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Plasmids are one of the key drivers of microbial adaptation and evolution. However, their diversity and role in adaptation, especially in extreme environments, remains largely unexplored. In this study, we aimed to identify, characterize, and compare plasmid sequences originating from samples collected from deep-sea hydrothermal vents located in Arctic Mid-Ocean Ridges. To achieve this, we employed, and benchmarked three recently developed plasmid identification tools-PlasX, GeNomad, and PLASMe-on metagenomic data from this unique ecosystem. To date, this is the first direct comparison of these computational methods in the context of data from extreme environments. Upon recovery of plasmid contigs, we performed a multiapproach analysis, focusing on identifying taxonomic and functional biases within datasets originating from each tool. Next, we implemented a majority voting system to identify high-confidence plasmid contigs, enhancing the reliability of our findings. By analysing the consensus plasmid sequences, we gained insights into their diversity, ecological roles, and adaptive significance. Within the high-confidence sequences, we identified a high abundance of Pseudomonadota and Campylobacterota, as well as multiple toxin-antitoxin systems. Our findings ensure a deeper understanding of how plasmids contribute to shaping microbial communities living under extreme conditions of hydrothermal vents, potentially uncovering novel adaptive mechanisms., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

In urbanized areas, extracellular DNA (exDNA) is suspected of carrying genes with undesirable traits like virulence genes (VGs) or antibiotic resistance genes (ARGs), which can spread through horizontal gene transfer (HGT). Hence, it is crucial to develop novel approaches for the mitigation of exDNA in the environment. Our research explores the role of goethite, a common iron mineral with high adsorption capabilities, in exDNA adsorption processes. We compare well-crystalline, semi-crystalline, and nano goethites with varying particle sizes to achieve various specific surface areas (SSAs) (18.7-161.6 m 2 /g) and porosities. We conducted batch adsorption experiments using DNA molecules of varying chain lengths (DNA sizes: <11 Kb, <6 Kb, and <3 Kb) and assessed the impact of Ca 2+ and biomacromolecules on the adsorption efficacy and mechanisms. Results show that porosity and pore structure significantly influence DNA adsorption capacity. Goethite with well-developed meso- and macroporosity demonstrated enhanced DNA adsorption. The accumulation of DNA on the goethite interface led to substantial aggregation in the system, thus the formation of DNA-goethite conjugates, indicating the bridging between mineral particles. DNA chain length, the presence of Ca 2+ , and the biomacromolecule matrix also affected the adsorption capacity and mechanism. Interactions between DNA and positively charged biomacromolecules or Ca 2+ led to DNA compaction, allowing greater DNA accumulation in pores. However, a high concentration of biomacromolecules led to the saturation of the goethite surface, inhibiting DNA adsorption. AFM imaging of goethite particles after adsorption suggested the formation of the DNA multilayer. The study advances understanding of the environmental behavior of exDNA and its interaction with iron oxyhydroxides, offering insights into developing more effective methods for ARGs removal in wastewater treatment plants. By manipulating the textural properties of goethite, it's possible to enhance exDNA removal, potentially reducing the spread of biocontamination in urban and industrial environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Wastewater treatment plants have been recognised as point sources of various antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG) which are considered recently emerging biological contaminants. So far, culture-based and molecular-based methods have been successfully applied to monitor antimicrobial resistance (AMR) in WWTPs. However, the methods applied do not permit the comprehensive identification of the true diversity of ARGs. In this study we applied next-generation sequencing for a metagenomic analysis of PCR amplicons of ARGs from the subsequent stages of the analysed WWTP. The presence of 14 genes conferring resistance to different antibiotic families was screened by PCR. In the next step, three genes were selected for detailed analysis of changes of the profile of ARG variants along the process. A relative abundance of 79 variants was analysed. The highest diversity was revealed in the ermF gene, with 52 variants. The relative abundance of some variants changed along the purification process, and some ARG variants might be present in novel hosts for which they were currently unassigned. Additionally, we identified a pool of novel ARG variants present in the studied WWTP. Overall, the results obtained indicated that the applied method is sufficient for analysing ARG variant diversity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gorecki, Ostapczuk and Dziewit.)

Gray mold, caused by Botrytis cinerea is a major cause of post-harvest rot of fresh fruits and vegetables. The utilization of selected microorganisms as biocontrol agents is a promising alternative to effectively control gray mold on tomatoes. The current study was conducted to explore potential biocontrol mechanisms of the Pseudomonas strain to control infections on post-harvest tomatoes. Among the 8 tested bacterial isolates, Pseudomonas protegens ML15 demonstrated antagonistic activity to Botrytis cinerea . Moreover, P. protegens ML15 exhibited the production of siderophores, hydrogen cyanide, ammonia, exopolysaccharides, lipase, biosurfactant, 2,4-diacetylphloroglucinol, and several other antifungal compounds, such as 1-tetradecanol, cyclododecane, 2,4-di-tert-butylphenol, and 2-methyl-1-hexadecanol. A comprehensive genomic analysis of P. protegens ML15 unravels 18 distinct genetic regions with the potential for biosynthesizing secondary metabolites, known for their pivotal role in biocontrol responses against plant pathogens. In vivo , experiments showed that both culture suspension and cell-free supernatant of P. protegens ML15 significantly reduced fungal growth (53.0 ± 0.63%) and mitigated disease development (52.8 ± 1.5%) in cherry tomatoes at four days post- B . cinerea inoculation. During the infection, the application of P. protegens ML15 resulted in the augmentation of total antioxidant, phenolic content, and ascorbic acids content. Thus, our results suggested that P. protegens ML15's role as a biocontrol agent against B. cinerea -induced postharvest tomato decay achieved through the secretion of antifungal substances, induction of tomato defense responses, and inhibition of mycelial growth of B. cinerea . These findings provide a significant contribution to the ongoing search for alternative, eco-friendly methods of controlling gray mold in fresh products. The utilization of P. protegens ML15 as a biocontrol agent could help to reduce the reliance on chemical fungicides and promote sustainable agriculture practices., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ajijah, Fiodor, Dziurzynski, Stasiuk, Pawlowska, Dziewit and Pranaw.)

Among Psychrobacter spp., there are several multireplicon strains, carrying more than two plasmids. Psychrobacter sp. ANT&#95;H3 carries as many as 11 extrachromosomal replicons, which is the highest number in Psychrobacter spp. Plasmids of this strain were subjected to detailed genomic analysis, which enables an insight into the structure and functioning of this multireplicon genome. The replication and conjugal transfer modules of ANT&#95;H3 plasmids were analyzed functionally to discover their potential for being used as building blocks for the construction of novel plasmid-vectors for cold-active bacteria. It was shown that two plasmids have a narrow host range as they were not able to replicate in species other than Psychrobacter, while remaining plasmids had a wider host range and were functional in various Alpha- and Gammaproteobacteria. Moreover, it was confirmed that mobilization modules of seven plasmids were functional, i.e., could be mobilized for conjugal transfer by the RK2 conjugation system. Auxiliary genes were also distinguished in ANT&#95;H3 plasmids, including these encoding putative DNA-protecting protein DprA, multidrug efflux SMR transporter of EmrE family, glycine cleavage system T protein, MscS small-conductance mechanosensitive channel protein, and two type II restriction-modification systems. Finally, all genome-retrieved plasmids of Psychrobacter spp. were subjected to complex genome- and proteome-based comparative analyses showing that Antarctic replicons are significantly different from plasmids from other locations., (© 2023. The Author(s).)

Several seed priming methods can be used to improve seed germination, seedling vigor, and to overcome abiotic stress. In addition to these benefits, only the biopriming method provides the additional benefit of biotic stress management, earning it special attention. Seed biopriming is useful in almost all crops around the world and is an environmentally friendly alternative to chemical fungicides. Biopriming usually refers to use of beneficial microorganisms, in particular plant growth-promoting bacteria (PGPB) able to survive under various harsh environmental conditions. In this study, various bacterial strains were isolated from samples of different origins, i.e., rhizospheric soil, desert sand, and sea mud. Preliminary screening of 156 bacterial isolates was conducted on the basis of their potassium (K), phosphorus (P) solubilization ability, and production of plant growth hormone, i.e., indole acetic acid (IAA). The most efficient bacteria were identified by 16S rRNA gene nucleotide sequences and further examined for their ACC deaminase activity, ammonia production, and biocontrol activity (defined via chitinolytic activity, HCN, and siderophores production). Finally, carrot seed germination assay was conducted with 10 shortlisted most potent isolates. 68.6, 58.3, and 66.7% of tested bacterial isolates were capable of P, K, and Zn solubilization, respectively. Klebsiella aerogenes AF3II1 showed the highest P and K solubilization, while isolate AF4II5, AF7II3, and PC3 showed the highest IAA synthesis ability. Serratia plymuthica EDC15 and Pseudomonas putida AF1I1 showed the strongest chitinolytic and siderophore production activity, respectively. Seven isolates demonstrated strong HCN production ability. Five isolates improved carrot seed germination. Only selected isolates with plant growth-promoting properties can improve carrot germination. The results of this study demonstrate that mainly auxins are involved in seed germination. Furthermore, the data suggest that phosphate solubilization ability may play an additional role in seed germination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fiodor, Ajijah, Dziewit and Pranaw.)

Arctic soils are constantly subjected to extreme environmental conditions such as low humidity, strong winds, high salinity, freeze-thaw cycles, UV exposition, and low nutrient availability, therefore, they have developed unique microbial ecosystems. These environments provide excellent opportunities to study microbial ecology and evolution within pristine (i.e. with limited anthropogenic influence) regions since the High Arctic is still considered one of the wildest and least explored environments on the planet. This environment is also of interest for the screening and recovery of unique microbial strains suitable for various biotechnological applications. In this study, a combination of culture-depended and culture-independent approaches was used to determine the cultivation bias in studies of the diversity of cold-active microorganisms. Cultivation bias is a reduction in recovered diversity, introduced when applying a classical culturing technique. Six different soil types, collected in the vicinity of the Polish Polar Station Hornsund (Spitsbergen, Norway), were tested. It was revealed that the used media allowed recovery of only 6.37 % of bacterial and 20 % of fungal genera when compared with a culture-independent approach. Moreover, it was shown that a combination of R2A and Marine Broth media recovered as much as 93.6 % of all cultivable bacterial genera detected in this study. Based on these results, a novel protocol for genome-guided bioprospecting, combining a culture-dependent approach, metabarcoding, next-generation sequencing, and genomic data reuse was developed. With this methodology, 14 psychrotolerant, multi-metal-resistant strains, including the highly promising Rhodococcus spp., were obtained. These strains, besides increased metal tolerance, have a petroleum hydrocarbon utilization capacity, and thus may be good candidates for future bioremediation technologies, also suited to permanently cold regions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Antarctica has one of the most extreme environments in the world. This region is inhabited by specifically adapted microorganisms that produce various unique secondary metabolites (e.g. pigments) enabling their survival under the harsh environmental conditions. It was already shown that these natural, biologically active molecules may find application in various fields of biotechnology., Results: In this study, a cold-active brown-pigment-producing Pseudomonas sp. ANT&#95;H4 strain was characterized. In-depth genomic analysis combined with the application of a fosmid expression system revealed two different pathways of melanin-like compounds biosynthesis by the ANT&#95;H4 strain. The chromatographic behavior and Fourier-transform infrared spectroscopic analyses allowed for the identification of the extracted melanin-like compound as a pyomelanin. Furthermore, optimization of the production and thorough functional analyses of the pyomelanin were performed to test its usability in biotechnology. It was confirmed that ANT&#95;H4-derived pyomelanin increases the sun protection factor, enables scavenging of free radicals, and interacts with the iron from minerals. Moreover, it was shown for the first time that pyomelanin exhibits priming properties toward Calendula officinalis hairy roots in in vitro cultures., Conclusions: Results of the study indicate the significant biotechnological potential of ANT&#95;H4-derived pyomelanin and open opportunities for future applications. Taking into account protective features of analyzed pyomelanin it may be potentially used in medical biotechnology and cosmetology. Especially interesting was showing that pyomelanin exhibits priming properties toward hairy roots, which creates a perspective for its usage for the development of novel and sustainable agrotechnical solutions., (© 2022. The Author(s).)

Fungi can be found in almost all ecosystems. Some of them can even survive in harsh, anthropogenically transformed environments, such as post-industrial soils. In order to verify how the soil fungal diversity may be changed by pollution, two soil samples from each of the 28 post-industrial sites were collected. Each soil sample was characterized in terms of concentration of heavy metals and petroleum derivatives. To identify soil fungal communities, fungal internal transcribed spacer 2 (ITS2) amplicon was sequenced for each sample using Illumina MiSeq platform. There were significant differences in the community structure and taxonomic diversity among the analysed samples. The highest taxon richness and evenness were observed in the non-polluted sites, and lower numbers of taxa were identified in multi-polluted soils. The presence of monocyclic aromatic hydrocarbons, gasoline and mineral oil was determined as the factors driving the differences in the mycobiome. Furthermore, in the culture-based selection experiment, two main groups of fungi growing on polluted media were identified - generalists able to live in the presence of pollution, and specialists adapted to the usage of BTEX as a sole source of energy. Our selection experiment proved that it is long-term soil contamination that shapes the community, rather than temporary addition of pollutant., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Extreme environmental conditions observed in polar regions create a unique ecological niche for microbial life. Bacteria living under these harsh, environmental conditions exhibit specific metabolic capabilities. In this report, we present multimetal-resistant Agrococcus sp. strain ARC&#95;14, isolated from soil samples collected in Spitsbergen, Svalbard, Norway.

Antibiotic resistance is considered one of the biggest threats to public health and has become a major concern for governments and international organizations. Combating this problem starts with improving global surveillance of antibiotic resistance genes (ARGs) and applying standardized protocols, both in a clinical and environmental context, in agreement with the One Health approach. Exceptional efforts should be directed to controlling ARGs conferring resistance to Critically Important Antimicrobials (CIA). In this study, a systematic literature review to synthesize data on the identification of mcr genes using a PCR technique was performed. Additionally, a novel set of PCR primers for mcr-1 - mcr-9 genes detection was proposed. The developed primers were in silico and experimentally validated by comparison with mcr-specific PCR primers reported in the literature. This validation, besides being a proof-of-concept for primers' usefulness, provided insight into the distribution of mcr genes in municipal wastewater, clay and river sediments, glacier moraine, manure, seagulls and auks feces and daphnids from four countries. This analysis proved that commonly used primers may deliver false results, and some mcr genes may be overlooked in tested samples. Newly-developed PCR primers turned out to be relevant for the screening of mcr genes in various environments., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Iron is the fourth most abundant element on earth. However, its low bioavailability is a key plant-growth limiting factor. Bacteria play an important role in plant growth promotion since they produce specific secondary metabolites that may increase macro- and micronutrient accessibility in soil. Therefore, bacterial-derived iron chelators, as well as surface-active compounds, are recognised as essential to plant welfare. In this study, three cold-active Antarctic bacterial strains, i.e. Pseudomonas sp. ANT&#95;H12B, Psychrobacter sp. ANT&#95;H59 and Bacillus sp. ANT&#95;WA51, were analysed. The physiological and genomic characterisation of these strains revealed their potential for plant growth promotion, reflected in the production of various biomolecules, including biosurfactants (that may lower the medium surface tension of even up to 53%) and siderophores (including ANT&#95;H12B-produced mixed-type siderophore that demonstrated the highest production, reaching the concentration of up to 1.065 mM), increasing the availability of nutrients in the environment and neutralising fungal pathogens. Tested bacteria demonstrated an ability to promote the growth of a model plant, alfalfa, increasing shoots' length and fresh biomass even up to 26 and 46% respectively; while their metabolites increased the bioavailability of iron in soil up to 40%. It was also revealed that the introduced strains did not disrupt physicochemical conditions and indigenous soil microbial composition, which suggests that they are promising amendments preserving the natural biodiversity of soil and increasing its fertility., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Styczynski, Biegniewski, Decewicz, Rewerski, Debiec-Andrzejewska and Dziewit.)

Antibiotic resistance (AR) is a global problem requiring international cooperation and coordinated action. Global monitoring must rely on methods available and comparable across nations to quantify AR occurrence and identify sources and reservoirs, as well as paths of AR dissemination. Numerous analytical tools that are gaining relevance in microbiology, have the potential to be applied to AR research. This review summarizes the state of the art of AR monitoring methods, considering distinct needs, objectives and available resources. Based on the overview of distinct approaches that are used or can be adapted to monitor AR, it is discussed the potential to establish reliable and useful monitoring schemes that can be implemented in distinct contexts. This discussion places the environmental monitoring within the One-Health approach, where two types of risk, dissemination across distinct environmental compartments, and transmission to humans, must be considered. The plethora of methodological approaches to monitor AR and the variable features of the monitored sites challenge the capacity of the scientific community and policy makers to reach a common understanding. However, the dialogue between different methods and the production of action-oriented data is a priority. The review aims to warm up this discussion., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Plasmids have the potential to transfer genetic traits within bacterial communities and thereby serve as a crucial tool for the rapid adaptation of bacteria in response to changing environmental conditions. Our knowledge of the environmental pool of plasmids (the metaplasmidome) and encoded functions is still limited due to a lack of sufficient extraction methods and tools for identifying and assembling plasmids from metagenomic datasets. Here, we present the first insights into the functional potential of the metaplasmidome of permafrost-affected active-layer soil-an environment with a relatively low biomass and seasonal freeze-thaw cycles that is strongly affected by global warming. The obtained results were compared with plasmid-derived sequences extracted from polar metagenomes. Metaplasmidomes from the Siberian active layer were enriched via cultivation, which resulted in a longer contig length as compared with plasmids that had been directly retrieved from the metagenomes of polar environments. The predicted hosts of plasmids belonged to Moraxellaceae, Pseudomonadaceae, Enterobacteriaceae, Pectobacteriaceae, Burkholderiaceae, and Firmicutes. Analysis of their genetic content revealed the presence of stress-response genes, including antibiotic and metal resistance determinants, as well as genes encoding protectants against the cold., (© 2021. The Author(s).)

Polar regions contain a wide variety of lentic ecosystems. These include periodic ponds that have a significant impact on carbon and nitrogen cycling in polar environments. This study was conducted to assess the taxonomic and metabolic diversity of bacteria found in Antarctic pond affected by penguins and sea elephants and to define their role in ongoing processes. Metabolic assays showed that of the 168 tested heterotrophic bacteria present in the Antarctic periodic pond, 96% are able to degrade lipids, 30% cellulose, 26% proteins, and 26% starch. The taxonomic classification of the obtained isolates differs from that based on the composition of the 16S rRNA relative abundances in the studied pond. The dominant Actinobacteria constituting 45% of isolates represents a low proportion of the community, around 4%. With the addition of run-off, the proportions of inhabiting bacteria changed, including a significant decrease in the abundance of Cyanobacteria , from 2.38 to 0.33%, increase of Firmicutes from 9.32 to 19.18%, and a decreasing richness (Chao1 index from 1299 to 889) and diversity (Shannon index from 4.73 to 4.20). Comparative studies of communities found in different Antarctic environments indicate a great role for penguins in shaping bacterial populations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Krucon, Dziewit and Drewniak.)

Over the last 15 years, the costs of DNA sequencing have sharply fallen, effectively shifting the costs of DNA analysis from sequencing to bioinformatic curation and storage. A huge number of available DNA sequences (including genomes and metagenomes) resulted in the development of various tools for sequence annotation. While much effort has been invested into the development of automatic annotation pipelines, manual curation of their results is still necessary in order to obtain a reliable and strictly validated data. Unfortunately, due to its time-consuming nature, manual annotation is now rarely used.In this chapter, a protocol for efficient manual annotation of prokaryotic DNA sequences using a novel bioinformatic tool-MAISEN ( http://maisen.ddlemb.com ), is presented. MAISEN is a free, web-based tool designed to accelerate manual annotation, by providing the user with simple interface and precomputed alignments for each predicted feature. It was designed to be available for every scientist, regardless of their bioinformatic proficiency.

Cultural heritage objects constitute a very diverse environment, inhabited by various bacteria and fungi. The impact of these microorganisms on the degradation of artworks is undeniable, but at the same time, some of them may be applied for the efficient biotreatment of cultural heritage assets. Interventions with microorganisms have been proven to be useful in restoration of artworks, when classical chemical and mechanical methods fail or produce poor or short-term effects. The path to understanding the impact of microbes on historical objects relies mostly on multidisciplinary approaches, combining novel meta-omic technologies with classical cultivation experiments, and physico-chemical characterization of artworks. In particular, the development of metabolomic- and metatranscriptomic-based analyses associated with metagenomic studies may significantly increase our understanding of the microbial processes occurring on different materials and under various environmental conditions. Moreover, the progress in environmental microbiology and biotechnology may enable more effective application of microorganisms in the biotreatment of historical objects, creating an alternative to highly invasive chemical and mechanical methods.

Antarctic regions are characterized by low temperatures and strong UV radiation. This harsh environment is inhabited by psychrophilic and psychrotolerant organisms, which have developed several adaptive features. In this study, we analyzed two Antarctic bacterial strains, Planococcus sp. ANT&#95;H30 and Rhodococcus sp. ANT&#95;H53B. The physiological analysis of these strains revealed their potential to produce various biotechnologically valuable secondary metabolites, including surfactants, siderophores, and orange pigments. The genomic characterization of ANT&#95;H30 and ANT&#95;H53B allowed the identification of genes responsible for the production of carotenoids and the in silico reconstruction of the pigment biosynthesis pathways. The complex manual annotation of the bacterial genomes revealed the metabolic potential to degrade a wide variety of compounds, including xenobiotics and waste materials. Carotenoids produced by these bacteria were analyzed chromatographically, and we proved their activity as scavengers of free radicals. The quantity of crude carotenoid extracts produced at two temperatures using various media was also determined. This was a step toward the optimization of carotenoid production by Antarctic bacteria on a larger scale.

As small, mobilizable replicons with a broad host range, IncQ plasmids are widely distributed among clinical and environmental bacteria. They carry antibiotic resistance genes, and it has been shown that they confer resistance to β-lactams, fluoroquinolones, aminoglycosides, trimethoprim, sulphonamides, and tetracycline. The previously proposed classification system divides the plasmid group into four subgroups, i.e., IncQ-1, IncQ-2, IncQ-3, and IncQ-4. The last two subgroups have been poorly described so far. The aim of this study was to analyze five newly identified IncQ-3 plasmids isolated from a wastewater treatment plant in Poland and to compare them with all known plasmids belonging to the IncQ-3 subgroup whose sequences were retrieved from the NCBI database. The complete nucleotide sequences of the novel plasmids were annotated and bioinformatic analyses were performed, including identification of core genes and auxiliary genetic load. Furthermore, functional experiments testing plasmid mobility were carried out. Phylogenetic analysis based on three core genes ( repA , mobA / repB, and mobC ) revealed the presence of three main clusters of IncQ-3 replicons. Apart from having a highly conserved core, the analyzed IncQ-3 plasmids were vectors of antibiotic resistance genes, including (I) the qnrS2 gene that encodes fluoroquinolone resistance and (II) β-lactam, trimethoprim, and aminoglycoside resistance genes within integron cassettes.

The study presents a comparison of the influence of a clinoptilolite-rich rock-zeolite (commonly used for improving anaerobic digestion processes)-and a highly porous clay mineral, halloysite (mainly used for gas purification), on the biogas production process. Batch experiments showed that the addition of each mineral increased the efficiency of mesophilic anaerobic digestion of both sewage sludge and maize silage. However, halloysite generated 15% higher biogas production during maize silage transformation. Halloysite also contributed to a much higher reduction of chemical oxygen demand for both substrates (by ~8% for maize silage and ~14% for sewage sludge) and a higher reduction of volatile solids and total ammonia for maize silage (by ~8% and ~4%, respectively). Metagenomic analysis of the microbial community structure showed that the addition of both mineral sorbents influenced the presence of key members of archaea and bacteria occurring in a well-operated biogas reactor. The significant difference between zeolite and halloysite is that the latter promoted the immobilization of key methanogenic archaea Methanolinea (belong to Methanomicrobia class). Based on this result, we postulate that halloysite could be useful not only as a sorbent for (bio)gas treatment methodologies but also as an agent for improving biogas production.

Background: Carotenoids are natural tetraterpene pigments widely utilized in the food, pharmaceutical and cosmetic industries. Currently, chemical synthesis of these compounds outperforms their production in Escherichia coli or yeast due to the limited efficiency of the latter. The use of natural microbial carotenoid producers, such as bacteria of the genus Paracoccus (Alphaproteobacteria), may help to optimize this process. In order to couple the ability to synthesize these pigments with the metabolic versatility of this genus, we explored the possibility of introducing carotenoid synthesis genes into strains capable of efficient growth on simple low-cost media., Results: We constructed two carotenoid-producing strains of Paracoccus carrying a new plasmid, pCRT01, which contains the carotenoid synthesis gene locus crt from Paracoccus marcusii OS22. The plasmid was created in vivo via illegitimate recombination between crt-carrying vector pABW1 and a natural "paracoccal" plasmid pAMI2. Consequently, the obtained fusion replicon is stably maintained in the bacterial population without the need for antibiotic selection. The introduction of pCRT01 into fast-growing "colorless" strains of Paracoccus aminophilus and Paracoccus kondratievae converted them into efficient producers of a range of both carotenes and xanthophylls. The exact profile of the produced pigments was dependent on the strain genetic background. To reduce the cost of carotenoid production in this system, we tested the growth and pigment synthesis efficiency of the two strains on various simple media, including raw industrial effluent (coal-fired power plant flue gas desulfurization wastewater) supplemented with molasses, an industrial by-product rich in sucrose., Conclusions: We demonstrated a new approach for the construction of carotenoid-producing bacterial strains which relies on a single plasmid-mediated transfer of a pigment synthesis gene locus between Paracoccus strains. This strategy facilitates screening for producer strains in terms of synthesis efficiency, pigment profile and ability to grow on low-cost industrial waste-based media, which should increase the cost-effectiveness of microbial production of carotenoids.

Methods of stool assessment are mostly focused on next-generation sequencing (NGS) or classical culturing, but only rarely both. We conducted a series of experiments using a multi-method approach to trace the stability of gut microbiota in various donors over time, to find the best method for the proper selection of fecal donors and to find "super-donor" indicators. Ten consecutive stools donated by each of three donors were used for the experiments (30 stools in total). The experiments assessed bacterial viability measured by flow cytometry, stool culturing on different media and in various conditions, and NGS (90 samples in total). There were no statistically significant differences between live and dead cell numbers; however, we found a group of cells classified as not-dead-not-alive, which may be possibly important in selection of "good" donors. Donor C, being a regular stool donor, was characterized by the largest number of cultivable species (64). Cultivable core microbiota (shared by all donors) was composed of only 16 species. ANCOM analysis of NGS data highlighted particular genera to be more abundant in one donor vs. the others. There was a correlation between the not-dead-not-alive group found in flow cytometry and Anaeroplasma found by NGS, and we could distinguish a regular stool donor from the others. In this work, we showed that combining various methods of microbiota assessment gives more information than each method separately.

The Ochrobactrum genus consists of an extensive repertoire of biotechnologically valuable bacterial strains but also opportunistic pathogens. In our previous study, a novel strain, Ochrobactrum sp. POC9, which enhances biogas production in wastewater treatment plants (WWTPs) was identified and thoroughly characterized. Despite an insightful analysis of that bacterium, its susceptibility to bacteriophages present in WWTPs has not been evaluated. Using raw sewage sample from WWTP and applying the enrichment method, two virulent phages, vB&#95;OspM&#95;OC and vB&#95;OspP&#95;OH, which infect the POC9 strain, were isolated. These are the first virulent phages infecting Ochrobactrum spp. identified so far. Both phages were subjected to thorough functional and genomic analyses, which allowed classification of the vB&#95;OspM&#95;OC virus as a novel jumbo phage, with a genome size of over 227 kb. This phage encodes DNA methyltransferase, which mimics the specificity of cell cycle regulated CcrM methylase, a component of the epigenetic regulatory circuits in Alphaproteobacteria . In this study, an analysis of the overall diversity of Ochrobactrum -specific (pro)phages retrieved from databases and extracted in silico from bacterial genomes was also performed. Complex genome mining allowed us to build similarity networks to compare 281 Ochrobactrum -specific viruses. Analyses of the obtained networks revealed a high diversity of Ochrobactrum phages and their dissimilarity to the viruses infecting other bacteria.

Pseudomonas spp. are widely distributed in various environments around the world. They are also common in the Antarctic regions. To date, almost 200 plasmids of Pseudomonas spp. have been sequenced, but only 12 of them were isolated from psychrotolerant strains. In this study, 15 novel plasmids of cold-active Pseudomonas spp. originating from the King George Island (Antarctica) were characterized using a combined, structural and functional approach, including thorough genomic analyses, functional analyses of selected genetic modules, and identification of active transposable elements localized within the plasmids and comparative genomics. The analyses performed in this study increased the understanding of the horizontal transfer of plasmids found within Pseudomonas populations inhabiting Antarctic soils. It was shown that the majority of the studied plasmids are narrow-host-range replicons, whose transfer across taxonomic boundaries may be limited. Moreover, structural and functional analyses enabled identification and characterization of various accessory genetic modules, including genes encoding major pilin protein (PilA), that enhance biofilm formation, as well as active transposable elements. Furthermore, comparative genomic analyses revealed that the studied plasmids of Antarctic Pseudomonas spp. are unique, as they are highly dissimilar to the other known plasmids of Pseudomonas spp.

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.

A literature-based, manually-curated database of PCR primers for the detection of antibiotic resistance genes in various environments was constructed (LCPDb-ARG; lcpdb.ddg.biol.uw.edu.pl and lcpdb.ddlemb.com). Currently, this database is comprised of 607 PCR primer pairs designed for the amplification of various genes conferring resistance to antibiotics representing 10 classes of antimicrobial agents. Three parameters were assigned for each primer pair: specificity, efficacy and taxonomic efficacy. These parameters were evaluated using a novel bioinformatic tool, UniPriVal, developed for this study. UniPriVal was used to validate each primer pair against various databases, including the Bacterial Antimicrobial Resistance Reference Gene Database (BARRGDB) and those of the National Center of Biotechnology Information (NCBI). Primer pairs specific for each antibiotic resistance gene were ranked based on their model success metric value. To validate the utility and correctness of the information collected in the LCPDb-ARG, selected primer pairs were tested in bioinformatic and experimental PCR surveys. To our knowledge, this is the first database designed to facilitate PCR monitoring of the occurrence and diversity of antibiotic resistance genes in environmental and clinical samples. The internal validation system of this user-friendly application enables the quantified ranking of PCR primer pairs, which assists selection of the best primers for each application., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, we used a multifaceted approach to select robust bioaugmentation candidates for enhancing biogas production and to demonstrate the usefulness of a genome-centric approach for strain selection for specific bioaugmentation purposes. We also investigated the influence of the isolation source of bacterial strains on their metabolic potential and their efficiency in enhancing anaerobic digestion. Whole genome sequencing, metabolic pathway reconstruction, and physiological analyses, including phenomics, of phylogenetically diverse strains, Rummeliibacillus sp. POC4, Ochrobactrum sp. POC9 (both isolated from sewage sludge) and Brevundimonas sp. LPMIX5 (isolated from an agricultural biogas plant) showed their diverse enzymatic activities, metabolic versatility and ability to survive under varied growth conditions. All tested strains display proteolytic, lipolytic, cellulolytic, amylolytic, and xylanolytic activities and are able to utilize a wide array of single carbon and energy sources, as well as more complex industrial by-products, such as dairy waste and molasses. The specific enzymatic activity expressed by the three strains studied was related to the type of substrate present in the original isolation source. Bioaugmentation with sewage sludge isolates-POC4 and POC9-was more effective for enhancing biogas production from sewage sludge (22% and 28%, respectively) than an approach based on LPMIX5 strain (biogas production boosted by 7%) that had been isolated from an agricultural biogas plant, where other type of substrate is used., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Cold-active bacteria are currently of great interest in biotechnology, and their genomic and physiological features have been extensively studied. One of the model psychrotolerant bacteria are Psychrobacter spp. Analysis of Arctic psychrophilic Psychrobacter sp. DAB&#95;AL32B genome content provided an insight into its overall stress response, and genes conferring protection against various life-limiting factors (i.e., low temperature, increased ultraviolet radiation, oxidative stress and osmotic pressure) were recognized and described. Moreover, it was revealed that the strain carries a large plasmid pP32BP2. Its replication system was used for the construction of two novel shuttle vectors (pPS-NR-Psychrobacter-Escherichia coli-specific plasmid and pPS-BR-Psychrobacter-various Proteobacteria-specific plasmid) of an increased carrying capacity, which may be used for genetic engineering of Psychrobacter spp.

Evolution of bacteria to selective chemical pressure (e.g. antibiotics) is well studied in contrast to the influence of physical stressors. Here we show that instantaneous physical stress in a homogeneous environment (without concentration gradient) induces fast adaptation of Escherichia coli. We exposed E. coli to a large number of collisions of around 10 5 per bacterium per second with sharp ZnO nanorods. The pressure exerted on the bacterial cell wall was up to 10 GPa and induced phenotype changes. The bacteria's shape became more spherical, the density of their periplasm increased by around 15% and the average thickness of the cell wall by 30%. Such E. coli cells appeared almost as Gram-positive bacteria in the standard Gram staining. Additionally, we observed a combination of changes occurring at the genomic level (mutations identified in form of single nucleotide polymorphisms) and down-regulation of expression of 61 genes encoding proteins involved in β-oxidation of fatty acids, glycolysis, the citric acid cycle, as well as uptake of amino acids and enzyme cofactors. Thus, we show that bacteria undergo phenotypic changes upon instantaneous, acute physical stress without any obviously available time for gradual adaptation.

Bacteria of the genus Paracoccus inhabit various pristine and anthropologically-shaped environments. Many Paracoccus spp. have biotechnological value and several are opportunistic human pathogens. Despite extensive knowledge of their metabolic potential and genome architecture, little is known about viruses of Paracoccus spp. So far, only three active phages infecting these bacteria have been identified. In this study, 16 Paracoccus strains were screened for the presence of active temperate phages, which resulted in the identification of five novel viruses. Mitomycin C-induced prophages were isolated, visualized and their genomes sequenced and thoroughly analyzed, including functional validation of their toxin-antitoxin systems. This led to the identification of the first active Myoviridae phage in Paracoccus spp. and four novel Siphoviridae phages. In addition, another 53 prophages were distinguished in silico within genomic sequences of Paracoccus spp. available in public databases. Thus, the Paracoccus virome was defined as being composed of 66 (pro)phages. Comparative analyses revealed the diversity and mosaicism of the (pro)phage genomes. Moreover, similarity networking analysis highlighted the uniqueness of Paracoccus (pro)phages among known bacterial viruses.

Psychrobacter sp. DAB&#95;AL32B, originating from Spitsbergen island (Arctic), carries the large plasmid pP32BP2 (54,438 bp). Analysis of the pP32BP2 nucleotide sequence revealed the presence of three predicted phenotypic modules that comprise nearly 30% of the plasmid genome. These modules appear to be involved in fimbriae synthesis via the chaperone-usher pathway (FIM module) and the aerobic and anaerobic metabolism of carnitine (CAR and CAI modules, respectively). The FIM module was found to be functional in diverse hosts since it facilitated the attachment of bacterial cells to abiotic surfaces, enhancing biofilm formation. The CAI module did not show measurable activity in any of the tested strains. Interestingly, the CAR module enabled the enzymatic breakdown of carnitine, but this led to the formation of the toxic by-product trimethylamine, which inhibited bacterial growth. Thus, on the one hand, pP32BP2 can enhance biofilm formation, a highly advantageous feature in cold environments, while on the other, it may prevent bacterial growth under certain environmental conditions. The detrimental effect of harboring pP32BP2 (and its CAR module) seems to be conditional, since this replicon may also confer the ability to use carnitine as an alternative carbon source, although a pathway to utilize trimethylamine is most probably necessary to make this beneficial. Therefore, the phenotype determined by this CAR-containing plasmid depends on the metabolic background of the host strain.

Arthrobacter spp. are coryneform Gram-positive aerobic bacteria, belonging to the class Actinobacteria . Representatives of this genus have mainly been isolated from soil, mud, sludge or sewage, and are usually mesophiles. In recent years, the presence of Arthrobacter spp. was also confirmed in various extreme, including permanently cold, environments. In this study, 36 psychrotolerant and metalotolerant Arthrobacter strains isolated from petroleum-contaminated soil from the King George Island (Antarctica), were screened for the presence of plasmids. The identified replicons were thoroughly characterized in order to assess their diversity and role in the adaptation of Arthrobacter spp. to harsh Antarctic conditions. The screening process identified 11 different plasmids, ranging in size from 8.4 to 90.6 kb. A thorough genomic analysis of these replicons detected the presence of numerous genes encoding proteins that potentially perform roles in adaptive processes such as (i) protection against ultraviolet (UV) radiation, (ii) resistance to heavy metals, (iii) transport and metabolism of organic compounds, (iv) sulfur metabolism, and (v) protection against exogenous DNA. Moreover, 10 of the plasmids carry genetic modules enabling conjugal transfer, which may facilitate their spread among bacteria in Antarctic soil. In addition, transposable elements were identified within the analyzed plasmids. Some of these elements carry passenger genes, which suggests that these replicons may be actively changing, and novel genetic modules of adaptive value could be acquired by transposition events. A comparative genomic analysis of plasmids identified in this study and other available Arthrobacter plasmids was performed. This showed only limited similarities between plasmids of Antarctic Arthrobacter strains and replicons of other, mostly mesophilic, isolates. This indicates that the plasmids identified in this study are novel and unique replicons. In addition, a thorough meta-analysis of 247 plasmids of psychrotolerant bacteria was performed, revealing the important role of these replicons in the adaptation of their hosts to extreme environments.

Rhizobia are bacteria that can form symbiotic associations with plants of the Fabaceae family, during which they reduce atmospheric di-nitrogen to ammonia. The symbiosis between rhizobia and leguminous plants is a fundamental contributor to nitrogen cycling in natural and agricultural ecosystems. Rhizobial microsymbionts are a major reason why legumes can colonize marginal lands and nitrogen-deficient soils. Several leguminous species have been found in metal-contaminated areas, and they often harbor metal-tolerant rhizobia. In recent years, there have been numerous efforts and discoveries related to the genetic determinants of metal resistance by rhizobia, and on the effectiveness of such rhizobia to increase the metal tolerance of host plants. Here, we review the main findings on the metal resistance of rhizobia: the physiological role, evolution, and genetic determinants, and the potential to use native and genetically-manipulated rhizobia as inoculants for legumes in phytoremediation practices.

Ensifer ( Sinorhizobium ) sp. M14 is an efficient arsenic-oxidizing bacterium (AOB) that displays high resistance to numerous metals and various stressors. Here, we report the draft genome sequence and genome-guided characterization of Ensifer sp. M14, and we describe a pilot-scale installation applying the M14 strain for remediation of arsenic-contaminated waters. The M14 genome contains 6874 protein coding sequences, including hundreds not found in related strains. Nearly all unique genes that are associated with metal resistance and arsenic oxidation are localized within the pSinA and pSinB megaplasmids. Comparative genomics revealed that multiple copies of high-affinity phosphate transport systems are common in AOBs, possibly as an As-resistance mechanism. Genome and antibiotic sensitivity analyses further suggested that the use of Ensifer sp. M14 in biotechnology does not pose serious biosafety risks. Therefore, a novel two-stage installation for remediation of arsenic-contaminated waters was developed. It consists of a microbiological module, where M14 oxidizes As(III) to As(V) ion, followed by an adsorption module for As(V) removal using granulated bog iron ores. During a 40-day pilot-scale test in an abandoned gold mine in Zloty Stok (Poland), water leaving the microbiological module generally contained trace amounts of As(III), and dramatic decreases in total arsenic concentrations were observed after passage through the adsorption module. These results demonstrate the usefulness of Ensifer sp. M14 in arsenic removal performed in environmental settings., Competing Interests: The authors declare no conflicts of interest.

Sewage sludge is an abundant source of microorganisms that are metabolically active against numerous contaminants, and thus possibly useful in environmental biotechnologies. However, amongst the sewage sludge isolates, pathogenic bacteria can potentially be found, and such isolates should therefore be carefully tested before their application. A novel bacterial strain, Ochrobactrum sp. POC9, was isolated from a sewage sludge sample collected from a wastewater treatment plant. The strain exhibited lipolytic, proteolytic, cellulolytic, and amylolytic activities, which supports its application in biodegradation of complex organic compounds. We demonstrated that bioaugmentation with this strain substantially improved the overall biogas production and methane content during anaerobic digestion of sewage sludge. The POC9 genome content analysis provided a deeper insight into the biotechnological potential of this bacterium and revealed that it is a metalotolerant and a biofilm-producing strain capable of utilizing various toxic compounds. The strain is resistant to rifampicin, chloramphenicol and β-lactams. The corresponding antibiotic resistance genes (including bla OCH and cmlA/floR ) were identified in the POC9 genome. Nevertheless, as only few genes in the POC9 genome might be linked to pathogenicity, and none of those genes is a critical virulence factor found in severe pathogens, the strain appears safe for application in environmental biotechnologies.

Cold-active bacteria of the genus Polaromonas (class Betaproteobacteria ) are important components of glacial microbiomes. In this study, extrachromosomal replicons of 26 psychrotolerant Polaromonas strains, isolated from Arctic and Antarctic glaciers, were identified, sequenced, and characterized. The plasmidome of these strains consists of 13 replicons, ranging in size from 3,378 to 101,077 bp. In silico sequence analyses identified the conserved backbones of these plasmids, composed of genes required for plasmid replication, stable maintenance, and conjugal transfer. Host range analysis revealed that all of the identified plasmids are narrow-host-range replicons, only able to replicate in bacteria of closely related genera ( Polaromonas and Variovorax ) of the Comamonadaceae family. Special attention was paid to the identification of plasmid auxiliary genetic information, which may contribute to the adaptation of bacteria to environmental conditions occurring in glaciers. Detailed analysis revealed the presence of genes encoding proteins potentially involved in (i) protection against reactive oxygen species, ultraviolet radiation, and low temperatures; (ii) transport and metabolism of organic compounds; (iii) transport of metal ions; and (iv) resistance to heavy metals. Some of the plasmids also carry genes required for the molecular assembly of iron-sulfur [Fe-S] clusters. Functional analysis of the predicted heavy metal resistance determinants demonstrated that their activity varies, depending on the host strain. This study provides the first molecular insight into the mobile DNA of Polaromonas spp. inhabiting polar glaciers. It has generated valuable data on the structure and properties of a pool of plasmids and highlighted their role in the biology of psychrotolerant Polaromonas strains and their adaptation to the environmental conditions of Arctic and Antarctic glaciers.

Psychrobacter sp. DAB&#95;AL43B, isolated from ornithogenic soil collected on the Arctic island of Spitsbergen, is a newly sequenced psychrophilic strain susceptible to conjugation and electrotransformation. Its genome consists of a circular chromosome (3.3 Mb) and four plasmids (4.4-6.4kb). In silico genome mining and microarray-based phenotypic analysis were performed to describe the metabolic potential of this strain and identify possible biotechnological applications. Metabolic reconstruction indicated that DAB&#95;AL43B prefers low-molecular-weight carboxylates and amino acids as carbon and energy sources. Genetic determinants of heavy-metal resistance, anthracene degradation and possible aerobic denitrification were also identified. Comparative analyses revealed a relatively close relationship between DAB&#95;AL43B and other sequenced Psychrobacter species. In addition, the plasmids of this strain were used as the basis for the construction of Escherichia coli-Psychrobacter spp. shuttle vectors. Taken together, the results of this work suggest that DAB&#95;AL43B is a promising candidate as a new model strain for studies on Psychrobacter spp., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Plasmids play an important role in the adaptation of bacteria to changeable environmental conditions. As the main vectors of horizontal gene transfer, they can spread genetic information among bacteria, sometimes even across taxonomic boundaries. Some plasmids carry genes involved in the utilization of particular carbon compounds, which can provide a competitive advantage to their hosts in particular ecological niches. Analysis of the multireplicon genome of the soil bacterium P. aminovorans JCM 7685 revealed the presence of an extrachromosomal replicon pAMV1 (185 kb) with a unique structure and properties. This lifestyle-determining plasmid carries genes facilitating the metabolism of many different carbon compounds including sugars, short-chain organic acids and C1 compounds. Plasmid pAMV1 contains a large methylotrophy island (MEI) that is essential not only for the utilization of particular C1 compounds but also for the central methylotrophic metabolism required for the assimilation of C1 units (serine cycle). We demonstrate that the expression of the main serine cycle genes is induced in the presence of C1 compounds by the transcriptional regulator ScyR. The extrachromosomal localization of the MEI and the distribution of related genes in Paracoccus spp. indicate that it could have been acquired by HGT by an ancestor of P. aminovorans., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The knowledge on plasmids of cold-active bacteria is highly limited. In this study, the molecular characterization of the pA3J1 plasmid of Antarctic psychrotolerant bacterium Pseudomonas sp. ANT&#95;J3 was performed. Within this plasmid, thirteen putative open reading frames were identified. Nine of them encoded proteins involved in replication, partitioning, postsegregational elimination of plasmid-less cells (via a toxin-antitoxin system activity), multimer resolution and mobilization by conjugal transfer. These genes constitute the plasmid backbone. The functional analysis of the pA3J1 maintenance region revealed that it is a narrow host range replicon, stably maintained in the host cells by the combined activities of the partitioning and relBE-type toxin-antitoxin systems. It was also suggested that the replication system of the pA3J1 plasmid may be temperature-sensitive. Comparative analyses revealed the presence of 16 Pseudomonas plasmids encoding homologous replication proteins and 5 plasmids carrying mobA genes homologous to the corresponding gene of pA3J1. The relaxase (MobA) of the pA3J1 plasmid was classified into MOB Q family, and the phylogenetic analysis suggested that this may be a representative of a novel group (or subgroup) within this family. The structural and comparative analyses revealed that the arrangement of genetic modules in the pA3J1 plasmid is unique., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Bartonella spp. are parasites of mammalian erythrocytes and endothelial cells, transmitted by blood-feeding arthropod ectoparasites. Different species of rodents may constitute the main hosts of Bartonella, including several zoonotic species of Bartonella. The aim of this study was to identify and compare Bartonella species and genotypes isolated from rodent hosts from the South Sinai, Egypt. Prevalence of Bartonella infection was assessed in rodents (837 Acomys dimidiatus, 73 Acomys russatus, 111 Dipodillus dasyurus, and 65 Sekeetamys calurus) trapped in 2000, 2004, 2008, and 2012 in four dry montane wadis around St. Katherine town in the Sinai Mountains. Total DNA was extracted from blood samples, and PCR amplification and sequencing of the Bartonella-specific 860-bp gene fragment of rpoB and the 810-bp gene fragment of gltA were used for molecular and phylogenetic analyses. The overall prevalence of Bartonella in rodents was 7.2%. Prevalence differed between host species, being 30.6%, 10.8%, 9.6%, and 3.6% in D. dasyurus, S. calurus, A. russatus, and A. dimidiatus, respectively. The phylogenetic analyses of six samples of Bartonella (five from D. dasyurus and one from S. calurus) based on a fragment of the rpoB gene, revealed the existence of two distinct genetic groups (with 95-96% reciprocal sequence identity), clustering with several unidentified isolates obtained earlier from the same rodent species, and distant from species that have already been described (90-92% of sequence identity to the closest match from the GenBank reference database). Thus, molecular and phylogenetic analyses led to the description of two species: Candidatus Bartonella fadhilae n. sp. and Candidatus Bartonella sanaae n. sp. The identification of their vectors and the medical significance of these species need further investigation.

The genus Sinorhizobium/Ensifer mostly groups nitrogen-fixing bacteria that create root or stem nodules on leguminous plants and transform atmospheric nitrogen into ammonia, which improves the productivity of the plants. Although these biotechnologically-important bacteria are commonly found in various soil environments, little is known about their phages. In this study, the genome of Sinorhizobium sp. LM21 isolated from a heavy-metal-contaminated copper mine in Poland was investigated for the presence of prophages and DNA methyltransferase-encoding genes. In addition to the previously identified temperate phage, ΦLM21, and the phage-plasmid, pLM21S1, the analysis revealed the presence of three prophage regions. Moreover, four novel phage-encoded DNA methyltransferase (MTase) genes were identified and the enzymes were characterized. It was shown that two of the identified viral MTases methylated the same target sequence (GANTC) as cell cycle-regulated methyltransferase (CcrM) of the bacterial host strain, LM21. This discovery was recognized as an example of the evolutionary convergence between enzymes of sinorhizobial viruses and their host, which may play an important role in virus cycle. In the last part of the study, thorough comparative analyses of 31 sinorhizobial (pro)phages (including active sinorhizobial phages and novel putative prophages retrieved and manually re-annotated from Sinorhizobium spp. genomes) were performed. The networking analysis revealed the presence of highly conserved proteins (e.g., holins and endolysins) and a high diversity of viral integrases. The analysis also revealed a large number of viral DNA MTases, whose genes were frequently located within the predicted replication modules of analyzed prophages, which may suggest their important regulatory role. Summarizing, complex analysis of the phage protein similarity network enabled a new insight into overall sinorhizobial virome diversity.

Aeromonas spp. are among the most ubiquitous microorganisms, as they have been isolated from different environmental niches including waters, soil, as well as wounds and digestive tracts of poikilothermic animals and humans. Although much attention has been paid to the pathogenicity of Aeromonads, the role of these bacteria in environmentally important processes, such as transformation of heavy metals, remains to be discovered. Therefore, the aim of this study was a detailed genomic characterization of Aeromonas sp. O23A, the first representative of this genus capable of dissimilatory arsenate reduction. The strain was isolated from microbial mats from the Zloty Stok mine (SW Poland), an environment strongly contaminated with arsenic. Previous physiological studies indicated that O23A may be involved in both mobilization and immobilization of this metalloid in the environment. To discover the molecular basis of the mechanisms behind the observed abilities, the genome of O23A (∼5.0 Mbp) was sequenced and annotated, and genes for arsenic respiration, heavy metal resistance ( hmr ) and other phenotypic traits, including siderophore production, were identified. The functionality of the indicated gene modules was assessed in a series of minimal inhibitory concentration analyses for various metals and metalloids, as well as mineral dissolution experiments. Interestingly, comparative analyses revealed that O23A is related to a fish pathogen Aeromonas salmonicida subsp. salmonicida A449 which, however, does not carry genes for arsenic respiration. This indicates that the dissimilatory arsenate reduction ability may have been lost during genome reduction in pathogenic strains, or acquired through horizontal gene transfer. Therefore, particular emphasis was placed upon the mobilome of O23A, consisting of four plasmids, a phage, and numerous transposable elements, which may play a role in the dissemination of hmr and arsenic metabolism genes in the environment. The obtained results indicate that Aeromonas sp. O23A is well-adapted to the extreme environmental conditions occurring in the Zloty Stok mine. The analysis of genome encoded traits allowed for a better understanding of the mechanisms of adaptation of the strain, also with respect to its presumable role in colonization and remediation of arsenic-contaminated waters, which may never have been discovered based on physiological analyses alone.

The composition of the oral microbiome in healthy individuals is complex and dynamic, and depends on many factors, such as anatomical location in the oral cavity, diet, oral hygiene habits or host immune responses. It is estimated at present that worldwide about 2 billion people suffer from diseases of the oral cavity, mainly periodontal disease and dental caries. Importantly, the oral microflora involved in local infections may spread and cause systemic, even life-threatening infections. In search for etiological agents of infections in dentistry, traditional approaches are not sufficient, as about 50% of oral bacteria are not cultivable. Instead, metagenomic analyses are particularly useful for studies of the complex oral microbiome - both in healthy individuals, and in patients with oral and dental diseases. In this paper we review the current and future applications of metagenomic studies in evaluation of both the composition of the oral microbiome as well as its potential pathogenic role in infections in dentistry.

The draft genome of multidrug-resistant Aeromonas sp. ARM81 isolated from a wastewater treatment plant in Warsaw (Poland) was obtained. Sequence analysis revealed multiple genes conferring resistance to aminoglycosides, β-lactams or tetracycline. Three different β-lactamase genes were identified, including an extended-spectrum β-lactamase gene bla PER-1 . The antibiotic susceptibility was experimentally tested. Genome sequencing also allowed us to investigate the plasmidome and transposable mobilome of ARM81. Four plasmids, of which two carry phenotypic modules (i.e., genes encoding a zinc transporter ZitB and a putative glucosyltransferase), and 28 putative transposase genes were identified. The mobility of three insertion sequences (isoforms of previously identified elements ISAs12, ISKpn9 and ISAs26) was confirmed using trap plasmids.

Sinorhizobium sp. M14 is an As(III)-oxidizing, psychrotolerant strain, capable of growth in the presence of extremely high concentrations of arsenic and many other heavy metals. Metallotolerant abilities of the M14 strain depend upon the presence of two extrachromosomal replicons: pSinA (∼ 109 kb) and pSinB (∼ 300 kb). The latter was subjected to complex analysis. The performed analysis demonstrated that the plasmid pSinB is a narrow-host-range repABC-type replicon, which is fully stabilized by the phd-vapC-like toxin-antitoxin stabilizing system. In silico analysis showed that among the phenotypic gene clusters of the plasmid pSinB, eight modules are potentially involved in heavy metals resistance (HMR). These modules carry genes encoding efflux pumps, permeases, transporters and copper oxidases, which provide resistance to arsenic, cadmium, cobalt, copper, iron, mercury, nickel, silver and zinc. The functional analysis revealed that the HMR modules are active and have an effect on the minimal inhibitory concentration (MIC) values observed for the heterological host cells. The phenotype was manifested by an increase or decrease of the MICs of heavy metals and it was strain specific. The analysis of distribution of the heavy metal resistance genes, i.e. resistome, in Sinorhizobium spp. plasmids, revealed that the HMR modules are common in these replicons., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA&#95;BRE&#95;C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas .

Aeromonas species are causative agents of a wide spectrum of diseases in animals and humans. Although these bacteria are commonly found in various environments, little is known about their phages. Thus far, only one temperate Aeromonas phage has been characterized. Whole-genome sequencing of an Aeromonas sp. strain ARM81 revealed the presence of two prophage clusters. One of them is integrated into the chromosome and the other was maintained as an extrachromosomal, linear plasmid-like prophage encoding a protelomerase. Both prophages were artificially and spontaneously inducible. We separately isolated both phages and compared their genomes with other known viruses. The novel phages show no similarity to the previously characterized Aeromonas phages and might represent new evolutionary lineages of viruses infecting Aeromonadaceae. Apart from the comparative genomic analyses of these phages, complemented with their structural and molecular characterization, a functional analysis of four DNA methyltransferases encoded by these viruses was conducted. One of the investigated N6-adenine-modifying enzymes shares sequence specificity with a Dam-like methyltransferase of its bacterial host, while another one is non-specific, as it catalyzes adenine methylation in various sequence contexts. The presented results shed new light on the diversity of Aeromonas temperate phages.

The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes.

Two novel prophages ФAH14a and ФAH14b of a psychrotolerant Antarctic bacterium Pseudomonas sp. ANT&#95;H14 have been characterized. They were simultaneously induced with mitomycin C and packed into capsids of the same size and protein composition. The genome sequences of ФAH14a and ФAH14b have been determined. ФAH14b, the phage with a smaller genome (16,812 bp) seems to parasitize ФAH14a (55,060 bp) and utilizes its capsids, as only the latter encodes a complete set of structural proteins. Both viruses probably constitute a phage helper-satellite system, analogous to the P2-P4 duo. This study describes the architecture and function of the ФAH14a and ФAH14b genomes. Moreover, a functional analysis of a ФAH14a-encoded lytic enzyme and a DNA methyltransferase was performed. In silico analysis revealed the presence of the homologs of ФAH14a and ФAH14b in other Pseudomonas genomes, which may suggest that helper-satellite systems related to the one described in this work are common in pseudomonads.