Clonality and α-a Recombination in the Australian Cryptococcus gattii VGII Population - An Emerging Outbreak in Australia

BACKGROUND: Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals. Within C. gattii, four molecular types are recognized (VGI to VGIV). The Australian VGII population has been in the spotlight since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island (British Columbia, Canada), with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population and determine processes shaping its population structure. METHODOLOGY/PRINCIPAL FINDINGS: A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America, Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international consensus multi-locus sequence typing (MLST) scheme, including seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1. Despite the overall clonality observed, the presence of MATa VGII isolates in Australia was demonstrated for the first time in association with recombination in MATα-MATa populations. Our results also support the hypothesis of a "smouldering" outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is possibly caused by a founder effect followed by a clonal expansion. CONCLUSIONS/SIGNIFICANCE: The detection of sexual recombination in MATα-MATa population in Australia is in accordance with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating strategy suggested elsewhere. The potential for an Australian wide outbreak highlights the crucial issue to develop active surveillance procedures.Fabian Carriconde, Félix Gilgado, Ian Arthur, David Ellis, Richard Malik, Nathalie van de Wiele, Vincent Robert, Bart J. Currie, Wieland Meye

How sulphate-reducing microorganisms cope with stress: lessons from systems biology

Sulphate-reducing microorganisms (SRMs) are a phylogenetically diverse group of anaerobes encompassing distinct physiologies with a broad ecological distribution. As SRMs have important roles in the biogeochemical cycling of carbon, nitrogen, sulphur and various metals, an understanding of how these organisms respond to environmental stresses is of fundamental and practical importance. In this Review, we highlight recent applications of systems biology tools in studying the stress responses of SRMs, particularly Desulfovibrio spp., at the cell, population, community and ecosystem levels. The syntrophic lifestyle of SRMs is also discussed, with a focus on system-level analyses of adaptive mechanisms. Such information is important for understanding the microbiology of the global sulphur cycle and for developing biotechnological applications of SRMs for environmental remediation, energy production, biocorrosion control, wastewater treatment and mineral recovery

The membrane-bound cytochrome cy of Rhodobacter capsulatus can serve as an electron donor to the photosynthetic reaction center of Rhodobacter sphaeroides

Rhodobacter capsulatus has two different pathways for reduction of the photo-oxidized reaction center, one using water-soluble cytochrome c2, the other via membrane-associated cytochrome cy. Rhodobacter sphaeroides differs in that it lacks a cytochrome cy homologue capable of functioning in photosynthetic electron transfer; cytochrome c2 is thus the sole electron carrier, and is required for photosynthetic (Ps+) growth. Genetic evidence indicates that cytochrome cy of R. capsulatus can complement a Ps- cytochrome-c2-deficient mutant of R. sphaeroides (Jenney, F.E. and Daldal, F. (1993) EMBO J. 12, 1283-1292). Here, we show that it transfers electrons from the cytochrome bc1 complex to the reaction center in R. sphaeroides, albeit at a lower rate than that catalyzed by the endogenous cytochrome c2. When cytochrome cy is expressed in R. sphaeroides in the presence of cytochrome c2, there is an increase in the amount of photo-oxidizible c-type cytochrome. In the absence of cytochrome c2, electron transfer via cytochrome cy shows significantly different kinetics for reaction center reduction and cytochrome c oxidation. These findings further establish that cytochrome cy, the electron carrier permitting soluble cytochrome c2-independent photosynthetic growth in R. capsulatus, can function in a similar capacity in R. sphaeroides

Anaerobic microbes: Oxygen detoxification without superoxide dismutase

Superoxide reductase from the hyperthermophilic anaerobe Pyrococcus furiosus uses electrons from reduced nicotinamide adenine dinucleotide phosphate, by way of rubredoxin and an oxidoreductase, to reduce superoxide to hydrogen peroxide, which is then reduced to water by peroxidases. Unlike superoxide dismutase, the enzyme that protects aerobes from the toxic effects of oxygen, SOR does not catalyze the production of oxygen from superoxide and therefore confers a selective advantage on anaerobes. Superoxide reductase and associated proteins are catalytically active 80 °C below the optimum growth temperature (100 °C) of P. furiosus, conditions under which the organism is likely to be exposed to oxygen

PF0610, a novel winged helix-turn-helix variant possessing a rubredoxin-like Zn ribbon motif from the hyperthermophilic archaeon, Pyrococcus furiosus

PF0610, a protein from the hyperthermophile Pyrococcus furiosus, has homologues only in other archaeal species and in three species of Fe(III)-reducing bacteria. It is thought to have a helixturn-helix (HTH) domain at the N-terminus and possesses two CXXC motifs characteristic of metal binding proteins. We have determined the solution structure of the Zn-bound protein using NMR. PF0610 is a novel winged helix-turn-helix (wHTH) protein with a rubredoxin-like Zn ribbon as its W1 segment. In addition, it possesses a large number of basic residues on its surface. Clusters of basic residues can be found on both helix H3 and the metal-binding loops of W1, suggesting that it might be a DNA-binding protein. Accordingly, gel shift assays using both linear and circular DNA showed that PF0610 does bind DNA, at least in a sequence-independent fashion. Modeling the PF0610-DNA interaction based on other wHTH protein-DNA structures revealed that besides helix H3, basic residues around the second CXXC motif in the metal-binding loop could make extensive contacts with DNA. However, the bulkiness of the W1 region implies that the DNA conformation may be distorted upon PF0610 binding. PF0610 is the first protein known to have a Zn ribbon-embedded wHTH fold and, as such, has potential roles both as a metal-dependent transcription regulator and as a component of the chromosome packing system in P. furiosus. The discovery of this novel structure represents the addition of another branch to the winged HTH protein family and could contribute to our understanding of transcription regulatory processes in P. furiosus. © 2007 American Chemical Society

Solvent Tuning of Electrochemical Potentials in the Active Sites of HiPIP Versus Ferredoxin

A persistent puzzle in the field of biological electron transfer is the conserved iron-sulfur cluster motif in both high potential iron-sulfur protein (HiPIP) and ferredoxin (Fd) active sites. Despite this structural similarity, HiPIPs react oxidatively at physiological potentials, whereas Fds are reduced. Sulfur K-edge x-ray absorption spectroscopy uncovers the substantial influence of hydration on this variation in reactivity. Fe-S covalency is much lower in natively hydrated Fd active sites than in HiPIPs but increases upon water removal; similarly, HiPIP covalency decreases when unfolding exposes an otherwise hydrophobically shielded active site to water. Studies on model compounds and accompanying density functional theory calculations support a correlation of Fe-S covalency with ease of oxidation and therefore suggest that hydration accounts for most of the difference between Fd and HiPIP reduction potentials

Comparison of small- and large-scale expression of selected Pyrococcus furiosus genes as an aid to high-throughput protein production

As the natural extension of the genomic sequencing projects, the goal of the various world-wide Structural Genomics projects is development of techniques for high throughput (HTP) cloning, protein overexpression, purification and structural determination, with the ultimate goal of determining all possible protein structures. Rapid (small-scale) screening of potential expression clones under different growth conditions is presumed to be possible and a viable way to increase throughput of protein expression. In order to test the utility of screening for soluble, heterologous protein expression, we have compared the production of recombinant proteins on a small scale (1 ml cultures in 96-well plates) in Escherichia coli under two growth conditions [a rich medium and a defined (minimal) medium] using an enzyme-linked immunosorbent assay (ELISA) against the affinity tag, with the amount of recombinant protein produced during the large-scale (500 ml) growth of E. coli. The large-scale expression products were examined after a single step affinity purification by visualization on SDS-PAGE gels. Of the open reading frames that were successfully expressed on the 1 ml scale as judged by immunodetection, 80% of them successfully scaled-up to 500 ml in a rich medium and 81% of them scaled-up in a defined medium. This is significantly higher than would be expected by a randomly selected expression condition and validates the use of small-scale expression as a screening tool for more efficient protein production. © Springer 2005

High-throughput production of Pyrococcus furiosus proteins: Considerations for metalloproteins

Free-living prokaryotic organisms contain all of the proteins required for the basic biochemical processes of life. As part of the Southeastern Collaboratory for Structural Genomics (SECSG), Pyrococcus furiosus is being used as a model system for developing a high-throughput protein expression and purification protocol. Its 1.9 million basepair genome encodes ∼2200 putative proteins, less than 25% of which show similarity to any structurally characterized protein in the Protein Data Bank. The overall goal of the structural genomics initiative is to determine, in total, all existing protein folds. The immediate objective of this work is to obtain recombinant forms of all P. furiosus proteins in their functional states for structural determination. Proteins successfully produced by over-expression in another organism such as the bacterium Escherichia coli typically contain a single subunit, are soluble and do not contain (complex) cofactors. Analyses of the P. furiosus genome suggest that perhaps only a quarter of the genes encode proteins that would fall into this category. The hypothesis is that lack of the appropriate cofactor or of the partner protein(s) necessary to form a complex are major reasons why many recombinant proteins are insoluble. This work describes development of the production pipeline with attention to prediction and incorporation of cofactors. © 2005 International Union of Crystallography Printed in Great Britain - all rights reserved

The first agmatine/cadaverine aminopropyl transferase: Biochemical and structural characterization of an enzyme involved in polyamine biosynthesis in the hyperthermophilic archaeon Pyrococcus furiosus

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the kcat/Km value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108°C that increases to 112°C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-Å resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. füriosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation. Copyright © 2007, American Society for Microbiology. All Rights Reserved