Desjardins, J. B. | Search bioRxiv

bioRxiv2026

An ART-fold Rhs toxin from Pluralibacter gergoviae defines Tne5, a new clade of NAD(P)⁺ glycohydrolases effectors

The type VI secretion system (T6SS) ia a widespread bacterial nanomachine that mediates interbacterial competition by delivering toxic effectors into neighboring cells. Among these, enzymes targeting nicotinamide adenine dinucleotide cofactors (NAD and NADP) are particularly potent because they rapidly disrupt redox homeostasis and central metabolism. Several families of T6SS-associated NAD(P)-consuming effectors (Tne1-Tne4) have been described. Here, we characterize a T6SS-associated Rhs toxin from Pluralibacter gergoviae. Competition assays show that P. gergoviae kills Escherichia coli in a T6SS-dependent manner. Heterologous production reveals that the Rhs C-terminal extension is toxic in the E. coli cytoplasm and that co-production with the protein encoded downstream neutralizes this activity. AlphaFold3 modeling predicts that the toxin adopts an ADP-ribosyltransferase (ART)-like /{beta} fold with a putative catalytic pocket accommodating NAD. By contrast to T6SS ART toxins described so far, the toxin does not inhibit transcription, translation or cell division, but instead depletes NAD and NADP. Phylogenetic analyses and structural modeling show that this effector defines a new ART-related family of NAD(P) glycohydrolases, which we propose to name Tne5, broadly distributed across antagonistic systems.

microbiology↗

bioRxiv2025

Molecular bases for the loss of type VI secretion system activity during enteroaggregative E. coli experimental evolution

The type VI secretion system (T6SS) is a nanoweapon deployed by Gram-negative to inject effectors into target cells and hence involved in pathogenesis and bacterial competition. While T6SS gene clusters are found in all recently isolated commensals or pathogenic Escherichia coli strains, they are absent from classical laboratory strains. These E. coli strains, which have been used since decades for bacterial genetics, were usually grown in pure cultures, suggesting that T6SS might have been lost during evolution in absence of competitors. Here, we conducted a 640-generation experimental evolution by passaging the enteroaggregative E. coli (EAEC) 17-2 strain under controlled competition conditions against susceptible or immune recipient cells. EAEC T6SS activity was almost abolished when grown in the presence of immune recipients, while no difference with the ancestral strain was observed for EAEC grown in the presence of susceptible cells. Whole genome sequencing of 18 clones identified adaptative mutations responsible for the loss of T6SS activity, including mutations of the T6SS promoter and within genes encoding T6SS subunits, and revealed a novel regulatory mechanism involving RfaH-dependent antitermination. We further found that the RfaH binding site ops element is present in T6SS gene clusters of several species, suggesting a conserved RfaH-dependent regulation of T6SSs across enterobacteria. This work exemplifies the power of experimental evolution to understand T6SS genetic adaptation and to unravel new players for its function.

microbiology↗