Directionality of substrate translocation of the hemolysin A Type I secretion System

Type 1 secretion systems (T1SS) of Gram-negative bacteria are responsible for the secretion of various proteases, lipases, S-layer proteins or toxins into the extracellular space. The paradigm of these systems is the hemolysin A (HlyA) T1SS of Escherichia coli. This multiple membrane protein complex is able to secrete the toxin HlyA in one step across both E. coli membranes. Common to all secreted T1SS substrates is a C-terminal secretion sequence being necessary as well as sufficient for secretion. However, it is not known whether transport occurs directionally, i.e. the N- or the C-terminus of T1SS substrates is secreted first. We have addressed this question by constructing HlyA fusions with the rapidly folding eGFP resulting in a stalled T1SS. Differential labeling and subsequent fluorescence microscopic detection of C- and N-terminal parts of the fusions allowed us to demonstrate vectorial transport of HlyA through the T1SS with the C-terminus appearing first outside the bacterial cells

Microbe sample from Escherichia coli

BioSample: SAMN11037806; Sample name: C41 delta ompF delta acrB; SRA: SRS442251

Improving the purification of membrane proteins from Escherichia coli C41(DE3)

The overexpression and purification of membrane proteins to high purity and homogeneity is a challenging task. Over time, several strains have been developed that decrease the toxic side-effects and thus result in higher bio mass and protein yield. However, two major contaminants have been identified in membrane protein preparations from E. coli: the outer membrane porin OmpF and AcrB, which is part of a tripartite efflux pump. Both proteins crystallise from low concentrations and diverse conditions, which make them a major problem, especially in membrane protein crystallography. In this study, we present a C41(DE3)-derived expression strains that is depleted of these two proteins

Deformation behavior of migmatites: insights from microstructural analysis of a garnet–sillimanite–mullite–quartz–feldspar-bearing anatectic migmatite at Rampura–Agucha, Aravalli–Delhi Fold Belt, NW India

In the present study we investigate the microstructural development in mullite, quartz and garnet in an anatectic migmatite hosted within a Grenvillian-age shear zone in the Aravalli–Delhi Fold Belt. The migmatite exhibits three main deformation structures and fabrics (S1, S2, S3). Elongated garnet porphyroblasts are aligned parallel to the metatexite S2 layers and contain crenulation hinges defined by biotite–sillimanite–mullite–quartz (with S1 axial planar foliation). Microstructural evidence and phase equilibrium relations establish the garnet as a peritectic phase of incongruent melting by breakdown of biotite, sillimanite ± mullite and quartz at peak P–T of ~ 8 kbar, 730 °C along a tight-loop, clockwise P–T path. Monazite dating establishes that the partial melting occurred between ~ 1000 and 870 Ma. The absence of subgrains and systematic crystal lattice distortions in these garnets despite their elongation suggests growth pseudomorphing pre-existing 3-D networks of S1 biotite aggregates rather than high-temperature crystal plastic deformation which is noted in the S1 quartz grains that exhibit strong crystallographic preferred orientation (CPO), undulatory extinction and subgrains. Mode-I fractures in these garnet porphyroblasts induced by high melt pressure during late stage of partial melt crystallization are filled by retrograde biotite–sillimanite. Weak CPO and non-systematic crystal lattice distortions in the coarse quartz grains within the S2 leucosome domains indicate these crystallized during melt solidification without later crystal plastic deformation overprint. In the later stages of deformation (D3), strain was mostly accommodated in the mullite–biotite–sillimanite-rich restite domains forming S3 which warps around garnet and leucosome domains; consequently, fine-grained S3 quartz does not exhibit strong CPOs