Structural insights into the membrane-extracted dimeric form of the ATPase TraB from the Escherichia coli pKM101 conjugation system

Background: Type IV secretion (T4S) systems are involved in secretion of virulence factors such as toxins or transforming molecules, or bacterial conjugation. T4S systems are composed of 12 proteins named VirB1-B11 and VirD4. Among them, three ATPases are involved in the assembly of the T4S system and/or provide energy for substrate transfer, VirB4, VirB11 and VirD4. The X-ray crystal structures of VirB11 and VirD4 have already been solved but VirB4 has proven to be reluctant to any structural investigation so far. Results: Here, we have used small-angle X-ray scattering to obtain the first structural models for the membrane-extracted, dimeric form of the TraB protein, the VirB4 homolog encoded by the E. coli pKM101 plasmid, and for the monomeric soluble form of the LvhB4 protein, the VirB4 homolog of the T4S system encoded by the Legionella pneumophila lvh operon. We have obtained the low resolution structures of the full-length TraB and of its N- and C-terminal halves. From these SAXS models, we derive the internal organisation of TraB. We also show that the two TraB N- and C-terminal domains are independently involved in the dimerisation of the full-length protein. Conclusions: These models provide the first structural insights into the architecture of VirB4 proteins. In particular, our results highlight the modular arrangement and functional relevance of the dimeric-membrane-bound form of TraB

Crystal Structure of the P Pilus Rod Subunit PapA

P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE) mechanism, whereby the chaperone's G1 β-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it. Author Summary. Bacterial adhesion to a host is a crucial step that determines the onset of bacterial infection. It is mediated through recognition of a receptor on the host cell surface by a protein called an adhesin displayed on the surface of the bacterium. Many adhesins are displayed at the tip of specialized organelles called pili, some of which are assembled by the ubiquitous chaperone-usher pathway. In this pathway, each pilus subunit is assisted in folding by a chaperone. The resulting chaperone-subunit complex is targeted to a pore located in the outer membrane, called the usher, that serves as assembly platform. There, pilus subunits dissociate from the chaperone and polymerize, resulting in a surface organelle, the pilus, that protrudes out of the usher. Here, we have determined the structure of the major subunit of the P pilus, PapA. The P pilus, produced in uropathogenic Escherichia coli, displays the adhesin PapG responsible for targeting the bacterium to the kidney epithelium. We have determined the structure of PapA either bound to its cognate chaperone, PapD, or bound to another PapA subunit. These structures provide a view of PapA before and after its assembly in the pilus and shed light on the mechanism of PapA assembly.National Institutes of Health (DE 09761, GM040388, DE 09161); Committee of Scientific Research (3 PO4A 003 24, 2 P05A 137 24); Foundation for Polish Science (SUBSYDIUM PROFESORSKIE award); Swedish Rheumatism Association; Nanna Svartz Foundation; King Gustaf V Foundatio

Comparative determination of miconazole, nystatin, hydrocortisone and neomycin by HPTLC/HPLC-DAD

Two new validated methods for the quantification of miconazole (MIC), nystatin (NYS), hydrocortisone acetate (HCA) and neomycin (NS) by high performance thin layer chromatography (HPTLC)-densitometry and reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector (RP-HPLC-DAD) were developed and compared. HPTLC separations were performed using a mobile phase, ethylacetate:methanol:glacial acetic acid (60:40:0.4, v:v:v) for MIC, NYS, HCA and ethylacetate for NS. For HPLC-DAD analysis, using an isocratic elution system, separation of all compounds was achieved. Good resolution and quantization were achieved, accuracy and precision, as well as detection and quantitation limits of the two methods, were evaluated and compared. Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9997. HPTLC limits of quantitation were 15.37×10-2, 9.8×10-2, 13.32×10-2 and 15.19×10-2 µg/mL for MIC, NYS, HCA and NS respectively, whereas HPLC limits were 6.80×10-2, 6.56×10-2, 1×10-1 and 10.16×10-2 µg/band for MIC, NYS, HCA and NS respectively. In comparison with HPLC, HPTLC is less expensive and faster procedure, requiring 2-3 h to analyse 10-12 samples on a single plate. The developed methods were proved to be specific, robust and accurate for the determination of cited drugs in pharmaceutical preparations

Techniques for the realization of ultrareliable spaceborne computers Interim scientific report

Reliability tests on spaceborne digital computer

The ways of the lustre: looking for the tunisian connection

Recent excavations at the Fatimid and Zirid site of Sabra al-Mansuriya near Kairouan (Tunisia) provide the first evidence of lustreware production in medieval Ifriqiya, in the 10th–11th centuries AD. As the Fatimid dynasty moved from Ifriqiya to Egypt to establish its capital in Fustat (Cairo), technological connections with the Egyptian lustreware could be expected. Tunisian lustreware may also be the link in the transmission of the technique towards Muslim Spain. It represents a new piece of the puzzle of understanding the diffusion of lustre technology from the East to the West of the Mediterranean. The composition and microstructure of the bodies and glazes, and the micro- and nano-structure of the lustre layer are compared in a preliminary approach to the technological relationships between Tunisian, Egyptian, and Spanish lustrewares.Peer ReviewedPostprint (published version

Structural and molecular biology of a protein-polymerizing nanomachine for pilus biogenesis

Bacteria produce protein polymers on their surface called pili or fimbriae that serve either as attachment devices or as conduits for secreted substrates. This review will focus on the chaperone–usher pathway of pilus biogenesis, a widespread assembly line for pilus production at the surface of Gram-negative bacteria and the archetypical protein-polymerizing nanomachine. Comparison with other nanomachines polymerizing other types of biological units, such as nucleotides during DNA replication, provides some unifying principles as to how multidomain proteins assemble biological polymers

Emma Baulch, Making Scenes: Reggae, Punk, and Death Metal in 1990s Bali

I should start this review with a caveat: I am not an expert on the music of Bali or of Southeast Asia more generally. My review of Emma Baulch’s Making Scenes, then, is written from the point of view of a reader less interested in the area studies component of the book than in its usefulness as a case study concerning the musical genres under discussion, especially death metal and punk. On this score, Baulch’s book stands out as one of relatively few that takes the relationship between disti..

Learner Persistence in English for Speakers of Other Languages (ESOL) Programs

Persistence – or continued, intense study – is a common challenge for adults in English for Speakers of Other Languages (ESOL) programs because of various institutional, situational, and dispositional factors. The current state and federal funding standards for adult ESOL programs are driven by human capital theory, and therefore most funders require demonstrated employment outcomes for students. These top-down objectives do not always align with English learners’ own motivations and goals. ESOL organizations must consider the complexities of these interacting forces to develop effective persistence strategies for their constituents. This case study of an ESOL organization in central Massachusetts is based on information from an English learner focus group, an educator focus group, an anonymous survey, and a quantitative analysis of attendance data. Two major findings emerged from the data. First, English learners’ social context and educational experiences are inseparable, and a special focus must be given to the influence of family and life stage on a student’s educational path. Second, the conflicting priorities of funders, organizations, teachers, and English learners are evident in the classroom. At the organizational level, several changes to instructional strategies and allocation of resources have been recommended to promote learner persistence. Policymakers must reconsider the purposes and desired outcomes of adult ESOL programs when shaping funding standards

Donor-strand exchange in chaperone-assisted pilus assembly revealed in atomic detail by molecular dynamics

Adhesive multi-subunit fibres are assembled on the surface of many pathogenic bacteria via the chaperone-usher pathway. In the periplasm, a chaperone donates a β-strand to a pilus subunit to complement its incomplete immunoglobulin-like fold. At the outer membrane, this is replaced with a β-strand formed from the N-terminal extension (Nte) of an incoming pilus subunit by a donorstrand exchange (DSE) mechanism. This reaction has previously been shown to proceed via a concerted mechanism, in which the Nte interacts with the chaperone:subunit complex before the chaperone has been displaced, forming a ternary intermediate. Thereafter, the pilus and chaperone β-strands have been postulated to undergo a strand swap by a ‘zip-in-zip-out’ mechanism, whereby the chaperone strand zips out, residue by residue, as the Nte simultaneously zips in. Here, molecular dynamics simulations have been used to probe the DSE mechanism during formation of the Salmonella enterica Saf pilus at an atomic level, allowing the direct investigation of the zip-inzip- out hypothesis. The simulations provide an explanation of how the incoming Nte is able to dock and initiate DSE due to inherent dynamic fluctuations within the chaperone:subunit complex. The chaperone donor-strand is shown to unbind from the pilus subunit residue by residue, in direct support of the zip-in-zip-out hypothesis. In addition, an interaction of a residue towards the Nterminus of the Nte with a specific binding pocket (P*) on the adjacent pilus subunit is shown to stabilise the DSE product against unbinding, which also proceeds by a zippering mechanism. Together, the study provides an in-depth picture of DSE, including the first insights into the molecular events occurring during the zip-in-zip-out mechanism