Genomic insights into the adaptation of Acinetobacter johnsonii RB2-047 to the heavy metal-contaminated subsurface mine environment.

The subsurface mine environments characterized by high levels of toxic metals and low nutrient availability represent an extreme threat to bacterial persistence. In recent study, the genomic analysis of the Acinetobacter johnsonii strain RB2-047 isolated from the Rozália Gold Mine in Slovakia was performed. As expected, the studied isolate showed a high level of heavy metal tolerance (minimum inhibitory concentrations were 500 mg/L for copper and nickel, 1,500 mg/L for lead, and 250 mg/L for zinc). The RB2-047 strain also showed noticeable resistance to several antibiotics (ampicillin, kanamycin, chloramphenicol, tetracycline and ciprofloxacin). The genomic composition analysis demonstrated a low number of antibiotic and metal resistance coding genes, but a high occurrence of efflux transporter genes located on the bacterial chromosome. The experimental inhibition of efflux pumps resulted in decreased tolerance to Zn and Ni (but not to Cu and Pb) and to all antibiotics tested. In addition, the H33342 dye-accumulation assay confirmed the high efflux activity in the RB2-047 isolate. These findings showed the important role of efflux pumps in the adaptation of Acinetobacter johsonii strain RB2-047 to metal polluted mine environment as well as in development of multi-antibiotic resistance.

Bacterial Community Structure of Two Cold Sulfur Springs in Slovakia (Central Europe).

Sulfur-oxidizing bacteria, especially those from hot springs, have attracted the attention of microbiologists for more than 150 years. In contrast, the microbial diversity of cold sulfur springs remains largely unrecognized. Culture-dependent and culture-independent approaches were used to study the diversity of sulfur-oxidizing bacterial communities in two cold sulfur springs in Slovakia. Geological conditions and resulting spring water chemistry appear to be major factors influencing the composition of the sulfur-oxidizing bacterial community. Bacterial communities in both springs were found to be dominated by Proteobacteria with Epsilonproteobacteria being prevalent in the high-salinity Stankovany spring and Alpha- and Gammaproteobacteria in the low-salinity Jovsa spring. Limited overlap was found between culture-dependent and culture-independent approaches with multiple taxa of cultivated sulfur-oxidizing bacteria not being detected by the culture-independent metagenomics approach. Moreover, four cultivated bacterial isolates could represent novel taxa based on the low similarity of their 16S rRNA gene sequence (similarity lower than 98%) to sequences of known bacteria. Our study supports the current view that multiple approaches are required to assess the bacterial diversity in natural habitats and indicates that sulfur springs in Slovakia harbor unique, yet-undescribed microorganisms.

Diversity, heavy metals, and antibiotic resistance in culturable heterotrophic bacteria isolated from former lead-silver-zinc mine heap in Tarnowskie Gory (Silesia, Poland).

Mine tailings represent a great environmental concern due to their high contents of heavy metals. Cultivation analysis of microbiota of Tarnowskie Góry (Poland) mine tailing showed the occurrence of bacteria with colony-forming units as low as 5.7 × 104 per one gram of dried substrate. Among 110 bacterial isolates identified by a combination of MALDI-TOF mass spectrometry and 16S rRNA gene sequencing, phylum Actinobacteria was dominant, followed by Firmicutes and Proteobacteria. Extremely high levels of heavy-metal resistance were observed in Arthrobacter spp., particularly for zinc (500 mg/L), lead (1500 mg/L), and cadmium (1000 mg/L). On the other hand, Staphylococcus spp. showed high tolerance to several antibiotics tested, especially ampicillin, partly due to blaZ gene presence. Due to the occurrence of antibiotic-resistant bacteria, mine tailings are not the cause of heavy-metal contamination only, but also a source of antibiotic-resistant bacteria and thus may represent a serious risk for public health.

Allosteric Inter-Domain Contacts in Bacterial Hsp70 Are Located in Regions That Avoid Insertion and Deletion Events.

Heat shock proteins 70 (Hsp70) are chaperones consisting of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD), the latter of which binds protein clients. After ATP binds to the NBD, the SBD α/ß subdomains' shared interface opens, and the open SBD docks to the NBD. Such allosteric effects are stabilized by the newly formed NBD-SBD interdomain contacts. In this paper, we examined how such an opening and formation of subdomain interfaces is affected during the evolution of Hsp70. In particular, insertion and deletion events (indels) can be highly disruptive for the mechanical events since such changes introduce a collective shift in the pairing interactions at communicating interfaces. Based on a multiple sequence alignment analysis of data collected from Swiss-Prot/UniProt database, we find several indel-free regions (IFR) in Hsp70. The two largest IFRs are located in interdomain regions that participate in allosteric structural changes. We speculate that the reason why the indels have a lower likelihood of occurrence in these regions is that indel events in these regions cause dysfunction in the protein due to perturbations of the mechanical balance. Thus, the development of functional allosteric machines requires including in the rational design a concept of the balance between structural elements.

CYP3A gene variability and cancer cells response to the treatment.

The treatment of cancer depends on the activity of the cytochrome P450 enzyme family, which is essentially carried out by the CYP3A4 and CYP3A5 enzymes. The aim of our study was to investigate whether the CYP3A4 polymorphism could contribute to protein activity and their influence to the response of cancer cells to treatment. The variability of CYP3A4 cDNA profiles between the cancer cell lines parental HT-29 and resistant HT-29-OxR adenocarcinoma was detected using denaturing gradient gel electrophoresis (DGGE). Subsequently, sequence analysis of CYP3A family members (CYP3A4, CYP3A5) confirmed polymorphism of the CYP3A4 gene in studied cancer cell lines. Variations at the gene expression level, the protein level and the activity of CYP3A4 protein in 12 cancer cell lines were observed, also different response to drug treatments between cell line HT-29 and oxaliplatin-resistant cell line HT-29-OxR. The variability of CYP3A might affect the efficiency of anti-cancer drugs in general and have an impact on metabolism.

New Endophytic Fusarium spp. from Fraxinus excelsior Leaves in Slovakia.

Fraxinus excelsior is currently suffering from ash dieback disease caused by the fungus Hymenoscyphus fraxineus. Co-occurrence of large numbers of other fungi with endophytic as well as pathogenic lifestyles has been observed on F. excelsior. In this study, new endophytic Fusarium spp. isolate Fraxinus excelsior Fusarium from Slovak territory, associated with F. excelsior, was characterised. Using morphology-based approaches, the fungus was assigned to the genus Fusarium. The isolate formed thick-walled hyaline, fusiform, slightly curved macroconidia, produced from monophialides in sporodochia, pointed at the tip, mostly 3 - 5 septate, occasionally 6 septate, and 40 - 68 × 3.8 - 5.0 µm in size with basal pedicellate cells. For more accurate taxonomic affiliation, molecular sequence analyses of the internal transcribed spacer, translation elongation factor 1-alpha sequences, and partial ß-tubulin gene were performed. While the internal transcribed spacer sequence of the isolate showed the highest similarity to the Fraxinus oxysporum species, translation elongation factor 1-alpha and partial ß-tubulin sequences were distantly related to the Fraxinus avenaceum species. Based on these data, the Fraxinus excelsior Fusarium isolate could probably be considered a new species of the Fusarium genus.

Correction to: The Bacterial Population of Neutral Mine Drainage Water of Elizabeth's Shaft (Slovinky, Slovakia).

The article "The Bacterial Population of Neutral Mine Drainage Water of Elizabeth's Shaft (Slovinky, Slovakia)", written by Jana Kisková, Zuzana Perháèová, Ladislav Vlèko, Jana Sedláková, Simona Kvasnová and Peter Pristas, was originally published electronically on the publisher's internet portal on 12 March 2018 without open access. With the author(s)' decision to opt for Open Choice the copyright of the article changed on 9 April 2020 to © The Author(s) 2018 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ ), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The original article has been corrected.

The Faecal Microbiome of Building-Dwelling Insectivorous Bats (Myotis myotis and Rhinolophus hipposideros) also Contains Antibiotic-Resistant Bacterial Representatives.

The bacteriome of bat faeces and bat guano is still not well known, despite the increasing knowledge of it as a potential source of pathogenic and/or antibiotic-resistant species. In this study, the faecal microbiome composition of two building-dwelling insectivorous bat species (Myotis myotis and Rhinolophus hipposideros) was analysed using cultivation-based and non-cultivation-based approaches. The cultivation analyses indicate the dominance of Gram-positive bacteria, represented by the genera Enterococcus, Lactococcus and Lactobacillus. Non-cultivation analysis based on 16S rRNA DGGE assays provided a different pattern, with the genus Rahnella found to predominate in the faecal microbiome. The analyses showed bat species to be the most important factor influencing the structure of the guano-associated microbial population. The presence of several possible human pathogens (Hafnia alvei, Serratia fonticola, S. liquefaciens) and antibiotic-resistant bacteria (e.g. vancomycin resistant Staphylococcus nepalensis) was detected in faeces samples, indicating possible human health risks associated with bat guano. Although the bat-human transmission of infection caused by pathogenic bacteria has not been reliably confirmed to date, this possibility should not be underestimated. Given the very important ecosystem services of bats, particular those studied herein, it is time to think about appropriate management of bat populations inhabiting man-made buildings and potential conflicts with humans.

Candidatus Cryptoplasma Associated with Green Lizards and Ixodes ricinus Ticks, Slovakia, 2004-2011.

During 2004-2011, we collected green lizards and Ixodes ricinus ticks in Slovak Karst National Park in Slovakia; 90% (36/40) of lizards and 37% of ticks removed from lizards were infected with family Anaplasmataceae bacteria. Only Candidatus Cryptoplasma sp. REP (reptile) was identified in these samples. Green lizards transmit this bacterium.

The Bacterial Population of Neutral Mine Drainage Water of Elizabeth's Shaft (Slovinky, Slovakia).

Although neutral mine drainage is the less frequent subject of the interest than acid mine drainage, it can have adverse environmental effects caused mainly by precipitation of dissolved Fe. The aim of the study was to characterize the composition of bacterial population in environment with high concentration of iron and sulfur compounds represented by neutral mine drainage water of Elizabeth's shaft, Slovinky (Slovakia). Direct microscopic observations, cultivation methods, and 454 pyrosequencing of the 16S rRNA gene amplicons were used to examine the bacterial population. Microscopic observations identified iron-oxidizing Proteobacteria of the genera Gallionella and Leptothrix which occurrence was not changed during the years 2008-2014. Using 454 pyrosequencing, there were identified members of 204 bacterial genera that belonged to 25 phyla. Proteobacteria (69.55%), followed by Chloroflexi (10.31%) and Actinobacteria (4.24%) dominated the bacterial community. Genera Azotobacter (24.52%) and Pseudomonas (14.15%), followed by iron-oxidizing Proteobacteria Dechloromonas (11%) and Methyloversatilis (8.53%) were most abundant within bacterial community. Typical sulfur bacteria were detected with lower frequency, e.g., Desulfobacteraceae (0.25%), Desulfovibrionaceae (0.16%), or Desulfobulbaceae (0.11%). Our data indicate that the composition of bacterial community of the Elizabeth's shaft drainage water reflects observed neutral pH, high level of iron and sulfur ions in this aquatic habitat.

Two rep genes in small cryptic plasmid pKST21 of Escherichia coli.

The complete nucleotide sequence of a small cryptic plasmid pKST21 from Escherichia coli was determined. This plasmid is 1,460 bp long with an overall GC content of 51 %. Based on sequence analysis, the presence of two segments with different average GC density was observed. The segment with higher GC content revealed 98-90 % similarity to several small plasmids of E. coli and to pCR1 from Gram-positive Corynebacterium renale. Plasmid pKST21 possesses two conversely oriented open reading frames encoding proteins with a high degree of amino acid identity to Rep proteins involved in replication. ORF1 encodes replication protein similar to RepA protein of Bartonella tribocorum or Bacillus cereus plasmids or to the putative plasmid Rep protein from ecologically close Selenomonas ruminantium. ORF2 similarly encodes a replication protein, which shares 97 % homology with Rep protein from C. renale. Genetic diversity observed in plasmid pKST21 indicates a mosaic structure of the plasmid with different segments acquired from different sources. Deletion analysis showed that both fragments carrying the repA and repB genes are necessary for the replication of pKST21 in E. coli. The presence of plasmid with the same gene composition was revealed in 14 % of tested E. coli isolates from the rumen of sheep. All these strains produced identical ERIC-PCR profiles indicating isogenic origin of the strain and lack of horizontal gene transfer of pKST21 plasmid.

Gene and domain shuffling in lytic cassettes of Enterococcus spp. bacteriophages.

The genomes of Enterococcus faecalis and Enterococcus faecium bacteriophages were analysed for gene shuffling in the lytic cassettes of bacteriophages infecting. It was found that enterococcal bacteriophages could be classified into well-defined groups based on the size of their genomes and each size group had its own conserved gene composition of lytic cassettes. Enterococcal bacteriophages use a relatively broad spectrum of holins and endolysins with variable cell-wall binding (CWB) and catalytic domains, and most of them utilise a lytic cassette with more than two genes. Enterococcal bacteriophages most commonly use endolysins with amidase catalytic domains and the CWB domain SH3_5. Some bacteriophages possess in their lytic cassette a holin-like gene with the XhlA domain protein, characteristic of hemolysin. Regardless of the shuffling of genes encoding holins and endolysins in lytic modules, a novel example of CWB domain shuffling within enterococcal endolysins was identified. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03775-w.

Cold Sulfur Springs-Neglected Niche for Autotrophic Sulfur-Oxidizing Bacteria.

Since the beginning of unicellular life, dissimilation reactions of autotrophic sulfur bacteria have been a crucial part of the biogeochemical sulfur cycle on Earth. A wide range of sulfur oxidation states is reflected in the diversity of metabolic pathways used by sulfur-oxidizing bacteria. This metabolically and phylogenetically diverse group of microorganisms inhabits a variety of environments, including extreme environments. Although they have been of interest to microbiologists for more than 150 years, meso- and psychrophilic chemolithoautotrophic sulfur-oxidizing microbiota are less studied compared to the microbiota of hot springs. Several recent studies suggested that cold sulfur waters harbor unique, yet not described, bacterial taxa.

Thiothrix and Sulfurovum genera dominate bacterial mats in Slovak cold sulfur springs.

Microbiota of sulfur-rich environments has been extensively studied due to the biotechnological potential of sulfur bacteria, or as a model of ancient life. Cold terrestrial sulfur springs are less studied compared to sulfur-oxidizing microbiota of hydrothermal vents, volcanic environments, or soda lakes. Despite that, several studies suggested that sulfur springs harbor diverse microbial communities because of the unique geochemical conditions of upwelling waters. In this study, the microbiota of five terrestrial sulfur springs was examined using a 16 S rRNA gene sequencing. The clear dominance of the Proteobacteria and Campylobacterota phyla of cold sulfur springs microbiota was observed. Contrary to that, the microbiota of the hot sulfur spring was dominated by the Aquificota and Firmicutes phylum respectively. Sulfur-oxidizing genera constituted a dominant part of the microbial populations with the Thiothrix and Sulfurovum genera identified as the core microbiota of cold sulfur terrestrial springs in Slovakia. Additionally, the study emphasizes that sulfur springs in Slovakia support unique, poorly characterized bacterial communities of sulfur-oxidizing bacteria.

Genetic Basis of Acinetobacter sp. K1 Adaptation Mechanisms to Extreme Environmental Conditions.

Anthropogenic pollution often leads to the generation of technosols, technogenic soils with inhospitable conditions for all living organisms including microbiota. Aluminum production near Ziar nad Hronom (Slovakia) resulted in the creation of a highly alkaline and heavy-metal-rich brown mud landfill, from which a bacterial strain of a likely new species of the genus Acinetobacter, Acinetobacter sp. K1, was isolated. The whole-genome sequence analysis of this strain confirmed the presence of operon units enabling tolerance to the heavy metals copper, zinc, cobalt, cadmium, chromium, and metalloid arsenic, which are functionally active. Despite the predominance of plasmid-related sequences in the K1 genome, the results indicate that most of the resistance genes are chromosomally encoded. No significant alkali tolerance of Acinetobacter sp. K1 was observed in vitro, suggesting that community level mechanisms are responsible for the survival of this strain in the highly alkaline, brown mud bacterial community.

Morphometric and Molecular Analysis of Five-Spine Epidinium Morphotypes Taken from the Rumen of European Bison, Bison bonasus.

An important feature of ruminal ciliates is their phenotypic plasticity, which makes their identification difficult. The common manifestation of the phenotypic plasticity in rumen ciliates is a change in their cell size and caudal spination. We analyzed various morphotypes of Epidinium with five caudal processes (spines) taken from the rumen of European bison (Bison bonasus). In the study, the cluster analysis and K-means analysis of morphometric data could not distinguish very similar morphotypes of Epidinium with five caudal processes. However, the morphotype of E. parvicaudatum prevailed (70%). The DNA of four individual E. parvicaudatum was isolated successfully from formaldehyde-preserved samples. The partial 18S rDNA gene sequences (about 350-400 bp) were identical to Epidinium sequences in GenBank (E. caudatum, a one-spine morphotype, and E. cattanei, a five-spine morphotype). It can be assumed that these short sequences cannot distinguish the differences between the Epidinium morphospecies. Complete gene sequences from various hosts and various molecular markers are necessary to reveal the validity of the Epidinium five-spine species. In conclusion, classical morphology should be supplemented with molecular data when more morphotypes of the rumen ciliate species are present in samples.

How Different Molecular Markers Estimate the Diversity of European Species of the Ganoderma Genus.

Based on published anatomical-morphological and ecological characteristics and phylogenetic evidence, six species of the Ganoderma genus are known to occur in Europe, namely, G. applanatum (Pers.) Pat., G. adspersum (Schulzer) Donk, G. pfeifferi Bres., G. resinaceum Boud., G. carnosum Pat., and G. lucidum (Curtis) P. Karst. Molecular markers (DNA sequences of selected genes or intergenic spacers) revolutionized our view of fungal variability. Every one of the four most frequently used molecular markers (ITS (internal transcribed spacer) and partial sequences of LSU (rRNA large subunit), tef1-α (translation elongation factor 1-alpha), and Rpb2 (RNA polymerase II second largest subunit)) provides a different view on the variability of European species of the Ganoderma genus. Both the lowest intraspecies variability and the best species differentiation (interspecies divergence) were observed for the LSU marker, and based on our data, this marker could be recommended for identification and species delineation in European Ganoderma spp. The sequences of the most frequently used ITS marker were unable to discriminate between G. lucidum and G. carnosum, and in general, this species pair showed the lowest interspecies divergence using all markers tested. Surprisingly, up to now, hidden variability has been detected in several Ganoderma spp., indicating the existence of possible cryptic taxa within the European Ganoderma morphospecies.

Antibiotic Resistance and Genetic Variability of Acinetobacter spp. from Wastewater Treatment Plant in Koksov-Baksa (Kosice, Slovakia).

This study investigated the genetic variability and antibiotic resistance of Acinetobacter community depending on the stage of wastewater treatment in Koksov-Baksa for the city of Kosice (Slovakia). After cultivation, bacterial isolates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and their sensitivity to ampicillin, kanamycin, tetracycline, chloramphenicol and ciprofloxacin was examined. Acinetobacter spp. and Aeromonas spp. dominated bacterial populations in all wastewater samples. We identified 12 different groups based on protein profiling, 14 genotypes by amplified ribosomal DNA restriction analysis and 11 Acinetobacter species using 16S rDNA sequence analysis within Acinetobacter community, which showed significant variability in their spatial distribution. While Acinetobacter population structure changed during the wastewater treatment, the prevalence of antibiotic-resistant strains did not significantly vary depending on the stage of wastewater treatment. The study highlights the role of a highly genetically diverse Acinetobacter community surviving in wastewater treatment plants as an important environmental reservoir assisting in the further dissemination of antibiotic resistance in aquatic systems.

Some Aspects of the Physiology of the Nyctotherus velox, a Commensal Ciliated Protozoon Taken from the Hindgut of the Tropical Millipede Archispirostreptus gigas.

In this paper, the growth requirements, fermentation pattern, and hydrolytic enzymatic activities of anaerobic ciliates collected from the hindgut of the African tropical millipede Archispirostreptus gigas are described. Single-cell molecular analysis showed that ciliates from the millipede hindgut could be assigned to the Nyctotherus velox and a new species named N. archispirostreptae n. sp. The ciliate N. velox can grow in vitro with unspecified prokaryotic populations and various plant polysaccharides (rice starch-RS, xylan, crystalline cellulose20-CC, carboxymethylcellulose-CMC, and inulin) or without polysaccharides (NoPOS) in complex reduced medium with soluble supplements (peptone, glucose, and vitamins). Specific catalytic activity (nkat/g of protein) of α amylase of 300, xylanase of 290, carboxymethylcellulase of 190, and inulinase of 170 was present in the crude protein extract of N. velox. The highest in vitro dry matter digestibility was observed in RS and inulin after 96 h of fermentation. The highest methane concentration was observed in xylan and inulin substrates. The highest short-chain fatty acid concentration was observed in RS, inulin, and xylan. In contrast, the highest ammonia concentration was observed in NoPOS, CMC, and CC. The results indicate that starch is the preferred substrate of the N. velox. Hydrolytic enzyme activities of N. velox showed that the ciliates contribute to the fermentation of plant polysaccharides in the gut of millipedes.

Water treatment using activated carbon supporting silver and magnetite.

Recent efforts in water purification have led to the development of novel materials whose unique properties can offer effective biocidal capabilities with greater ease of use and at lower cost. In this study, we introduce a novel procedure for the preparation of activated carbon (charcoal) composite in which magnetite and silver are incorporated (MCAG); we also describe the use of this material for the disinfection of surface water. The formation process of magnetic MCAG composite was studied using ultraviolet-visible spectroscopy. The results demonstrated the high sorption efficiency of AgNO3 to magnetic activated carbon. The antimicrobial capabilities of the prepared MCAG were examined and the results clearly demonstrate their inhibitory effect on total river water bacteria and on Pseudomonas koreensis and Bacillus mycoides cultures isolated from river water. The bacterial counts in river water samples were reduced by five orders of magnitude following 30 min of treatment using 1 g l⁻¹ of MCAG at room temperature. The removal of all bacteria from the surface water samples implies that the MCAG material would be a suitable disinfectant for such waters. In combination with its magnetic character, MCAG would be an excellent candidate for the simple ambulatory disinfection of surface water.