Homologous recombination (HR) is a central process of genome biology driven by a conserved recombinase, which catalyses the pairing of single-stranded DNA (ssDNA) with double-stranded DNA to generate a D-loop intermediate. Bacterial RadA is a conserved HR effector acting with RecA recombinase to promote ssDNA integration. The mechanism of this RadA-mediated assistance to RecA is unknown. Here, we report functional and structural analyses of RadA from the human pathogen Streptococcus pneumoniae. RadA is found to facilitate RecA-driven ssDNA recombination over long genomic distances during natural transformation. RadA is revealed as a hexameric DnaB-type helicase, which interacts with RecA to promote orientated unwinding of branched DNA molecules mimicking D-loop boundaries. These findings support a model of DNA branch migration in HR, relying on RecA-mediated loading of RadA hexamers on each strand of the recipient dsDNA in the D-loop, from which they migrate divergently to facilitate incorporation of invading ssDNA.

Bacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is related to Myoviridae phages. It is composed of a phage tail‐like structure inserted in the bacterial cell envelope by a membrane complex (MC) comprising the TssJ, TssL and TssM proteins. We previously reported the low‐resolution negative‐stain electron microscopy structure of the enteroaggregative Escherichia coli MC and proposed a rotational 5‐fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo‐electron tomography analyses of the T6SS MC confirm the 5‐fold symmetry in situ and identify the regions of the structure that insert into the bacterial membranes. A high‐resolution model obtained by single‐particle cryo‐electron microscopy highlights new features: five additional copies of TssJ, yielding a TssJ:TssL:TssM stoichiometry of 3:2:2, an 11‐residue loop in TssM, protruding inside the lumen of the MC and constituting a functionally important periplasmic gate, and hinge regions. Based on these data, we propose an updated model on MC structure and dynamics during T6SS assembly and function.

Data relating to the structural basis of ligand recognition by integrins are limited. Here we describe the physical requirements for high affinity binding of ligands to αvβ6. By combining a series of structural analyses with functional testing, we show that 20-mer peptide ligands, derived from high affinity ligands of αvβ6 (foot-and-mouth-disease virus, latency associated peptide), have a common structure comprising an Arg-Gly-Asp motif at the tip of a hairpin turn followed immediately by a C-terminal helix. This arrangement allows two conserved Leu/Ile residues at Asp⁺¹ and Asp⁺⁴ to be presented on the outside face of the helix enabling a potential hydrophobic interaction with the αvβ6 integrin, in addition to the Arg-Gly-Asp interaction. The extent of the helix determines peptide affinity for αvβ6 and potency as an αvβ6 antagonist. A major role of this C-terminal helix is likely to be the correct positioning of the Asp⁺¹ and Asp⁺⁴ residues. These data suggest an explanation for several biological functions of αvβ6 and provide a structural platform for design of αvβ6 antagonists.

Acetaldehyde–alcohol dehydrogenase (AdhE) enzymes are a key metabolic enzyme in bacterial physiology and pathogenicity. They convert acetyl-CoA to ethanol via an acetaldehyde intermediate during ethanol fermentation in an anaerobic environment. This two-step reaction is associated to NAD⁺ regeneration, essential for glycolysis. The bifunctional AdhE enzyme is conserved in all bacterial kingdoms but also in more phylogenetically distant microorganisms such as green microalgae. It is found as an oligomeric form called spirosomes, for which the function remains elusive. Here, we use cryo-electron microscopy to obtain structures of Escherichia coli spirosomes in different conformational states. We show that spirosomes contain active AdhE monomers, and that AdhE filamentation is essential for its activity in vitro and function in vivo. The detailed analysis of these structures provides insight showing that AdhE filamentation is essential for substrate channeling within the filament and for the regulation of enzyme activity.