Structure of a bacterial type IV secretion core complex at subnanometre resolution

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane-spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O-layer inserted in the outer membrane and the I-layer inserted in the inner membrane. While the structure of the O-layer has been solved by X-ray crystallography, there is no detailed structural information on the I-layer. Using high-resolution cryo-electron microscopy and molecular modelling combined with biochemical approaches, we determined the I-layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived

Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones

Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for,10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e. g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 A crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5

Bond-randomness-induced Neel order in weakly coupled antiferromagnetic spin chains

Quasi-one-dimensional antiferromagnetic (AF) quantum spin systems show a wide range of interesting phenomena such as the spin-Peierls transition and disorder driven long range ordering. While there is no magnetic long range order in strictly one-dimensional systems, in real systems some amount of interchain coupling is always present and AF long range order may appear below a Neel ordering temperature T_N. We study the effect of bond randomness on Neel ordering in weakly coupled random AF S=1/2 chains both with and without dimerization (or spin-Peierls order). We use the real space renormalization group method to tackle the intrachain couplings, and a mean-field approximation to treat the interchain coupling. We show that in the non-dimerized chain, disorder (represented by bond randomness) enhances the Neel order parameter; in the dimerized chain which shows no magnetic ordering in the weak interchain coupling limit without randomness, disorder can actually lead to long range order. Thus disorder is shown to lead to, or enhance the tendency toward long range order, providing another example of the order-by-disorder phenomenon. We make a qualitative comparison of our results with the observed phenomenon of doping induced long range ordering in quasi-one-dimensional spin systems such as CuGeO_3.Comment: 8 pages, 4 figure

Magnetic Resonance of the Intrinsic Defects of the Spin-Peierls Magnet CuGeO3

ESR of the pure monocrystals of CuGeO3 is studied in the frequency range 9-75 GHz and in the temperature interval 1.2-25 K. The splitting of the ESR line into several spectral components is observed below 5 K, in the temperature range where the magnetic susceptibility is suppressed by the spin-Peierls dimerization. The analysis of the magnetic resonance signals allows one to separate the signals of the S=1/2- and S=1 defects of the spin-Peierls phase. The value of g-factor of these signals is close to that of the Cu-ion. The additional line of the magnetic resonance is characterized by an anomalous value of the g-factor and by the threshold-like increase of the microwave susceptibility when the microwave power is increasing. The ESR signals are supposingly attributed to two types of the planar magnetic defects, arising at the boundaries of the domains of the spin-Peierls state with the different values of the phase of the dimerization.Comment: LATEX-text, 12 PS-figures, typos corrected, LATEX-style change

Étude du composé spin-Peierls CuGeO₃ DOPÉ

La physique des systèmes unidimensionnels étant très riche et sa description théorique relativement accessible, de nombreux systèmes appartenant à cette famille sont étudiés pour leurs propriétés exotiques. Dans les métaux, par exemple, la transition de Peierls transforme un métal en un isolant, d'autre part, dans les systèmes magnétiques, les effets quantiques des interactions entre les spins conduisent à de nombreuses transitions de phase. Notamment, les matériaux isolants antiferromagnétiques quasi unidimensionnels de spin S=½ (chaîne de spins ½) présentent, lorsqu'on diminue la température, une transition du type magnéto-élastique, une dimérisation de la chaîne, due aux interactions à une dimension entre les spins ½ de la chaîne et les phonons du réseau tridimensionnel. Cette transition est appelée transition spin-Peierls par analogie avec la transition de Peierls. En effet, lors de cette transition, il y a formation d'un gap magnétique entre l'état fondamental et le triplet d'états excités le plus bas en énergie. Les calculs théoriques prévoient que la transition spin-Peierls a des effets importants sur plusieurs propriétés physiques. La susceptibilité connaît une discontinuité à la température de transition Tsp, puis une décroissance exponentielle pour finalement tendre vers zéro lorsque T tend vers zéro. La chaleur spécifique présente elle aussi une anomalie caractéristique lors de la transition. Enfin, la diffraction de rayons X montre l'apparition de nouvelles réflexions de Bragg correspondant à un doublement de la dimension de la cellule unité le long des chaînes en dessous de Tsp (Bray, Interrante, Jacobs et Bonner, 1982)

Suppression of type 1 pilus assembly in uropathogenic Escherichia coli by chemical inhibition of subunit polymerization

OBJECTIVES: To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). METHODS: Using an SDS-PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone-usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined. RESULTS: N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili. CONCLUSIONS: We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone-usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens

Curr Issues Mol Biol

In this review we examine the use of secretion systems by bacteria to subvert host functions. Bacteria have evolved multiple systems to interact with and overcome their eukaryotic host and other prokaryotes. Secretion systems are required for the release of several effectors through the bacterial membrane(s) into the extracellular space or directly into the cytoplasm of the host. We review the secretion systems of Gram-positive and Gram-negative bacteria and describe briefly the structural composition of the seven secretion systems that have been associated with increased virulence through subversion of host functions. Some of the effects of such systems on eukaryotic host processes have been studied extensively. We also describe the best-characterized effectors of each secretion system to give an overview of the molecular mechanisms employed by bacteria to hide from the immune system and convert eukaryotic cells into optimal ecological niches for their replication

Structural studies of T4S systems by electron microscopy

Abstract: Type IV secretion (T4S) systems are large dynamic nanomachines that transport DNA and/or proteins through the membranes of bacteria. Analysis of T4S system architecture is an extremely challenging task taking into account their multi protein organisation and lack of overall global symmetry. Nonetheless the last decade demonstrated an amazing progress achieved by X-ray crystallography and cryo-electron microscopy. In this review we present a structural analysis of this dynamic complex based on recent advances in biochemical, biophysical and structural studies

EMBO J

Some DNA helicases play central and specific roles in genome maintenance and plasticity through their branch migration activity in different pathways of homologous recombination. RadA is a highly conserved bacterial helicase involved in DNA repair throughout all bacterial species. In Gram-positive Firmicutes, it also has a role in natural transformation, while in Gram-negative bacteria, ComM is the canonical transformation-specific helicase. Both RadA and ComM helicases form hexameric rings and use ATP hydrolysis as an energy source to propel themselves along DNA. In this study, we present the cryoEM structures of RadA and ComM interacting with DNA and ATP analogs. These structures reveal important molecular interactions that couple ATP hydrolysis and DNA binding in RadA, as well as the role of the Lon protease-like domain, shared by RadA and ComM, in this process. Taken together, these results provide new molecular insights into the mechanisms of DNA branch migration in different pathways of homologous recombination