Thermithiobacillus plumbiphilus AAFK—Arsenic-Resistant Bacteria Isolated from Arsenopyrite Material

Abstract

Autotrophic sulfur-oxidizing bacteria can play a key role in the metal bioleaching from low-grade sulfide-containing ores. The most commonly used bioleaching group is presented with acidophilic bacteria of the order Acidithiobacillales. We studied the diversity of bacteria in the arsenopyrite gold-bearing ore and also discovered a wide distribution of neutrophilic non-thermophilic bacteria Thermithiobacillus plumbiphilus in this ore, as well as its drainage and flotation concentrate. For the first time, T. plumbiphilus was isolated from the natural arsenic-containing mineral material. The first description of complete genome for the species T. plumbiphilus was also carried out and discovered genes providing the As resistance. Culturing the isolated strain T. plumbiphilus AAFK confirmed the found bacterial resistance to arsenite and cocadylate during the effective thiosulfate oxidation. Experiments on the arsenopyrite bioleaching showed that T. plumbiphilus AAFK can be used as an auxiliary bacterial culture capable of oxidizing reduced / intermediate sulfur compounds. The genetic basis of the T. plumbiphilus AAFK resistance to the arsenic compounds is discussed; the mechanisms are similar with the ones known for acidophilic thiobacilli. The biofilm formation is shown for the first time for T. plumbiphilus; presumably, it could provide some protection and immobilization of the cells. Structures of the T. plumbiphilus AAFK cells and their production of outer membrane vesicles are described and discussed.

1. Introduction

In recent times, the bioleaching of refractory ores, especially arsenopyrite minerals, is mainly based on the application of autotrophic acidophilic bacteria Acidithiobacillus ferrooxidans [1,2]. This industrial application is related to a strong acidophilicity of the group which permits combining the bioleaching with chemical acid leaching. Meanwhile, the use of molecular methods for identification since the end of the 20th century made it possible to show that representatives of the initial genus Thiobacillus should be transferred to many various genera, families, and orders [3,4], so the order Acidithiobacillales includes just two genera: acidophilic bacteria Acidithiobacillus and neutrophilic Thermithiobacillus. The latter genus received its name “Thermi-” after the first described thermophilic species of the genus, while later, a mesophilic strain was described as the second species of the genus: T. plumbiphillus [5]. Currently, this type of strain is the only representative of the species in official collections, although its presence has been found in many natural habitats (https://bacdive.dsmz.de/strain/132526 (accessed on 5 October 2024)).

During the study of the microbiota of the refractory gold-bearing arsenopyrite, we discovered the presence of T. plumbiphillus bacteria in numerous ore specimens. In the continuation of these studies, we isolated a culture of neutrophilic autotrophic bacteria from the flotation concentrate of the arsenopyrite ore. The discovery of this species in arsenopyrite ore required an investigation into its survival mechanisms and the possibility of its application for the bioleaching.

The aim of the presented research was to study the new isolated strain (later identified as T. plumbiphillus AAFK), paying particular attention to its high resistance to arsenic. The research objectives included: testing the effect of different arsenic compounds on bacterial oxidative activity, analyzing the genome and genes responsible for arsenic resistance, and studying the resulting biofilm and its possible role.

2. Materials and Methods

2.1. Mineral Materials

The samples of the arsenopyrite gold-bearing ore and arsenopyrite flotation concentrate were presented by the Bioprom Technologies Ltd., Stepnogorsk, the Republic of Kazakhstan. Concentrations of the key chemical elements in the specimens are shown in Table 1.

2.2. Study of Bacterial Diversity in the Ore by Molecular Methods

DNA from arsenopyrite ore, arsenopyrite mineral raw materials, and arsenopyrite mining drainage water was isolated using a Zimoclean (Zymoclean™ Gel DNA Recovery Kit, Irvine, CA, USA) DNA extraction kit according to the manufacturer’s recommendations. Bacterial 16S ribosomal RNA (rRNA) genes were amplified using unique primers flanking the V3–V4 hypervariable regions (341F–785R) with MiSeq adapters. Sequencing was performed on an Illumina MiSeq.

Sequence analysis was performed using QIIME2 v.2022.11 software [6]. Sequence quality control was performed using the Deblur plugin [7] in positive mode as the following. First, chimeric sequences were removed by the consensus method, which could arise after merging paired reads to prevent false positive results during taxonomic data processing. Then, SortMeRNA was used, in which all raw reads were compared with the GreenGenes reference database [8], and only sequences with an e-value ≤ 10 were kept. After this step, the reads were truncated by length. This way, error correction in amplicon sequences and noise reduction with ASV formation were performed. The remaining sequences were assigned taxonomy using a pre-trained classifier (Naive Bayes classifiers) that was assembled from the complete 16S rRNA gene sequence using the GreenGenes reference database [8].

The taxonomic composition of bacterial populations was also determined using the restriction analysis of the amplified 16S rRNA gene (molecular analysis). The isolated DNA samples were amplified by PCR using bacterial universal primers for the 16S rRNA gene: forward primer 27F (5′-AGAGTTTGATCMTGGCTCAG) and reverse primer 1492R (5′-TACGGYTACCTTGTTACGACTT). To remove deoxyadenosine residues from the 3′ ends formed due to Taq polymerase amplification, the purified PCR products were treated with T4 DNA polymerase (Thermo Scientific, Waltham, MA, USA), which has 3′–5′ exonuclease activity. Then, the DNA fragments were ligated at the blunt ends into the pZeRO vector (Novagen, Madison, WI, USA) opened with the EcoRV restriction endonuclease. Then, the strain E. coli Top10 (Novagen) was transformed with the ligation mixture. Transformed E. coli Top10 cells were grown using the selective LB medium supplemented with 50 μg/μL kanamycin and 20 μg/mL streptomycin.

After that, 100 colonies from each library were analyzed by PCR analysis with primers of the vector ZeroF2 (5′-CTATGACCATGATTACGCCAAGC) and ZeroR2 (5′-GAATTGTAATACGACTCACTATAGGGC).

After reprecipitation, the amplified PCR product (approximately 1.7 kb) was digested with restriction endonuclease AluI. The resulting restriction products were separated by gel electrophoresis in 3.0% agarose gel to select samples with different restriction profiles. Plasmids were isolated from selected clones using GeneJet Plasmid Miniprep Kit (Thermo Scientific), from which the insert was amplified using primers 27F and 1492R, followed by retreatment with AluI restriction endonuclease. This procedure made it possible to detect plasmids with the reverse insertion of the PCR product and in turn reduce the number of samples by half. Plasmids, in which the inserts differed by restriction analysis, were sequenced. The resulting sequences were analyzed using the NCBI BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST, accessed on 1 August 2024) to identify similar 16S rRNA sequences in the NCBI database.

2.3. Culturing

The culture of autotrophic bacteria capable of thiosulfate oxidation was isolated in the medium DSMZ 71 (https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium71.pdf (accessed on 5 October 2024)) with pH 4.5. This isolated strain was designated as AAFK. As long as the selected pH 4.5 allowed the detection of various acidophilic, moderately acidophilic, and neutrophilic thiobacilli, the optimal pH value for the isolated strain AAFK was estimated in additional culturing experiments. The later culturing was carried out in the ATCC medium 290 “S6 Medium for Thiobacilli” (https://www.atcc.org/-/media/product-assets/documents/microbial-media-formulations/2/9/0/atcc-medium-290.pdf (accessed on 5 October 2024)) with the found optimal pH 7.0. The constituent solutions of the medium (basic salts, thiosulfate, and microelements) were autoclaved separately at 121 °C for 15 min and mixed after sterilization. As long as the optimal temperature was estimated, the strain was grown at 24–28 °C.

To study bacterial resistance to arsenic, the strain was cultured in the ATCC medium ATCC 290 supplemented with the As-compounds (sodium arsenite, sodium arsenate, and sodium cacodylate. Bacterial growth with sodium arsenite was studied at the following concentrations: 0.5, 1.0, 2.0, and 4.0 g/L. The final selected concentrations of the As-bearing supplements for this study were 2 g/L, which is equal to approximately 15 mM for sodium arsenite and 10 mM for sodium arsenate. These concentrations significantly exceed the naturally occurring levels of dissolved arsenic in mine drainage and groundwater [9,10] and have also been used previously in model experiments with acidophilic leaching bacteria [11]. Bacterial thiosulfate oxidation in the mentioned liquid medium ATCC 290 was measured by sulfate production (Section 2.7 “Chemical analyses”). Protein concentrations were determined using the classical Bradford method [12]. Measurements were performed using a Shimadzu UV-1400 spectrophotometer (Shimadzu, Kyoto, Japan) at a wavelength of 595 nm.

2.4. Identification of the Strain AAFK

The bacterial identification of the strain AAFK was based on the classic 16S rRNA gene analysis. The DNA was isolated according to [9] and then treated with the NanoDrop 2000c Kit (Thermo Scientific). The universal primers of 16S rRNA prokaryotes were used for the amplification: 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-TACGGYTACCTTGTTACGACTT-3′) [13]. The sequencing of the resulting fragments was performed with the Applied Biosystems Genetic Analyzer automatic sequencer. The phylogenetic screening of the obtained sequences was carried out with the EzBioCloud database (www.ezbiocloud.net, accessed on 13 December 2024) and BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 13 December 2024). For phylogenetic analysis, the nucleotide sequences of the 16S rRNA gene for the AAFK strain were manually aligned with the sequences of the closely related reference strains. The 16S rRNA gene of the AAFK strain is deposited in the NCBI GenBank database under the accession number PP111118.

Sequencing for whole genomic DNA of the AAFK strain was carried out with Illumina HiSeq500 at the BioSpark biotechnological laboratory (Troitsk, Moscow, Russia). Average nucleotide identity was calculated with the EzBioCloud web service [14]. The value of digital DNA–DNA hybridization (DDH) was calculated using the Genome-to-Genome Distance Calculator 2.1 as previously described by Kanehisa et al. [15].

2.5. Light Microscopy

The morphology of the bacterial cells and structure of biofilms were studied using both phase contrast and epifluorescence microscopy. The standard phase contrast microscopic studies were conducted using a Nikon Eclipse Ci microscope (Nikon, Tokyo, Japan) with a ProgRes SpeedXT camera (Jenoptic, Jena, Germany). The fluorescence microscopy needed specific dyers. So, to stain both cells and polysaccharides of biofilms, the specimens of biofilm were incubated for 20 min at room temperature with the CF^®488A Concanavalin A (Biotium 29016, Fremont, CA, USA) and Propidium Iodide—FluoroPure™ Grade (Invitrogen P1304MP) in the same buffer. The cells were examined using AXIO Imager A1 (Carl Zeiss AG, Oberkochen, Germany) with a set of filters 56HE at the wavelength (λ) 470 nm (excitation) and 512 + 630 nm (emission). An Axiocam 506 (Carl Zeiss AG, Oberkochen, Germany) camera was used to obtain the images.

2.6. Transmission and Scanning Electron Microscopy

The thin structure of bacterial cells was studied by the transmission electron microscopy (TEM). For negative staining, diluted cell suspensions were treated with 3% phosphotungstic acid, pH 5.0, for 3–5 min at room temperature.

Cell biomass was used to prepare ultrathin sections. Cell biomass of pure cultures was prefixed with 1.5% (v/v) glutaraldehyde solution in the 0.05 M cacodylate buffer (pH 7.2) at 4 °C for 1 h. After three washings with the same buffer, the material was additionally fixed with 1% OsO₄ in 0.05 M cacodylate buffer at room temperature for 3 h. To contrast the cell wall polysaccharides, sheaths, and capsule substances, glutaraldehyde–osmium fixation in the presence of ruthenium red was applied [16]. After dehydration, the material was embedded into Epon 812, and ultrathin sections were made on an LKB III ultratome (Stockholm, Sweden). The sections were mounted on copper grids covered with Formvar film, contrasted with uranyl acetate (3% solution in 70% ethanol) for 30 min, and then stained with lead citrate [17] at room temperature for 4–5 min. The preparations were examined with the JEM-1400Plus (JEOL Ltd., Tokyo, Japan) microscope.

The surface morphology of the biofilms was examined using scanning electron microscopy (SEM). Samples of the cells placed on membrane filters were fixed in glutaraldehyde vapor for 24 h at 4 °C and post fixed in OsO₄ vapor for 3 h at 20 °C. After dehydration in propylene oxide vapor, the samples were coated with gold (Fine Coat Ion Sputter JFC-1100, Tokyo, Japan) and examined with the scanning microscope JSM-6510LV (JEOL, Tokyo, Japan).

2.7. Chemical Analyses

Sulfate concentration was estimated by high-pressure ion chromatography using the Metrohm 761 Compact IC (Metrohm, Herisau, Switzerland), equipped with a conductometric detector and a chemical anion suppression module. The separation column Metrohm Metrostep A Supp 5 100/4.0 mm (Switzerland) was used. The eluent was carbonate buffer with 3.2 mM Na₂CO₃ and 1.0 mM NaHCO₃. The analysis time was 30 min. The MultiChrom 3.4 software package (Ampersend, Russia) was installed to control the process.

2.8. Statistics

Experimental analyses were performed in triplicate. Data were presented as arithmetic means where the confidence intervals did not exceed ±5%. Statistical analysis was performed using the standard methods provided by Excel (https://support.microsoft.com/en-us/office/use-the-analysis-toolpak-to-perform-complex-data-analysis-6c67ccf0-f4a9-487c-8dec-bdb5a2cefab6 (accessed on 5 October 2024)). ANOVA was used to estimate the significance of the presented data. All the presented data were significant (p > 99%).

3. Results

3.1. Diversity of Bacteria in the Arsenopyrite Ore

Today, the standard for highly informative analysis of the composition of microbial communities is the 16S rRNA gene profiling method using high-throughput sequencing technologies. We performed 16S rRNA gene profiling of arsenopyrite ore (KZ), arsenopyrite mining drainage waters (WSUZ), and arsenopyrite flotation concentrate (FK).

The samples showed the presence of 14 bacterial types: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Planctomycetes, Gemmatimonadetes, Parcubacteria, Verrucomicrobia, Dependentiae, Nitrospirae, Chlamydiae, Acidobacteria, Elusimicrobia, and Poribacteria.

Of these, 51 taxa were identified at the genus level; the dominated genera/groups are shown in Figure 1. They are listed as follows. In the KZ sample, Pseudomonas (52.64%) and Arthrobacter (16.83%) dominated; in the KF sample, Pseudomonas made up 85% of all identified genera in the sample; and in the WSUZ sample, the group HB2-32-21, which combines representatives of Alteromonadaceae, a family of Pseudomonadota (8.14%) and Parvibaculum (3.18%), dominated. The WSUZ sample is characterized by the highest diversity of identified genera (33 genera), and the KF sample is the lowest (12 genera). Therefore, the WSUZ community is more taxonomically diverse, while, in the KZ and KF samples, we observed a clear shift in the abundance of microorganisms towards 1–2 taxa.

At the species level, only 12 microorganisms were identified, namely Pseudomonas fragi, P. stutzeri, Sulfuricurvum kujiense, Bacillus muralis, B. selenatarsenatis, Pedobacter composti, Pseudoburkholderia malthae, Sediminibacter furfurosus, Rhodococcus fascians, Nevskia ramosa, Nitrosococcus mobilis, and Reyranella massiliensis.

Data on genera/groups present in minor amounts (Figure 1) do not only allow us to assess their role but may also be useful for a general understanding of the microbiota. Nevertheless, we can conclude that pseudomanadotes, which include asidithiobacilli, predominated in microbiota. This conclusion was also confirmed by the methods of isolation and clone library.

Due to the lack of organic substrate and extremely low pH, the studied habitats are characterized by low bacterial abundance and concentration. Molecular genetic analyses of bacterial diversity in the flotation concentrate and ore samples showed that the total bacterial DNA content was insufficient for full-genome profiling analysis. Thus, the 16S gene profiling method was not enough informative, while full-genome profiling was impossible due to this small amount of DNA. Attempts to isolate bacterial DNA showed that there were so few bacteria in the ore samples that either demonstrated preliminary bacterial growth, which would distort the results, or the use of another molecular biological method, the clone library method, was required to perform the analysis.

In this study, we used the technology of clone library due to the small amount of DNA in the original environmental samples. Our studies of small populations (less than 100 cells/mL) showed that they are representative in identifying dominant forms, determining the important processes of S- and Fe-transformation in the samples [18].

Profiling through a clone library revealed the presence of common and well-known bacterial species responsible for bioleaching processes; the following species were found, which are typical in environments with sulfide minerals: Acidithiobacillus caldus, A. ferrivorans, A. ferrooxidans, and A. thiooxidans. As a new discovery, this approach also detected the presence of neutrophilic bacteria T. plumbiphillus in the samples from all three habitats, a bacterial species not previously reported to be involved in bioleaching. As described above in Section 2.3, the strain of neutrophilic autotrophic bacteria AAFK was isolated from the arsenopyrite mineral material.

3.2. Identification of the Isolated Strain and Its Genetic Similarity

According to the analysis of the nucleotide sequence of the 16S rRNA gene, the strain AAFK was assigned to the class Acidithiobacillia, order Acidithiobacillales, family Thermithiobacillaceae, genus Thermithiobacillus, species T. plumbiphilus (Figure 2). The 16S rRNA gene of T. plumbiphilus AAFK was entered into the NCBI GenBank database under the number PP111118. For the identification of thiobacilli, the similarity analysis of bacterial 16S rRNA genes appears to be sufficient. Meanwhile, due to the lack of data on the complete genome of T. plumbiphilus in the official genetic libraries, this paper was also the first report on the complete genome of the bacterium and its features. The genome of T. plumbiphilus AAFK was entered into the NCBI GenBank database under the number PRJNA1094888. According to the entire nucleotide sequence of the T. plumbiphilus AAFK genome, the phylogenetically closest species was T. tepidarius (Figure 3). These data on the similarity of the genomes of the two species of the genus are not necessary for species identification, since this was already demonstrated using 16S rRNA gene analysis. However, these results are extremely interesting, since they show the similarity of the genomes of thermophilic and mesophilic species, sufficient for their assignment to the same genus Thermithiobacillus.

3.3. Experiments on the T. plumbiphilus AAFK Resistance to the Arsenic Compounds

Since the strain AAFK was isolated from the mineral material with high arsenic content (7.7%), we conducted experiments to estimate the effects of different arsenic compounds on the bacterial activity measured as the sulfate production during thiosulfate oxidation in the mineral medium (Figure 4). The concentration of the produced sulfate was measured every 3 days using ion chromatography.

The obtained data showed that neither arsenite nor cacodylate, 2 g/L, decreased thiosulfate oxidation. In contrast, the same concentration of arsenate caused the inhibition of thiosulfate oxidation. These results indicated the presence of different mechanisms of bacterial resistance to various arsenic compounds. Accordingly, an analysis of the genetic basis of the bacterial resistance to arsenic compounds is essential.

Calculations of the cell number and/or optical density were complicated with the formation of a biofilm produced by T. plumbiphilus AAFK. Strong mixing of the culture medium only resulted in the sedimentation of the biofilm parts/torn pieces to the bottom, without any essential transition of the cells into a dispersed suspension. For this reason, the protein concentration in the culturing medium could only serve as some indicator of a trend. Nevertheless, these data also confirmed estimated As-resistance; protein production measured by optical density is shown at Figure 5.

3.4. Cellular Structure and Biofilm of T. plumbiphilus AAFK

Our studies with phase-contrast light microscopy revealed the formation of extracellular droplets of colloidal sulfur by T. plumbiphilus AAFK during the oxidation of thiosulfate (Figure 6a), which is well known and typical for all classical thiobacilli. The new data for this group of autotrophic neutrophilic sulfur-oxidizing bacteria included the discovery of the biofilm and some hyper-secretion of outer membrane vesicles (OMVs) (Figure 6b). Transmission electron microscopy (TEM) of the thin cell sections showed an accumulation of inclusions typical for polyphosphates (Figure 6c). Equally, these ultrathin sections showed the presence of carboxysomes (polyhedral bodies), they compose multifaceted nanocompartments with a single-layer protein shell and contain the RubisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) of the cell, i.e., the presence of carbon-fixing enzymes. Similar organelles were already found in various autotrophic CO₂-fixing bacteria [19].

This study presented the first demonstration of the biofilm formation by T. plumbiphilus. Using specific cytochemical reactions with concanavalin A (ConA) and ruthenium, it was established that the biofilm had a polysaccharide nature (Figure 7a,c). In addition, fixation with ruthenium red resulted in charge detachment: the ruthenium red cation [Ru₃O₂(NH₃)₁₄]⁶⁺ reacted with acidic mucopolysaccharides. In this case, the polysaccharide capsule was so strongly negatively charged that it detached from the cell wall. The biofilm structure had a reticular structure, which is visible when examined using both fluorescence and scanning microscopy (Figure 7a,b). It is worth noting that the cells were not rigidly fixed in the biofilm, and there is some space between the exocellular substance and the cell membrane (Figure 7a,c,e). Light fluorescence microscopy with ConA at high magnification showed a specific heterogeneity of the biofilm (Figure 7d). A powerful secretory process of OMV formation is visible both with the scanning electron microscopy (Figure 7e) and with the negative-contrast transmission electron microscopy, as well as when examining ultrathin sections (Figure 7f). Using ultrathin sections, we also showed that these vesicles had a double membrane (Figure 7f). Negative contrast showed that the vesicles were filled.

OMVs in Gram-negative bacteria are commonly formed as extracellular sacs derived from the outer membrane, and they perform a wide range of physiological functions. Overall, OMVs are multifunctional and were previously described for host colonization and pathogenesis, adaptive responses to stress, resistance to bacteriophage infection, resistance to antibiotics and antibodies, bacterial mortality, gene transfer, and nutrient cycling [20,21,22,23,24,25,26]. The specific roles of OMVs for, in our case, the autotrophic neutrophilic sulfur-oxidizing bacteria are still open for discussion.

The present research is the first description of OMV and biofilm produced by T. plumbiphilus and by neutrophilic thiobacilli. In this regard, the functions of the biofilm and OMV produced by T. plumbiphilus are only considered as hypothetical and discussed in comparison with known published data for related groups of bacteria.

Under natural conditions, the formation of bacterial biofilm suggests a close interaction of the cells with mineral surfaces to support microbial catabolism. This mineral–bacteria interface was already demonstrated for the autotrophic acidophilic sulfur-oxidizing bacteria A. ferrooxidans, A. thiooxidans, and Leptospirillum ferrooxidans [27,28,29]. The strain T. plumbiphilus AAFK was isolated from mineral materials, so it can be assumed that its biofilm formation serves for the cell adhesion. It is also possible that the biofilm protects the bacterial cells from toxic compounds coming from minerals. It has been published that biofilm components can increase mineral extraction [30]. The main leaching component of the biofilm was a lipoprotein called lycanantase. The addition of lycanantase to the solution increased the rate of chalcopyrite leaching.

It should be assumed that the polysaccharide film can also prevent any chaotic migration of cells into the external environment and allows the immobilization of the secreted vesicles on/into the film (Figure 7d).

The data obtained are new for neutrophilic thiobacilli and open up a wide area for some scientific speculation and discussion. The biofilm can provide adhesion on the mineral surfaces, interface for “mineral–bacterial cells” reactions, and probably environment for the produced OMV as an intermediate in quorum sensing. The OMV probably contains some substances which affect quorum sensing. It is known for several bacterial species, as OMVs play a structural and regulatory role in the biofilm formation and are involved in the quorum sensing, intercellular association, and interactions with exopolysaccharides and extracellular DNA [31,32,33,34,35,36].

3.5. Genetic Basis of the T. plumbiphilus AAFK Resistance to the Arsenic Compounds and Sulfur Transformation

The study on the isolated arsenic-resistant strain included the first description of complete genome for the species T. plumbiphilus. The analysis of the T. plumbiphilus AAFK genome revealed the presence of the ars (arsenic resistance system) gene group (Figure 8).

It has already been known that some bacteria have genes providing their resistance both to organic and inorganic arsenic compounds. The most common, and, therefore, the best-studied, mechanism of bacterial tolerance to arsenic is the system encoded by the ars (arsenic resistance system) operon, which is responsible for the detoxification of arsenate to arsenite via its reduction [37]. The genome analysis of T. plumbiphilus AAFK revealed genes arsA, arsB, arsC, arsD, and arsR (Figure 8). Arsenate reductase, a very small protein (~13–16 kDa), is encoded with the arsC gene, it is responsible for arsenate reduction, i.e., the reduction of As⁵⁺ to As³⁺. Arsenite/antimonite: H+ antiporter-expulsion pump is encoded with arsB, which acts synergistically with ArsA (ATPase), while the last one is responsible for arsenite efflux [38,39,40]. ArsD, a metallochaperone, increases the affinity of arsenite for ArsA [40,41]. A protein of the metalloregulator family, ArsR, negatively regulates the ars operon in the presence of arsenite. The enlarged arsRDABC operon was found in fewer bacterial genomes than arsRBC, but it confers a higher level of arsenic resistance [42]. A gene encoding the ACR3 protein was also found in the genome. This gene is a homolog of ArsB, but the expression of arsenical resistance protein ACR3 induced high-level arsenite resistance. Members of the Acr3 family of arsenite permeases conferred resistance to trivalent arsenic by its extrusion from cells. Thus, the strain AAFK showed that all genetic determinants for the operation of the arsenic resistance system in several variants, as well as a gene ACR3 with higher expression, was found.

Bacteria can also perform arsenic oxidation via the aioA and aioB genes, which encode arsenite oxidase, a key enzyme for the oxidation of arsenite to arsenate. The sequences of the arsenite oxidase large subunit (aioA) and arsenite oxidase small subunit (aioB) genes were found in the T. plumbiphilus AAFK genome. Arsenite oxidase, an important enzyme for arsenite oxidation, is encoded by the aioA gene. The AioA protein has two subunits, which are encoded by the aioA and aioB genes (Figure 8).

In addition, the respiratory arsenate reductase (Mo-binding subunit, ArrA) and respiratory arsentate reductase (FeS subunit, ArrB) were found in the T. plumbiphilus AAFK genome. These genes participate in the arr operon. Arsenic respiration using the arr operon was discovered many years ago. But, there are still many uncertainties regarding the specific mechanism of respiration [43]. Bacteria use the arr operon to obtain energy by mediating the dissimilatory reduction of As(V) by encoding a membrane-bound arsenate reductase that is responsible for the periplasmic anaerobic respiration of As(V) using different electron sources.

The arr and arx systems show several homologies, despite the existence of some differences between their patterns of genetic organization. The sequence similarity observed between arr and arx suggests that Arx may be a variant of Arr that performs the reverse reaction [44]. Regardless, we have to mention that the T. plumbiphilus AAFK culturing was only carried out under aerobic conditions.

The absorption of arsenate and arsenite by bacteria can depend on the phosphate transporter [45], and the intracellular deposition of insoluble polyphosphates takes part in the precipitated complex. The large amounts of polyphosphates found in the T. plumbiphilus AAFK cells may indicate an additional mechanism. Finally, the T. plumbiphilus AAFK genome contains arsenate reductase, which is capable of reducing arsenate to arsenite, and a pump that removes arsenite from cells. Thus, according to our genome analysis, Figure 8 presents an overview of the existing arsenic resistance systems in the strain T. plumbiphilus AAFK, which seems to be similar with one found in acidophilic A. ferrooxidans [2,11,46].

As long as the first description of complete genome for the species T. plumbiphilus was also carried out, it also permitted the presentation of the whole scheme of possible sulfur transformations in T. plumbiphilus AAFK (Figure 9). According to this scheme, T. plumbiphilus is capable of oxidizing reduced and intermediate sulfur compounds, which is in good agreement with known data for all thiobacilli.

4. Conclusions

For the first time, the neutrophilic autotrophic sulfur-oxidizing bacteria T. plumbiphilus were isolated from a natural mineral source with a high concentration of arsenic. The study on the genome of the T. plumbiphilus, which was also the first one for this species, showed a complex system that can provide defense against arsenic compounds. Experiments on growing T. plumbiphilus AAFK with various arsenic compounds confirmed this resistance.

The high As-resistance of the strain is an object of special interest. The first study on the species genome discovered numerous various genes that provide this complex defense system, which permits at least a few ways for defense. Overall, the system of the As-resistance is similar with its analogs in strongly acidophilic bacteria A. ferrooxidans; however, as long as T. plumbiphilus is neutrophilic, in contrast to A. ferrooxidans, it can serve as a donor for the transfer of the resistance genes.

It was also discovered that autotrophic strain T. plumbiphilus AAFK can produce an abundant biofilm of polysaccharide origin. For the first time, this study showed formation of the OMV accompanied with the biofilm production by these bacteria. Roles of the described biofilm and OMV are open for discussion.

Author Contributions

Conceptualization, T.A. and M.V.; methodology, T.A., A.S., and A.Z.; validation, T.A.; investigation, T.A., A.S., and A.Z.; resources, T.A.; data curation, T.A. and M.V.; writing and editing, T.A. and M.V.; supervision, T.A.; project administration, T.A.; funding acquisition, T.A. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Acknowledgments

Shared Core Facilities of the Pushchino Scientific Center for Biological Research (http://www.ckp-rf.ru/ckp/670266/, accessed on 13 December 2024).

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Table

Figure 1. Taxonomic profiles for the 16S rRNA gene of arsenopyrite ore (KZ), arsenopyrite mining drainage waters (WSUZ), and arsenopyrite flotation concentrate (FK). The ordinate axis presents percentage of the detected bacterial taxa. The group HB2-32-21 combines representatives of the family Alteromonadaceae.

Figure 2. Phylogenetic tree constructed from complete 16S rRNA gene sequences of T. plumbiphilus AAFK.

Figure 3. Phylogenetic tree constructed from the complete genome sequences of T. plumbiphilus AAFK.

Figure 4. Sulfate production by T. plumbiphilus AAFK grown in the thiosulfate–containing medium ATCC 290 during 15 days of cultivation in the presence of various arsenic compounds. Arsenic compounds, 2 g/L, are shown in the figure.

Figure 5. Protein production by T. plumbiphilus AAFK grown in the thiosulfate-containing medium ATCC 290 with various concentrations of arsenite. The arsenite concentrations and culturing time are shown in the figure. Data on protein production are presented as changes in the optical density (OD).

Figure 6. Micrographs of cells and biofilm of T. plumbiphilus AAFK. (a) Light phase-contrast microscopy: biofilm with bacterial cells and droplets of colloidal sulfur. (b) Transmission electron microscopy: negative stained bacterial cells and outer membrane vesicles. (c) Transmission electron microscopy: ultrathin sections of the cells; C—carboxysome, PP—polyphosphate.

Figure 7. Photomicrographs of T. plumbiphilus AAFK cells and biofilm. Scale bars are shown in the figure. (a) Light fluorescence microscopy of polysaccharide biofilm and bacteria: green emission of ConA is bound to bacterial polysaccharide, and red emission of propidium iodide is bound to nucleic acids inside bacteria. (b) Scanning electron microscopy of bacterial biofilm. (c) Cytochemical reaction with ruthenium red on polysaccharide, ultrathin section. (d) Light fluorescence microscopy of ConA-bound biofilm fragment; irregularities are presumably formed by secreted vesicles. (e) Scanning electron microscopy of bacterial cells with biofilm. (f) Transmission electron microscopy of ultrathin section of vesicles secreted into the external environment.

Figure 8. Overview of bacterial interactions with arsenic for strain T. plumbiphilus AAFK based on the presented research data.

Figure 9. The full scheme of sulfur oxidation and reduction processes provided by the T. plumbiphilus AAFK genome. Abbreviations in the figure: Apr—adenylyl-sulfate reductase; Cys—sulfate/thiosulfate transport system ATP−binding protein; Dox—thiosulfate dehydrogenase (quinone); Fcc—sulfide dehydrogenase [flavocytochrome c]; PAPSS—3′-phosphoadenosine 5′-phosphosulfate synthase; Phs—polysulfide reductase; Sir—sulfite reductase (ferredoxin); Sor—sulfite dehydrogenase (cytochrome); Soe—sulfite dehydrogenase (quinone); Sox—sulfur oxidizing system; Sqr—sulfide:quinone oxidoreductase; Ssu—sulfonate transport system substrate-binding protein; Tau—taurine transport system substrate-binding protein; TST—thiosulfate/3-mercaptopyruvate sulfur transferase.

Table 1

Key chemical elements in the arsenopyrite gold-bearing ore and arsenopyrite flotation concentrate, Bioprom Technologies Ltd.

Elements	Concentration, mg/kg
	Ore	Flotation Concentrate
As	12,700	77,279
Fe	33,600	24,740
Au	150	100

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