Isolation and characterisation of KP34—a novel φKMV-like bacteriophage for Klebsiella pneumoniae

Bacteriophage KP34 is a novel virus belonging to the subfamily Autographivirinae lytic for extended-spectrum β-lactamase-producing Klebsiella pneumoniae strains. Its biological features, morphology, susceptibility to chemical and physical agents, burst size, host specificity and activity spectrum were determined. As a potential antibacterial agent used in therapy, KP34 molecular features including genome sequence and protein composition were examined. Phylogenetic analyses and clustering of KP34 phage genome sequences revealed its clear relationships with “phiKMV-like viruses”. Simultaneously, whole-genome analyses permitted clustering and classification of all phages, with completely sequenced genomes, belonging to the Podoviridae

Arsenic and Antimony Transporters in Eukaryotes

Arsenic and antimony are toxic metalloids, naturally present in the environment and all organisms have developed pathways for their detoxification. The most effective metalloid tolerance systems in eukaryotes include downregulation of metalloid uptake, efflux out of the cell, and complexation with phytochelatin or glutathione followed by sequestration into the vacuole. Understanding of arsenic and antimony transport system is of high importance due to the increasing usage of arsenic-based drugs in the treatment of certain types of cancer and diseases caused by protozoan parasites as well as for the development of bio- and phytoremediation strategies for metalloid polluted areas. However, in contrast to prokaryotes, the knowledge about specific transporters of arsenic and antimony and the mechanisms of metalloid transport in eukaryotes has been very limited for a long time. Here, we review the recent advances in understanding of arsenic and antimony transport pathways in eukaryotes, including a dual role of aquaglyceroporins in uptake and efflux of metalloids, elucidation of arsenic transport mechanism by the yeast Acr3 transporter and its role in arsenic hyperaccumulation in ferns, identification of vacuolar transporters of arsenic-phytochelatin complexes in plants and forms of arsenic substrates recognized by mammalian ABC transporters

Disentangling genetic and epigenetic determinants of ultrafast adaptation

A major rationale for the advocacy of epigenetically mediated adaptive responses is that they facilitate faster adaptation to environmental challenges. This motivated us to develop a theoretical–experimental framework for disclosing the presence of such adaptation‐speeding mechanisms in an experimental evolution setting circumventing the need for pursuing costly mutation–accumulation experiments. To this end, we exposed clonal populations of budding yeast to a whole range of stressors. By growth phenotyping, we found that almost complete adaptation to arsenic emerged after a few mitotic cell divisions without involving any phenotypic plasticity. Causative mutations were identified by deep sequencing of the arsenic‐adapted populations and reconstructed for validation. Mutation effects on growth phenotypes, and the associated mutational target sizes were quantified and embedded in data‐driven individual‐based evolutionary population models. We found that the experimentally observed homogeneity of adaptation speed and heterogeneity of molecular solutions could only be accounted for if the mutation rate had been near estimates of the basal mutation rate. The ultrafast adaptation could be fully explained by extensive positive pleiotropy such that all beneficial mutations dramatically enhanced multiple fitness components in concert. As our approach can be exploited across a range of model organisms exposed to a variety of environmental challenges, it may be used for determining the importance of epigenetic adaptation‐speeding mechanisms in general.publishedVersio

Multiple cysteine residues are necessary for sorting and transport activity of the arsenite permease Acr3p from Saccharomyces cerevisiae

AbstractThe yeast transporter Acr3p is a low affinity As(III)/H+ and Sb(III)/H+ antiporter located in the plasma membrane. It has been shown for bacterial Acr3 proteins that just a single cysteine residue, which is located in the middle of the fourth transmembrane region and conserved in all members of the Acr3 family, is essential for As(III) transport activity. Here, we report a systematic mutational analysis of all nine cysteine residues present in the Saccharomyces cerevisiae Acr3p. We found that mutagenesis of highly conserved Cys151 resulted in a complete loss of metalloid transport function. In addition, lack of Cys90 and Cys169, which are conserved in eukaryotic members of Acr3 family, impaired Acr3p trafficking to the plasma membrane and greatly reduced As(III) efflux, respectively. Mutagenesis of five other cysteines in Acr3p resulted in moderate reduction of As(III) transport capacities and sorting perturbations. Our data suggest that interaction of As(III) with multiple thiol groups in the yeast Acr3p may facilitate As(III) translocation across the plasma membrane

Data from: Metal tolerance protein MTP6 affects mitochondrial iron and manganese homeostasis in cucumber

Members of the cation diffusion facilitator family have been identified in all kingdoms of life. They have been divided into three subgroups – Zn-CDF, Fe/Zn-CDF and Mn-CDF – based on their putative specificity to transported metal ions. The plant metal tolerance 6 (MTP6) proteins fall into the Fe/Zn-CDF subgroup, however their function in iron/zinc transport has not been confirmed yet. Here we characterize the MTP6 protein from cucumber. When expressed in yeast and protoplasts isolated from A. thaliana cells, CsMTP6 localized in mitochondria and contributed to the efflux of iron and manganese from the mitochondria. The immunolocalization of CsMTP6 in cucumber membranes confirmed that CsMTP6 is associated with mitochondria. The root expression and protein level of CsMTP6 were significantly up-regulated in conditions of iron deficiency and iron excess but were not affected by Mn availability. These results indicate that plant MTP6 proteins contribute to the distribution of iron and manganese between the cytosol and mitochondria of plant cells and are regulated by Fe to maintain the mitochondrial and cytosolic iron homeostasis under various Fe availability

Metal content in yeast data

Mitochondrial metals (Fe, Cu, Co, Zn, Mn, Cd) content in yeast transformed with empty vector or expressing CsMTP6. The yeast were grown in the presence of 100 microM concentration of each metal for 12 hours prior the preparation of mitochondria. metals content was determined by Atomic Absorption Spectrophotometer (AAS 3300, Perkin Elmer). The data are expressed in nmol x mg-1 protein and come from three separate experiments

CsMTP6 expression data

Organ expression pattern of CsMTP6 in cucumber. Real-time expression analyses of the level of CsMTP6 transcript in the roots, hypocotyls, cotyledons, leaf petioles and leaves of 2-week-old cucumber seedlings growing in standard nutrition media. The CsMTP6 transcript levels relative to the constitutively expressed reference gene CACS were calculated (Lightcycler 480, Roche software) from the arithmetic means of ΔCp values obtained in three independent experiments. each experiment was performed in triplicate

Metal content in protoplasts data

Mitochondrial metals (Fe, Mn) content in protoplasts prepared from Arabidopsis thaliana suspension cells and transformed with empty vector pA7 or with the vector carrying cucumber CsMTP6 gene. Following transformation, protoplasts were incubated with 100 mcroM FeSO4-EDTA or 100 microM MnSO4 with slow agitation. Metal content was determined by AAS.The data are expressed in nmol of metal x mg-1 protein and were obtained in three separate experiment

Supplementary Figures and Tables

PDF document with supplementary tables and figures: Supplementary Table S1. Yeast strains used in this work; Supplementary Fig. S1. Organ expression pattern of CsMTP6 in cucumber; Supplementary Fig. S2. ClustalW alignment of the CsMTP6 nucleic acid and CsMTP6 amino acid sequences; Supplementary Fig. S3. Western blot analysis of mitochondria isolated from yeast expressing CsMTP6; Supplementary Fig. S4. Localization of CsMTP6 in Δzrc1, K667 and Δsmf1 cells; Supplementary Fig. S5. Effect of CsMTP6 expression on yeast sensitivity to Zn, Ni and Cd

GDO activity in yeast data

Cytosolic gentisate dioxigenase (c-GDO) activity in the wild type (WT) or yeast mutant cells (mmt1/2) expressing c-GDO or c-GDO and cucumber CsMTP6 protein. c-GDO activity is expressed as nanomoles of substrate converted per minute per mg of protein. The data obtained come from the analysis of four individual yeast transformants