rich oral cavity, including periodontal pockets that retain residual oxygen. Upon exposure to oxygen, reactive oxygen species (ROS) are generated within the bacterial cell, posing a severe threat to its survival. Additional ROS are produced by host immune responses and neighboring streptococci, subjecting Fn to persistent oxidative stress during early plaque formation, mature biofilm development, and tissue invasion. However, how Fn senses, responds to, and withstands oxidative stress remains poorly understood, representing a critical gap in understanding its persistence and pathogenesis. Unexpectedly, we discovered that Fn can grow robustly in ambient air when the redox potential of the medium is lowered using cysteine, thereby overcoming its presumed strict anaerobic barrier. This enabled, for the first time, transcriptomic comparisons of Fn grown under aerobic and anaerobic conditions. Cultures were grown to stationary phase (OD  1.2), harvested by centrifugation, resuspended in 1 mL of TRIzol reagent (Ambion), and lysed 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 instructiRNA 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 75-cycle sequencing on an Illumina NextSeq 550 System (Illumina, Inc., USA) using High Output Kit v2.5 (#20024907, Illumina, Inc., USA).