Relevance of FeoAB system in Rhodanobacter sp. B2A1Ga4 resistance to heavy metals, aluminium, gallium, and indium.

Aluminium (Al), gallium (Ga), and indium (In) are metals widely used in diverse applications in industry, which consequently result in a source of environmental contamination. In this study, strain Rhodanobacter sp. B2A1Ga4, highly resistant to Al, Ga, and In, was studied to reveal the main effects of these metals on the strain and the bacterial mechanisms linked to the ability to cope with them. An indium-sensitive mutant obtained by random transposon mutagenesis has the feoA gene interrupted. This gene together with the feoB gene is part of the feo operon which encodes a ferrous uptake system (FeoAB). The mutant strain exhibited higher oxidative stress supported by a high concentration of reactive oxygen species (ROS) and low levels of reduced glutathione (GSH) in the presence of metals. The iron supplementation of the growth medium reverted the growth inhibition of the mutant strain caused by Ga and In, significantly reduced the ROS amounts in mutant cells grown in all conditions, and increased its GSH/total glutathione ratio to values similar to those of the native strain. Moreover, the mutant strain when submitted to In increased the production of siderophores. The genome sequence analysis of strain B2A1Ga4 showed a large number of genes encoding putative proteins involved in iron uptake from the cell surface to the cytoplasm. Understanding the bacteria-metal interactions linked to resistance to high-tech metals is relevant to future application of microorganisms in bioremediation and/or biorecovery processes of these metals. KEY POINTS: • The disruption of FeoAB system compromises the bacterial resistance to Al, Ga, and In. • The iron acquisition in Rhodanobacter sp. B2A1Ga4 controls the oxidative stress. • Genome mining of strain B2A1Ga4 reveals several iron transport related genes.

Glaciimonas singularis sp. nov., isolated from a uranium mine wastewater treatment plant.

A bacterial strain, A2-57(T), recovered from a water sample collected in a uranium mine was taxonomically studied in detail. This strain was a Gram-reaction-negative, rod-shaped bacterium that grew optimally at 25 °C and at pH 6.0-7.0 and had a DNA G+C content of 55.0 mol%. Ubiquinone 8 (UQ-8) was the predominant respiratory quinone and the major fatty acids were C16:0, C17:0 cyclo, summed feature 3 (C16:1ω6c and/or ω7c and/or C15:0 iso 2-OH) and C18:1ω7c. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain A2-57(T) belonged to the family Oxalobacteraceae and formed a distinct branch with Glaciimonas immobilis Cr9-30(T). Strain A2-57(T) shared approximately 97.3% 16S rRNA sequence similarity with G. immobilis Cr9-30(T) and also showed high sequence similarity with members of the genera Herbaspirillum (96.3-97.0%) and Collimonas (96.2-97.0%). Although phylogenetically closely related to the type strain of G. immobilis, the low level of DNA-DNA hybridization between the two strains (21.6%) and several physiological and biochemical properties indicated that the novel strain could be clearly distinguished from G. immobilis LMG 25547(T). Therefore, it is concluded that strain A2-57(T) represents a novel species of the genus Glaciimonas, for which the name Glaciimonas singularis sp. nov. is proposed. The type strain is A2-57(T) (=CIP 110539(T)=LMG 27070(T)).

Genome mining to unravel potential metabolic pathways linked to gallium bioleaching ability of bacterial mine isolates.

Gallium (Ga) is considered a high-tech Critical Metal, used in the manufacture of several microelectronic components containing either gallium arsenide (GaAs) or gallium nitride (GaN). The current high demand for this critical metal urges the development of effective recovery processes from secondary resources such as mine tailings or electronic recycling material. The importance of bioleaching as a biotechnological process to recover metals prompted this study, where an integrative approach combining experimental and genomic analysis was undertaken to identify potential mechanisms involved in bioleaching ability and strategies to cope with high metal(loid)s concentrations in five mine isolates. The Clusters of Orthologous Group (COG) annotation showed that the "amino acid transport and metabolism" [E] was the most predominant functional category in all genomes. In addition, the KEEG pathways analysis also showed predicted genes for the biosynthetic pathways of most amino acids, indicating that amino acids could have an important role in the Ga leaching mechanism. The presence of effective resistance mechanisms to Ga and arsenic (As) was particularly important in GaAs bioleaching batch assays, and might explain the divergence in bioleaching efficiency among the bacterial strains. Rhodanobacter sp. B2A1Ga4 and Sphingomonas sp. A2-49 with higher resistance, mainly to As, were the most efficient bioleaching strains under these conditions. In bioleaching assays using cell-free spent medium Arthrobacter silviterrae A2-55 with lower As resistance outperformed all the other stains. Overall, higher efficiency in Ga leaching was obtained in bioleaching assays using GaAs when compared to GaN.

Roseomonas pecuniae sp. nov., isolated from the surface of a copper-alloy coin.

Strain N75(T) was isolated from the surface of a copper-alloy 50 Euro cent coin collected from general circulation. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain N75(T) formed a distinct branch within the genus Roseomonas and placed it in the Alphaproteobacteria. Strain N75(T) showed 16S rRNA gene sequence similarities of 92.4-97.1  % to type strains of species of the genus Roseomonas. Strain N75(T) was a Gram-negative, non-spore-forming, non-motile coccoid, with an optimum growth temperature of about 30 °C; the strain did not grow at 5 or 37 °C. Strain N75(T) did not grow in medium containing NaCl. The major respiratory quinone was ubiquinone 10 (Q-10). The major fatty acids were unsaturated C16:1ω7c/C16:1ω6c and C18:1ω7c (around 70 % of the total fatty acids); the third most abundant fatty acid was the hydroxylated C18:1 2-OH. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine and an unknown aminolipid. The DNA G+C content was 72.8 mol%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics, we conclude that strain N75(T) represents a novel species of the genus Roseomonas, for which we propose the name Roseomonas pecuniae sp. nov. (type strain N75(T) =LMG 25481(T) =CIP 110074(T)).

A DedA Family Membrane Protein in Indium Extrusion in Rhodanobacter sp. B2A1Ga4.

Indium (In) is a critical metal widely used in electronic equipment, and the supply of this precious metal is a major challenge for sustainable development. The use of microorganisms for the recovery of this critical high-tech element has been considered an excellent eco-friendly strategy. The Rhodanobacter sp. B2A1Ga4 strain, highly resistant to In, was studied in order to disclose the bacterial mechanisms closely linked to the ability to cope with this metal. The mutation of the gene encoding for a DedA protein homolog, YqaA, affected drastically the In resistance and the cellular metabolic activity of strain Rhodanobacter sp. B2A1Ga4 in presence of this metal. This indicates that this protein plays an important role in its In resistance phenotype. The negative impact of In might be related to the high accumulation of the metal into the mutant cells showing In concentration up to approximately 4-fold higher than the native strain. In addition, the expression of the yqaA gene in this mutant reverted the bacterial phenotype with a significant decrease of In accumulation levels into the cells and an increase of In resistance. Membrane potential measurements showed similar values for native and mutant cells, suggesting that there was no loss of proton-motive force in the mutant cells. The results from this study suggest a potential role of this DedA family protein as a membrane transporter involved in the In efflux process. The mutant strain also has the potential to be used as a biotool in bioaccumulation strategies, for the recovery of In in biomining activities.

Identification of an aox system that requires cytochrome c in the highly arsenic-resistant bacterium Ochrobactrum tritici SCII24.

Microbial biotransformations have a major impact on environments contaminated with toxic elements, including arsenic, resulting in an increasing interest in strategies responsible for how bacteria cope with arsenic. In the present work, we investigated the metabolism of this metalloid in the bacterium Ochrobactrum tritici SCII24. This heterotrophic organism contains two different ars operons and is able to oxidize arsenite to arsenate. The presence of arsenite oxidase genes in this organism was evaluated, and sequence analysis revealed structural genes for an As(III) oxidase (aoxAB), a c-type cytochrome (cytC), and molybdopterin biosynthesis (moeA). Two other genes coding for a two-component signal transduction pair (aoxRS) were also identified upstream from the previous gene cluster. The involvement of aox genes in As(III) oxidation was confirmed by functionally expressing them into O. tritici 5bvl1, a non-As(III) oxidizer. Experiments showed that the As(III) oxidation process in O. tritici requires not only the enzyme arsenite oxidase but also the cytochrome c encoded in the operon. The fundamental role of this cytochrome c, reduced in the presence of arsenite in strain SCII24 but not in an O. tritici DeltaaoxB mutant, is surprising, since to date this feature has not been found in other organisms. In this strain the presence of an aox system does not seem to confer an additional arsenite resistance capability; however, it might act as part of an As(III)-detoxifying strategy. Such mechanisms may have played a crucial role in the development of early stages of life on Earth and may one day be exploited as part of a potential bioremediation strategy in toxic environments.

Tailings microbial community profile and prediction of its functionality in basins of tungsten mine.

In a circular economy concept, where more than 300 million tons of mining and quarrying wastes are produced annually, those are valuable resources, supplying metals that are extracted today by other processes, if innovative methods and processes for efficient extraction of these elements are applied. This work aims to assess microbiological and chemical spatial distribution within two tailing basins from a tungsten mine, using a MiSeq approach targeting the 16S rRNA gene, to relate microbial composition and function with chemical variability, thus, providing information to enhance the efficiency of the exploitation of these secondary sources. The tailings sediments core microbiome comprised members of family Anaerolineacea and genera Acinetobacter, Bacillus, Cellulomonas, Pseudomonas, Streptococcus and Rothia, despite marked differences in tailings physicochemical properties. The higher contents of Al and K shaped the community of Basin 1, while As-S-Fe contents were correlated with the microbiome composition of Basin 2. The predicted metabolic functions of the microbiome were rich in genes related to metabolism pathways and environmental information processing pathways. An in-depth understanding of the tailings microbiome and its metabolic capabilities can provide a direction for the management of tailings disposal sites and maximize their potential as secondary resources.

The chromate-inducible chrBACF operon from the transposable element TnOtChr confers resistance to chromium(VI) and superoxide.

Large-scale industrial use of chromium(VI) has resulted in widespread contamination with carcinogenic chromium(VI). The abilities of microorganisms to survive in these environments and to detoxify chromate require the presence of specific resistance systems. Here we report identification of the transposon-located (TnOtChr) chromate resistance genes from the highly tolerant strain Ochrobactrum tritici 5bvl1 surviving chromate concentrations of >50 mM. The 7,189-bp-long TnOtChr of the mixed Tn21/Tn3 transposon subfamily contains a group of chrB, chrA, chrC, and chrF genes situated between divergently transcribed resolvase and transposase genes. The chrB and chrA genes, but not chrF or chrC, were essential for establishment of high resistance in chromium-sensitive O. tritici. The chr promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of chr expression. Induction of the chr operon suppressed accumulation of cellular Cr through the activity of a chromate efflux pump encoded by chrA. Expression of chrB, chrC, or chrF in an Escherichia coli sodA sodB double mutant restored its aerobic growth in minimal medium and conferred resistance to superoxide-generating agents menadione and paraquat. Nitroblue tetrazolium staining on native gels showed that ChrC protein had superoxide dismutase activity. TnOtChr appears to represent a mobile genetic system for the distribution of the chromate-regulated resistance operon. The presence of three genes protecting against superoxide toxicity should provide an additional survival advantage to TnOtChr-containing cells in the environments with multiple redox-active contaminants.

Sequencing and expression of two arsenic resistance operons with different functions in the highly arsenic-resistant strain Ochrobactrum tritici SCII24T.

BACKGROUND: Arsenic (As) is a natural metalloid, widely used in anthropogenic activities, that can exist in different oxidation states. Throughout the world, there are several environments contaminated with high amounts of arsenic where many organisms can survive. The most stable arsenical species are arsenate and arsenite that can be subject to chemically and microbiologically oxidation, reduction and methylation reactions. Organisms surviving in arsenic contaminated environments can have a diversity of mechanisms to resist to the harmful effects of arsenical compounds. RESULTS: The highly metal resistant Ochrobactrum tritici SCII24 was able to grow in media with arsenite (50 mM), arsenate (up to 200 mM) and antimonite (10 mM). This strain contains two arsenic and antimony resistance operons (ars1 and ars2), which were cloned and sequenced. Sequence analysis indicated that ars1 operon contains five genes encoding the following proteins: ArsR, ArsD, ArsA, CBS-domain-containing protein and ArsB. The ars2 operon is composed of six genes that encode two other ArsR, two ArsC (belonging to different families of arsenate reductases), one ACR3 and one ArsH-like protein. The involvement of ars operons in arsenic resistance was confirmed by cloning both of them in an Escherichia coli ars-mutant. The ars1 operon conferred resistance to arsenite and antimonite on E. coli cells, whereas the ars2 operon was also responsible for resistance to arsenite and arsenate. Although arsH was not required for arsenate resistance, this gene seems to be important to confer high levels of arsenite resistance. None of ars1 genes were detected in the other type strains of genus Ochrobactrum, but sequences homologous with ars2 operon were identified in some strains. CONCLUSION: A new strategy for bacterial arsenic resistance is described in this work. Two operons involved in arsenic resistance, one giving resistance to arsenite and antimonite and the other giving resistance to arsenate were found in the same bacterial strain.

Impact of chromium-contaminated wastewaters on the microbial community of a river.

The influence of chromium on the microbial community structure was analyzed in a river system subjected to long-term chromium contamination, by plating and by sequencing 16S rRNA genes cloned from DNA extracted from the river sediments. We also analyzed the influence of chromium on the ability of the microbial community to resist and reduce Cr(VI) and on its resistance to antibiotics. Shifts in the microbial community structure were analyzed by amplified ribosomal DNA restriction analysis fingerprinting. The isolates obtained were phylogenetically related to Actinobacteria, Firmicutes, Bacteroidetes and Proteobacteria, whereas Acidobacteria and Deltaproteobacteria were only revealed by clone analyses. Cr(VI)-resistant and Cr(VI)-reducing strains were isolated in all sites examined. However, each sample site had a microbial community with a different antibiotic resistance pattern. Our study seems to indicate that in this river ecosystem chromium influenced the microbial communities, altering some of their functional characteristics, such as the percentage of the microbial community able to resist or to reduce Cr(VI) and the phylogenetic groups isolated, but it did not affect the structural diversity. Furthermore, the concentration of Cr(VI) in the sediments could not be correlated with a lower number of bacteria or lower index of generic diversity, neither with the ability of the microbial community to resist or to reduce higher Cr(VI) concentrations.

Leucobacter chromiireducens sp. nov, and Leucobacter aridicollis sp. nov., two new species isolated from a chromium contaminated environment.

Two strains designated strains L-1T and L-9T were isolated from activated sludge of a treatment plant that receives wastewater from the tannery industry contaminated with chromium. Phylogenetic analysis showed that the organisms represented two new species of the genus Leucobacter. Strains L-1T and L-9T could be distinguished from the type strain of L. komagatae and from the type strain of "L. albus" by the B-type peptidoglycan composition, fatty acid composition, several phenotypic and physiological characteristics. The major fatty acids of the organisms were iso- and anteiso-branched C15:0 and C17:0, straight-chain C16:0 was also found in relatively high proportions. The organisms were halotolerant, grew in medium containing 9% NaCl, and all strains, including the type strain of L. komagatae grew in medium containing 5 mM Cr(VI). On the basis of the distinct peptidoglycan composition, 16S ribosomal DNA sequence analysis, percentage of DNA-DNA reassociation values, and phenotypic characteristics we are of the opinion that strain L-1T represents a new species of the genus Leucobacter for which we propose the name Leucobacter chromiireducens and that strain L-9T represents an additional new species of the same genus for which we propose the name Leucobacter aridicollis.

Aerobic uranium immobilization by Rhodanobacter A2-61 through formation of intracellular uranium-phosphate complexes.

Severe environmental problems arise from old uranium mines, which continue to discharge uranium (U) via acid mine drainage water, resulting in soil, subsoil and groundwater contamination. Bioremediation of U contaminated environments has been attempted, but most of the conceptual models propose U removal by cell suspensions of anaerobic bacteria. In this study, strain Rhodanobacter A2-61, isolated from Urgeiriça Mine, Portugal, was shown to resist up to 2 mM of U(vi). The conditions used (low nutrient content and pH 5) potentiated the interaction of the toxic uranyl ion with the tested strain. The strain was able to remove approximately 120 µM of U(vi) when grown aerobically in the presence of 500 µM U. Under these conditions, this strain was also able to lower the phosphate concentration in the medium and increased its capacity to take up inorganic phosphate, accumulating up to 0.52 µmol phosphate per optical density unit of the medium at 600 nm, after 24 hours, corresponding approximately to the late log phase of the bacterial culture. Microscopically dense intracellular structures with nanometer size were visible. The extent of U inside the cells was quantified by LS counting. EDS analysis of heated cells showed the presence of complexes composed of phosphate and uranium, suggesting the simultaneous precipitation of U and phosphate within the cells. XRD analysis of the cells containing the U-phosphate complexes suggested the presence of a meta-autunite-like mineral structure. SEM identified, in pyrolyzed cells, crystalline nanoparticles with shape in the tetragonal system characteristic of the meta-autunite-like mineral structures. U removal has been reported previously but mainly by cell suspensions and through release of phosphate. The innovative Rhodanobacter A2-61 can actively grow aerobically, in the presence of U, and can efficiently remove U(vi) from the environment, accumulating it in a structural form consistent with that of the mineral meta-autunite inside the cell, corresponding to effective metal immobilization. This work supports previous findings that U bioremediation could be achieved via the biomineralization of U(vi) in phosphate minerals.

Hymenobacter perfusus sp. nov., Hymenobacter flocculans sp. nov. and Hymenobacter metalli sp. nov. three new species isolated from an uranium mine waste water treatment system.

Three red-pink pigmented strains, designated A1-12(T), A2-50A(T) and A2-91(T), were recovered from two different sites in a uranium mine. For all strains, the optimum growth temperature was 25°C, the optimum pH was 6.0-6.5 and the DNA G+C contents were between 60 and 63.4 mol%. The major respiratory quinone was menaquinone 7 (MK-7) and the fatty acid profiles contained iso- and anteiso-branched C15 fatty acids, summed feature 3 (16:1 ω6c and/or ω7c and/or 15:0 iso 2-OH), summed feature 4 (17:1 anteiso B and/or iso I) and the unsaturated fatty acid 16:1 ω5c as the major components. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented three distinct branches within the family Flexibacteraceae most closely related to the members of the genus Hymenobacter. Strain A1-12(T) formed a distinct phylogenetic line along with H. rigui KCTC 12533(T) and they shared approximately 98.9% 16S rRNA gene sequence similarity. However, these two strains shared only 14.7% pairwise similarity in their genomic DNA. Strains A2-50A(T) and A2-91(T) formed two distinct lineages, related to the species H. soli KCTC 12607(T), sharing about 95.5% 16S rRNA gene sequence similarity between themselves, and 88.3 and 92.0% with other members of the genus Hymenobacter. Based on the phylogenetic analysis and physiological and biochemical characteristics, these isolates were considered to represent three novel species for which we propose the names Hymenobacter perfusus for strain A1-12(T) (=CIP 110166=LMG 26000), Hymenobacter flocculans for strain A2-50A(T) (=CIP 110139=LMG 25699) and Hymenobacter metalli for strain A2-91(T) (=CIP 110140=LMG 25700).

Nosocomiicoccus ampullae gen. nov., sp. nov., isolated from the surface of bottles of saline solution used in wound cleansing.

Strains TRF-1(T) and TC-9 were isolated from transfer spikes of two separate bottles containing 0.9 % NaCl (physiological saline) solution used repeatedly to wash wounds in hospital wards, months apart. Phylogenetic analysis of 16S rRNA gene sequences revealed that strains TRF-1(T) and TC-9 formed a distinct branch within the family Bacillaceae most closely related to the members of the genus Jeotgalicoccus. The two strains, with identical 16S rRNA gene sequences, showed sequence similarities of 89.8-93.9 % with species of Jeotgalicoccus, 91.1-91.8 % with species of Salinicoccus and 89.1-89.7 % with species of Staphylococcus. Strains TRF-1(T) and TC-9 are Gram-positive, non-spore-forming and non-motile cocci, with an optimum growth temperature of about 37 degrees C. Strain TRF-1(T) grew optimally in medium containing 3 % (w/v) NaCl (maximum about 14 % NaCl), while strain TC-9 grew optimally in medium with 1 % (w/v) NaCl. Both strains produce a brown pigment when grown in the presence of NaCl. The cell-wall peptidoglycan is of the A3alpha type with a cross-linkage containing the peptide l-Lys-Gly(4)-l-Ser-Gly. The major respiratory quinones are menaquinone 7 (MK-7) and menaquinone 8 (MK-8), the major fatty acids are straight-chain C(14 : 0) and C(16 : 0) (more than 85 % of the total) and the major polar lipid is an unknown aminophospholipid. The DNA G+C content is 33.5 mol%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics, we are of the opinion that strains TRF-1(T) and TC-9 represent a novel species of a new genus, for which we propose the name Nosocomiicoccus ampullae gen. nov., sp. nov. The type strain of Nosocomiicoccus ampullae is strain TRF-1(T) (=LMG 24060(T) =CIP 109506(T)).

Bacterial diversity in a nonsaline alkaline environment: heterotrophic aerobic populations.

Heterotrophic populations were isolated and characterized from an alkaline groundwater environment generated by active serpentinization, which results in a Ca(OH)2-enriched, extremely diluted groundwater with pH 11.4. One hundred eighty-five strains were isolated in different media at different pH values during two sampling periods. To assess the degree of diversity present in the environment and to select representative strains for further characterization of the populations, we screened the isolates by using random amplified polymorphic DNA-PCR profiles and grouped them based on similarities determined by fatty acid methyl ester analysis. Phenotypic characterization, determinations of G+C content, phylogenetic analyses by direct sequencing of 16S rRNA genes, and determinations of pH tolerance were performed with the selected isolates. Although 38 different populations were identified and characterized, the vast majority of the isolates were gram positive with high G+C contents and were affiliated with three distinct groups, namely, strains closely related to the species Dietzia natrolimnae (32% of the isolates), to Frigoribacterium/Clavibacter lineages (29% of the isolates), and to the type strain of Microbacterium kitamiense (20% of the isolates). Other isolates were phylogenetically related to strains of the genera Agrococcus, Leifsonia, Kytococcus, Janibacter, Kocuria, Rothia, Nesterenkonia, Citrococcus, Micrococcus, Actinomyces, Rhodococcus, Bacillus, and Staphylococcus. Only five isolates were gram negative: one was related to the Sphingobacteria lineage and the other four were related to the alpha-Proteobacteria lineage. Despite the pH of the environment, the vast majority of the populations were alkali tolerant, and only two strains were able to grow at pH 11.