Analyses of the vrl gene cluster in Desulfococcus multivorans: Homologous to the virulence-associated locus of the ovine footrot pathogen Dichelobacter nodosus strain A198

Major parts of the virulence-associated vrl locus known from the gammaproteobacterium Dichelobacter nodosus, the causative agent of ovine footrot, were analyzed in the genome of the sulfate-reducing deltaproteobacterium Desulfococcus multivorans. In the genome of D. multivorans 13 of the 19 vrl genes described for D. nodosus are present and highly conserved with respect to gene sequence and order. The vrl locus and its flanking regions suggest a bacteriophage-mediated transfer into the genome of D. multivorans. Comparative analysis of the deduced Vrl proteins reveals a wide distribution of parts of the virulence-associated vrl locus in distantly related bacteria. Horizontal transfer is suggested as driving mechanism for the circulation of the vrl genes in bacteria. Except for the vrlBMN genes D. multivorans and Desulfovibrio desulfuricans G20 together contain all vrl genes displaying a high degree of similarity. For D. multivorans it could be shown that guanine plus cytosine (GC) content, GC skew, di-, tri- or tetranucleotide distribution did not differ between the vrl locus and its flanking sequences. This could be a hint that the vrl locus originated from a related organism or at least a genome with similar characteristics. The conspicuous high degree of conservation of the analyzed vrl genes may result from a recent transfer event or reflect a function of the vrl genes, which is still unknown and not necessarily disease associated. The latter is supported by the evidence for expression of the vrl genes in D. multivorans, which has not been described as pathogen or to be associated to any disease pattern before

Identification of soluble protein fragments by gene fragmentation and genetic selection

We describe a new method, which identifies protein fragments for soluble expression in Escherichia coli from a randomly fragmented gene library. Inhibition of E. coli dihydrofolate reductase (DHFR) by trimethoprim (TMP) prevents growth, but this can be relieved by murine DHFR (mDHFR). Bacterial strains expressing mDHFR fusions with the soluble proteins green fluroscent protein (GFP) or EphB2 (SAM domain) displayed markedly increased growth rates with TMP compared to strains expressing insoluble EphB2 (TK domain) or ketosteroid isomerase (KSI). Therefore, mDHFR is affected by the solubility of fusion partners and can act as a reporter of soluble protein expression. Random fragment libraries of the transcription factor Fli1 were generated by deoxyuridine incorporation and endonuclease V cleavage. The fragments were cloned upstream of mDHFR and TMP resistant clones expressing soluble protein were identified. These were found to cluster around the DNA binding ETS domain. A selected Fli1 fragment was expressed independently of mDHFR and was judged to be correctly folded by various biophysical methods including NMR. Soluble fragments of the cell-surface receptor Pecam1 were also identified. This genetic selection method was shown to generate expression clones useful for both structural studies and antibody generation and does not require a priori knowledge of domain architecture

Small-scale, semi-automated purification of eukaryotic proteins for structure determination

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 μg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 μg/ml, and of purified selenomethione-labeled AIA–GFP (His8 removed by treatment with TEV protease) was 172 μg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10–50 ml) cell growth and automated purification. 1H–15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA–GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 Å. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination

Contourite depositional system after the exit of a strait: Case study from the late Miocene South Rifian Corridor, Morocco

Idealized facies of bottom current deposits (contourites) have been established for fine-grained contourite drifts in modern deep-marine sedimentary environments. Their equivalent facies in the ancient record however are only scarcely recognized due to the weathered nature of most fine-grained deposits in outcrop. Facies related to the erosional elements (i.e. contourite channels) of contourite depositional systems have not yet been properly established and related deposits in outcrop appear non-existent. To better understand the sedimentary facies and facies sequences of contourites, the upper Miocene contourite depositional systems of the South Rifian Corridor (Morocco) is investigated. This contourite depositional system formed by the dense palaeo-Mediterranean Outflow Water. Foraminifera assemblages were used for age-constraints (7.51 to 7.35 Ma) and to determine the continental slope depositional domains. Nine sedimentary facies have been recognized based on lithology, grain-size, sedimentary structures and biogenic structures. These facies were subsequently grouped into five facies associations related to the main interpreted depositional processes (hemipelagic settling, contour currents and gravity flows). The vertical sedimentary facies succession records the tectonically induced, southward migration of the contourite depositional systems and the intermittent behaviour of the palaeo-Mediterranean Outflow Water, which is mainly driven by precession and millennial-scale climate variations. Tides substantially modulated the palaeo-Mediterranean Outflow Water on a sub-annual scale. This work shows exceptional examples of muddy and sandy contourite deposits in outcrop by which a facies distribution model from the proximal continental slope, the contourite channel to its adjacent contourite drift, is proposed. This model serves as a reference for contourite recognition both in modern environments and the ancient record. Furthermore, by establishing the hydrodynamics of overflow behaviour a framework is provided that improves process-based interpretation of deep-water bottom current deposits

Palaeoenvironment of Eocene prodelta in Spitsbergen recorded by the trace fossil Phycosiphon incertum

Ichnological, sedimentological and geochemical analyses were conducted on the Eocene Frysjaodden Formation in order to interpret palaeoenvironment prodelta sediments in the Central Basin of Spitsbergen. Phycosiphon incertum is the exclusive ichnotaxon showing differences in size, distribution, abundance and density, and relation to laminated/bioturbated intervals. Large P. incertum mainly occur dispersed, isolated and randomly distributed throughout the weakly laminated/non-laminated intervals. Small P. incertum occur occasionally in patches of several burrows within laminated intervals or as densely packed burrows in thin horizons in laminated intervals or constituting fully bioturbated intervals that are several centimetres thick. Ichnological changes are mainly controlled by oxygenation, although the availability of benthic food cannot be discarded. Changes in oxygenation and rate of sedimentation can be correlated with the registered variations in the Bouma sequence of the distal turbiditic beds within prodeltal shelf sediments.Funding for this research was provided by Project CGL2012-33281 (Secretaría de Estado de Investigación, Desarrollo e Innovación, Spain), Project RYC-2009-04316 (Ramón y Cajal Programme) and Projects RNM-3715 and RNM-7408 and Research Group RNM-178 (Junta de Andalucía). The authors benefited from a bilateral agreement between the universities of Granada and Oslo, supported by the University of Granada

Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

BACKGROUND:The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS), resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface. METHODOLOGY:The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen. CONCLUSIONS:The results of this study provide evidence for the cooperative role of multiple virulence determinants in C. neoformans pathogenesis and suggest new avenues for the development of anti-infective agents in the prevention of fungal tissue invasion

Residues critical for formylglycine formation and/or catalytic activity of arylsulfatase A

Knaust A, Schmidt B, Dierks T, Bulow von R, Figura von K. Residues critical for formylglycine formation and/or catalytic activity of arylsulfatase A. BIOCHEMISTRY. 1998;37(40):13941-13946.Sulfatases contain a unique posttranslational modification in their active site, a formylglycine residue generated from a cysteine or a serine residue. The formylglycine residue is part of a sequence that is highly conserved among sulfatases, suggesting that it might direct the generation of this unique amino acid derivative. In the present study residues 68-86 flanking formylglycine 69 in arylsulfatase A were subjected to an alanine/glycine scanning mutagenesis. The mutants were analyzed for the conversion of cysteine 69 to formylglycine and their kinetic properties. Only cysteine 69 turned out to be essential for formation of the formylglycine residue, while substitution of leucine 68, proline 71, and alanine 74 within the heptapeptide LCTPSRA reduced the formylglycine formation to about 30-50%. Several residues that are part of or directly adjacent to an alpha-helix presenting the formylglycine 69 at the bottom of the active site pocket were found to be critical for catalysis. A surprising outcome of this study was that a number of residues fully or highly conserved between all known eukaryotic and prokaryotic sulfatases turned out to be essential neither for generation of formylglycine nor for catalysis