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Currently, due to the misuse of antibiotics, we are facing a major public health problem. The resistance to antibiotics of certain bacterial strains makes the treatment of infections very complex.
In this context, the present thesis project concerns the study of a bacterial efflux complex capable of transporting antibiotics from the cytoplasm to the outside of the cell. This complex is composed of an inner-membrane Major Facilitator Superfamily (MFS) transporter (EmrB, E. coli multidrug resistance), a channel of the outer membrane TolC (Tolerance to Colicin E1) and a periplasmic adapter (EmrA, E. coli multidrug resistance). Unlike RND-type efflux systems (such as AcrAB-TolC), little is known about the MFS-type EmrAB-TolC system. It is therefore important to study the entire complex on a structural and functional level, to analyse the marked differences between these two types of transport systems. The goal of my thesis project was to study at least one EmrAB-TolC complex from a structural point of view. For my studies the aim was to isolate the complex directly from bacteria overexpressing the three protein partners. In a first step, 15 homologous EmrAB-TolC systems were identified and their corresponding genes amplified from genomic DNA of different Gram-negative bacteria. Among the genes of the 15 systems, the genes coding for the E. coli and V. cholerae systems were further studied. The expression vectors encoded fluorescent markers for the monitoring of the expression levels of different proteins and for studying the formation of complexes. In a first step, the different protein expression levels (EmrB-mRFP1 and EmrA-sfGFP) were studied for several expression strains of E. coli by measuring the red and green fluorescence levels and by Western blot (anti-His, Myc, and Strep for EmrB, EmrA, and TolC). The E. coli strain C41(DE3) was best suited for co-expression of EmrAB-TolC. In a second step, the FSEC (Fluorescence detection Size Exclusion Chromatography) methodology was used to identify a complex suitable for structural study. Thus this method enabled the observation that the EmrAB-TolC complex of E. coli was produced in higher amount than that of V. cholerae. The final co-purification protocol consists in perfoming a gentle lysis of the bacteria using lysozyme, then after solubilization with DDM, the purification is started by a Ni2+-NTA affinity chromatography step followed by a size exclusion chromatography step. Finally, the fractions containing the three protein partners are used for the detergent-exchange by amphipol A8-35 before the structural study by electron microscopy. Negative stain EM-micrographs displayed elongated objects with a length of 33 nm in side view. An average image of EmrAB-TolC shows similarities to that of the AcrAB-TolC complex observed under similar conditions. Similarities included the characteristic densities of TolC. Whereas differences were found in the lower part of EmrAB which is thinner than the lower part of AcrAB. The densities visible above the amphipol-ring correspond to EmrA, which displays a channel-like structure as in AcrA. The channel however seems to extend further towards the amphipol belt. Since EmrB does not have an extended periplasmic domain as the RND proteins have, these densities are therefore solely assigned to EmrA. EmrA, on the other side, contacts TolC akin to the interaction of AcrA/MexA to their cognate outer membrane channels (TolC/OprM) in a ‘tip-to-tip’ fashion.
Cheilostome Bryozoa Anoteropora latirostris, a colonial marine invertebrate, constructs its skeleton from calcite and aragonite. This study presents firstly correlated multi-scale electron microscopy, micro-computed tomography, electron backscatter diffraction and NanoSIMS mapping. We show that all primary, coarse-grained platy calcitic lateral walls are covered by fine-grained fibrous aragonite. Vertical lateral walls separating autozooid chambers have aragonite only on their distal side. This type of asymmetric mineralization of lateral walls results from the vertical arrangement of the zooids at the growth margins of the colony and represents a type of biomineralization previously unknown in cheilostome bryozoans. NanoSIMS mapping across the aragonite-calcite interface indicates an organic layer between both mineral phases, likely representing an organic template for biomineralization of aragonite on the calcite layer. Analysis of crystallographic orientations show a moderately strong crystallographic preferred orientation (CPO) for calcite (7.4 times random orientation) and an overall weaker CPO for aragonite (2.4 times random orientation) with a high degree of twinning (45%) of the aragonite grains. The calculated Young’s modulus for the CPO map shows a weak mechanical direction perpendicular to the colony’s upper surface facilitating this organism’s strategy of clonal reproduction by fragmentation along the vertical zooid walls.
Nerve tissue contains a high density of chemical synapses, about 1 per µm3 in the mammalian cerebral cortex. Thus, even for small blocks of nerve tissue, dense connectomic mapping requires the identification of millions to billions of synapses. While the focus of connectomic data analysis has been on neurite reconstruction, synapse detection becomes limiting when datasets grow in size and dense mapping is required. Here, we report SynEM, a method for automated detection of synapses from conventionally en-bloc stained 3D electron microscopy image stacks. The approach is based on a segmentation of the image data and focuses on classifying borders between neuronal processes as synaptic or non-synaptic. SynEM yields 97% precision and recall in binary cortical connectomes with no user interaction. It scales to large volumes of cortical neuropil, plausibly even whole-brain datasets. SynEM removes the burden of manual synapse annotation for large densely mapped connectomes.