Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic representation of the activated methyl cycle (AMC) illustrating methyl group transfer and methionine recycling. MetK (S-adenosylmethionine synthetase) converts methionine to S-adenosylmethionine (SAM), the universal methyl donor. Following methyl transfer by S-adenosylmethionine-dependent methyltransferases (SDMs), SAM is converted to S-adenosylhomocysteine (SAH). In the LuxS-dependent pathway, SAH is processed by Pfs (5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase) to form S-ribosylhomocysteine (SRH), which is subsequently cleaved by LuxS to generate homocysteine and the AI-2 precursor DPD. In alternative pathways found in other bacteria, SAH can be directly converted to homocysteine by SahH (S-adenosylhomocysteine hydrolase). (B) Schematic diagram of the metK deletion construct (pCM-galK-Δ metK ) used to generate an in-frame chromosomal deletion in FNN ATCC 23726. Approximately 1.5 kb of upstream and downstream homologous regions flank the deleted metK coding sequence to facilitate double-crossover recombination. ( C) PCR screening of more than 100 counterselected colonies following allelic exchange showed retention of the wild-type metK allele, with no Δ metK mutants recovered, indicating that metK is essential under the tested conditions. Representative PCR results from 10 independent colonies are shown. (D) Strategy for the construction of a conditional metK mutant. Because metK is essential, chromosomal deletion was performed in the presence of a plasmid-borne copy of metK expressed under the control of a theophylline- inducible riboswitch, allowing complementation in trans. (E) PCR confirmation of successful chromosomal deletion of metK in the presence of plasmid-mediated complementation, demonstrating that deletion is possible only when metK expression is provided in trans. (F) Growth analysis of the conditional metK mutant showing strict dependence on theophylline for viability. Bacterial growth exhibited a dose-dependent response to the inducer, and no growth was observed in its absence, confirming that metK is essential for survival in F. nucleatum . (G) Transmission electron microscopy (TEM) of the conditional Δ metK strain. Cells grown in the presence of 3 mM theophylline displayed normal morphology comparable to wild type. In contrast, depletion of metK (no inducer; cells precultured with 2 mM theophylline and then grown for 12 h without inducer) resulted in pronounced morphological abnormalities, including curved cells ( H1 ), surface-associated tubular-like structures ( H2 ), and marked cell elongation (H3; enlarged view shown).

Article Snippet: The deletion plasmid pCM-galK-Δ metK was introduced into competent cells of strain cw1, a Δ galK derivative of ATCC 23726, by electroporation.

Techniques: Bacteria, Construct, Sequencing, Mutagenesis, Plasmid Preparation, Control, Expressing, Transmission Assay, Electron Microscopy