Xylella fastidiosa modulates exopolysaccharide polymer length and the dynamics of biofilm development with a ß-1,4-endoglucanase.

IMPORTANCE: It is well established that exopolysaccharide (EPS) is an integral structural component of bacterial biofilms necessary for assembly and maintenance of the three-dimensional architecture of the biofilm. However, the process and role of EPS turnover within a developing biofilm is not fully understood. Here, we demonstrated that Xylella fastidiosa uses a self-produced endoglucanase to enzymatically process its own EPS to modulate EPS polymer length. This enzymatic processing of EPS dictates the early stages of X. fastidiosa's biofilm development, which, in turn, affects its behavior in planta. A deletion mutant that cannot produce the endoglucanase was hypervirulent, thereby linking enzymatic processing of EPS to attenuation of virulence in symptomatic hosts, which may be a vestige of X. fastidiosa's commensal behavior in many of its other non-symptomatic hosts.

Single-Molecule Dynamics of SARS-CoV-2 5' Cap Recognition by Human eIF4F.

Coronaviruses initiate translation through recognition of the viral RNA 5' m 7 GpppA m cap by translation factor eIF4F. eIF4F is a heterotrimeric protein complex with cap-binding, RNA-binding, and RNA helicase activities. Modulating eIF4F function through cellular regulation or small-molecule inhibition impacts coronavirus replication, including for SARS-CoV-2. Translation initiation involves highly coordinated dynamics of translation factors with messenger or viral RNA. However, how the eIF4F subunits coordinate on the initiation timescale to define cap-binding efficiency remains incompletely understood. Here we report that translation supported by the SARS-CoV-2 5'-UTR is highly sensitive to eIF4A inhibition by rocaglamide. Through a single-molecule fluorescence approach that reports on eIF4E-cap interaction, we dissect how eIF4F subunits contribute to cap-recognition efficiency on the SARS-CoV-2 5' UTR. We find that free eIF4A enhances cap accessibility for eIF4E binding, but eIF4G alone does not change the kinetics of eIF4E-RNA interaction. Conversely, formation of the full eIF4F complex significantly alters eIF4E-cap interaction, suggesting that coordinated eIF4E and eIF4A activities establish the net eIF4F-cap recognition efficiency. Moreover, the eIF4F complex formed with phosphomimetic eIF4E(S209D) binds the viral UTR more efficiently than with wild-type eIF4E. These results highlight a dynamic interplay of eIF4F subunits and mRNA that determines cap-recognition efficiency.