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Vine growing is one of the most economically important sectors of Mediterranean agriculture, but its cultivation practices are highly detrimental to the environment and the associated biota. The application of both natural products inducing endogenous plant defense mechanisms and natural soil management systems represents a potential solution for the preservation and improvement of soil health and biodiversity. The Life Green Grapes project aimed at evaluating the effects of different natural and sustainable vine protection strategies and soil management on vineyard edaphic communities. Soil TOC, TN, C:N ratio, CaCO3 content and pH were measured. Microbial communities (bacteria and fungi) were characterized through NGS, while nematodes and microarthropods were isolated and identified. Obtained data highlighted a relationshipbetween bacteria, fungi and nematodes with soil chemistry, and an effect of the different soil management on the single and total edaphic communities. Network analysis evidenced a positive effect of the application of sustainable soil managements on the relationships among the different investigated soil trophic levels, suggesting that more natural soil managements allow a better interaction between soil organisms. In conclusion, this work confirms the importance of the application of sustainable and natural soil management practices in agricultural ecosystems, with the aim of conserving and improving soil biodiversity.

Abstract Membrane vesicles (MVs) are spherical particles with nanoscale dimensions and characterized by the presence of diverse cargos, such as nucleic acids, proteins, lipids, and cellular metabolites. Many examples of (micro)organisms producing MVs are reported in literature. Among them, bacterial MVs are of particular interest because they are now considered as the fourth mechanism of horizontal gene transfer. Streptomyces bacteria are well-known for their ecological roles and ability to synthesize bioactive compounds, with Streptomyces coelicolor being the model organism. It was previously demonstrated that it can produce distinct populations of MVs characterized by different protein and metabolite cargos. In this work we demonstrated for the first time that MVs of S. coelicolor carry both DNA and RNA and that their DNA content represents the entire chromosome of the bacterium. These findings suggest that MV DNA could have a role in the evolution of Streptomyces genomes and that MVs could be exploited in new strain engineering strategies.

Understanding how microbial communities survive in extreme environmental pressure is critical for interpreting ecological patterns and microbial diversity. Great Gobi A Strictly Protected Area represents an intriguing model for studying the bacterial community since it is a protected and intact wild area of the Mongolian desert. In this work, the composition of a bacterial community of the soil from four oases was characterized by extracting total DNA and sequencing through the Illumina NovaSeq platform. In addition, the soil’s chemical and physical properties were determined, and their influence on shaping the microbial communities was evaluated. The results showed a high variability of bacterial composition among oases. Moreover, combining specific chemical and physical parameters significantly shapes the bacterial community among oases. Data obtained suggested that the oases were highly variable in physiochemical parameters and bacterial communities despite the similar extreme climate conditions. Moreover, core functional microbiome were constituted by aerobic chemoheterotrophy and chemoheterotrophy, mainly contributed by the most abundant bacteria, such as Actinobacteriota, Pseudomonadota, and Firmicutes. This result supposes a metabolic flexibility for sustaining life in deserts. Furthermore, as the inhabitants of the extreme regions are likely to produce new chemical compounds, isolation of key taxa is thus encouraged.

The Sasso Fratino Integral Nature Reserve (Italy) aims to protect nature and territory. Since no anthropic activities are allowed, it represents a good model to study the bacterial community of a wild environment. The aim of this work was to characterise the cultivable and the total bacterial community of soil samples from the reserve in terms of taxonomy, composition, and structure. Seven soil samples were collected at different altitudes, and the chemical composition, the total and the cultivable microbiota, and the antibiotic resistance profiles of isolates were investigated. Total bacterial communities, studied through Next Generation Sequences analysis, included 390 genera. Samples differed in terms of microbial composition basing on the different altitude/vegetation of collection points. Random Amplified Polymorphic DNA Analysis (RAPD) allowed to identify 82 haplotypes out of 158 bacterial isolates. The taxonomic identification through 16S rDNA sequencing revealed that the strains were affiliated to 21 genera. Antibiotic resistance profiles of bacteria were also investigated, highlighting a high resistance against streptomycin and kanamycin. This work represents the first description of the soil bacterial community from the Natural Reserve of Sasso Fratino, and it is the first study considering the soil microbiota of an Italian integral nature reserve.

Essential oils (EOs) from medicinal plants have long been used in traditional medicine for their widely known antimicrobial properties and represent a promising reservoir of bioactive compounds against multidrug-resistant pathogens. Endophytes may contribute to the yield and composition of EOs, representing a useful tool for biotechnological applications. In this work, we investigated the genomic basis of this potential contribution. The annotated genomes of four endophytic strains isolated from Origanum vulgare L. were used to obtain KEGG ortholog codes, which were used for the annotation of different pathways in KEGG, and to evaluate whether endophytes might harbor the (complete) gene sets for terpene and/or plant hormone biosynthesis. All strains possessed ortholog genes for the mevalonate-independent pathway (MEP/DOXP), allowing for the production of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) precursors. Ortholog genes for the next steps in terpenoid biosynthesis were scarce. All the strains possess potential plant growth promotion (PGP) ability, as shown by the presence of orthologous genes involved in the biosynthesis of indoleacetic acid. The main contribution of endophytes to the yield and composition of O. vulgare EO very likely resides in their PGP activities and in the biosynthesis of precursors of bioactive compounds.

Medicinal plants play an important role in the discovery of new bioactive compounds with antimicrobial activity, thanks to their pharmacological properties. However, members of their microbiota can also synthesize bioactive molecules. Among these, strains belonging to the genera Arthrobacter are commonly found associated with the plant’s microenvironments, showing plant growth-promoting (PGP) activity and bioremediation properties. However, their role as antimicrobial secondary metabolite producers has not been fully explored. The aim of this work was to characterize the Arthrobacter sp. OVS8 endophytic strain, isolated from the medicinal plant Origanum vulgare L., from molecular and phenotypic viewpoints to evaluate its adaptation and influence on the plant internal microenvironments and its potential as a producer of antibacterial volatile molecules (VOCs). Results obtained from the phenotypic and genomic characterization highlight its ability to produce volatile antimicrobials effective against multidrug-resistant (MDR) human pathogens and its putative PGP role as a producer of siderophores and degrader of organic and inorganic pollutants. The outcomes presented in this work identify Arthrobacter sp. OVS8 as an excellent starting point toward the exploitation of bacterial endophytes as antibiotics sources.

The propagation of foreign DNA in Escherichia coli is central to molecular biology. Recent advances have dramatically expanded the ability to engineer (bacterial) cells; however, most of these techniques remain time-consuming. The aim of the present work was to explore the possibility to use the cloning-free genome editing (CFGE) approach, proposed by Döhlemann and coworkers (2016), for E. coli genetics, and to deepen the knowledge about the homologous recombination mechanism. The E. coli auxotrophic mutant strains FB182 (hisF892) and FB181 (hisI903) were transformed with the circularized wild-type E. coli (i) hisF gene and hisF gene fragments of decreasing length, and (ii) hisIE gene, respectively. His+ clones were selected based on their ability to grow in the absence of histidine, and their hisF/hisIE gene sequences were characterized. CFGE method allowed the recombination of wild-type his genes (or fragments of them) within the mutated chromosomal copy, with a different recombination frequency based on the fragment length, and the generation of clones with a variable number of in tandem his genes copies. Data obtained pave the way to further evolutionary studies concerning the homologous recombination mechanism and the fate of in tandem duplicated genes.

Phytodepuration occurs in the plant-mediated remediation processes exploited to remove pollutants from wastewater, and Phragmites australis is one of the most used plants. This goal is achieved using constructed wetlands (CW), which are engineered systems designed to mimic the natural processes of pollutants removal. The aim of this work was to characterize the bacterial communities associated to P. australis, soils, and permeates of the CW of Calice (Prato, Italy), to evaluate the possible effect of wastewaters on the CW bacterial communities, through a next-generation sequencing-based approach. A total of 122 samples were collected from different tissues of P. australis (i.e., roots, aerial parts, and stem), soil (i.e., rhizospheric and bulk soil), and permeates, and analyzed. All samples were collected during five sampling campaigns, with the first one performed before the activation of the plant. Obtained results highlighted a specific microbiota of P. australis, conserved among the different plant tissues and during time, showing a lower alpha diversity than the other samples and not influenced by the more complex and variable environmental (soils and permeates) bacterial communities. These data suggest that P. australis is able to select and maintain a defined microbiota, a capacity that could allow the plant to survive in hostile environments, such as that of CW.

Seed-associated microbiota are believed to play a crucial role in seed germination, seedling establishment, and plant growth and fitness stimulation, due to the vertical transmission of a core microbiota from seeds to the next generations. It might be hypothesized that medicinal and aromatic plants could use the seeds as vectors to vertically transfer beneficial endophytes, providing plants with metabolic pathways that could influence phytochemicals production. Here, we investigated the localization, the structure and the composition of the bacterial endophytic population that resides in Origanum heracleoticum L. seeds. Endocellular bacteria, surrounded by a wall, were localized close to the aleurone layer when using light and transmission electron microscopy. From surface-sterilized seeds, cultivable endophytes were isolated and characterized through RAPD analysis and 16S RNA gene sequencing, which revealed the existence of a high degree of biodiversity at the strain level and the predominance of the genus Pseudomonas. Most of the isolates grew in the presence of six selected antibiotics and were able to inhibit the growth of clinical and environmental strains that belong to the Burkholderia cepacia complex. The endophytes production of antimicrobial compounds could suggest their involvement in plant secondary metabolites production and might pave the way to endophytes exploitation in the pharmaceutical field.

Medicinal aromatic plants’ essential oils (EOs) are mixtures of volatile compounds showing antimicrobial activity, which could be exploited to face the emerging problem of multi-drug resistance. Their chemical composition can depend on the interactions between the plant and its endophytic microbiota, which is known to synthesize volatile organic compounds (VOCs). However, it is still not clear whether those volatile metabolites can contribute to the composition of the aroma profile of plants’ EOs. The aims of this study were to characterize medicinal plant O. vulgare ssp. vulgare bacterial endophyte VOCs, evaluating their ability to antagonize the growth of opportunistic human pathogens belonging to the Burkholderia cepacia complex (Bcc) and compare them with O. vulgare EO composition. Many of the tested endophytic strains showed (i) a bactericidal and/or bacteriostatic activity against most of Bcc strains and (ii) the production of VOCs with widely recognized antimicrobial properties, such as dimethyl disulfide, dimethyl trisulfide, and monoterpenes. Moreover, these monoterpenes were also detected in the EOs extracted from the same O. vulgare plants from which endophytes were isolated. Obtained results suggest that endophytes could also play a role in the antibacterial properties of O. vulgare ssp. vulgare and, potentially, in determining its aromatic composition.

Although proto-evolutionary ideas date back to the time of the ancient Greeks, the idea that organisms evolve was not considered a basic element of scientific knowledge until Charles Darwin published his “On the Origin of Species” in 1859 [...]

Multidrug-resistant pathogens represent a serious threat to human health. The inefficacy of traditional antibiotic drugs could be surmounted through the exploitation of natural bioactive compounds of which medicinal plants are a great reservoir. The finding that bacteria living inside plant tissues, (i.e., the endophytic bacterial microbiome) can influence the synthesis of the aforementioned compounds leads to the necessity of unraveling the mechanisms involved in the determination of this symbiotic relationship. Here, we report the genome sequence of four endophytic bacterial strains isolated from the medicinal plant Origanum vulgare L. and able to antagonize the growth of opportunistic pathogens of cystic fibrosis patients. The in silico analysis revealed the presence of gene clusters involved in the production of antimicrobial compounds, such as paeninodin, paenilarvins, polymyxin, and paenicidin A. Endophytes’ adaptation to the plant microenvironment was evaluated through the analysis of the presence of antibiotic resistance genes in the four genomes. The diesel fuel degrading potential was also tested. Strains grew in minimum media supplemented with diesel fuel, but no n-alkanes degradation genes were found in their genomes, suggesting that diesel fuel degradation might occur through other steps involving enzymes catalyzing the oxidation of aromatic compounds.

Microorganisms possess the potential to adapt to fluctuations in environmental parameters, and their evolution is driven by the continuous generation of mutations. The reversion of auxotrophic mutations has been widely studied; however, little is known about the reversion of frameshift mutations resulting in amino acid auxotrophy and on the structure and functioning of the protein encoded by the revertant mutated gene. The aims of this work were to analyze the appearance of reverse mutations over time and under different selective pressures and to investigate revertant enzymes’ three-dimensional structures and their correlation with a different growth ability. Escherichia coli FB182 strain, carrying the hisF892 single nucleotide deletion resulting in histidine auxotrophy, was subjected to different selective pressures, and revertant mutants were isolated and characterized. The obtained results allowed us to identify different indels of different lengths located in different positions in the hisF gene, and relations with the incubation time and the selective pressure applied were observed. Moreover, the structure of the different mutant proteins was consistent with the respective revertant ability to grow in absence of histidine, highlighting a correlation between the mutations and the catalytic activity of the mutated HisF enzyme.

Improvement of industrial productions through more environment-friendly processes is a hot topic. In particular, land and marine environment pollution is a main concern, considering that recalcitrant compounds can be spread and persist for a long time. In this context, an efficient and cost-effective treatment of wastewater derived from industrial applications is crucial. Phytodepuration has been considered as a possible solution and it is based on the use of plants and their associated microorganisms to remove and/or transform pollutants. In this work we investigated the culturable microbiota of Phragmites australis roots, sampled from the constructed wetlands (CWs) pilot plant in the G.I.D.A. SpA wastewater treatment plant (WWTP) of Calice (Prato, Tuscany, Italy) before and after the CW activation in order to check how the influx of wastewater might affect the resident bacterial community. P. australis specimens were sampled and a panel of 294 culturable bacteria were isolated and characterized. This allowed to identify the dynamics of the microbiota composition triggered by the presence of wastewater. 27 out of 37 bacterial genera detected were exclusively associated to wastewater, and Pseudomonas was constantly the most represented genus. Moreover, isolates were assayed for their resistance against eight different antibiotics and synthetic wastewater (SWW). Data obtained revealed the presence of resistant phenotypes, including multi-drug resistant bacteria, and a general trend regarding the temporal evolution of resistance patterns: indeed, a direct correlation linking the appearance of antibiotic- and SWW-resistance with the time of exposure to wastewater was observed. In particular, nine isolates showed an interesting behavior since their growth was positively affected by the highest concentrations of SWW. Noteworthy, this study is among the few investigating the P. australis microbiota prior to the plant activation.

The insurgence of antibiotic resistance and emergence of multidrug-resistant (MDR) pathogens prioritize research to discover new antimicrobials. In this context, medicinal plants produce bioactive compounds of pharmacological interest: some extracts have antimicrobial properties that can contrast different pathogens. For such a purpose, Origanum vulgare L. (Lamiaceae family) is a medicinal aromatic plant, whose essential oil (EO) is recognized for its antiseptic, antimicrobial and antiviral activities. The cultivable bacteria from different compartments (i.e., flower, leaf, stem and soil) were isolated in order to: (i) characterize the bacterial microbiota associated to the plant, determining the forces responsible for the structuring of its composition (by evaluation of cross inhibition); (ii) investigate if bacterial endophytes demonstrate antimicrobial activities against human pathogens. A pool of plants belonging to O. vulgare species was collected and the specimen chemotype was defined by hydrodistillation of its essential oil. The isolation of plant associated bacteria was performed from the four compartments. Microbiota was further characterized through a culture-independent approach and next-generation sequencing analysis, as well. Isolates were molecularly typed by Random Amplified Polymorphic DNA (RAPD) profiling and taxonomically assigned by 16S rRNA gene sequencing. Antibiotic resistance profiles of isolates and pairwise cross-inhibition of isolates on agar plates (i.e., antagonistic interactions) were also assessed. High level of diversity of bacterial isolates was detected at both genus and strain level in all different compartments. Most strains were tolerant against common antibiotics; moreover, they produced antagonistic patterns of interactions mainly with strains from different compartments with respect to that of original isolation. Strains that exhibited high inhibitory properties were further tested against human pathogens, revealing a strong capacity to inhibit the growth of strains resistant to several antibiotics. In conclusion, this study regarded the characterization of O. vulgare L. chemotype and of the bacterial communities associated to this medicinal plant, also allowing the evaluation of antibiotic resistance and antagonistic interactions. This study provided the bases for further analyses on the possible involvement of endophytic bacteria in the production of antimicrobial molecules that could have an important role in clinical and therapeutic applications.

One of the most studied metabolic routes is the biosynthesis of histidine, especially in enterobacteria where a single compact operon composed of eight adjacent genes encodes the complete set of biosynthetic enzymes. It is still not clear how his genes were organized in the genome of the last universal common ancestor community. The aim of this work was to analyze the structure, organization, phylogenetic distribution, and degree of horizontal gene transfer (HGT) of his genes in the Bacteroidota-Rhodothermota-Balneolota-Chlorobiota superphylum, a group of phylogenetically close bacteria with different surviving strategies. The analysis of the large variety of his gene structures and organizations revealed different scenarios with genes organized in more or less compact—heterogeneous or homogeneous—operons, in suboperons, or in regulons. The organization of his genes in the extant members of the superphylum suggests that in the common ancestor of this group, genes were scattered throughout the chromosome and that different forces have driven the assembly of his genes in compact operons. Gene fusion events and/or paralog formation, HGT of single genes or entire operons between strains of the same or different taxonomic groups, and other molecular rearrangements shaped the his gene structure in this superphylum.

This study aimed to characterise the gut microbiome composition of European hares (Lepus europaeus) and its potential changes after a short-term diet modification. The high sensitivity of European hare to habitat changes makes this species a good model to analyse possible alterations in gut microbiome after the introduction of additional nourishment into the diet. In total, 20 pairs were chosen for the experiments; 10 pairs formed the control group and were fed with standard fodder. The other 10 pairs represented the experimental group, whose diet was integrated with apples and carrots. The DNA from fresh faecal pellets collected after 4 days from the start of the experiment was extracted and the V3-V4 hypervariable regions were amplified and sequenced using the Illumina MiSeq® platform. The obtained amplicon sequence variants were classified into 735 bacterial genera belonging to 285 families and 36 phyla. The control and the experimental groups appeared to have a homogenous dispersion for the two taxonomic levels analysed with the most abundant phyla represented by Bacteroidetes and Firmicutes. No difference between control and experimental samples was detected, suggesting that the short-term variation in food availability did not alter the hares’ gut microbiome. Further research is needed to estimate significant time threshold.

Gene elongation is a molecular mechanism consisting of an in-tandem duplication of a gene and divergence and fusion of the two copies, resulting in a gene constituted by two divergent paralogous modules. The aim of this work was to evaluate the importance of gene elongation in the evolution of histidine biosynthetic genes and to propose a possible evolutionary model for some of them. Concerning the genes hisA and hisF, which code for two homologous (β/α)8-barrels, it has been proposed that the two extant genes could be the result of a cascade of gene elongation/domain shuffling events starting from an ancestor gene coding for just one (β/α) module. A gene elongation event has also been proposed for the evolution of hisB and hisD; structural analyses revealed the possibility of an early elongation event, resulting in the repetition of modules. Furthermore, it is quite possible that the gene elongations responsible for the evolution of the four proteins occurred before the earliest phylogenetic divergence. In conclusion, gene elongation events seem to have played a crucial role in the evolution of the histidine biosynthetic pathway, and they may have shaped the structures of many genes during the first steps of their evolution.

Although volcanoes represent extreme environments for life, they harbour bacterial communities. Vulcano Island (Aeolian Islands, Sicily) presents an intense fumarolic activity and widespread soil degassing, fed by variable amounts of magmatic gases (dominant at La Fossa Crater) and hydrothermal fluids (dominant at Levante Bay). The aim of this study is to analyse the microbial communities from the different environments of Vulcano Island and to evaluate their possible correlation with the composition of the gas emissions. Microbial analyses were carried out on soils and pioneer plants from both La Fossa Crater and Levante Bay. Total DNA has been extracted from all the samples and sequenced through Illumina MiSeq platform. The analysis of microbiome composition and the gases sampled in the same sites could suggest a possible correlation between the two parameters. We can suggest that the ability of different bacterial genera/species to survive in the same area might be due to the selection of particular genetic traits allowing the survival of these microorganisms. On the other side, the finding that microbial communities inhabiting different sites exhibiting different emission profiles are similar might be explained on the basis of a possible sharing of metabolic abilities related to the gas composition.