Tetraether-linked membrane monolayers in <Emphasis Type="Italic">Ferroplasma</Emphasis> spp: a key to survival in acid

Ferroplasma acidarmanus thrives in hot, extremely low pH, metal-rich solutions associated with dissolving metal sulfide ore deposits. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thin layer chromatography analyses of F. acidarmanus membranes indicate that tetraether lipids predominate, with at least three core lipid structures. NMR measurements indicate that the cytoplasmic pH of F. acidarmanus is ~5.6. The optimal growth pH is ~1.2, and the lowest growth pH is ~0.0. Thus, these organisms maintain pH gradients across their membranes that approach 5 pH units. Tetraether lipids were originally thought to be specifically associated with thermophiles but are now known to be widely distributed within the archaeal domain. Our data, in combination with recently published results for thermophilic and mesothermophilic acidophilic archaea, indicate that there may be a stronger association between tetraether lipids and tolerance to acid and/or large metal ion gradients.     

Characterization of Ferroplasma Isolates and Ferroplasma acidarmanus sp. nov., Extreme Acidophiles from Acid Mine Drainage and Industrial Bioleaching Environments

Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum Y^T by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate “Ferroplasma acidarmanus” Fer1^T required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42°C and the pH optima were 1.0 to 1.7, and “F. acidarmanus” Fer1^T was capable of growing at pH 0. The optimum yeast extract concentration for “F. acidarmanus” Fer1^T was higher than that for the other three isolates. Phenotypic results suggested that isolate “F. acidarmanus” Fer1^T is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and Y^T group as a single species. “F. acidarmanus” Fer1^T groups separately, and we propose the new species “F. acidarmanus” Fer1^T sp. nov.     

Pseudomonas aeruginosa zinc homeostasis: Key issues for an opportunistic pathogen

Zinc is an essential trace element for almost all living organisms. In the opportunistic bacterial pathogen Pseudomonas aeruginosa, zinc has been shown to play an important role in virulence, in colonization of the host organism and has also been shown to be involved in antibiotic resistance. P. aeruginosa possesses numerous systems enabling it to thrive in zinc-depleted conditions as well as high-zinc situations, two environments that are encountered during human infection. These capabilities account for its pathogenic strength. The main aim of this review is to focus on zinc homeostasis in P. aeruginosa and the genetic regulation of the systems involved. The interconnection with virulence, as well as the mechanism of co-regulation between metal and antibiotic resistance, are of prime interest for understanding the molecular mechanisms allowing P. aeruginosa to switch from its existence as a common environmental bacterium to a severe opportunistic pathogen. This article is part of a Special Issue entitled: Dynamic gene expression, edited by Prof. Patrick Viollier.     

Microbial iron management mechanisms in extremely acidic environments: comparative genomics evidence for diversity and versatility

Background  

Iron is an essential nutrient but can be toxic at high intracellular concentrations and organisms have evolved tightly regulated mechanisms for iron uptake and homeostasis. Information on iron management mechanisms is available for organisms living at circumneutral pH. However, very little is known about how acidophilic bacteria, especially those used for industrial copper bioleaching, cope with environmental iron loads that can be 10¹⁸ times the concentration found in pH neutral environments. This study was motivated by the need to fill this lacuna in knowledge. An understanding of how microorganisms thrive in acidic ecosystems with high iron loads requires a comprehensive investigation of the strategies to acquire iron and to coordinate this acquisition with utilization, storage and oxidation of iron through metal responsive regulation. In silico prediction of iron management genes and Fur regulation was carried out for three Acidithiobacilli: Acidithiobacillus ferrooxidans (iron and sulfur oxidizer) A. thiooxidans and A. caldus (sulfur oxidizers) that can live between pH 1 and pH 5 and for three strict iron oxidizers of the Leptospirillum genus that live at pH 1 or below.     

Effect of the aeration mode and yeast extract on the oxidation of high-pyrrhotite sulfide ore flotation concentrate and on the composition of the acidophilic chemolithotrophic microbial community

Optimal aeration conditions were determined and the effect of yeast extract on biooxidation of high-pyrrhotite sulfide ore flotation concentrate in the course of continuous cultivation of an acidophilic chemolithotrophic microbial community was studied in a line of four sequential laboratory reactors; the aeration rate was 3 L/(L min), yeast extract concentration was 0.02%. The gold recovery level was 96.45% at 2.23% elemental sulfur content in the solid residue. The dominant strains identified in the community responsible for biooxidation were Acidithiobacillus caldus OL13-1, At. caldus OL13-3 = At. caldus OL12-3, and an ‘Acidiferrobacter’ strain. Strains Sulfobacillus thermosulfidooxidans OL13-2 = S. thermosulfidooxidans OL12-1 and Ferroplasma acidiphilum OL13-4 = F. acidiphilum OL12-4 were isolated in pure culture and identified.     

Construction of arsB and tetH Mutants of the Sulfur-Oxidizing Bacterium Acidithiobacillus caldus by Marker Exchange

Acidithiobacillus caldus is a moderately thermophilic, acidophilic bacterium that has been reported to be the dominant sulfur oxidizer in stirred-tank processes used to treat gold-bearing arsenopyrite ores. It is also widely distributed in heap reactors used for the extraction of metals from ores. Not only are these bacteria commercially important, they have an interesting physiology, the study of which has been restricted by the nonavailability of defined mutants. A recently reported conjugation system based on the broad-host-range IncW plasmids pSa and R388 was used to transfer mobilizable narrow-host-range suicide plasmid vectors containing inactivated and partially deleted chromosomal genes from Escherichia coli to A. caldus. Through the dual use of a selectable kanamycin resistance gene and a hybridization probe made from a deleted portion of the target chromosomal gene, single- and double-recombinant mutants of A. caldus were isolated. The functionality of the gene inactivation system was shown by the construction of A. caldus arsB and tetH mutants, and the effects of these mutations on cell growth in the presence of arsenic and by means of tetrathionate oxidation were demonstrated.     

Succession of Acidophilic Bacterial Community During Bio-oxidation of Refractory Gold-Containing Sulfides

To optimize the rate of bio-oxidation to recover gold from sulfide minerals, it is important to understand the dynamic change of acidophilic bacteria involved in this process. In this study, a batch bio-oxidation experiment was set up to bioleach Au from refractory pyrite and arsenopyrite using a mixed acidophilic culture over the duration of eight days. The 16S rRNA gene clone library and denaturing gradient gel electrophoresis approaches (DGGE) were used to monitor the dynamic succession of the acidophilic bacterial population. The results showed that there were five bacteria in the bio-oxidation reactor: Leptospirillum ferriphilum, Acidithiobacillus caldus, Sulfobacillus thermotolerans, Alicyclobacillus sp. and a heterotrophic iron-oxidizing bacterium. The overall succession pattern was that Acidithiobacillus caldus, a sulfur oxidizer, and Sulfobacillus thermotolerans, a sulfur-iron oxidizer, were predominant at the beginning of the bio-oxidation process, but they were replaced by iron oxidizer L. ferriphilum at a later stage. The competitive advantage of At. caldus and Sb. thermotolerans over L. ferriphilum at the early stage was availability of abundant inorganic sulfur compounds, but lower pH, higher redox potential, and ferrous iron favored L. ferriphilum growth at a later stage. These results have important implications for understanding the role of acidophilic bacterial population in bio-oxidation of refractory gold-containing sulfides.     

Draft Genome Sequence of the Sulfobacillus thermosulfidooxidans Cutipay Strain,an Indigenous Bacterium Isolated from a Naturally Extreme Mining Environment in Northern Chile

Sulfobacillus thermosulfidooxidans strain Cutipay is a mixotrophic, acidophilic, moderately thermophilic bacterium isolated from mining environments of the north of Chile, making it an interesting subject for studying the bioleaching of copper. We introduce the draft genome sequence and annotation of this strain, which provide insights into its mechanisms for heavy metal resistance.     

Alternative Activation of Macrophages and Induction of Arginase Are Not Components of Pathogenesis Mediated by Francisella Species

Virulent Francisella tularensis ssp tularensis is an intracellular, Gram negative bacterium that causes acute lethal disease following inhalation of fewer than 15 organisms. Pathogenicity of Francisella infections is tied to its unique ability to evade and suppress inflammatory responses in host cells. It has been proposed that induction of alternative activation of infected macrophages is a mechanism by which attenuated Francisella species modulate host responses. In this report we reveal that neither attenuated F. tularensis Live Vaccine Strain (LVS) nor virulent F. tularensis strain SchuS4 induce alternative activation of macrophages in vitro or in vivo. LVS, but not SchuS4, provoked production of arginase1 independent of alternative activation in vitro and in vivo. However, absence of arginase1 did not significantly impact intracellular replication of LVS or SchuS4. Together our data establish that neither induction of alternative activation nor expression of arginase1 are critical features of disease mediated by attenuated or virulent Francisella species.     

Transition metal homeostasis: from yeast to human disease

Transition metal ions are essential nutrients to all forms of life. Iron, copper, zinc, manganese, cobalt and nickel all have unique chemical and physical properties that make them attractive molecules for use in biological systems. Many of these same properties that allow these metals to provide essential biochemical activities and structural motifs to a multitude of proteins including enzymes and other cellular constituents also lead to a potential for cytotoxicity. Organisms have been required to evolve a number of systems for the efficient uptake, intracellular transport, protein loading and storage of metal ions to ensure that the needs of the cells can be met while minimizing the associated toxic effects. Disruptions in the cellular systems for handling transition metals are observed as a number of diseases ranging from hemochromatosis and anemias to neurodegenerative disorders including Alzheimer??s and Parkinson??s disease. The yeast Saccharomyces cerevisiae has proved useful as a model organism for the investigation of these processes and many of the genes and biological systems that function in yeast metal homeostasis are conserved throughout eukaryotes to humans. This review focuses on the biological roles of iron, copper, zinc, manganese, nickel and cobalt, the homeostatic mechanisms that function in S. cerevisiae and the human diseases in which these metals have been implicated.     

Biofilm development in the extremely acidophilic archaeon ‘Ferroplasma acidarmanus’ Fer1

‘Ferroplasma acidarmanus’ Fer1 is an iron-oxidizing extreme acidophile isolated from the Iron Mountain mine, California, USA. This archaeon is predominantly found in biofilm-associated structures in the environment, and produces two distinct biofilm morphologies. Bioinformatic analysis of the ‘F. acidarmanus’ Fer1 genome identified genes annotated as involved in attachment and biofilm formation. No putative quorum sensing signaling genes were identified and no N-acyl homoserine lactone-like compounds were found in ‘F. acidarmanus’ Fer1 biofilm supernatant. Scanning confocal microscopy analysis of biofilm development on the surface of pyrite demonstrated the temporal and spatial development of biofilm growth. Furthermore, two-dimensional polyacrylamide gel electrophoresis was used to examine differential protein expression patterns between biofilm and planktonic populations. Ten up-regulated proteins were identified that included six enzymes associated with anaerobic growth, suggesting that the dominating phenotype in the mature biofilm was associated with anaerobic modes of growth. This report increases our knowledge of the genetic and proteomic basis of biofilm formation in an extreme acidophilic archaeon.     

Bacillithiol,a new role in buffering intracellular zinc

Zinc is a catalytic or structural cofactor of numerous proteins but can also be toxic if cells accumulate too much of this essential metal. Therefore, mechanisms of zinc homeostasis are needed to maintain a low but adequate amount of free zinc so that newly translated zinc‐dependent proteins can bind their cofactor without confounding issues of toxicity. These mechanisms include the regulation of uptake and efflux transporters and buffering of the free metal concentration by low‐molecular‐weight ligands in the cytosol. While many of the transporters involved in zinc homeostasis have been discovered in recent years, the molecules that buffer zinc have remained largely a mystery. In the new report highlighted by this commentary, Ma et al. (2014) provide convincing evidence that bacillithiol, the major low‐molecular‐weight thiol compound in Bacillus subtilis, serves as an important zinc buffer in those cells. Their discovery provides an important piece to the puzzle of how zinc buffering occurs in a large number of microbes and provides new clues about the role and relative importance of zinc buffering in all organisms.     

Heterodimerization,Altered Subcellular Localization,and Function of Multiple Zinc Transporters in Viable Cells Using Bimolecular Fluorescence Complementation

Zinc plays a crucial role in numerous key physiological functions. Zinc transporters (ZnTs) mediate zinc efflux and compartmentalization in intracellular organelles; thus, ZnTs play a central role in zinc homeostasis. We have recently shown the in situ dimerization and function of multiple normal and mutant ZnTs using bimolecular fluorescence complementation (BiFC). Prompted by these findings, we here uncovered the heterodimerization, altered subcellular localization, and function of multiple ZnTs in live cells using this sensitive BiFC technique. We show that ZnT1, -2, -3, and -4 form stable heterodimers at distinct intracellular compartments, some of which are completely different from their homodimer localization. Specifically, unlike the plasma membrane (PM) localization of ZnT1 homodimers, ZnT1-ZnT3 heterodimers localized at intracellular vesicles. Furthermore, upon heterodimerization with ZnT1, the zinc transporters ZnT2 and ZnT4 surprisingly localized at the PM, as opposed to their vesicular homodimer localization. We further demonstrate the deleterious effect that the G87R-ZnT2 mutation, associated with transient neonatal zinc deficiency, has on ZnT1, ZnT3, and ZnT4 upon heterodimerization. The functionality of the various ZnTs was assessed by the dual BiFC-Zinquin assay. We also undertook a novel transfection competition assay with ZnT cDNAs to confirm that the driving force for heterodimer formation is the core structure of ZnTs and not the BiFC tags. These findings uncover a novel network of homo- and heterodimers of ZnTs with distinct subcellular localizations and function, hence highlighting their possible role in zinc homeostasis under physiological and pathological conditions.     

The effect of the introduction of exogenous strain Acidithiobacillus thiooxidans A01 on functional gene expression, structure and function of indigenous consortium during pyrite bioleaching

Acidithiobacillus thiooxidans A01 was added to a consortium of bioleaching bacteria including Acidithiobacilluscaldus, Leptospirillumferriphilum, Acidithiobacillus ferrooxidans, Sulfobacillus thermosulfidooxidans, Acidiphilium spp., and Ferroplasma thermophilum cultured in modified 9 K medium containing 0.5% (w/v) pyrite, and 10.7% increase of bioleaching rate was observed. Changes in community structure and gene expression were monitored with real-time PCR and functional gene arrays (FGAs). Real-time PCR showed that addition of At. thiooxidans caused increased numbers of all consortium members except At. caldus, and At. caldus, L. ferriphilum, and F. thermophilum remained dominant in this community. FGAs results showed that after addition of At. thiooxidans, most genes involved in iron, sulfur, carbon, and nitrogen metabolisms, metal resistance, electron transport, and extracellular polymeric substances of L. ferriphilum, F. thermophilum, and Acidiphilium spp., were up-regulated while most of these genes were down-regulated at 70-78 h in At. caldus and up-regulated in At. ferrooxidans, then down-regulated at 82-86 h.     

Isolation of a strain of <Emphasis Type="Italic">Acidithiobacillus caldus</Emphasis> and its role in bioleaching of chalcopyrite

A moderately thermophilic and acidophilic sulfur-oxidizing bacterium named S₂, was isolated from coal heap drainage. The bacterium was motile, Gram-negative, rod-shaped, measured 0.4 to 0.6 by 1 to 2 μm, and grew optimally at 42–45°C and an initial pH of 2.5. The strain S₂ grew autotrophically by using elemental sulfur, sodium thiosulfate and potassium tetrathionate as energy sources. The strain did not use organic matter and inorganic minerals including ferrous sulfate, pyrite and chalcopyrite as energy sources. The morphological, biochemical, physiological characterization and analysis based on 16S rRNA gene sequence indicated that the strain S₂ is most closely related to Acidithiobacillus caldus (>99% similarity in gene sequence). The combination of the strain S₂ with Leptospirillum ferriphilum or Acidithiobacillus ferrooxidans in chalcopyrite bioleaching improved the copper-leaching efficiency. Scanning electron microscope (SEM) analysis revealed that the chalcopyrite surface in a mixed culture of Leptospirillum ferriphilum and Acidithiobacillus caldus was heavily etched. The energy dispersive X-ray (EDX) analysis indicated that Acidithiobacillus caldus has the potential role to enhance the recovery of copper from chalcopyrite by oxidizing the sulfur formed during the bioleaching progress.     

A study on the dynamics of the zraP gene expression profile and its application to the construction of zinc adsorption bacteria

Zinc ion plays essential roles in biological chemistry. Bacteria acquire Zn(2+) from the environment, and cellular concentration levels are controlled by zinc homeostasis systems. In comparison with other homeostatic systems, the ZraSR two-component system was found to be more efficient in responding to exogenous zinc concentrations. To understand the dynamic response of the bacterium ZraSR two-component system with respect to exogenous zinc concentrations, the genetic circuit of the ZraSR system was integrated with a reporter protein. This study was helpful in the construction of an E. coli system that can display selective metal binding peptides on the surface of the cell in response to exogenous zinc. The engineered bacterial system for monitoring exogenous zinc was successfully employed to detect levels of zinc as low as 0.001 mM, which directly activates the expression of chimeric ompC(t)--zinc binding peptide gene to remove zinc by adsorbing a maximum of 163.6 μmol of zinc per gram of dry cell weight. These results indicate that the engineered bacterial strain developed in the present study can sense the specific heavy metal and activates a cell surface display system that acts to remove the metal.