Shedding light on the electron transfer chain of a moderately acidophilic iron oxidizer: characterization of recombinant HiPIP-41, CytC-18 and CytC-78 derived from Ferrovum sp. PN-J47-F6.

Efficient electron transfer from the donor to the acceptor couple presents a necessary requirement for acidophilic and neutrophilic iron oxidizers due to the low energy yield of aerobic ferrous iron oxidation. Involved periplasmic electron carriers are very diverse in these bacteria and show adaptations to the respective thermodynamic constraints such as a more positive redox potential reported for extreme acidophilic Acidithiobacillus spp. Respiratory chain candidates of moderately acidophilic members of the genus Ferrovum share similarities with both their neutrophilic iron oxidizing relatives and the more distantly related Acidithiobacillus spp. We examined our previous omics-based conclusions on the potential electron transfer chain in Ferrovum spp. by characterizing the three redox protein candidates CytC-18, CytC-78 and HiPIP-41 of strain PN-J47-F6 which were produced as recombinant proteins in Eschericha coli. UV/Vis-based redox assays suggested that HiPIP-41 has a very positive redox potential while redox potentials of CytC-18 and CytC-78 are more negative than their counterparts in Acidithiobacillus spp. Far Western dot blotting demonstrated interactions between all three recombinant redox proteins while redox assays showed the electron transfer from HiPIP-41 to either of the cytochromes. Altogether, CytC-18, CytC-78 and HiPIP-41 indeed represent very likely candidates of the electron transfer in Ferrovum sp. PN-J4-F6.

Alicyclobacillus sp. SO9, a novel halophilic acidophilic iron-oxidizing bacterium isolated from a tailings-contaminated beach, and its effect on copper extraction from chalcopyrite in the presence of high chloride concentration.

Many acidophilic iron-oxidizing bacteria used in the mining industry for the bioleaching of sulfidic minerals are intolerant to high chloride concentrations, resulting in problems where chloride occurs in the deposit at high concentrations or only seawater is available. In search for strains tolerating such conditions a tetrathionate- and iron-oxidizing bacterium was isolated from a tailings-contaminated beach sample at Portman Bay, Cartagena-La Union mining district, Spain, in the presence of 20 g l-1 (0.34 M) sodium chloride. The isolate was able to form spores, did not grow in the absence of NaCl, and oxidized ferrous iron in the presence of up to 1.5 M (∼87 g l-1) NaCl. Genome sequencing based on a combination of Illumina and PacBio reads revealed two contigs, a circular bacterial chromosome of 5.2 Mbp and a plasmid of 90 kbp, respectively. The chromosome comprised seven different 16S rRNA genes. Submission of the chromosome to the Type (Strain) Genome Server (TYGS) without preselection of similar sequences revealed exclusively type strains of the genus Alicyclobacillus. In the TYGS analyses the respective most similar species were dependent on whether the final tree was derived from just 16S rRNA, from the genomes, or from the proteomes. Thus, TYGS analysis clearly showed that isolate SO9 represents a novel species of the genus Alicyclobacillus. In the presence of artificial seawater with almost 0.6 M chloride, the addition of Alicyclobacillus sp. SO9 improved copper dissolution from chalcopyrite (CuFeS2) compared to abiotic leaching without bacteria. The new isolate SO9, therefore, has potential for bioleaching at elevated chloride concentrations.

Transcriptomic analysis of chloride tolerance in Leptospirillum ferriphilum DSM 14647 adapted to NaCl.

Chloride ions are toxic for most acidophilic microorganisms. In this study, the chloride tolerance mechanisms in the acidophilic iron-oxidizing bacterium Leptospirillum ferriphilum DSM 14647 adapted to 180 mM NaCl were investigated by a transcriptomic approach. Results showed that 99 genes were differentially expressed in the adapted versus the non-adapted cultures, of which 69 and 30 were significantly up-regulated or down-regulated, respectively. Genes that were up-regulated include carbonic anhydrase, cytochrome c oxidase (ccoN) and sulfide:quinone reductase (sqr), likely involved in intracellular pH regulation. Towards the same end, the cation/proton antiporter CzcA (czcA) was down-regulated. Adapted cells showed a higher oxygen consumption rate (2.2 x 10-9 ppm O2 s-1cell-1) than non-adapted cells (1.2 x 10-9 ppm O2 s-1cell-1). Genes coding for the antioxidants flavohemoprotein and cytochrome c peroxidase were also up-regulated. Measurements of the intracellular reactive oxygen species (ROS) level revealed that adapted cells had a lower level than non-adapted cells, suggesting that detoxification of ROS could be an important strategy to withstand NaCl. In addition, data analysis revealed the up-regulation of genes for Fe-S cluster biosynthesis (iscR), metal reduction (merA) and activation of a cellular response mediated by diffusible signal factors (DSFs) and the second messenger c-di-GMP. Several genes related to the synthesis of lipopolysaccharide and peptidoglycan were consistently down-regulated. Unexpectedly, the genes ectB, ectC and ectD involved in the biosynthesis of the compatible solutes (hydroxy)ectoine were also down-regulated. In line with these findings, although hydroxyectoine reached 20 nmol mg-1 of wet biomass in non-adapted cells, it was not detected in L. ferriphilum adapted to NaCl, suggesting that this canonical osmotic stress response was dispensable for salt adaptation. Differentially expressed transcripts and experimental validations suggest that adaptation to chloride in acidophilic microorganisms involves a multifactorial response that is different from the response in other bacteria studied.

Metabolic history and metabolic fitness as drivers of anabolic heterogeneity in isogenic microbial populations.

Microbial populations often display different degrees of heterogeneity in their substrate assimilation, that is, anabolic heterogeneity. It has been shown that nutrient limitations are a relevant trigger for this behaviour. Here we explore the dynamics of anabolic heterogeneity under nutrient replete conditions. We applied time-resolved stable isotope probing and nanoscale secondary ion mass spectrometry to quantify substrate assimilation by individual cells of Pseudomonas putida, P. stutzeri and Thauera aromatica. Acetate and benzoate at different concentrations were used as substrates. Anabolic heterogeneity was quantified by the cumulative differentiation tendency index. We observed two major, opposing trends of anabolic heterogeneity over time. Most often, microbial populations started as highly heterogeneous, with heterogeneity decreasing by various degrees over time. The second, less frequently observed trend, saw microbial populations starting at low or very low heterogeneity, and remaining largely stable over time. We explain these trends as an interplay of metabolic history (e.g. former growth substrate or other nutrient limitations) and metabolic fitness (i.e. the fine-tuning of metabolic pathways to process a defined growth substrate). Our results offer a new viewpoint on the intra-population functional diversification often encountered in the environment, and suggests that some microbial populations may be intrinsically heterogeneous.

Secondary metabolites released by the rhizosphere bacteria Arthrobacter oxydans and Kocuria rosea enhance plant availability and soil-plant transfer of germanium (Ge) and rare earth elements (REEs).

Here, we explore effects of metallophore-producing rhizobacteria on the plant availability of germanium (Ge) and rare earth elements (REEs). Five isolates of the four species Rhodococcus erythropolis, Arthrobacter oxydans, Kocuria rosea and Chryseobacterium koreense were characterized regarding their production of element-chelators using genome-mining, LC-MS/MS analysis and solid CAS-assay. Additionally, a soil elution experiment was conducted in order to identify isolates that increase solubility of Ge and REEs in soil solution. A. oxydans ATW2 and K. rosea ATW4 released desferrioxamine-, bacillibactin- and surfactin-like compounds that mobilized Ge and REEs as well as P, Fe, Si and Ca in soil. Subsequently, oat, rapeseed and reed canary grass were cultivated on soil and sand and treated with cells and iron depleted culture supernatants of A. oxydans ATW2 and K. rosea ATW4. Inoculation increased plant yield and shoot phosphorus (P), manganese (Mn), Ge and REE concentrations. However, effects of the inoculation varied substantially between the growth substrates and plant species. On sand, A. oxydans ATW2 increased accumulation of REEs in all plant species and root-shoot translocation in rapeseed, while K. rosea ATW4 enhanced REE accumulation in rapeseed only, without effects on other plant species. Sand-cultured oat plants showed increased Ge accumulation and root-shoot translocation in presence of A. oxydans ATW2 cells and K. rosea ATW4 supernatant; however, there was no effect on other plant species, irrespective the growth substrate used. In contrast, soil-cultured rapeseed showed enhanced REE accumulation in presence of cells of A. oxydans ATW2 while there were no effects on other plant species and Ge. The processes involved are not yet fully understood. Nevertheless, we demonstrated that chemical microbe-soil-plant relationships influence plant availability of nutrients together with Ge and REEs, which has major implications on our understanding of biogeochemical element cycling and development of sustainable bioremediation and biomining technologies.

Isolation and characterization of arsenic-binding siderophores from Rhodococcus erythropolis S43: role of heterobactin B and other heterobactin variants.

Rhodococcus erythropolis S43 is an arsenic-tolerant actinobacterium isolated from an arsenic contaminated soil. It has been shown to produce siderophores when exposed to iron-depleting conditions. In this work, strain S43 was shown to have the putative heterobactin production cluster htbABCDEFGHIJ(K). To induce siderophore production, the strain was cultured in iron-depleted medium in presence and absence of sodium arsenite. The metabolites produced by S43 in the colorimetric CAS and As-mCAS assays, respectively, showed iron- and arsenic-binding properties reaching a chelating activity equivalent to 1.6 mM of desferroxamine B in the supernatant of the culture without arsenite. By solid-phase extraction and two subsequent HPLC separations from both cultures, several fractions were obtained, which contained CAS and As-mCAS activity and which were submitted to LC-MS analyses including fragmentation of the major peaks. The mixed-type siderophore heterobactin B occurred in all analyzed fractions, and the mass of the "Carrano heterobactin A" was detected as well. In addition, generation of a molecular network based on fragment spectra revealed the occurrence of several other compounds with heterobactin-like structures, among them a heterobactin B variant with an additional CH2O moiety. 1H NMR analyses obtained for preparations from the first HPLC step showed signals of heterobactin B and of "Carrano heterobactin A" with different relative amounts in all three samples. In summary, our results reveal that in R. erythropolis S43, a pool of heterobactin variants is responsible for the iron- and arsenic-binding activities. KEY POINTS: • Several heterobactin variants are the arsenic-binding compounds in Rhodococcus erythropolis S43. • Heterobactin B and the compound designated heterobactin A by Carrano are of importance. • In addition, other heterobactins with ornithine in the backbone exist, e.g., the new heterobactin C.

Biochemical Characterization of Phenylacetaldehyde Dehydrogenases from Styrene-degrading Soil Bacteria.

Four phenylacetaldehyde dehydrogenases (designated as FeaB or StyD) originating from styrene-degrading soil bacteria were biochemically investigated. In this study, we focused on the Michaelis-Menten kinetics towards the presumed native substrate phenylacetaldehyde and the obviously preferred co-substrate NAD+. Furthermore, the substrate specificity on four substituted phenylacetaldehydes and the co-substrate preference were studied. Moreover, these enzymes were characterized with respect to their temperature as well as long-term stability. Since aldehyde dehydrogenases are known to show often dehydrogenase as well as esterase activity, we tested this capacity, too. Almost all results showed clearly different characteristics between the FeaB and StyD enzymes. Furthermore, FeaB from Sphingopyxis fribergensis Kp5.2 turned out to be the most active enzyme with an apparent specific activity of 17.8 ± 2.1 U mg-1. Compared with that, both StyDs showed only activities less than 0.2 U mg-1 except the overwhelming esterase activity of StyD-CWB2 (1.4 ± 0.1 U mg-1). The clustering of both FeaB and StyD enzymes with respect to their characteristics could also be mirrored in the phylogenetic analysis of twelve dehydrogenases originating from different soil bacteria.

Effect of Sodium Chloride on Pyrite Bioleaching and Initial Attachment by Sulfobacillus thermosulfidooxidans.

Biomining applies microorganisms to extract valuable metals from usually sulfidic ores. However, acidophilic iron (Fe)-oxidizing bacteria tend to be sensitive to chloride ions which may be present in biomining operations. This study investigates the bioleaching of pyrite (FeS2), as well as the attachment to FeS2 by Sulfobacillus thermosulfidooxidans DSM 9293T in the presence of elevated sodium chloride (NaCl) concentrations. The bacteria were still able to oxidize iron in the presence of up to 0.6M NaCl (35 g/L), and the addition of NaCl in concentrations up to 0.2M (~12 g/L) did not inhibit iron oxidation and growth of S. thermosulfidooxidans in leaching cultures within the first 7 days. However, after approximately 7 days of incubation, ferrous iron (Fe2+) concentrations were gradually increased in leaching assays with NaCl, indicating that iron oxidation activity over time was reduced in those assays. Although the inhibition by 0.1M NaCl (~6 g/L) of bacterial growth and iron oxidation activity was not evident at the beginning of the experiment, over extended leaching duration NaCl was likely to have an inhibitory effect. Thus, after 36 days of the experiment, bioleaching of FeS2 with 0.1M NaCl was reduced significantly in comparison to control assays without NaCl. Pyrite dissolution decreased with the increase of NaCl. Nevertheless, pyrite bioleaching by S. thermosulfidooxidans was still possible at NaCl concentrations as high as 0.4M (~23 g/L NaCl). Besides, cell attachment in the presence of different concentrations of NaCl was investigated. Cells of S. thermosulfidooxidans attached heterogeneously on pyrite surfaces regardless of NaCl concentration. Noticeably, bacteria were able to adhere to pyrite surfaces in the presence of NaCl as high as 0.4M. Although NaCl addition inhibited iron oxidation activity and bioleaching of FeS2, the presence of 0.2M seemed to enhance bacterial attachment of S. thermosulfidooxidans on pyrite surfaces in comparison to attachment without NaCl.

Effect of inoculum history, growth substrates and yeast extract addition on inhibition of Sulfobacillus thermosulfidooxidans by NaCl.

This study reports on the effect of inoculum history, growth substrates, and yeast extract on sodium chloride tolerance of Sulfobacillus thermosulfidooxidans DSM 9293T. The concentrations of NaCl for complete inhibition of Fe2+ oxidation by cells initially grown with ferrous iron sulfate, or tetrathionate, or pyrite as energy sources were 525 mM, 725 mM, and 800 mM, respectively. Noticeably, regardless of NaCl concentrations, oxygen consumption rates of S. thermosulfidooxidans with 20 mM tetrathionate were higher than with 50 mM FeSO4. NaCl concentrations of higher than 400 mM strongly inhibited the iron respiration of S. thermosulfidooxidans. In contrast, the presence of NaCl was shown to stimulate tetrathionate oxidation. This trend was especially pronounced in NaCl-adapted cells where respiration rates at 200 mM NaCl were threefold of those in the absence of NaCl. In NaCl-adapted cultures greater respiration rates for tetrathionate were observed than in non-NaCl-adapted cultures, especially at concentrations ≥ 200 mM NaCl. At concentrations of ≤ 200 mM NaCl, cell growth and iron oxidation were enhanced with the addition of increasing concentrations of yeast extract. Thus, cell numbers in cultures with 0.05% yeast extract were ∼5 times higher than without yeast extract addition. At NaCl concentration as high as 400 mM, however, iron oxidation rates improved compared to control assays without yeast extract, but there was no clear dependence on yeast extract concentrations. The initial growth of bacteria with and without yeast extract in the presence of different NaCl concentrations was shown to impact leaching of copper from chalcopyrite. Copper dissolution was enhanced in the presence of 200 mM NaCl and absence of yeast extract, while the addition of 0.02% yeast extract was shown to promote copper solubilization in the presence of 500 mM NaCl.

Cultivation dependent formation of siderophores by Gordonia rubripertincta CWB2.

Herein we demonstrate cultivation-dependent siderophore production by the actinomycete Gordonia rubripertincta CWB2. The strain produces mostly citrate, but also desferrioxamine E (DFOE) and new hydroxamate-type siderophores. The production of hydroxamate-like siderophores is influenced by cultivation conditions, for example available carbon sources or presence of metals, such as the rare earth erbium or the heavy metal lead. By cultivation with succinate and extraction with an adsorbing resin (XAD) we purified the G. rubripertincta CWB2 siderophores (yield up to 178 mg L-1). The respective workflow comprises genome mining, cultivation, and overproduction strategies, a rapid screening procedure, as well as traditional structure enrichment and structure elucidation methods. This combination of methods allows the discovery of new natural products with metal complexation capacity, also for lanthanides of commercial value. G. rubripertincta CWB2 carries a desferrioxamine-like biosynthetic gene cluster. Its transcription was proven by a transcriptomic approach comparing expression levels of the selected gene cluster during cultivation in iron-depleted and repleted media. Further investigation of the siderophores of this desferrioxamine producing Actinobacterium could lead to new structures.

Data on metal-chelating, -immobilisation and biosorption properties by Gordonia rubripertincta CWB2 in dependency on rare earth adaptation.

Recent studies have shown that the metal adaptation of Actinobacteria offers a rich source of metal inducible environmentally relevant bio-compounds and molecules. These interact through biosorption towards the unique cell walls or via metal chelating activity of metallophors with trace elements, heavy metals and even with lanthanides to overcome limitations and toxic concentrations. Herein, the purpose is to investigate the adaptation potential of Gordonia rubripertincta CWB2 in dependence of the rare earths and to determine if we can utilize promising metallophore metal affinities for metal separation from aquatic solutions. For details on data interpretation and applicability of siderophores we refer to the related article entitled "Cultivation dependent formation of siderophores by Gordonia rubripertincta CWB2" [1]. The respective workflow comprises a metal adaptation method to demonstrate effects on bacterial growth, pH, metallophore production, and metabolic change. All this was evaluated by LC-MS/MS and effects on biosorption of rare earths was verified by ICP-MS. Furthermore, we were able to carry out batch metal adsorption and desorption studies of metallophores entrapped in inorganic polymers of tetramethoxysilane (TMOS) to determine metal chelating capacities and selective enrichment effects from model solutions. The adaptation potential of strain CWB2 at increased erbium and manganese concentrations was verified by increased chelating activity on agar plates, in liquid assays and demonstrated by the successful enrichment of erbium by metallophore-functionalized TMOS-polymers from an aquatic model solution. Furthermore, the number of detected compounds in dependency of rare earths differ in spectral counts and diversity compared to the wild type. Finally, the biosorption of rare earths for the selected adaptation was increased significantly up to 2-fold compared to the wild-type. Overall a holistic approach to metal stress was utilised, integrating a bacterial erbium adaptation, metal chelating, biosorption of lanthanides and immobilization as well as enrichment of metals using metallophore functionalized inorganic TMOS polymers for separation of metals from aquatic model solutions.

Osmotic Imbalance, Cytoplasm Acidification and Oxidative Stress Induction Support the High Toxicity of Chloride in Acidophilic Bacteria.

In acidophilic microorganisms, anions like chloride have higher toxicity than their neutrophilic counterparts. In addition to the osmotic imbalance, chloride can also induce acidification of the cytoplasm. We predicted that intracellular acidification produces an increase in respiratory rate and generation of reactive oxygen species, and so oxidative stress can also be induced. In this study, the multifactorial effect as inducing osmotic imbalance, cytoplasm acidification and oxidative stress in the iron-oxidizing bacterium Leptospirillum ferriphilum DSM 14647 exposed to up to 150 mM NaCl was investigated. Results showed that chloride stress up-regulated genes for synthesis of potassium transporters (kdpC and kdpD), and biosynthesis of the compatible solutes (hydroxy)ectoine (ectC and ectD) and trehalose (otsB). As a consequence, the intracellular levels of both hydroxyectoine and trehalose increased significantly, suggesting a strong response to keep osmotic homeostasis. On the other hand, the intracellular pH significantly decreased from 6.7 to pH 5.5 and oxygen consumption increased significantly when the cells were exposed to NaCl stress. Furthermore, this stress condition led to a significant increase of the intracellular content of reactive oxygen species, and to a rise of the antioxidative cytochrome c peroxidase (CcP) and thioredoxin (Trx) activities. In agreement, ccp and trx genes were up-regulated under this condition, suggesting that this bacterium displayed a transcriptionally regulated response against oxidative stress induced by chloride. Altogether, these data reveal that chloride has a dramatic multifaceted effect on acidophile physiology that involves osmotic, acidic and oxidative stresses. Exploration of the adaptive mechanisms to anion stress in iron-oxidizing acidophilic microorganisms may result in new strategies that facilitate the bioleaching of ores for recovery of precious metals in presence of chloride.

Quantitation and Comparison of Phenotypic Heterogeneity Among Single Cells of Monoclonal Microbial Populations.

Phenotypic heterogeneity within microbial populations arises even when the cells are exposed to putatively constant and homogeneous conditions. The outcome of this phenomenon can affect the whole function of the population, resulting in, for example, new "adapted" metabolic strategies and impacting its fitness at given environmental conditions. Accounting for phenotypic heterogeneity becomes thus necessary, due to its relevance in medical and applied microbiology as well as in environmental processes. Still, a comprehensive evaluation of this phenomenon requires a common and unique method of quantitation, which allows for the comparison between different studies carried out with different approaches. Consequently, in this study, two widely applicable indices for quantitation of heterogeneity were developed. The heterogeneity coefficient (HC) is valid when the population follows unimodal activity, while the differentiation tendency index (DTI) accounts for heterogeneity implying outbreak of subpopulations and multimodal activity. We demonstrated the applicability of HC and DTI for heterogeneity quantitation on stable isotope probing with nanoscale secondary ion mass spectrometry (SIP-nanoSIMS), flow cytometry, and optical microscopy datasets. The HC was found to provide a more accurate and precise measure of heterogeneity, being at the same time consistent with the coefficient of variation (CV) applied so far. The DTI is able to describe the differentiation in single-cell activity within monoclonal populations resolving subpopulations with low cell abundance, individual cells with similar phenotypic features (e.g., isotopic content close to natural abundance, as detected with nanoSIMS). The developed quantitation approach allows for a better understanding on the impact and the implications of phenotypic heterogeneity in environmental, medical and applied microbiology, microbial ecology, cell biology, and biotechnology.

Investigation of the co-metabolic transformation of 4-chlorostyrene into 4-chlorophenylacetic acid in Pseudomonas fluorescens ST.

The side-chain oxygenation of styrene is able to yield substituted phenylacetic acids from corresponding styrenes by co-metabolic transformation. This co-metabolization was investigated in Pseudomonas fluorescens ST using 4-chlorostyrene as co-substrate. It was shown that non-substituted styrene is necessary to ensure the co-metabolic process. Furthermore, aspects affecting the co-transformation were studied, e.g. cell density, amount of inducer, pH, effects of co-substrate/co-product. It was demonstrated that 4-chlorophenylacetic acid and 4-chlorostyrene are able to inhibit the reaction. But, these inhibitions are influenced by salt and trace elements. Finally, a protocol was established which considers all findings. Therewith, about 6.7 g L-1 co-product were obtained after 451 h. Compared to previous studies, the co-product concentration was improved by the factor 1.4 while the reaction time was decreased by the factor 18.5. The study offers also aspects for prospective improvements in order to establish an efficient way to gain substituted acids without genetic manipulation.

Draft genome sequence of Rhodococcus erythropolis B7g, a biosurfactant producing actinobacterium.

Biosurfactants are amphipathic molecules with relevance in biotechnology due to their structural diversity, low toxicity and biodegradability. The genus Rhodococcus has extensively been studied because of its capacity to produce trehalose-containing surfactants as well as trehalose lipids as potential pathogenic factor. Here we present the draft genome sequence of Rhodococcus erythropolis B7g isolated with toluene from fuel-contaminated soil. The genome comprises 7,175,690 bp in 121 contigs, a G + C content of 62,4% and 7,153 coding DNA sequences (CDSs), and it contains genes for trehalose biosynthesis and surfactant production. Additionally, genes for the production of trehalose-tetraester biosurfactant were identified, whose function was experimentally verified making the strain B7g a potential candidate for use in bioremediation applications or in biosurfactant exploration.

Iron targeted transcriptome study draws attention to novel redox protein candidates involved in ferrous iron oxidation in "Ferrovum" sp. JA12.

The response of the acidophilic iron oxidizer "Ferrovum" sp. JA12 to elevated concentrations of ferrous iron was targeted at transcriptome level in order to assess models on oxidative stress management and ferrous iron oxidation. Overall transcriptome profiles indicate a high cellular activity of "Ferrovum" sp. JA12 up to 50 mM of ferrous iron with genes predicted to be involved in iron oxidation, carbon fixation and ribosome formation showing the highest transcript levels. The data support the iron oxidation pathway inferred from genome analysis and draws attention to further redox proteins potentially associated with iron oxidation. The restriction of homologous proteins to iron oxidizing beta- and zetaproteobacteria underlines the previous notion of a common origin of iron oxidation in these phyla. Detoxification of reactive oxygen species and primary products of oxidative damage of membrane lipids appears to be of permanent relevance under conditions mimicking those of the original habitat of "Ferrovum" sp. JA12. Also the maintenance of a reverse membrane potential appears to be its most important strategy to withstand the acidic external pH.

Detection of arsenic-binding siderophores in arsenic-tolerating Actinobacteria by a modified CAS assay.

The metalloid arsenic is highly toxic to all forms of life, and in many countries decontamination of water and soil is still required. Some bacteria have mechanisms to detoxify arsenic and can live in its presence. Actinobacteria are well known for their ability to produce a myriad of biologically-active compounds. In the present study, we isolated arsenic-tolerant Actinobacteria from contaminated water in Saxony, Germany, and determined their ability to produce siderophores able to bind arsenic. The binding capacity of different siderophore-like compounds was determined by a modified chrome azurol S (As-mCAS) assay with As(III) at high pH and using CAS decolorization as a readout. Arsenic-tolerant isolates from three actinobacterial genera were identified by 16 S rRNA gene sequence analysis: Rhodococcus, Arthrobacter and Kocuria. The isolated Actinobacteria showed a high As(III)-binding activity by siderophore-like compounds, resulting in 82-100% CAS decolorization, as compared to the results with EDTA. The interaction between As(III) and siderophore-like compounds was also detected at neutral pH. In summary, our results suggest that the isolated arsenic-tolerant Actinobacteria produce siderophores that bind arsenic, and open new perspectives on potential candidates for decontaminating environments with arsenic and for other biotechnological applications.

Heterologous production of different styrene oxide isomerases for the highly efficient synthesis of phenylacetaldehyde.

The styrene oxide isomerase (SOI, StyC) represents a key enzyme of the styrene-degrading pathway and has been discussed as promising biocatalyst during recent studies. The enzyme enables the synthesis of pure phenylacetaldehyde from styrene oxide. In this study the native as well as the corresponding codon-optimized genes of three different SOIs from Rhodococcus opacus 1CP (StyC-1CP), Sphingopyxis fribergensis Kp5.2 (StyC-Kp5.2), and Pseudomonas fluorescens ST (StyC-ST) were investigated for the expression in Escherichia coli BL21(DE3)pLysS. Specific enzyme activities of 61.9±7.5Umg-1, 23.2±2.8Umg-1, and 10.9±1.2Umg-1 were achieved after 6-9h for the codon-optimized gene of strain 1CP and the native genes of Kp5.2 and ST, respectively. Afterwards, these enzymes were enriched and applied for biotransformation studies. A complete conversion of 150mM styrene oxide to phenylacetaldehyde was observed for the enzyme StyC-Kp5.2 indicating a significantly improved stability towards product inactivation. Remarkably, more than 300mM product (>36gL-1, yield of about 80%) were finally synthesized from 400mM substrate with 150U of this enzyme within 60-120min. This represents the highest product concentration which has been reached with this type of enzymes, so far.

Draft Genome of the Heterotrophic Iron-Oxidizing Bacterium "Acidibacillus ferroxidans" Huett2, Isolated from a Mine Drainage Ditch in Freiberg, Germany.

Here, we communicate the draft genome of "Acidibacillus ferrooxidans" Huett2, a novel strain of an acidophilic, heterotrophic, iron-oxidizing bacterium belonging to the phylum Firmicutes It was isolated from a water drainage system of a former minefield in Freiberg, Germany.

Microbial diversity of the hypersaline and lithium-rich Salar de Uyuni, Bolivia.

Salar de Uyuni, situated in the Southwest of the Bolivian Altiplano, is the largest salt flat on Earth. Brines of this athalassohaline hypersaline environment are rich in lithium and boron. Due to the ever- increasing commodity demand, the industrial exploitation of brines for metal recovery from the world's biggest lithium reservoir is likely to increase substantially in the near future. Studies on the composition of halophilic microbial communities in brines of the salar have not been published yet. Here we report for the first time on the prokaryotic diversity of four brine habitats across the salar. The brine is characterized by salinity values between 132 and 177 PSU, slightly acidic to near-neutral pH and lithium and boron concentrations of up to 2.0 and 1.4g/L, respectively. Community analysis was performed after sequencing the V3-V4 region of the 16S rRNA genes employing the Illumina MiSeq technology. The mothur software package was used for sequence processing and data analysis. Metagenomic analysis revealed the occurrence of an exclusively archaeal community comprising 26 halobacterial genera including only recently identified genera like Halapricum, Halorubellus and Salinarchaeum. Despite the high diversity of the halobacteria-dominated community in sample P3 (Shannon-Weaver index H'=3.12 at 3% OTU cutoff) almost 40% of the Halobacteriaceae-assigned sequences could not be classified on the genus level under stringent filtering conditions. Even if the limited taxonomic resolution of the V3-V4 region for halobacteria is considered, it seems likely to discover new, hitherto undescribed genera of the family halobacteriaceae in this particular habitat of Salar de Uyuni in future.