Disorder and amino acid composition in proteins: their potential role in the adaptation of extracellular pilins to the acidic media, where Acidithiobacillus thiooxidans grows.

There are few biophysical studies or structural characterizations of the type IV pilin system of extremophile bacteria, such as the acidophilic Acidithiobacillus thiooxidans. We set out to analyze their pili-comprising proteins, pilins, because these extracellular proteins are in constant interaction with protons of the acidic medium in which At. thiooxidans grows. We used the web server Operon Mapper to analyze and identify the cluster codified by the minor pilin of At. thiooxidans. In addition, we carried an in-silico characterization of such pilins using the VL-XT algorithm of PONDR® server. Our results showed that structural disorder prevails more in pilins of At. thiooxidans than in non-acidophilic bacteria. Further computational characterization showed that the pilins of At. thiooxidans are significantly enriched in hydroxy (serine and threonine) and amide (glutamine and asparagine) residues, and significantly reduced in charged residues (aspartic acid, glutamic acid, arginine and lysine). Similar results were obtained when comparing pilins from other Acidithiobacillus and other acidophilic bacteria from another genus versus neutrophilic bacteria, suggesting that these properties are intrinsic to pilins from acidic environments, most likely by maintaining solubility and stability in harsh conditions. These results give guidelines for the application of extracellular proteins of acidophiles in protein engineering.

Frustules of Amphora sp. as a photonic crystal with photoluminescent CdS nanoparticles.

Diatom frustules have species-specific patterns of pores, striae, pores, and nanopores, periodically arranged on its silica surface, as sets of cavities that modify the vacuum electromagnetic density of states. Therefore, frustules may be considered photonic crystals; the interaction with light-emitting sources inside the pores may potentially result in enhancement or inhibition of their spontaneous radiative emission rate and frequencies. In this work, we studied the photoluminescence of cadmium sulfide nanoparticles (CdS-NP) deposited inside frustule cavities that conveyed evidence of cavity-NP interaction. We synthesized CdS-NP, a semiconductor compound achieving quantum dots small enough to impose confinement effects to the electronic states. CdS-NP and their clusters were physiosorbed onto the surface, striae, and predominantly inside the pores of the cleansed frustules of Amphora sp. A broad peak with a maximum intensity at 437 nm (2.84 eV) was recorded after excitation with a 375 nm light source, showing a large blue shift and signal amplification of the CdS-NP photoluminescence when these were embedded inside the pores of the silica frustule. Using the Brus equation, we estimated a NP size of 4.1 ± 0.2 nm for the CdS-NP snuggly packed inside the smaller pores of the frustule, of 10 ± 0.7 nm in average diameter, The emission Purcell enhancement factor for an emitting atom in a cavity was calculated. The obtained Q factor (c. 5) was smaller than typical Q factors for designed semiconductor cavities of similar dimensions, an expected situation if it is assumed that the pores are open-ended cavities.

Bioelectrochemical system for the biooxidation of a chalcopyrite concentrate by acidophilic bacteria coupled to energy current generation and cathodic copper recovery.

OBJECTIVES: To develop a bioelectrochemical system (BES) to couple the biooxidation of chalcopyrite (CuFeS2), bioelectrogenesis, and the cathodic Cu2+ reduction, bioanodes of acidophilic (pH < 2) and aerobic chemolithoautotrophic bacteria Acidithiobacillus thiooxidans (sulfur oxidizing) and Leptospirillum sp. (Fe2+ oxidizing) were used. RESULTS: CuFeS2 biooxidation increases the charge transfer from the media due to the bioleaching of Cu and Fe. The biofilm on a graphite bar endows a more electropositive (anodic) character to the bioelectrode. By adding the bioleachate generated by both bacteria into the anodic chamber, the acidic bioleachate provides the faradaic intensity. The maximum current density was 0.86 ± 19 mA cm-2 due to the low potential of the BES of 0.18 ± 0.02 V. Such low potential was sufficient for the cathodic deposit of Cu2+. CONCLUSIONS: This work demonstrates a proof of concept for energy savings for mining industries: bioanodes of A. thiooxidans and Leptospirillum sp. are electroactive during the biooxidation of CuFeS2.

Deciphering the enigmatic PilY1 of Acidithiobacillus thiooxidans: An in silico analysis.

Thirty years since the first report on the PilY1 protein in bacteria, only the C-terminal domain has been crystallized; there is no study in which the N-terminal domain, let alone the complete protein, has been crystallized. In our laboratory, we are interested in characterizing the Type IV Pili (T4P) of Acidithiobacillus thiooxidans. We performed an in silico characterization of PilY1 and other pilins of the T4P of this acidophilic bacterium. In silico characterization is crucial for understanding how proteins adapt and function under extreme conditions. By analyzing the primary and secondary structures of proteins through computational methods, researchers can gain valuable insights into protein stability, key structural features, and unique amino acid compositions that contribute to resilience in harsh environments. Here, it is presented a description of the particularities of At. thiooxidans PilY1 through predictor software and homology data. Our results suggest that PilY1 from At. thiooxidans may have the same role as has been described for other PilY1 associated with T4P in neutrophilic bacteria; also, its C-terminal interacts (interface interaction) with the minor pilins PilX, PilW and PilV. The N-terminal region comprises domains such as the vWA and the MIDAS, involved in signaling, ligand-binding, and protein-protein interaction. In fact, the vWA domain has intrinsically disordered regions that enable it to maintain its structure over a wide pH range, not only at extreme acidity to which At. thiooxidans is adapted. The results obtained helped us design the correct methodology for its heterologous expression. This allowed us partially experimentally characterize it by obtaining the N-terminal domain recombinantly and evaluating its acid stability through fluorescence spectroscopy. The data suggest that it remains stable across pH changes. This work thus provides guidance for the characterization of extracellular proteins from extremophilic organisms.

Understanding a Core Pilin of the Type IVa Pili of Acidithiobacillus thiooxidans, PilV.

Pilins are protein subunits of pili. The pilins of type IV pili (T4P) in pathogenic bacteria are well characterized, but anything is known about the T4P proteins in acidophilic chemolithoautotrophic microorganisms such as the genus Acidithiobacillus. The interest in T4P of A. thiooxidans is because of their possible role in cell recruitment and bacterial aggregation on the surface of minerals during biooxidation of sulfide minerals. In this study we present a successful ad hoc methodology for the heterologous expression and purification of extracellular proteins such as the minor pilin PilV of the T4P of A. thiooxidans, a pilin exposed to extreme conditions of acidity and high oxidation-reduction potentials, and that interact with metal sulfides in an environment rich in dissolved minerals. Once obtained, the model structure of A. thiooxidans PilV revealed the core basic architecture of T4P pilins. Because of the acidophilic condition, we carried out in silico characterization of the protonation status of acidic and basic residues of PilV in order to calculate the ionization state at specific pH values and evaluated their pH stability. Further biophysical characterization was done using UV-visible and fluorescence spectroscopy and the results showed that PilV remains soluble and stable even after exposure to significant changes of pH. PilV has a unique amino acid composition that exhibits acid stability, with significant biotechnology implications such as biooxidation of sulfide minerals. The biophysics profiles of PilV open new paradigms about resilient proteins and stimulate the study of other pilins from extremophiles.

Influence of the surface speciation on biofilm attachment to chalcopyrite by Acidithiobacillus thiooxidans.

Surfaces of massive chalcopyrite (CuFeS2) electrodes were modified by applying variable oxidation potential pulses under growth media in order to induce the formation of different secondary phases (e.g., copper-rich polysulfides, S n(2-); elemental sulfur, S(0); and covellite, CuS). The evolution of reactivity (oxidation capacity) of the resulting chalcopyrite surfaces considers a transition from passive or inactive (containing CuS and S n(2-)) to active (containing increasing amounts of S(0)) phases. Modified surfaces were incubated with cells of sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans) for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the density of cells attached to chalcopyrite surfaces, the structure of the formed biofilm, and their exopolysaccharides and nucleic acids were analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy coupled to dispersive X-ray analysis (SEM-EDS). Additionally, CuS and S n(2-)/S(0) speciation, as well as secondary phase evolution, was carried out on biooxidized and abiotic chalcopyrite surfaces using Raman spectroscopy and SEM-EDS. Our results indicate that oxidized chalcopyrite surfaces initially containing inactive S n(2-) and S n(2-)/CuS phases were less colonized by A. thiooxidans as compared with surfaces containing active phases (mainly S(0)). Furthermore, it was observed that cells were partially covered by CuS and S(0) phases during biooxidation, especially at highly oxidized chalcopyrite surfaces, suggesting the innocuous effect of CuS phases during A. thiooxidans performance. These results may contribute to understanding the effect of the concomitant formation of refractory secondary phases (as CuS and inactive S n(2-)) during the biooxidation of chalcopyrite by sulfur-oxidizing microorganisms in bioleaching systems.

Influence of the sulfur species reactivity on biofilm conformation during pyrite colonization by Acidithiobacillus thiooxidans.

Massive pyrite (FeS2) electrodes were potentiostatically modified by means of variable oxidation pulse to induce formation of diverse surface sulfur species (S(n)²â», S°). The evolution of reactivity of the resulting surfaces considers transition from passive (e.g., Fe(1-x )S2) to active sulfur species (e.g., Fe(1-x )S(2-y ), S°). Selected modified pyrite surfaces were incubated with cells of sulfur-oxidizing Acidithiobacillus thiooxidans for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the attached cells density and their exopolysaccharides were analyzed by confocal laser scanning microscopy (CLMS) and atomic force microscopy (AFM) on bio-oxidized surfaces; additionally, S(n)²â»/S° speciation was carried out on bio-oxidized and abiotic pyrite surfaces using Raman spectroscopy. Our results indicate an important correlation between the evolution of S(n)²â»/S° surface species ratio and biofilm formation. Hence, pyrite surfaces with mainly passive-sulfur species were less colonized by A. thiooxidans as compared to surfaces with active sulfur species. These results provide knowledge that may contribute to establishing interfacial conditions that enhance or delay metal sulfide (MS) dissolution, as a function of the biofilm formed by sulfur-oxidizing bacteria.

Electrochemical monitoring of Acidithiobacillus thiooxidans biofilm formation on graphite surface with elemental sulfur.

Inorganic wastewaters and sediments from the mining industry and mineral bioleaching processes have not been fully explored in bioelectrochemical systems (BES). Knowledge of interfacial changes due to biofilm evolution under acidic conditions may improve applications in electrochemical processes, specifically those related to sulfur compounds. Biofilm evolution of Acidithiobacillus thiooxidans on a graphite plate was monitored by electrochemical techniques, using the graphite plate as biofilm support and elemental sulfur as the only energy source. Even though the elemental sulfur was in suspension, S0 particles adhered to the graphite surface favoring biofilm development. The biofilms grown at different incubation times (without electric perturbation) were characterized in a classical three electrode electrochemical cell (sulfur and bacteria free culture medium) by non-invasive electrochemical impedance spectroscopy (EIS) and cyclic voltammetry. The biofilm structure was confirmed by Environmental Scanning Electrode Microscopy, while the relative fractions of exopolysaccharides and extracellular hydrophobic compounds at different incubation times were evaluated by Confocal Laser Scanning Microscopy. The experimental conditions chosen in this work allowed the EIS monitoring of the biofilm growth as well as the modification of Extracellular Polymeric Substances (EPS) composition (hydrophobic/ exopolysaccharides EPS ratio). This strategy could be useful to control biofilms for BES operation under acidic conditions.

Sequence analysis and confirmation of the type IV pili-associated proteins PilY1, PilW and PilV in Acidithiobacillus thiooxidans.

Acidithiobacillus thiooxidans is an acidophilic chemolithoautotrophic bacterium widely used in the mining industry due to its metabolic sulfur-oxidizing capability. The biooxidation of sulfide minerals is enhanced through the attachment of At. thiooxidans cells to the mineral surface. The Type IV pili (TfP) of At. thiooxidans may play an important role in the bacteria attachment since TfP play a key adhesive role in the attachment and colonization of different surfaces. In this work, we report for the first time the mRNA sequence of three TfP proteins from At. thiooxidans, the adhesin protein PilY1 and the TfP pilins PilW and PilV. The nucleotide sequences of these TfP proteins show changes in some nucleotide positions with respect to the corresponding annotated sequences. The bioinformatic analyses and 3D-modeling of protein structures sustain their classification as TfP proteins, as structural homologs of the corresponding proteins of Ps. aeruginosa, results that sustain the role of PilY1, PilW and PilV in pili assembly. Also, that PilY1 comprises the conserved Neisseria-PilC (superfamily) domain of the tip-associated adhesin, while PilW of the superfamily of putative TfP assembly proteins and PilV belongs to the superfamily of TfP assembly protein. In addition, the analyses suggested the presence of specific functional domains involved in adhesion, energy transduction and signaling functions. The phylogenetic analysis indicated that the PilY1 of Acidithiobacillus genus forms a cohesive group linked with iron- and/or sulfur-oxidizing microorganisms from acid mine drainage or mine tailings.

Biofilm formation by algae as a mechanism for surviving on mine tailings.

Photosynthetic biofilms successfully colonize the sediments of a mine tailings reservoir (Guanajuato, Mexico) despite the high metal concentrations that are present. To elucidate the mechanisms of biofilm survival despite metal ores, experiments were performed to evaluate the response of seminatural biofilms to Cu, Zn, and a combination of both metals at concentrations observed in the field. The biofilms were composed mostly of the chlorophyte Chlorococcum sp. and the cyanobacterium Phormidium sp., and their response to the two added metals was described by measurements of extracellular polymeric substances (EPS) and in vivo fluorescence. The photosynthetic efficiency and the minimal chlorophyll fluorescence of dark-adapted cells were measured by multiwavelength pulse amplitude-modulated fluorometry. The photosynthetic efficiency of light-adapted cells (phi(PSII)) also was measured. Metal exposure increased the EPS production of biofilms, as visualized with confocal laser-scanning microscopy. Extracellular polymeric substances enhanced the extracellular metal accumulation from the first day of metal exposure. Metals provoked changes in the relative abundance of the dominant taxa because of a species-specific response to the metals when added individually. Metals affected the phi(PSII) less than the total biomass, suggesting ongoing activity of the surviving biofilms. Survival of individual biofilm photosynthetic cells was found to resume from the embedding in the mucilaginous structure, which immobilizes the metals extracellularly. The survival of biofilms under mixed-metal exposure has practical applications in the remediation of mine tailings.