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Several serious gastrointestinal diseases, which are widespread all over the world, are caused by enteropathogenic Escherichia coli . The monomeric autotransporter AIDA-I ( adhesin involved in diffuse adherence ) represents an important virulence factor of these strains and is involved in adhesion, biofilm formation, aggregation and invasion into host cells. Here, we present the crystal structure of the transport unit of AIDA-I at 3.0 A resolution, which forms a 12-stranded β-barrel harboring the linker domain in its pore. Mutagenesis studies of the C-terminal amino acid demonstrated the great impact of this terminal residue on membrane integration of AIDA-I and passenger translocation.

The prokaryotic immune system, CRISPR, confers an adaptive and inheritable defense mechanism against invasion by mobile genetic elements. Guided by small CRISPR RNAs (crRNAs), a diverse family of CRISPR-associated (Cas) proteins mediates the targeting and inactivation of foreign DNA. Here, we demonstrate that Csn2, a Cas protein likely involved in spacer integration, forms a tetramer in solution and structurally possesses a ring-like structure. Furthermore, co-purified Ca 2+ was found important for the DNA binding property of Csn2, which contains a helicase fold, with highly conserved DxD and RR motifs found throughout Csn2 proteins. We could verify that Csn2 binds ds-DNA. In addition molecular dynamics simulations suggested a Csn2 conformation that can “sit” on the DNA helix and binds DNA in a groove on the outside of the ring.

Bacillus subtilis can synthesize the compatible solute glycine betaine as an osmoprotectant from an exogenous supply of the precursor choline. Import of choline is mediated by two osmotically inducible ABC transport systems: OpuB and OpuC. OpuC catalyzes the import of various osmoprotectants, whereas OpuB is highly specific for choline. OpuBC is the substrate-binding protein of the OpuB transporter, and we have analyzed the affinity of the OpuBC/choline complex by intrinsic tryptophan fluorescence and determined a K(d) value of about 30 μM. The X-ray crystal structure of the OpuBC/choline complex was solved at a resolution of 1.6 Å and revealed a fold typical of class II substrate-binding proteins. The positively charged trimethylammonium head group of choline is wedged into an aromatic cage formed by four tyrosine residues and is bound via cation-pi interactions. The hydroxyl group of choline protrudes out of this aromatic cage and makes a single interaction with residue Gln19. The substitution of this residue by Ala decreases choline binding affinity by approximately 15-fold. A water network stabilizes choline within its substrate-binding site and promotes indirect interactions between the two lobes of the OpuBC protein. Disruption of this intricate water network by site-directed mutagenesis of selected residues in OpuBC either strongly reduces choline binding affinity (between 18-fold and 25-fold) or abrogates ligand binding. The crystal structure of the OpuBC/choline complex provides a rational for the observed choline specificity of the OpuB ABC importer in vivo and explains its inability to catalyze the import of glycine betaine into osmotically stressed B. subtilis cells.

The ABC transporter (ATP-binding-cassette transporter) OpuA is one of five membrane transport systems in Bacillus subtilis that mediate osmoprotection by importing compatible solutes. Just like all bacterial and archaeal ABC transporters that catalyse the import of substrates, OpuA (where Opu is osmoprotectant uptake) is composed of an ATPase subunit (OpuAA), a transmembrane subunit (OpuAB) and an extracellular substrate-binding protein (OpuAC). In contrast with many well-known ABC-ATPases, OpuAA is composed not only of a catalytic and a helical domain but also of an accessory domain located at its C-terminus. The paradigm of such an architecture is MalK, the ABC-ATPase of the maltose importer of Escherichia coli, for which detailed structural and functional information is available. In the present study, we have applied solution FRET (Förster resonance energy transfer) techniques using two single cysteine mutants to obtain initial structural information on the architecture of the OpuAA dimer in solution. Analysing our results in detail and comparing them with the existing MalK structures revealed that the catalytic and helical domains adopted an arrangement similar to those of MalK, whereas profound differences in the three-dimensional orientation of the accessory domain, which contains two CBS (cystathionine β-synthetase) domains, were observed. These results shed new light on the role of this accessory domain present in a certain subset of ABC-ATPase in the fine-tuning of three-dimensional structure and biological function.

The adhesin involved in diffuse adherence (AIDA-I) from Escherichia coli belongs to the group of autotransporters, specifically the type Va secretion system (T5aSS). All autotransporter systems contain a C-terminal β-domain, which forms a barrel-like structure in the outer membrane with a hydrophilic pore allowing passenger translocation across the outer membrane. The passenger domain harbours the biological activity in the extracellular space and functions, for example, as an adhesin, an enzyme and a toxin. The exact transport mechanism of passenger translocation across the outer membrane is not clear at present. Thus, structure determination of the transport unit of AIDA-I could provide new insights into the transport mechanism. Here, the purification, crystallization and preliminary X-ray crystallographic studies of the transport unit of AIDA-I are reported.

International audience; The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB.