Recent progress in biohydrometallurgy and microbial characterisation

Since the discovery of microbiological metal dissolution, numerous biohydrometallurgical approaches have been developed to use microbially assisted aqueous extractive metallurgy for the recovery of metals from ores, concentrates, and recycled or residual materials. Biohydrometallurgy has helped to alleviate the challenges related to continually declining ore grades by transforming uneconomic ore resources to reserves. Engineering techniques used for biohydrometallurgy span from above ground reactor, vat, pond, heap and dump leaching to underground in situ leaching. Traditionally biohydrometallurgy has been applied to the bioleaching of base metals and uranium from sulfides and biooxidation of sulfidic refractory gold ores and concentrates before cyanidation. More recently the interest in using bioleaching for oxide ore and waste processing, as well as extracting other commodities such as rare earth elements has been growing. Bioprospecting, adaptation, engineering and storing of microorganisms has increased the availability of suitable biocatalysts for biohydrometallurgical applications. Moreover, the advancement of microbial characterisation methods has increased the understanding of microbial communities and their capabilities in the processes. This paper reviews recent progress in biohydrometallurgy and microbial characterisatio

Klebsiella aerogenes adhesion behaviour during biofilm formation on monazite

The adsorption behaviour of micro-organisms during the initial attachment stage of biofilm formation affects subsequent stages. The available area for attachment and the chemophysical properties of a surface affect microbial attachment performance. This study focused on the initial attachment behaviour of Klebsiella aerogenes on monazite by measuring the ratio of planktonic against sessile subpopulations (P:S ratio), and the potential role of extracellular DNA (eDNA). eDNA production, effects of physicochemical properties of the surface, particle size, total available area for attachment, and the initial inoculation size on the attachment behaviour were tested. K. aerogenes attached to monazite immediately after exposure to the ore; however, the P:S ratio significantly (p = 0.05) changed in response to the particle size, available area, and inoculation size. Attachment occurred preferentially on larger-sized (~50 µm) particles, and either decreasing the inoculation size or increasing the available area further promoted attachment. Nevertheless, a portion of the inoculated cells always remained in a planktonic state. K. aerogenes produced lower eDNA in response to the changed surface chemical properties when monazite was replaced by xenotime. Using pure eDNA to cover the monazite surface significantly (p ≤ 0.05) hindered bacterial attachment due to the repulsive interaction between the eDNA layer and bacteria

Model-Based Security Testing

Security testing aims at validating software system requirements related to security properties like confidentiality, integrity, authentication, authorization, availability, and non-repudiation. Although security testing techniques are available for many years, there has been little approaches that allow for specification of test cases at a higher level of abstraction, for enabling guidance on test identification and specification as well as for automated test generation. Model-based security testing (MBST) is a relatively new field and especially dedicated to the systematic and efficient specification and documentation of security test objectives, security test cases and test suites, as well as to their automated or semi-automated generation. In particular, the combination of security modelling and test generation approaches is still a challenge in research and of high interest for industrial applications. MBST includes e.g. security functional testing, model-based fuzzing, risk- and threat-oriented testing, and the usage of security test patterns. This paper provides a survey on MBST techniques and the related models as well as samples of new methods and tools that are under development in the European ITEA2-project DIAMONDS.Comment: In Proceedings MBT 2012, arXiv:1202.582

Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis

We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch–related process, and propose that Prk1p negatively regulates the actin assembly–stimulating activity of endocytic proteins

Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis

During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis

Vesicular Stomatitis Virus Enters Cells through Vesicles Incompletely Coated with Clathrin That Depend upon Actin for Internalization

Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical clathrin-coated vesicle. The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain. Here we examined how clathrin-coated vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially clathrin-coated vesicles. We found that on average, virus-containing vesicles contain more clathrin and clathrin adaptor molecules than conventional vesicles, but this increase is insufficient to permit full coating of the vesicle. We further show that virus-containing vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing vesicles, and chemical inhibition of actin polymerization trapped viral particles in vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery

Differential requirements for actin during yeast and mammalian endocytosis

Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination

The Mechanochemistry of Endocytosis

An integrated theoretical model reveals how the chemical and the mechanical aspects of endocytosis are coordinated coherently in yeast cells, driving progression through the endocytic pathway and ensuring efficient vesicle scission in vivo