In this work, we focus on the identification of novel fungal peroxygenase gene belonging to the peroxidase-peroxygenase superfamily. We applied a metagenomic approach on soil samples from primeval forest and appropriate bioinformatics tools for analysis of obtained genomic DNA sequence. Peroxidases are ubiquitous metalloenzymes that are able to reduce reactive peroxides, mainly hydrogen peroxide, into water, whereas several substrates can be concomitantly oxidized during their catalytic reaction. Our purpose was to collect unique peroxygenase sequence data originating from a preserved biotope for a robust phylogenetic reconstruction of a particular gene family coding for highly versatile heme-thiolate peroxidases that has peculiar yet undiscovered representatives among ectomycorrhizal fungi. We identified unique DNA sequence, 812 bp long, from ectomycorrhizal Suillus species coding for a heme-thiolate peroxidase with 1 typical intron that appears distinctive for Carpathian forests. After translation in corresponding protein sequence 251 amino acids long we could identify typical signatures of this peroxygenase. On the proximal side of heme we found the conserved P-C-P triad responsible for efficient ligation of heme iron thus influencing the reactivity of this peroxidase. On the distal side we recognized the E-H-D-X-S-L motif for interaction with a stabilizing magnesium ion. Maximum likelihood reconstruction of protein phylogeny revealed with a high bootstrap support the presence of a monophyletic HTP4 clade originating in numerous Suillus representatives. Together with sister clades of edible Boletus and poisonous Paxillus containing diverse peroxygenases these newly discovered heme catalyst can be considered for application of oxyfunctionalization of organic molecules.

Deep evolutionary origin of the conserved animal serum amyloid A (SAA) apolipoprotein family leading to yet unknown highly similar SAA-like sequences occurring in certain bacterial genomes is demonstrated in this contribution. Horizontal gene transfer event of corresponding genes between gut bacteria and non-vertebrate animals was discovered in the reconstructed phylogenetic tree obtained with maximum likelihood and neighbor-joining methods, respectively. This detailed phylogeny based on totally 128 complete sequences comprised diverse serum amyloid A isoforms from various animal vertebrate and non-vertebrate phyla and also corresponding genes coding for highly similar proteins from animal gut bacteria. Typical largely conserved sequence motifs and a peculiar structural fold consisting mainly of four α-helices in a bundle within all reconstructed clades of the SAA protein family are discussed with respect to their supposed biological functions in various organisms that contain corresponding genes.

In this study, we focus on a detailed bioinformatics analysis of hyBpox genes, mainly within the genomes of Sclerotiniaceae (Ascomycota, Leotiomycetes), which is a specifically evolved fungal family of necrotrophic host generalists and saprophytic or biotrophic host specialists. Members of the genus Sclerotium produce only sclerotia and no fruiting bodies or spores. Thus, their physiological role for peroxidases remains open. A representative species, S. cepivorum, is a dangerous plant pathogen causing white rot in Allium species, particularly in onions, leeks, and garlic. On a worldwide basis, the white rot caused by this soil-borne fungus is apparently the most serious threat to Allium-crop production. We have also found very similar peroxidase sequences in the related fungus S. sclerotiorum, although with minor yet important modifications in the architecture of its active centre. The presence of ScephyBpox1-specific mRNA was confirmed by transcriptomic analysis. The presence of Hybrid B peroxidase at the protein level as the sole extracellular peroxidase of this fungus was confirmed in the secretome of S. cepivorum through detailed proteomic analyses. This prompted us to systematically search for all available genes coding for Hybrid B heme peroxidases in the whole fungal family of Sclerotiniaceae. We present here a reconstruction of their molecular phylogeny and analyse the unique aspects of their conserved-sequence features and structural folds in corresponding ancestral sequences.

A soil bacterium MR-CH-I15-I was on the base of phylogenetic analysis of almost the whole (1,492 bp) 16S rRNA sequence and sequences of selected 9 marker genes identified as Pseudomonas putida strain NM-CH-I15-I.The bacterium exhibited typical morphological features and biochemical properties for this species, the highest resistance to nickel and copper and multidrug resistance to different antibiotic groups.In addition, the whole czcA-NM15I heavy-metal resistance gene sequence (3,126 bp, 1,042 amino acids, MW 112, 138 Da) was obtained and on the base of phylogenetic analysis was assigned to CzcA protein from Pseudomonas reidholzensis with 93% similarity.This gene was significantly induced mainly by the addition of zinc, cadmium and cobalt and in a lesser extent of nickel.Furthermore, an increased expression of the CzcA-NM15I protein was confirmed by immunoblot analysis after heterologous expression of the czcA-NM15I synthetic variant gene in E. coli BL21 (DE3).Finally, the location of amino acids (R83, R673, D402, D408, D619, E415, E568) in the homology model of the CzcA-NM15I protein suggested that these amino acids may play an important role in the transport of cations such as cobalt, zinc or cadmium.This soil bacterium can represent a new type strain of P. putida NM-CH-I15-I.

Natural or anthropogenic disturbances in the forest ecosystem alter ecological conditions and lead to shifts in microbial diversity. We focused on the topsoil properties of specific sites in Tatra National Park (Slovakia) that were affected by windthrow, wildfire and wood extraction. We analyzed soil organic matter content (SOM), dry weight (DW), enzymatic activity including dehydrogenase activity (DHA), fluorescein diacetate hydrolysis (FDH) and phosphomonoesterase (PME). Bacterial community profiles were analyzed using the PCR-DGGE approach, and Ellenberg’s indicator values (EIV) were used as eco-indices of studied sites. Dystric Cambisol was sampled at sites distinguished by management approaches established after windthrow in 2004 and wildfire in 2005. We focused on the windthrow site where wooden debris was extracted (EXT); the site left for self-recovery with no intervention (NEX); the site affected by wildfire (FIR), a former reference site damaged by the windstorm in 2014 followed by the extraction of wooden debris (REX), and a non-affected reference spruce stand (REF). The windthrow sites with different management (EXT, NEX) showed relative similarities based on enzymatic activity, bacterial community profiling (85%) and EIV comparison. The FIR site exhibited no similarities (0%) with EXT and NEX in the bacterial community structure. The SOM content correlated only with PME (r2 = 0.42) and soil moisture (r2 = 0.64). Based on RDA analysis, certain EIVs seem to be suitable indicators of selected soil properties. The lack of differences between NEX and EXT could be caused by the diminishing of post-disturbance effects on microbial communities. The effect of wildfire on microbial activity and bacterial community structure seems to be longer than different management approach.

A bacterial isolate MR-CH-I2 [KC809939] isolated from soil contaminated mainly by high nickel concentrations in southwest Slovakia was previously found carrying nccA-like heavy-metal resistance determinant, marked as MR-CH-I2-HMR [KF218096]. According to phylogenetic analysis of short (696 bp) 16S rDNA (16S rRNA) sequences this bacterium was tentatively assigned to Uncultured beta proteobacterium clone GC0AA7ZA05PP1 [JQ913301]. nccA-like gene product was on the same base of its partial (581 bp) sequences tentatively assigned to CzcA family heavy metal efflux pump [YP_001899332] from Ralstonia picketii 12J with 99% similarity. In this study the bacterium MR-CH-I2 and its heavy-metal resistance determinant were more precisely identified. This bacterial isolate was on the base of phylogenetic analysis of almost the whole (1,500 bp) 16S rDNA (16S rRNA) sequence, MR-CH-I2 [MF102046], and sequence for gyrB gene and its product respectively, MR-CH-I2-gyrB [MF134666], assigned to R. picketii 12J [CP001068] with 99 and 100% similarities, respectively. In addition, the whole nccA-like heavy-metal resistance gene sequence (3,192 bp), marked as MR-CH-I2-nccA [KR476581], was obtained and on the base of phylogenetic analysis its assignment was confirmed to MULTISPECIES: cation efflux system protein CzcA [WP_004635342] from Burkholderiaceae with 98% similarity. Furthermore, although the bacterium carried one high molecular plasmid of about 50 kb in size, nccA-like gene was not located on this plasmid. Finally, the results from RT-PCR analysis showed that MR-CH-I2-nccA gene was significantly induced only by the addition of nickel.

Technosols or technogenic substrates contaminated by potentially toxic elements as a result of iron mining causes not only contamination of the surrounding ecosystem but may also lead to changes of the extent, abundance, structure and activity of soil microbial community. Microbial biomass were significantly inhibited mainly by exceeding limits of potentially toxic metals as arsenic (in the range of 343 511 mg/kg), copper (in the range of 7980-9227 mg/kg), manganese (in the range of 2417-2670 mg/kg), alkaline and strong alkaline pH conditions and minimal contents of organic nutrients. All of the 14 bacterial isolates, belonged to 4 bacterial phyla, Actinobacteria, Firmicutes; beta-and gamma-Proteobacteria. Thirteen genera and 20 species of microscopic filamentous fungi were recovered. The most frequently found species belonged to genera Aspergillus (A. clavatus, A. niger, A. flavus, A. versicolor, Aspergillus sp.) with the dominating A. niger in all samples, and Penicillium (P. canescens, P. chrysogenum, P. spinulosum, Penicillium sp.). Fungal plant pathogens occurred in all surface samples. These included Bjerkandera adustata, Bionectria ochioleuca with anamorph state Clonostachys pseudochloleuca, Lewia infectoria, Phoma macrostoma and Rhizoctonia sp. (C) 2016 The Authors. Published by Elsevier Ltd.

The sampling sites situated in southwest Slovakia are according to environmental monitoring of Slovakia a part of strongly disturbed environment by heavy metals, mainly by high nickel concentrations. The aim of the present study was to characterise a complete microbial assemblage from a dump containing heavy-metal-contaminated waste as well as from farmland situated nearby this dump by using shotgun sequencing of 16S rDNA amplicons. It was found that nickel influenced both species richness and diversity and that microbiota of both samples differed significantly (Bray-Curtis dissimilarity 0.73) at genus level mainly by abundances of sequences from particular genera and occurrences of the unique genera in individual bacterial communities. In spite of these differences between microbial assemblages, both samples shared many bacterial genera that might constitute the specific nickel-resistant bacterial niche, and it was possible to delineate the core microbiome of our two samples at species level. The core set of 30 species, represented by the phyla Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria, suggest that these species might form a “core microbiome” of the specific nickel-resistant bacterial niche.

Abstract In this study we aimed to analyse the structure and diversity of overall bacterial community and its resistance determinants from nickel-contaminated soil in Slovakia by both, cultivation-dependent and independent approaches. The phylogeny was reconstructed using partial sequences of 16S rRNA (16S rDNA) and heavy-metal resistance genes from separated isolates and bacterial clones. A total of 518 bacterial sequences obtained from both, isolates and clones, represented 266 species belonging to 8 bacterial phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Gemmatimonadetes, Proteobacteria (α-, β- and γ-classes), Verrucomicrobia, and one yet unclassified group. In addition, among isolates and clones, 49 different nccA-like genes were found in the final output. Majority of them were assigned to a system of transmembrane metal pumps. Our results demonstrate the fact that the nickel-contaminated soil is able to present very specific heavy-metal resistant bacterial community which can be used in different bioremediation processes.

Studied technosols represent a unique system of a 50-year-old environmental burden after dam failure of coal-ash pond. The released ashes rich in arsenic with a thickness of 1–2 m were covered by a 40-cm thick layer of soil. Long-term exposure and selection pressure of elevated concentrations of arsenic (a range of 93–634 μg/g) induced the formation of the specific adapted autochthonous microorganisms. The phylum Proteobacteria was identified as a dominant phylum in the soils and represented only by one class—Gammaproteobacteria with six species. The species of phylum Firmicutes, Bacteroidetes and Actinobacteria were also identified. Thirty-three species of identified autochthonous microscopic fungi belong to 18 genera with the most abundant Mortierella alpina (Zygomycota). The most frequent identified mycobiota belongs to genera Penicillium, Aspergillus, Trichoderma and Alternaria. The isolates of Alternaria triticina, Bionectria ochroleuca, Chrysosporium queenslandicum, Exophiala psychrophila, Metarhizium robertsii, Trichoderma rossicum and Phlebia acerina were identified for the first time in Slovakia. Despite the stimulation of autochthonous community by nutrient medium and augmentation by native species, As leachability was relatively low—on average 5.63 wt.%, 9.23 wt.% and 17.04 wt.% of the total As for inoculated Pseudomonas chlororaphis ZK-1, Pseudomonas putida ZK-5 and Aspergillus niger, respectively. The highest As leachability was achieved through biostimulation of autochthonous microbiota using liquid SAB medium (34.73 wt.% of total As content). Additionally, microbial activity was efficient in the biovolatilization of As from soils (∼70 wt.% of the total As volatilized). It appears that bioremediation using microorganisms represents one of the possible ways of As removal from soils containing coal-combustion ashes with elevated concentrations of As.

A cultivable portion of the bacterial community exhibits important metabolic activities in metal-contaminated soils. We aimed to isolate, identify and characterise hardly cultivable and previously uncultured bacteria (with similarity<97% to closest relatives) from Ni-contaminated soil in Slovakia by a diffusion-chamber-based approach. Screened bacteria were either directly grown on minimal solid media or passaged through four generations of diffusion chamber. The phylogeny was reconstructed using partial sequences of 16S rDNA genes from separated isolates inoculum and PCR-DGGE analysis. A total of 260 isolates obtained from both, diffusion-chamber-based approach and traditional cultivation techniques, represented 108 species belonging to 6 bacterial phyla: Actinobacteria, Bacteroidetes, Firmicutes, α-, β- and γ-Proteobacteria, and one yet unclassified species. Up to 51 discrete bands from PCR-DGGE analysis, representing different taxa were assigned to same phyla. 29.6% of the isolates and 56.9% of the bands were found as previously uncultivable bacteria, and 8.5% of the isolates and 19.6% of the bands were found with similarity<97% to their closest relatives. The phylogeny was also reconstructed with partial sequences of selected heavy-metal resistance genes. In 14 of previously uncultured β- or γ-Proteobacteria nccA-like gene products were identified. Majority of them were assigned to a system of transmembrane metal pumps. All these isolates were resistant to Ni, Co, Zn, Cu and Cd ions. The comparison of diffusion-chamber approach with traditional cultivation revealed that diffusion chamber cultivation produced more isolates of a real phylogenetic novelty and led to in vitro cultivation of poorly cultivable species.

Heavy metals are a significant source of pollution in soils that have been demonstrated to exert significant toxic effect on soil microbial assemblages. Here we investigate the occurrence and metabolic characteristics of actinobacteria, which form a predominated component of farmland bacterial community near the town of Sereď in southwest Slovakia, contaminated by close nickel ore facility. Actinobacteria occurred in this environment with high concentrations of nickel (2.109 mg/kg), slightly above the natural occurrence of cobalt (355 mg/kg) and zinc (177 mg/kg), even too low concentration of iron (35.75 mg/kg) for a normal soil and not a toxic amount of copper (32.2 mg/kg) and cadmium (<0.25 mg/kg). The phylogeny was reconstructed using partial sequences of 16S rDNA genes of both, actinobacterial isolates and clones. A total of 105 actinobacterial representatives were divided into 66 species belonging to one order, 7 genera and 5 uncategorized groups. The selected 14 morphologically distinct isolates were able to produce drop collapsing, haemolytic and lipase activities. Whereas only 4 isolates produced dark brown melanin pigment and only 5 of them produced decolourization of the azo dye, all isolates tested were capable of assimilating all 11 sugars tested. All these actinobacterial isolates were resistant to nickel, cobalt, zinc, cadmium, copper, but the level of resistance differed between the individual isolates, and the resistance profiles of antibiotics (gentamycin, ampicillin, ciprofloxacin, chloramphenicol, erythromycin, rifampicin, penicillin-G) varied among them to a certain extent. Our results suggested that actinobacteria in soil contaminated by nickel present a relatively divergent group inside of microbial assemblage.

This study aims to isolate and identify autochthonous bacteria from arsenic contaminated soils collected in the locality of Zemianske Kostol'any. After the coal ash pond spill in 1965, soils, sediments, and ground water were all significantly contaminated with arsenic exceeding 1500 ppm and with other potentially toxic elements. Firstly, sixty-three morphologically different bacterial isolates were obtained from samples of contaminated substrates. Consequently, identification of 16S rRNA sequences showed identities to genus Pseudomonas, Rhodococcus, Bacillus, Streptomyces and Chryseobacterium. Out of these, Pseudomonas was the genus of the greatest proportion of isolates in the studied ashy-soil substrates (62.5 %). The genus was represented by the species Pseudomonas sp., P. chlororaphis, P. putida, P. baetica and P. reinekei. The results showed that the genus Bacillus had the second highest abundance (20.83 %) and was represented by species B. cereus and B. pumilus. Two isolates of the genus Bacillus were classified as uncultured soil bacterium clone. There were further recorded two isolates of the genus Chryseobacterium. The genus Streptomyces and Rhodococcus were represented by only one species. Last but not least, 20 % of isolates were not identified to their genus. Results of studied bacterial species diversity, which was able to adapt to living in contaminated environment, are the basis for the application of selected indigenous bacterial species in bioleaching process as one of the potential methods for bioremediation of arsenic-contaminated soils.

The use of indigenous bacterial strains is a valuable bioremediation strategy for cleaning the environment from hydrocarbon pollutants. The isolation and selection of hydrocarbon-degrading bacteria is therefore crucial for obtaining the most promising strains for site decontamination. Two different media, a minimal medium supplemented with a mixture of polycyclic aromatic hydrocarbons and a MS medium supplemented with triphenyltetrazolium chloride, were used for the isolation of bacterial strains from two hydrocarbon contaminated soils and from their enrichment phases. The hydrocarbon degradation abilities of these bacterial isolates were easily and rapidly assessed using the 2,6-dichlorophenol indophenol assay. The diversity of the bacterial communities isolated from these two soil samples and from their enrichment phases was evaluated by the combination of a bacterial clustering method, fluorescence ITS-PCR, and bacterial identification by 16S rRNA sequencing. Different PCR-based assays were performed in order to detect the genes responsible for hydrocarbon degradation. The best hydrocarbon-degrading bacteria, including Arthrobacter sp., Enterobacter sp., Sphingomonas sp., Pseudomonas koreensis, Pseudomonas putida and Pseudomonas plecoglossicida, were isolated directly from the soil samples on minimal medium. The nahAc gene was detected only in 13 Gram-negative isolates and the sequences of nahAc-like genes were obtained from Enterobacter, Stenotrophomonas, Pseudomonas brenneri, Pseudomonas entomophila and P. koreensis strains. The combination of isolation on minimal medium with the 2,6-dichlorophenol indophenol assay was effective in selecting different hydrocarbon-degrading strains from 353 isolates.

Different abandoned industrial areas contaminated by polycyclic aromatic hydrocarbons (PAHs) are present in Slovakia. These environmental burdens are very dangerous to the health of human and environment. The bioremediation, based on the use of hydrocarbons degrading microorganisms, is a promising strategy to sanitize these polluted sites. The aim of this investigation was to assess the bacterial diversity of a PAHs-contaminated soil and to select the potential hydrocarbonoclastic bacteria which can be used for different bioremediation approaches. The bacterial strains were isolated on minimal medium agar supplemented with a mixture of PAHs. Seventy-three isolated strains were grouped by ribosomal interspacer analysis in 15 different clusters and representatives of each cluster were identified by 16S rRNA sequencing. The PAHs degradation abilities of all bacterial isolates were estimated by the 2,6-dichlorophenol indophenol assay and by their growth on minimal broth amended with a mixture of PAHs. Different kinds of strains, members of the genus Pseudomonas, Enterobacter, Bacillus, Arthrobacter, Acinetobacter and Sphingomonas, were isolated from the contaminated soil. Four isolates (Pseudomonas putida, Arthrobacter oxydans, Sphingomonas sp. and S. paucimobilis) showed promising PAHs-degrading abilities and therefore their possible employing in bioremediation strategies.

The occurrence of Vibrio cholerae, an important aquatic pathogen, was assessed in the surface water of the Danube River near Bratislava. The isolates were distinguished by biochemical tests and grouped by ARDRA to three clusters corresponding to three species (V. cholerae, Vibrio metschnikovii, and Aeromonas spp.). The identification of V. cholerae was confirmed by multiplex PCR using primer pairs targeted to ompW gene (membrane protein), ctxA gene (toxicity gene), and toxR gene (regulatory gene). None from the isolated V. cholerae from surface water contained ctxA gene; seven of them possessed toxR gene. Serotyping of V. cholerae isolates with polyvalent O antiserum and O/139 antiserum was negative. All isolates of V. cholerae were susceptible to chloramphenicol, rifampicin, tetracycline, variable to ampicillin, and resistant to kanamycin and streptomycin.

In this study we performed the phylogenetic analysis of non-cultivable bacteria from anthropogenically disturbed soil using partial sequences of the 16S rRNA (16S rDNA) and the heavy-metal resistance genes. This soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg), smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg), and also a trace amount of cadmium (<0.25 mg/kg). The 16S rDNA sequences from a total of 74 bacterial clones were distributed into four broad taxonomic groups, Acidobacteria, Actinobacteria, Bacteroidetes and Gemmatimonadetes, and some of them were unidentified. Comparing our clone sequences with those from the GenBank database, only 9 clones displayed high similarity to known bacteria belongig to actinomycetes; others were identified as uncultured ones. Among clones evidently Actinobacteria predominated. Sixteen clones from soil sample carried only the nccA-like heavy-metal-resistance genes and all sequences showed too low similarity to known proteins encoded by these genes. However, our results suggested that the heavy-metal-contaminated soil is able to present very important reservoir of the new and until now unknown partly bacteria, partly heavy-metal-resistance determinants and their products. Bacteria and nccA-like genes identified in this study could represent the objects of interest as bioremediation agents because they can be potentially used in different transformation and immobilization processes.

In this study we performed a phylogenetic analysis of a culturable bacterial community isolated from heavymetal-contaminated soil from southwest Slovakia using 16S rRNA (16S rDNA) and heavy-metal resistance genes. The soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg), smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg), and a trace amount of cadmium (<0.25 mg/kg). A total of 100 isolates were grown on rich (Nutrient agar No. 2) or minimal (soil-extract agar medium) medium. The isolates were identified by phylogenetic analysis using partial sequences of their 16S rRNA (16S rDNA) genes. Representatives of two broad taxonomic groups, Firmicutes and Proteobacteria, were found on rich medium, whereas four taxonomic groups, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, were represented on minimal medium. Forty-two isolates grown on rich medium were assigned to 20 bacterial species, while 58 bacteria grown on minimal medium belonged to 49 species. Twenty-three isolates carried czcA- and/or nccA-like heavy-metal-resistance determinants. The heavy-metalresistance genes of nine isolates were identified by phylogenetic analysis of their protein sequences.