Ferric Uptake Regulator Provides a New Strategy for Acidophile Adaptation to Acidic Ecosystems.

Acidophiles play a dominant role in driving elemental cycling in natural acid mine drainage (AMD) habitats and exhibit important application value in bioleaching and bioremediation. Acidity is an inevitable environmental stress and a key factor that affects the survival of acidophiles in their acidified natural habitats; however, the regulatory strategies applied by acidophilic bacteria to withstand low pH are unclear. We identified the significance of the ferric uptake regulator (Fur) in acidophiles adapting to acidic environments and discovered that Fur is ubiquitous as well as highly conserved in acidophilic bacteria. Mutagenesis of the fur gene of Acidithiobacillus caldus, a prototypical acidophilic sulfur-oxidizing bacterium found in AMD, revealed that Fur is required for the acid resistance of this acidophilic bacterium. Phenotypic characterization, transcriptome sequencing (RNA-seq), mutagenesis, and biochemical assays indicated that the Acidithiobacillus caldus ferric uptake regulator (AcFur) is involved in extreme acid resistance by regulating the expression of several key genes of certain cellular activities, such as iron transport, biofilm formation, sulfur metabolism, chemotaxis, and flagellar biosynthesis. Finally, a Fur-dependent acid resistance regulatory strategy in A. caldus was proposed to illustrate the ecological behavior of acidophilic bacteria under low pH. This study provides new insights into the adaptation strategies of acidophiles to AMD ecosystems and will promote the design and development of engineered biological systems for the environmental adaptation of acidophiles.IMPORTANCE This study advances our understanding of the acid tolerance mechanism of A. caldus, identifies the key fur gene responsible for acid resistance, and elucidates the correlation between fur and acid resistance, thus contributing to an understanding of the ecological behavior of acidophilic bacteria. These findings provide new insights into the acid resistance process in Acidithiobacillus species, thereby promoting the study of the environmental adaptation of acidophilic bacteria and the design of engineered biological systems.

Active fluid with Acidithiobacillus ferrooxidans: correlations between swimming and the oxidation route.

To explore engineering platforms towards 'active bacterial baths', we grow and characterize native and commercial strains of Acidithiobacillus ferrooxidans to promote swimming locomotion. Three different energy sources were used, namely elemental sulfur, ferrous sulfate, and pyrite. The characteristics of the culture, such as pH, Eh, and the concentration of cells and ions, are monitored to seek correlations between the oxidation route and the transport mechanism. We found that only elemental sulfur induces swimming mobility in the commercial DSMZ - 24,419 strain, while ferrous sulfate and the sulfide mineral, pyrite, did not activate swimming on any strain. The bacterial mean squared displacement and the mean velocity are measured to provide a quantitative description of the bacterial mobility. We found that, even if the A. ferrooxidans strain is grown in a sulfur-rich environment, it preferentially oxidizes iron when an iron-based material is included in the media. Similar to other species, once the culture pH decreases below 1.2, the active locomotion is inhibited. The engineering control and activation of swimming in bacterial cultures offer fertile grounds towards applications of active suspensions such as energy-efficient bioleaching, mixing, drug delivery, and bio-sensing.

Severe microbiologically influenced corrosion of S32654 super austenitic stainless steel by acid producing bacterium Acidithiobacillus caldus SM-1.

Microbiologically influenced corrosion (MIC) of S32654 (654SMO) super austenitic stainless steel (SASS) by acid producing bacterium (APB), Acidithiobacillus caldus SM-1, a strain of sulfur-oxidizing bacteria (SOB) used in biohydrometallurgy field, was investigated using electrochemical measurements and surface characterizations during a 14-day immersion test. The results indicated that S32654 SASS was susceptible to MIC by APB, and A. caldus SM-1 was capable of producing an aggressive acidic environment underneath the biofilm, resulting in the dissolution of the passive film and severe pitting attacks against S32654 SASS, which is commonly regarded as a corrosion resistant material.

Strand-specific RNA-seq analysis of the Acidithiobacillus ferrooxidans transcriptome in response to magnesium stress.

Bioleaching is a promising process for 350 million tons Jinchuan low-grade pentlandite. But, Jinchuan pentlandite has lots of magnesium and high concentration of Mg2+ is harmful to bioleaching microorganisms. Thus, finding a way to improve the adaption of microorganisms to Mg2+ is a key for bioleaching. In the study, we found that oxidizing activity, bioleaching ability and biofilm formation of A.f were inhibited by Mg2+ stress. In addition, we analyzed mRNA and small RNA (sRNA) of Acidithiobacillus ferrooxidans (A.f) under Mg2+ stress by strand-specific RNA-sequencing (ssRNA-seq). After the bioinformatics process, 2475 coding genes were obtained, and there were 33 differential expression genes (DEGs) in 0.1 M-VS-Con, including 28 down-regulated and 5 up-regulated, whereas 52 DEGs were obtained in 0.5 M-VS-Con, including 28 down-regulated and 24 up-regulated. Gene ontology analysis showed most of DEGs were involved in catalytic activity, metabolic process and single-organism process. Furthermore, we identified 636 sRNA and some differential expression sRNA that may respond to Mg2+ stress. Further analysis of DEGs suggested that Mg2+ stress reduced biofilm formation perhaps through inhibiting Type IV Pili-related gene expression and inhibited bacterial activity perhaps through affecting carbon fixation. The study provided the foundation to understand the mechanisms of Mg2+ resistance in A.f and may be helpful to improve bioleaching ability for pentlandit.

The effect of pH on the dynamics of natural membranes.

Pure phospholipids and membrane fragments from bacterial cells living under various conditions were studied against the influence of the surrounding acidity on the internal dynamics. For that we compared mean square displacements extracted from elastic incoherent neutron scattering data, measured both at low and at neutral pH, of the phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine and of samples from neutralophilic and acidophilic micro-organisms (some being hyperthermophilic and others mesophilic). The lipids showed a slight shift in the phase transition temperature of about 4 degrees under pH variation and became slightly more mobile at lower pH. The membrane fragments not used to extreme acidic conditions were significantly more sensitive to variations in the pH values, whereas the acidophilic and -tolerant samples were much less influenced by this parameter. They presented the higher softness at low pH, which was closer to their native condition. Such findings might be a hint for adaptation mechanisms to different acidity conditions.

Mg2+ reduces biofilm quantity in Acidithiobacillus ferrooxidans through inhibiting Type IV pili formation.

Bioleaching is a promising process for 350 million tons of Jinchuan low-grade pentlandite. But high concentration of Mg2+ is harmful to bioleaching microorganisms. Interestingly, biofilm formation can improve leaching rate. Thus, it is actually necessary to investigate the effect of Mg2+ stress on Acidithiobacillus ferrooxidans biofilms formation. In this study, we found that 0.1 and 0.5 M Mg2+ stress significantly reduced the total biomass of biofilm in a dose-dependent manner. The observation results of extracellular polymeric substances and bacteria using confocal laser scanning microscopy showed that the biofilm became thinner and looser under Mg2+ stress. Whereas 0.1 and 0.5 M Mg2+ stress had no remarkable effect on the bacterial viability, the attachment rate of Acidithiobacillus ferrooxidans to pentlandite was reduced by Mg2+ stress. Furthermore, sliding motility, twitching motility and the gene expression level of pilV and pilW were inhibited under Mg2+ stress. These results suggested that Mg2+ reduced biofilm formation through inhibiting pilV and pilW gene expression, decreasing Type IV pili formation and then attenuating the ability of attachment, subduing the active expansion of biofilms mediated by twitching motility. This study provided more information about the effect of Mg2+ stress on biofilm formation and may be useful for increasing the leaching rate in low-grade pentlandit.

Salt Stress-Induced Loss of Iron Oxidoreduction Activities and Reacquisition of That Phenotype Depend on rus Operon Transcription in Acidithiobacillus ferridurans.

The type strain of the mineral-oxidizing acidophilic bacterium Acidithiobacillus ferridurans was grown in liquid medium containing elevated concentrations of sodium chloride with hydrogen as electron donor. While it became more tolerant to chloride, after about 1 year, the salt-stressed acidophile was found to have lost its ability to oxidize iron, though not sulfur or hydrogen. Detailed molecular examination revealed that this was due to an insertion sequence, ISAfd1, which belongs to the ISPepr1 subgroup of the IS4 family, having been inserted downstream of the two promoters PI and PII of the rus operon (which codes for the iron oxidation pathway in this acidophile), thereby preventing its transcription. The ability to oxidize iron was regained on protracted incubation of the culture inoculated onto salt-free solid medium containing ferrous iron and incubated under hydrogen. Two revertant strains were obtained. In one, the insertion sequence ISAfd1 had been excised, leaving an 11-bp signature, while in the other an ∼2,500-bp insertion sequence (belonging to the IS66 family) was detected in the downstream inverted repeat of ISAfd1 The transcriptional start site of the rus operon in the second revertant strain was downstream of the two ISs, due to the creation of a new "hybrid" promoter. The loss and subsequent regaining of the ability of A. ferriduransT to reduce ferric iron were concurrent with those observed for ferrous iron oxidation, suggesting that these two traits are closely linked in this acidophile.IMPORTANCE Iron-oxidizing acidophilic bacteria have primary roles in the oxidative dissolution of sulfide minerals, a process that underpins commercial mineral-processing biotechnologies ("biomining"). Most of these prokaryotes have relatively low tolerance to chloride, which limits their activities when only saline or brackish waters are available. The study showed that it was possible to adapt a typical iron-oxidizing acidophile to grow in the presence of salt concentrations similar to those in seawater, but in so doing they lost their ability to oxidize iron, though not sulfur or hydrogen. The bacterium regained its capacity for oxidizing iron when the salt stress was removed but simultaneously reverted to tolerating lower concentrations of salt. These results suggest that the bacteria that have the main roles in biomining operations could survive but become ineffective in cases where saline or brackish waters are used for irrigation.

Quorum sensing improves current output with Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is a strict acidophilic chemolithoautotrophic bacterium that obtains its energy from reduced inorganic sulfur species or ferrous iron oxidation under aerobic conditions. Carbon felt electrodes were pre-colonized by A. ferrooxidansATCC 23270T using ferrous iron or sulfur as electron donors, via the addition (or not) of a mixture of C14 acyl-homoserine lactones (C14-AHLs). Electrode coverage during pre-colonization was sparse regardless of the electron donor source, whereas activation of quorum sensing significantly enhanced it. Microbial fuel cells (MFCs) inoculated with pre-colonized electrodes (which behaved as biocathodes) were more efficient in terms of current production when iron was used as an electron donor. Biocathode coverage and current output were remarkably increased to -0.56 A m-2 by concomitantly using iron-based metabolism and C14-AHLs. Cyclic voltammetry displayed different electrochemical reactions in relation to the nature of the electron donor, underlying the implication of different electron transfer mechanisms.

Physiological and comparative genomic analysis of Acidithiobacillus ferrivorans PQ33 provides psychrotolerant fitness evidence for oxidation at low temperature.

Friendly environmental hydrometallurgy at low temperatures is principally promoted by Acidithiobacillus ferrivorans. Until recently, the synergy between cold tolerance and the molecular mechanism of ferrous iron (Fe2+) oxidation was unknown. In the present paper, we conducted a physiological and comparative genomics analysis of the new strain A. ferrivorans PQ33 to elucidate the oxidation mechanism at low temperatures, with emphasis placed on trehalose and the Rus operon. PQ33 exhibited a doubling time of 66.6 h in Fe2+ at pH 1.6 and 63.6 h in CuS at 5 °C. Genomic island (GI) identification and comparative genome analysis were performed with four available genomes of Acidithiobacillus sp. The genome comprised 3,298,172 bp and 56.55% GC content. In contrast to ATCC Acidithiobacillus ferrooxidans strains, the genome of A. ferrivorans PQ33 harbors one GI, which contains a RusB gene. Moreover, five genes of peptidyl-prolyl cis-trans isomerase (PPIases) were observed. Furthermore, comparative analysis of the trehalose operon suggested the presence of a horizontal transfer event. In addition, comparison of rusticyanin proteins revealed that RusB has better intrinsic flexibility than RusA. This comparison suggests psychrotolerant fitness and supports the genetic canalization of A. ferrivorans PQ33 for oxidation at low temperature.

Biogeography of sulfur-oxidizing Acidithiobacillus populations in extremely acidic cave biofilms.

Extremely acidic (pH 0-1.5) Acidithiobacillus-dominated biofilms known as snottites are found in sulfide-rich caves around the world. Given the extreme geochemistry and subsurface location of the biofilms, we hypothesized that snottite Acidithiobacillus populations would be genetically isolated. We therefore investigated biogeographic relationships among snottite Acidithiobacillus spp. separated by geographic distances ranging from meters to 1000s of kilometers. We determined genetic relationships among the populations using techniques with three levels of resolution: (i) 16S rRNA gene sequencing, (ii) 16S-23S intergenic transcribed spacer (ITS) region sequencing and (iii) multi-locus sequencing typing (MLST). We also used metagenomics to compare functional gene characteristics of select populations. Based on 16S rRNA genes, snottites in Italy and Mexico are dominated by different sulfur-oxidizing Acidithiobacillus spp. Based on ITS sequences, Acidithiobacillus thiooxidans strains from different cave systems in Italy are genetically distinct. Based on MLST of isolates from Italy, genetic distance is positively correlated with geographic distance both among and within caves. However, metagenomics revealed that At. thiooxidans populations from different cave systems in Italy have different sulfur oxidation pathways and potentially other significant differences in metabolic capabilities. In light of those genomic differences, we argue that the observed correlation between genetic and geographic distance among snottite Acidithiobacillus populations is partially explained by an evolutionary model in which separate cave systems were stochastically colonized by different ancestral surface populations, which then continued to diverge and adapt in situ.

A direct observation of bacterial coverage and biofilm formation by Acidithiobacillus ferrooxidans on chalcopyrite and pyrite surfaces.

To obtain a fundamental understanding of the population behaviour of Acidithiobacillus ferrooxidans at chalcopyrite and pyrite surfaces, the early stage attachment behaviour and biofilm formation by this bacterium on chalcopyrite (CuFeS2) and pyrite (FeS2) were studied by optical microscopy, Raman spectroscopy, time-of-flight secondary ion mass spectrometry (ToF-SIMS) and electron backscatter diffraction (EBSD). The results indicate there was no significant difference in selectivity of bacterial attachment between chalcopyrite and pyrite. However, the result of ToF-SIMS analysis suggests that the surface of the pyrite was covered more extensively by biofilm than that of the chalcopyrite, which may indicate more extracellular polymeric substances (EPS) formation by bacterial cells growing on pyrite. EBSD and optical image analysis indicated that selectivity of bacterial attachment to chalcopyrite was not significantly affected by crystal orientation. The results also suggest that the bacterial population in defective areas of chalcopyrite was significantly higher than on the polished surfaces.

Model-based evaluation of ferrous iron oxidation by acidophilic bacteria in chemostat and biofilm airlift reactors.

This article presents a model-based evaluation of ferrous iron oxidation in chemostat and biofilm airlift reactors inoculated with a mixed culture of Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans bacteria. The competition between the two types of bacteria in the chemostat and in the biofilm airlift reactors together with the distribution of both bacteria along the biofilm thickness at different time sections has been studied. The bacterial distribution profiles along the biofilm in the airlift reactor at different time scales show that in the beginning A. ferrooxidans bacteria are dominant, but when the reactor operates for a long time the desirable L. ferrooxidans species outcompete A. ferrooxidans as a result of the low Fe(2+) and high Fe(3+) concentrations. The results obtained from the simulation were compared with the experimental data of continuously operated internal loop airlift biofilm reactor. The model results are in good agreement with the experimental results.

Diguanylate cyclase null mutant reveals that C-Di-GMP pathway regulates the motility and adherence of the extremophile bacterium Acidithiobacillus caldus.

An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319) that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process.

Isolation and characterization of a novel Acidithiobacillus ferrivorans strain from the Chilean Altiplano: attachment and biofilm formation on pyrite at low temperature.

Microorganisms are used to aid the extraction of valuable metals from low-grade sulfide ores in mines worldwide, but relatively little is known about this process in cold environments. This study comprises a preliminary analysis of the bacterial diversity of the polyextremophilic acid River Aroma located in the Chilean Altiplano, and revealed that Betaproteobacteria was the most dominant bacterial group (Gallionella-like and Thiobacillus-like). Taxa characteristic of leaching environments, such Acidithiobacillus and Leptospirillum, were detected at low abundances. Also, bacteria not associated with extremely acidic, metal-rich environments were found. After enrichment in iron- and sulfur-oxidizing media, we isolated and identified a novel psychrotolerant Acidithiobacillus ferrivorans strain ACH. This strain can grow using ferrous iron, sulfur, thiosulfate, tetrathionate and pyrite, as energy sources. Optimal growth was observed in the presence of pyrite, where cultures reached a cell number of 6.5 · 10(7) cells mL(-1). Planktonic cells grown with pyrite showed the presence of extracellular polymeric substances (10 °C and 28 °C), and a high density of cells attached to pyrite grains were observed at 10 °C by electron microscopy. The attachment of cells to pyrite coupons and the presence of capsular polysaccharides were visualized by using epifluorescence microscopy, through nucleic acid and lectin staining with Syto(®)9 and TRITC-Con A, respectively. Interestingly, we observed high cell adhesion including the formation of microcolonies within 21 days of incubation at 4 °C, which was correlated with a clear induction of capsular polysaccharides production. Our data suggests that attachment to pyrite is not temperature-dependent in At. ferrivorans ACH. The results of this study highlight the potential of this novel psychrotolerant strain in oxidation and attachment to minerals under low-temperature conditions.

Compensation phenomena found in Acidithiobacillus ferrooxidans after starvation stress.

Acidithiobacillus ferrooxidans showed the compensate growth and oxidation after re-feeding with sufficient ferrous materials after starvation. Compensatory phenomena were first detected in chemoautotrophic organisms. Starvation stress of Acidithiobacillus ferrooxidans was achieved via culturing in low concentrations of iron. During compensation, growth and ferrous oxidation took place faster than in controls. In addition, some genes related to ferrous oxidation (such as rus) and carbon assimilation (cbbR, csoS3) were expressed in different patterns in the low energy environments. Their expression patterns can account for this increased growth and oxidation. Other groups of genes (cspAB, feoAB, fur) were suppressed in response to starvation stress. The presence of pyrite and joint cold stress can render compensation nearly undetectable. This may be why the compensation phenomena observed under these conditions was not the same as that observed under single starvation stress conditions. Gene expression reflected a possible mechanism of tolerance to starvation in Acidithiobacillus ferrooxidans, which would allow the organism to adapt and survive in ferrous-limited environments.

Type IV pili of Acidithiobacillus ferrooxidans can transfer electrons from extracellular electron donors.

Studies on Acidithiobacillus ferrooxidans accepting electrons from Fe(II) have previously focused on cytochrome c. However, we have discovered that, besides cytochrome c, type IV pili (Tfp) can transfer electrons. Here, we report conduction by Tfp of A. ferrooxidans analyzed with a conducting-probe atomic force microscope (AFM). The results indicate that the Tfp of A. ferrooxidans are highly conductive. The genome sequence of A. ferrooxidans ATCC 23270 contains two genes, pilV and pilW, which code for pilin domain proteins with the conserved amino acids characteristic of Tfp. Multiple alignment analysis of the PilV and PilW (pilin) proteins indicated that pilV is the adhesin gene while pilW codes for the major protein element of Tfp. The likely function of Tfp is to complete the circuit between the cell surface and Fe(II) oxides. These results indicate that Tfp of A. ferrooxidans might serve as biological nanowires transferring electrons from the surface of Fe(II) oxides to the cell surface.

Diversity and ecophysiology of new isolates of extremely acidophilic CS2-converting Acidithiobacillus strains.

Biofiltration of industrial carbon disulfide (CS2)-contaminated waste air streams results in the acidification of biofilters and therefore reduced performance, high water use, and increased costs. To address these issues, we isolated 16 extremely acidophilic CS2-converting Acidithiobacillus thiooxidans strains that tolerated up to 6% (vol/vol) sulfuric acid. The ecophysiological properties of five selected strains (2Bp, Sts 4-3, S1p, G8, and BBW1) were compared. These five strains had pH optima between 1 (2Bp) and 2 (S1p). Their affinities for CS2 ranged between 80 (G8) and 130 (2Bp) µM. Strains S1p, G8, and BBW1 had more hydrophobic cell surfaces and produced less extracellular polymeric substance than did strains 2Bp and Sts 4-3. All five strains converted about 80% of the S added as CS2 to S(0) when CS2 was supplied in excess. The rate of S(0) consumption varied between 7 (Sts 4-3) and 63 (S1p) nmol O2 min(-1) ml culture(-1). Low S(0) consumption rates correlated partly with low levels of cell attachment to externally produced S(0) globules. During chemostat growth, the relative amount of CS2 hydrolase in the cell increased with decreasing growth rates. This resulted in more S(0) accumulation during CS2 overloads at low growth rates. Intermittent interruptions of the CS2 supply affected all five strains. Strains S1p, G8, and BBW1 recovered from 24 h of starvation within 4 h, and strains 2Bp and Sts 4-3 recovered within 24 h after CS2 was resupplied. We recommend the use of mixtures of Acidithiobacillus strains in industrial biofilters.

Ferrous iron oxidation by sulfur-oxidizing Acidithiobacillus ferrooxidans and analysis of the process at the levels of transcription and protein synthesis.

In contrast to iron-oxidizing Acidithiobacillus ferrooxidans, A. ferrooxidans from a stationary phase elemental sulfur-oxidizing culture exhibited a lag phase in pyrite oxidation, which is similar to its behaviour during ferrous iron oxidation. The ability of elemental sulfur-oxidizing A. ferrooxidans to immediately oxidize ferrous iron or pyrite without a lag phase was only observed in bacteria obtained from growing cultures with elemental sulfur. However, these cultures that shifted to ferrous iron oxidation showed a low rate of ferrous iron oxidation while no growth was observed. Two-dimensional gel electrophoresis was used for a quantitative proteomic analysis of the adaptation process when bacteria were switched from elemental sulfur to ferrous iron. A comparison of total cell lysates revealed 39 proteins whose increase or decrease in abundance was related to this phenotypic switching. However, only a few proteins were closely related to iron and sulfur metabolism. Reverse-transcription quantitative PCR was used to further characterize the bacterial adaptation process. The expression profiles of selected genes primarily involved in the ferrous iron oxidation indicated that phenotypic switching is a complex process that includes the activation of genes encoding a membrane protein, maturation proteins, electron transport proteins and their regulators.

Shotgun proteomics study of early biofilm formation process of Acidithiobacillus ferrooxidans ATCC 23270 on pyrite.

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.

AHL signaling molecules with a large acyl chain enhance biofilm formation on sulfur and metal sulfides by the bioleaching bacterium Acidithiobacillus ferrooxidans.

Biofilm formation plays a pivotal role in bioleaching activities of bacteria in both industrial and natural environments. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacterium Acidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using Quorum Sensing molecules of the N-acylhomoserine lactone type (AHLs). Our results revealed that AHL-signaling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of A. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous A. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances. Our results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/rock drainage.