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Acidithiobacillus species are fundamental players in biofilm formation by acidophile bioleaching communities. It has been previously reported that Acidithiobacillus ferrooxidans possesses a functional quorum sensing mediated by acyl-homoserine lactones (AHL), involved in biofilm formation, and AHLs naturally produced by Acidithiobacillus species also induce biofilm formation in Acidithiobacillus thiooxidans. A c-di-GMP pathway has been characterized in Acidithiobacillus species but it has been pointed out that the c-di-GMP effector PelD and pel-like operon are only present in the sulfur oxidizers such as A. thiooxidans. PEL exopolysaccharide has been recently involved in biofilm formation in this Acidithiobacillus species. Here, by comparing wild type and &Delta, pelD strains through mechanical analysis of biofilm-cells detachment, fluorescence microscopy and qPCR experiments, the structural role of PEL exopolysaccharide and the molecular network involved for its biosynthesis by A. thiooxidans were tackled. Besides, the effect of AHLs on PEL exopolysaccharide production was assessed. Mechanical resistance experiments indicated that the loss of PEL exopolysaccharide produces fragile A. thiooxidans biofilms. qRT-PCR analysis established that AHLs induce the transcription of pelA and pelD genes while epifluorescence microscopy studies revealed that PEL exopolysaccharide was required for the development of AHL-induced biofilms. Altogether these results reveal for the first time that AHLs positively regulate pel genes and participate in the molecular network for PEL exopolysaccharide biosynthesis by A. thiooxidans.

Acidithiobacillus ferrooxidans is an acidophilic bacterium able to grow in environments with high concentrations of metals. It is a chemolithoautotroph able to form biofilms on the surface of solid minerals to obtain its energy. The response of both planktonic and sessile cells of A. ferrooxidans ATCC 23270 grown in elemental sulfur and adapted to high copper concentration was analyzed by quantitative proteomics. It was found that 137 proteins varied their abundance when comparing both lifestyles. Copper effllux proteins, some subunits of the ATP synthase complex, porins, and proteins involved in cell wall modification increased their abundance in copper-adapted sessile lifestyle cells. On the other hand, planktonic copper-adapted cells showed increased levels of proteins such as: cupreredoxins involved in copper cell sequestration, some proteins related to sulfur metabolism, those involved in biosynthesis and transport of lipopolysaccharides, and in assembly of type IV pili. During copper adaptation a decreased formation of biofilms was measured as determined by epifluorescence microscopy. This was apparently due not only to a diminished number of sessile cells but also to their exopolysaccharides production. This is the first study showing that copper, a prevalent metal in biomining environments causes dispersion of A. ferrooxidans biofilms., Supported by FONDECYT Chile, (Science and Technology Chilean Fund) grant n° 1150791 to C.A. Jerez. The Proteomics Unit of the Spanish National Biotechnology Centre belongs to ProteoRed (PRB2 -ISCIII) and is supported by FIS grant PT13/0001.

Deep continental subsurface is defined as oligotrophic environments where microorganisms present a very low metabolic rate. To date, due to the energetic cost of production and maintenance of biofilms, their existence has not been considered in poor porous subsurface rocks. We applied fluorescence in situ hybridization techniques and confocal laser scanning microscopy in samples from a continental deep drilling project to analyze the prokaryotic diversity and distribution and the possible existence of biofilms. Our results show the existence of natural microbial biofilms at all checked depths of the Iberian Pyrite Belt (IPB) subsurface and the co-occurrence of bacteria and archaea in this environment. This observation suggests that multi-species biofilms may be a common and widespread lifestyle in subsurface environments., MINECO

Industrial biomining processes are currently focused on metal sulfides and their dissolution, which is catalyzed by acidophilic iron(II)- and/or sulfur-oxidizing microorganisms. Cell attachment on metal sulfides is important for this process. Biofilm formation is necessary for seeding and persistence of the active microbial community in industrial biomining heaps and tank reactors, and it enhances metal release. In this study, we used a method for direct quantification of the mineral-attached cell population on pyrite or chalcopyrite particles in bioleaching experiments by coupling high-throughput, automated epifluorescence microscopy imaging of mineral particles with algorithms for image analysis and cell quantification, thus avoiding human bias in cell counting. The method was validated by quantifying cell attachment on pyrite and chalcopyrite surfaces with axenic cultures of Acidithiobacillus caldus, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans. The method confirmed the high affinity of L. ferriphilum cells to colonize pyrite and chalcopyrite surfaces and indicated that biofilm dispersal occurs in mature pyrite batch cultures of this species. Deep neural networks were also applied to analyze biofilms of different microbial consortia. Recent analysis of the L. ferriphilum genome revealed the presence of a diffusible soluble factor (DSF) family quorum sensing system. The respective signal compounds are known as biofilm dispersal agents. Biofilm dispersal was confirmed to occur in batch cultures of L. ferriphilum and S. thermosulfidooxidans upon the addition of DSF family signal compounds.IMPORTANCE The presented method for the assessment of mineral colonization allows accurate relative comparisons of the microbial colonization of metal sulfide concentrate particles in a time-resolved manner. Quantitative assessment of the mineral colonization development is important for the compilation of improved mathematical models for metal sulfide dissolution. In addition, deep-learning algorithms proved that axenic or mixed cultures of the three species exhibited characteristic biofilm patterns and predicted the biofilm species composition. The method may be extended to the assessment of microbial colonization on other solid particles and may serve in the optimization of bioleaching processes in laboratory scale experiments with industrially relevant metal sulfide concentrates. Furthermore, the method was used to demonstrate that DSF quorum sensing signals directly influence colonization and dissolution of metal sulfides by mineral-oxidizing bacteria, such as L. ferriphilum and S. thermosulfidooxidans.

Society’s demand for metals is ever increasing while stocks of high-grade minerals are being depleted. Biomining, for example of chalcopyrite for copper recovery, is a more sustainable biotechnological process that exploits the capacity of acidophilic microbes to catalyze solid metal sulfide dissolution to soluble metal sulfates. A key early stage in biomining is cell attachment and biofilm formation on the mineral surface that results in elevated mineral oxidation rates. Industrial biomining of chalcopyrite is typically carried out in large scale heaps that suffer from the downsides of slow and poor metal recoveries. In an effort to mitigate these drawbacks, this study investigated planktonic and biofilm cells of acidophilic (optimal growth pH, Measurement(s)biofilm formation • Proteome • protein expression data • RNA • transcriptomeTechnology Type(s)fluorescence microscopy • mass spectrometry • RNA sequencingSample Characteristic - OrganismLeptospirillum ferriphilum • Sulfobacillus thermosulfidooxidans • Acidithiobacillus caldusSample Characteristic - Environmentmine Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12173637

In this chapter we provide an overview of the biofilm lifestyle of (hyper) thermophilic and acidophilic archaea. In contrast to the bacterial domain, biofilm formation of archaea is far less studied and understood. Recent evidence has shown that upon biofilm formation, archaeal cells are able to secrete extracellular polymeric substances (EPS), whose composition changes according to available energy sources, environmental fluctuations and biofilm maturity. Also, several type IV pili-like surface cell appendages (e.g. the archaellum and the aap pili) have been shown to be involved in the initial attachment of archaea to surfaces. Little is known about cell-to-cell interactions and potential cell-to-cell communication mechanisms in archaea. Therefore, molecules and signaling pathways involved in these processes might be substantially different from what it is already described in bacteria. Recently, the first archaeal transcriptional regulators involved in biofilm formation have been discovered in Sulfolobus species. Future avenues in archaeal biofilm research include: (i) the EPS biosynthesis pathways and biofilm-specific bio-molecules, (ii) cell-to-cell communication mechanisms and microbial interactions within multispecies archaeal biofilms, as well as (iii) potential interaction with bacterial and eukaryotic microorganisms. Finally, biotechnological applications of thermophilic and acidophilic archaeal biofilms are discussed.

Bioleaching is the extraction of metals, such as copper or gold, from sulfidic ores by microorganisms. Their energy for growth is obtained by oxidation of ferrous iron and/or reduced inorganic sulfur compounds. Bacterial attachment to ores increase leaching activities through the formation of a special microenvironment between the bacterium and the metal sulfide surface, filled by extracellular polymeric substances (EPS). Recently, a high-throughput proteomic comparison from biofilm cells attached to pyrite (FeS2) and planktonic cells of the mesophilic iron and/or sulfur oxidizer Acidithiobacillus ferrooxidans ATCC 23270 was done. Several proteins were found to be up-regulated in biofilm cells. Among them, membrane and outer membrane proteins probably involved in osmotic regulation, polysaccharide biosynthesis and protein secretion, as well as proteins probably involved in cofactor metabolism were present. In order to extend our knowledge of the genus Acidithiobacilli, we started a high-throughput proteomic analysis of the sulfur oxidizer Acidithiobacillus caldus ATCC 51756 by comparing cells grown with an insoluble energy substrate such as elemental sulfur (S°) against cells grown on a soluble energy substrate, such as thiosulfate. The results revealed several differences in proteins related to sulfur metabolism, potential EPS biosynthesis pathways as well as membrane and transport functions. In both microorganisms several conserved hypothetical proteins were found. Some of them were also found to be induced in sessile cells, suggesting their potential involvement in biofilm formation. This study will provide new insights into the biology of Acidithiobacilli and will probably help assigning functions to poorly characterized and unknown proteins. Keywords: Biofilm, Proteomics, Acidithiobacilli

A functional luxIR-type Quorum Sensing (QS) system is present in Acidithiobacillus ferrooxidans. However, cell-cell communication among various acidophilic chemolithoautotrophs growing on pyrite has not been studied in detail. These aspects are the scope of this study with emphasis on the effects exerted by the N-acyl-homoserine lactone (AHL) type signaling molecules which are produced by Acidithiobacillus ferrooxidans. Their effects on attachment and leaching efficiency by other leaching bacteria, such as Acidithiobacillus ferrivorans, Acidiferrobacter spp. SPIII/3 and Leptospirillum ferrooxidans in pure and mixed cultures growing on pyrite is shown.

Bioleaching of metal sulfides is performed by a diverse group of microorganisms. The dissolution chemistry of metal sulfides follows two pathways, which are determined by the mineralogy and the acid solubility of the metal sulfides: the thiosulfate and the polysulfide pathways. Bacterial cells can effect this metal sulfide dissolution via iron(II) ion and sulfur compound oxidation. Thereby, iron(III) ions and protons, the metal sulfide-attacking agents, are available. Cells can be active either in planktonic state or in forming biofilms on the mineral surface; however, the latter is much more efficient in terms of bioleaching kinetics. In the case of Acidithiobacillus ferrooxidans, bacterial exopolymers contain iron(III) ions, each complexed by two uronic acid residues. The resulting positive charge allows an electrostatic attachment to the negatively charged pyrite. Thus, the first function of complexed iron(III) ions is the mediation of cell attachment, while their second function is oxidative dissolution of the metal sulfide, similar to the role of free iron(III) ions in non-contact leaching. In both cases, the electrons extracted from the metal sulfide reduce molecular oxygen via a redox chain forming a supercomplex spanning the periplasmic space and connecting both outer and inner membranes. In this review, we summarize some recent discoveries relevant to leaching bacteria which contribute to a better understanding of these fascinating microorganisms. These include surface science, biochemistry of iron and sulfur metabolism, anaerobic metabolism, and biofilm formation. The study of microbial interactions among multispecies leaching consortia, including cell-to-cell communication mechanisms, must be considered in order to reveal more insights into the biology of bioleaching microorganisms and their potential biotechnological use.

Acidithiobacillus ferrooxidans is a chemolithoautotrophic, mesophilic Gram-negative bacterium able to oxidize ferrous iron, sulfur, and metal sulfides. It forms monolayer biofilms where extracellular polymeric substances are essential for cell attachment and metal sulfide leaching. High-throughput proteomics has been applied to study the early process of biofilm formation on pyrite by At. ferrooxidans ATCC 23270. After 24 h contact with the mineral, planktonic and sessile (biofilm) cell subpopulations were separated and proteins extracted. In total, 1319 proteins were detected in both samples. Sixty-two of these were found to be increased in biofilms. Additionally, 25 proteins were found to be decreased in the biofilm cell subpopulation. Three transcriptional factors were found to be increased or decreased among both cell subpopulations, suggesting their potential involvement in the regulation of these processes. Although no significant differences were observed for the known proteins related to ferrous iron and sulfur oxidation pathways among both cell subpopulations, the results presented here show that the early steps of At. ferrooxidans biofilm formation consist of a set of metabolic adaptations following cell attachment to the mineral surface. Functions such as extracellular polymeric substances biosynthesis seem to be pivotal. This first high-throughput proteomic study may also contribute to the annotation of several unknown At. ferrooxidans proteins found.

Microrganisms are well known for their unique ability to thrive in different lifestyles (e.g. planktonic or sessile) and environments, even within extreme ones. The most common and widespread lifestyle of microbes on earth is in form of biofilms, associated colonies of microorganisms embedded in a matrix of extracellular polymeric substances (EPS). Extremely acidophilic metal/sulfur-oxidizing microorganisms (AMOM) thrive in special ecological niches characterized by harsh conditions such as low pH (below 3) and high concentration of heavy metals across a broad range of temperatures. The molecular mechanisms controlling biofilm formation in acidophilic leaching bacteria are starting to be elucidated while these operating in archaea are far less explored. In this chapter we provide an overview about the biofilm lifestyle of AMOM. This includes surface sciences, microscopy, cell-cell communication, interspecies interactions as well as molecular and high-throughput studies. Current knowledge on the EPS composition and biofilm visualization of acidophiles is also included. Future perspectives in this field include the elucidation of EPS biosynthesis pathways and a comprehensive analysis of the chemical nature of the EPS polymers. Cell-cell communication and microbial interactions within multispecies biofilms of acidophiles are considered to be crucial determinants in controlling the metabolic activity of AMOM. Either for their biotechnological applications in biomining or in mitigation of acid mine drainage (AMD) generation, further studies in both fields may presumably reveal key perspectives to influence and control bioleaching of sulfide minerals.

The elemental sulfur oxidising enzyme Sulfur Oxygenase Reductase (SOR) is very well investigated in acidothermophilic archaea, such as Acidianus brierleyi and Sulfolobus metallicus. In contrast, not much is known about the biochemistry of elemental sulfur oxidation in acidophilic bacteria. Recently, however, the SOR-encoding gene has been found also in a bacterial strain closely related to the moderate thermophile Acidithiobacillus caldus. Confusingly, for the latter species, also the involvement of the SOX system as well as thiosulfate:quinone oxidoreductase (TQO) and tetrathionate hydrolase (TTH) in sulfur compound oxidation has been proposed based on genome analysis. In this study, we have detected the sor-gene in other Acidithiobacillus caldus-like strains, isolated from various bioleaching habitats, indicating that SOR plays an important role in sulfur oxidation in this species. Based on sequence comparison, the new bacterial sor-genes are closely related and distant from the known archaeal sequences as well as from the SOR found in the neutrophilic bacterium Aquifex aeolicus. In addition, SOR activity has been detected in crude cell extracts from all Acidithiobacillus caldus-like strains tested. The enzyme is truly thermophilic as highest activities were achieved at 65 °C, which is far beyond the growth optimum of Acidithiobacillus caldus. This finding may give rise to the question whether the presence of SOR in Acidithiobacillus caldus is only relevant while growing at elevated temperatures. Currently, experiments are performed for testing this hypothesis (comparing growth and enzyme activities at 30 vs. 45 °C).

The aim of this study was to investigate interspecies interaction by quantifying pyrite dissolution and to visualize the colonization of metal sulfides in pure and mixed cultures of leaching bacteria. Strains, such as chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 23270 (type strain) and chemoorganoheterotrophic Acidiphilium cryptum JF-5, were used. Sessile, pyrite-detached and planktonic cells were visualized by epifluorescence microscopy using DAPI staining and FISH. Additionally, atomic force microscopy was used for investigations on cell morphology, bacterial distribution on pyrite and mineral surface topography. In pure At. ferrooxidans cultures and in mixed cultures with Ap. cryptum JF-5, it could be shown that the bacterial morphology of sessile cells differed significantly from those of planktonic cells by decreased cell sizes and enhanced production of extracellular polymeric substances in case of sessile cells. Interspecies interaction in mixed cultures resulted in increased pyrite leaching and production of extracellular polymeric substances and consequently, enhanced biofilm formation.

Bioleaching is the extraction of metals, such as copper or gold, from ore by microorganisms. Bacterial attachment increases leaching activities due to the formation of a "reaction space" between the metal sulfide surface and the cell. This process depends on abiotic characteristics such as purity and degree of crystallization of the metal sulfide, as well as biotic ones such as the capacity of the bacteria for detecting favourable attachment sites and synthesizing a suitable cell envelope (EPS), for adhesion. Planktonic and sessile cells should differ significantly in their metabolic activities and therefore in their gene expression patterns. To help to understand At. ferrooxidans biofilm formation, microarray transcript profiling was carried out to compare planktonic and sessile cells. The high contents of EPS and ferric iron of the biofilms are interfering with RNA extraction, causing inhibition of DNAse, reverse transcriptase and/or polymerase activities required to get labelled target cDNA. In order to have sufficient high quality RNA suitable for transcriptomic analysis, we have optimized the biofilm formation of At. ferrooxidans on pyrite (FeS2) and the RNA extraction from the sessile cell population. DNA microarrays have been hybridized with labelled cDNAs from sessile and planktonic cells and preliminary data suggest that some genes are differently expressed between these two subpopulations. The understanding of these differences may help us to shift populations of leaching bacteria from the planktonic state towards the sessile state in order to influence bioleaching.

The sulfur oxygenase reductase (Sor) catalyzes the oxygen dependent disproportionation of elemental sulfur, producing sulfite, thiosulfate and sulfide. Being considered an "archaeal like" enzyme, it is also encoded in the genomes of some acidophilic leaching bacteria such as Acidithiobacillus caldus, Acidithiobacillus thiooxidans, Acidithiobacillus ferrivorans and Sulfobacillus thermosulfidooxidans, among others. We measured Sor activity in crude extracts from Sb. thermosulfidooxidans DSM 9293(T). The optimum temperature for its oxygenase activity was achieved at 75 °C, confirming the "thermophilic" nature of this enzyme. Additionally, a search for genes probably involved in sulfur metabolism in the genome sequence of Sb. thermosulfidooxidans DSM 9293(T) was done. Interestingly, no sox genes were found. Two sor genes, a complete heterodisulfidereductase (hdr) gene cluster, three tetrathionate hydrolase (tth) genes, three sulfide quinonereductase (sqr), as well as the doxD component of a thiosulfate quinonereductase (tqo) were found. Seven At. caldus strains were tested for Sor activity, which was not detected in any of them. We provide evidence that an earlier reported Sor activity from At. caldus S1 and S2 strains most likely was due to the presence of a Sulfobacillus contaminant.

The exceptional diversity of ecogeographical conditions of Armenia and the richness of non-ferrous metals represent a great and valuable potential for the investigation of the biodiversity of acidophilic chemolithotrophic bacteria as well as for the isolation of new highly efficient strains and their communities. The studies of physiological and biotechnological properties of these relevant strains from the bioleaching communities will allow us to achieve comprehensive insight of interspecies relationships as a substantial prerequisite for the enhancement of the efficiency of biohydrometallurgical processes. In this study, novel strains of acidophilic chemolithotrophic bacteria belonging to genera Acidithiobacillus as well as Leptospirillum and Sulfobacillus have been isolated from sulfide ores of different geoformations in Armenia. The results showed that: (1) high efficiency of the isolated bacteria in bioleaching of pyrite was determined by their biochemical properties, especially enzyme activities involved in oxidation of ferrous iron and reduced inorganic sulfur compounds (RISCs). (2) Bioleaching of pyrite was directly connected with the ability of isolated strains to adhere on pyrite surfaces. Moreover, the efficiency of bioleaching of pyrite was correlated with the amount of adhered/attached bacterial cells of the isolated strains.

Bioleaching of metal sulfides is an interfacial process where biofilm formation is considered to be important in the initial steps of this process. Among the factors regulating biofilm formation, molecular cell-to-cell communication such as quorum sensing is involved. A functional LuxIR-type I quorum sensing system is present in Acidithiobacillus ferrooxidans. However, cell-to-cell communication among different species of acidophilic mineral-oxidizing bacteria has not been studied in detail. These aspects were the scope of this study with emphasis on the effects exerted by the external addition of mixtures of synthetic N-acyl-homoserine-lactones on pure and binary cultures. Results revealed that some mixtures had inhibitory effects on pyrite leaching. Some of them correlated with changes in biofilm formation patterns on pyrite coupons. We also provide evidence that A. thiooxidans and Acidiferrobacter spp. produce N-acyl-homoserine-lactones. In addition, the observation that A. thiooxidans cells attached more readily to pyrite pre-colonized by living iron-oxidizing acidophiles than to heat-inactivated or biofilm-free pyrite grains suggests that other interactions also occur. Our experiments show that pre-cultivation conditions influence A. ferrooxidans attachment to pre-colonized pyrite surfaces. The understanding of cell-to-cell communication may consequently be used to develop attempts to influence biomining/bioremediation processes.

Extracellular Polymeric Substances (EPS) are of fundamental importance for attachment to metal sulfides, biofilm formation and leaching efficiency of Acidithiobacillus ferrooxidans. In this work we have visualized the capsular polysaccharides (CPS) of A. ferrooxidans ATCC 23270 using the fluorescently-labeled lectin Concanavalin A (ConA) and confocal laser scanning microscopy (CLSM). Ferrous iron-grown cells exhibited low amounts of CPS. A strong induction of CPS production was observed for planktonic and mineralattached cells 24 h after transfer to pyrite (FeS2). Additionally, CPS production was inducible in the presence of metal sulfide degradation products, even when cells had no direct contact to metal sulfide surfaces. Despite being an autotrophic bacterium, it has been found that sugars, presumably serving as polysaccharide building blocks stimulate CPS production in A. ferrooxidans. A strong induction of CPS was found after growth of cells on floating filters in ferrous iron medium supplemented with 2.5 mg/mL glucose or galactose, while the presence of 0.25 mg/mL casaminoacids did not show effects on induction of CPS. A similar phenotype was also found after growth under phosphate (Pi) limited growth conditions. Our results strongly suggest that CPS expression is inducible by several factors, including bacterial contact with metal sulfide degradation products, while directmineral contact seems not to be a prerequisite. The molecular mechanisms regulating expression of genes encoding CPS biosynthesis and export systems in A. ferrooxidans remain to be elucidated.

The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans is of great importance in biomining operations. During the bioleaching of ores, microorganisms are subjected to a variety of environmental stresses and to the limitations of some nutrients, such as inorganic phosphate (P i ), which is an essential component for all living cells. Although the primary source of phosphorus for microorganisms is P i , some bacteria are also able to metabolize P i esters (with a C-O-P bond) and phosphonates (with a very inert C-P bond). By using bioinformatic analysis of genomic sequences of the type strain of A. ferrooxidans (ATCC 23270), we found that as part of a Pho regulon, this bacterium has a complete gene cluster encoding C-P lyase, which is the main bacterial enzyme involved in phosphonate (Pn) degradation in other microorganisms. A. ferrooxidans was able to grow in the presence of methyl-Pn or ethyl-Pn as an alternative phosphorus source. Under these growth conditions, a great reduction in inorganic polyphosphate (polyP) levels was seen compared with the level for cells grown in the presence of P i . By means of reverse transcription-PCR (RT-PCR), DNA macroarrays, and real-time RT-PCR experiments, it was found that A. ferrooxidans phn genes were cotranscribed and their expression was induced when the microorganism was grown in methyl-Pn as the only phosphorus source. This is the first report of phosphonate utilization in a chemolithoautotrophic microorganism. The existence of a functional C-P lyase system is a clear advantage for the survival under P i limitation, a condition that may greatly affect the bioleaching of ores.

Surface-enhanced Raman spectroscopy (SERS) was demonstrated to be a useful and sensitive structural spectroscopic tool for the characterization of different types of supramolecular architectures, ideally suited for trace analysis. SERS spectra allowed to distinguish cell membrane components of the biomining bacterium Acidithiobacillus ferrooxidans and their physical and chemical variations caused by different growth media. Several SERS spectral modifications were arguments to propose different structures for two isolated model blue cooper proteins, i.e., hemocyanins belonging to mollusc Concholepas concholepas (CCH) and Megathura crenulata (KLH). The complex structure of a humic acid was demonstrated to be highly sensitive to the pH of the medium; a globular conformation predominated at lower pH, while a decurled structure was observed at higher pH. Finally, the SERS technique using different excitation laser lines allowed to characterizing a carbon nanotube as a single-walled species, with metallic electronic characteristics; these species coexisted with different diameters

Acidithiobacillus ferrooxidans is one of the main acidophilic chemolithotrophic bacteria involved in the bioleaching of metal sulfide ores. The bacterium-mineral interaction requires the development of biofilms, whose formation is regulated in many microorganisms by type AI-1 quorum sensing. Here, we report the existence and characterization of a functional type AI-1 quorum-sensing system in A. ferrooxidans . This microorganism produced mainly acyl-homoserine lactones (AHL) with medium and large acyl chains and different C-3 substitutions, including 3-hydroxy-C 8 -AHL, 3-hydroxy-C 10 -AHL, C 12 -AHL, 3-oxo-C 12 -AHL, 3-hydroxy-C 12 -AHL, C 14 -AHL, 3-oxo-C 14 -AHL, 3-hydroxy-C 14 -AHL, and 3-hydroxy-C 16 -AHL. A quorum-sensing genetic locus that includes two open reading frames, afeI and afeR , which have opposite orientations and code for proteins with high levels of similarity to members of the acyl synthase (I) and transcriptional regulator (R) protein families, respectively, was identified. Overexpression of AfeI in Escherichia coli and the associated synthesis of AHLs confirmed that AfeI is an AHL synthase. As determined by reverse transcription-PCR, the afeI and afeR genes were transcribed in A. ferrooxidans . The transcription levels of the afeI gene were higher in cells grown in sulfur and thiosulfate media than in iron-grown cells. Phosphate starvation induced an increase in the transcription levels of afeI which correlated with an increase in AHL levels. Two afe boxes which could correspond to the AfeR binding sites were identified upstream of the afeI gene. This is the first report of a functional type AI-1 quorum-sensing system in an acidophilic chemolithotrophic microorganism, and our results provide a very interesting opportunity to explore the control and regulation of biofilm formation during the bioleaching process.