Furusawa, G. | Search bioRxiv

bioRxiv2024

Mechanism of bacterial predation via ixotrophy

Predation allows bacteria to access alternative substrates in low-nutrient conditions. Ixotrophy has been proposed as a predatory lifestyle of multicellular filamentous bacteria in aquatic environments; however, the molecular mechanism remains unknown. Here we uncover by a multidisciplinary approach that ixotrophy requires the interplay of multiple cellular machineries and a regulatory mechanism. Attacker-prey contacts are established by gliding motility and extracellular grappling hook-like structures that bind prey flagella. Cryo-electron microscopy identifies the grappling hooks as a heptameric assembly of a Type 9 Secretion System substrate. Cryo-electron tomography and functional assays show that killing is mediated by puncturing of the prey cell using a Type 6 Secretion System, possibly triggered by extracellular antennae. Single-cell analyses with stable isotope-labeled prey demonstrate that prey components are taken up by the attacker. Depending on nutrient availability, ixotrophy is switched off by endogenous Insertion Sequence Elements and re-activated through their excision. A marine metagenomic time series provides evidence for coupled dynamics of ixotrophic bacteria and their prey. Our study reveals the complex mechanism of a conserved microbial predatory lifestyle and indicates the need for its regulation in conditions where the expression of costly pathways is dispensable.

microbiology↗

bioRxiv2017

Characterisation and modelling of a polyhydroxyalkanoate synthase from Aquitalea sp. USM4 reveals a mechanism for polymer elongation

Polyhydroxyalkanoate synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace conventional petroleum-based plastics. While two PhaC structures have been determined recently, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. The PhaC from Aquitalea sp. USM4, PhaCAq, was characterised and showed a Km of 394 {micro}M and a kcat of 476.4 s-1 on the 3HB-CoA substrate. A model based on the structure of the closely related PhaC from Cupriavidus necator, PhaCCn revealed a three-branched tunnel at the dimeric interface. Two of the branches open to the solvent and serve as the putative routes for substrate entrance and product exit, while the third branch is elongated in a PhaC1 model from Pseudomonas aeruginosa, indicating a function of accommodating the hydroxyalkanoate (HA) moiety of the HA-CoA substrate. Docking of the two tetrahedral intermediates formed during catalysis suggests a PHA elongation mechanism that requires the HA moiety of the ligand to rotate ~180{degrees}. Both classes I and II PhaCs share a common mechanism for polymer elongation, and substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates. The PhaCAq model provides fresh insights into a general PhaC mechanism, pinpointing key residues for potential engineering of PhaCs with desirable characteristics.

molecular biology↗