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.

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.

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.

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.

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.

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.

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.

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.

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.

The Application of MALDI-TOF MS for a Variability Study of Paenibacillus larvae.

In recent decades, the significant deterioration of the health status of honey bees has been observed throughout the world. One of the most severe factors affecting the health of bee colonies worldwide is American foulbrood disease. This devastating disease, with no known cure, is caused by the Gram-positive spore-forming bacteria of Paenibacillus larvae species. At present, DNA-based methods are being used for P. larvae identification and typing. In our study, we compare two of the most advanced DNA-based technologies (rep-PCR and 16S rRNA analyses) with MALDI-TOF MS fingerprinting to evaluate P. larvae variability in Central Europe. While 16S rRNA analysis presents a very limited variation among the strains, MALDI-TOF MS is observed to be more efficient at differentiating P. larvae. Remarkably, no clear correlation is observed between whole-genome rep-PCR fingerprinting and MALDI-TOF MS-based typing. Our data indicate that MALDI-TOF protein profiling provides accurate and cost-effective methods for the rapid identification of P. larvae strains and provides novel perspectives on strain diversity compared to conventional DNA-based genotyping approaches. The current study provides a good foundation for future studies.

Intragenomic Variability of ITS Sequences in Bjerkandera adusta.

Bjerkandera adusta is a species of common white rot polyporoid fungi found worldwide. Despite playing an important role in deadwood decay, the species strains are used in bioremediation due to its ability to degrade polycyclic hydrocarbons and some of them are important etiological agents of chronic coughs and are associated with lung inflammations. In our experiments, diversity within the species was investigated using molecular approaches and we found that sequence diversity seen at ITS sequence level is not due to cryptic speciation but to intragenomic variability of ITS sequences in this species.

Deep Subsurface Hypersaline Environment as a Source of Novel Species of Halophilic Sulfur-Oxidizing Bacteria.

The sulfur cycle participates significantly in life evolution. Some facultatively autotrophic microorganisms are able to thrive in extreme environments with limited nutrient availability where they specialize in obtaining energy by oxidation of reduced sulfur compounds. In our experiments focused on the characterization of halophilic bacteria from a former salt mine in Solivar (Presov, Slovakia), a high diversity of cultivable bacteria was observed. Based on ARDRA (Amplified Ribosomal DNA Restriction Analysis), at least six groups of strains were identified with four of them showing similarity levels of 16S rRNA gene sequences lower than 98.5% when compared against the GenBank rRNA/ITS database. Heterotrophic sulfur oxidizers represented ~34% of strains and were dominated by Halomonas and Marinobacter genera. Autotrophic sulfur oxidizers represented ~66% and were dominated by Guyparkeria and Hydrogenovibrio genera. Overall, our results indicate that the spatially isolated hypersaline deep subsurface habitat in Solivar harbors novel and diverse extremophilic sulfur-oxidizing bacteria.

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.

Biodiversity of Actinomycetes from Heavy Metal Contaminated Technosols.

Technosols are artificial soils generated by diverse human activities and frequently contain toxic substances resulting from industrial processes. Due to lack of nutrients and extreme physico-chemical properties, they represent environments with limited bacterial colonization. Bacterial populations of technosols are dominated usually by Actinobacteria, including streptomycetes, known as a tremendous source of biotechnologically important molecules. In this study, the biodiversity of streptomycete-like isolates from several technosols, mainly mine soils and wastes (landfills and sludge) in Slovakia, was investigated. The combination of basic morphological and biochemical characterisations, including heavy metal resistance determination, and molecular approaches based on 16S rRNA gene analysis were used for the identification of the bacterial strains. From nine isolates of Actinobacteria collected from different habitats, one was found to represent a new species within the Crossiella genus. Eight other isolates were assigned to the genus Streptomyces, of which at least one could represent a new bacterial species. Some isolates showed high resistance to Pb, Zn, Cu or Ni. The most tolerated metal was Pb. The results obtained in this study indicate that technosols are a prospective source of new actinomycete species resistant to heavy metals what underlines their bioremediation potential.

Can wood-decaying urban macrofungi be identified by using fuzzy interference system? An example in Central European Ganoderma species.

Ganoderma is a cosmopolitan genus of wood-decaying basidiomycetous macrofungi that can rot the roots and/or lower trunk. Among the standing trees, their presence often indicates that a hazard assessment may be necessary. These bracket fungi are commonly known for the crust-like upper surfaces of their basidiocarps and formation of white rot. Six species occur in central European urban habitats. Several of them, such as Ganoderma adspersum, G. applanatum, G. resinaceum and G. pfeifferi, are most hazardous fungi causing extensive horizontal stem decay in urban trees. Therefore, their early identification is crucial for correct management of trees. In this paper, a fast technique is tested for the determination of phytopathologically important urban macrofungi using fuzzy interference system of Sugeno type based on 13 selected traits of 72 basidiocarps of six Ganoderma species and compared to the ITS sequence based determination. Basidiocarps features were processed for the following situations: At first, the FIS of Sugeno 2 type (without basidiospore sizes) was used and 57 Ganoderma basidiocarps (79.17%) were correctly determined. Determination success increased to 96.61% after selecting basidiocarps with critical values (15 basidiocarps). These undeterminable basidiocarps must be analyzed by molecular methods. In a case, that basidiospore sizes of some basidiocarps were known, a combination of Sugeno 1 (31 basidiocarps with known basidiospore size) and Sugeno 2 (41 basidiocarps with unknown basidiospore size) was used. 84.72% of Ganoderma basidiocarps were correctly identified. Determination success increased to 96.83% after selecting basidiocarps with critical values (11 basidiocarps).

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.

High frequency of antibiotic tolerance in deep subsurface heterotrophic cultivable bacteria from the Rozália Gold Mine, Slovakia.

The Rozália Mine, with its long mining history, could represent an environmental threat connected with metal contamination and associated antibiotic tolerance. Metal and antibiotic tolerance profiles of heterotrophic, cultivable bacteria isolated from the Rozália Gold Mine in Hodrusa-Hámre, Slovakia, and the surrounding area were analysed. Subsurface samples were collected from different mine levels or an ore storage dump. As expected, heterotrophic cultivable bacteria showed high minimum inhibitory concentrations for metals (up to 1000 mg/l for zinc and nickel, 2000 mg/l for lead and 500 mg/l for copper). Surprisingly, very high minimum inhibitory concentrations of selected antibiotics were observed, e.g. > 10,000 µg/ml for ampicillin, up to 4800 µg/ml for kanamycin, 800 µg/ml for chloramphenicol and 50 µg/ml for tetracycline. Correlation analysis revealed a linkage between increased tolerance to the antibiotics ampicillin and chloramphenicol and metal tolerance to nickel and copper. A correlation was also observed between tetracycline-kanamycin tolerance and zinc-lead tolerance. Our data indicate that high levels of antibiotic tolerance occur in deep subsurface microbiota, which is probably connected with the increased level of metal concentrations in the mine environment.

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.