Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance.DOI: http://dx.doi.org/10.7554/eLife.00792.001

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Summary The assembly of the type III secretion injectisome culminates in the formation of the needle. In Yersinia, this step requires not only the needle subunit (YscF), but also the small components YscI, YscO, YscX and YscY. We found that these elements act after the completion of the transmembrane export apparatus. YscX and YscY co‐purified with the export apparatus protein YscV, even in the absence of any other protein. YscY‐EGFP formed fluorescent spots, suggesting its presence in multiple copies. YscO and YscX were required for export of the early substrates YscF, YscI and YscP, but were only exported themselves after the substrate specificity switch had occurred. Unlike its flagellar homologue FliJ, YscO was not required for the assembly of the ATPase YscN. Finally, we investigated the role of the small proteins in export across the inner membrane. No export of the reporter substrate YscP1–137‐PhoA into the periplasm was observed in absence of YscI, YscO or YscX, confirming that these proteins are required for export of the first substrates. In contrast, YscP1–137‐PhoA accumulated in the periplasm in the absence of YscF, suggesting that YscF is not required for the function of the export apparatus, but that its polymerization opens the secretin YscC.

Abbreviations:YscU C = C-terminal domain of YscU comprising amino acids 211-354 T3SS = type III secretion system Wiesand et al., 2008 Structure of YscU 2 AbstractThe inner-membrane protein YscU has an important role during the assembly of the

The crystal structure of the dimeric O‐acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 Å resolution by X‐ray diffraction analysis. The C1‐carboxylate of citrate was bound at the carboxylate position of O‐acetylserine, whereas the C6‐carboxylate adopted two conformations. The activity of the enzyme and of several active center mutants was determined using an assay based on O‐acetylserine and thio‐nitrobenzoate (TNB). The unnatural substrate TNB was modeled into the reported structure. The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spectrum of the enzyme. A comparison of the reported structure with previously published CysM structures revealed large conformational changes. One of the crystal forms contained two dimers, each of which comprised one subunit in a closed and one in an open conformation. Although the homodimer asymmetry was most probably caused by crystal packing, it indicates that the enzyme can adopt such a state in solution, which may be relevant for the catalytic reaction.