Moving toward the Inclusion of Epigenomics in Bacterial Genome Evolution: Perspectives and Challenges.

The universality of DNA methylation as an epigenetic regulatory mechanism belongs to all biological kingdoms. However, while eukaryotic systems have been the primary focus of DNA methylation studies, the molecular mechanisms in prokaryotes are less known. Nevertheless, DNA methylation in prokaryotes plays a pivotal role in many cellular processes such as defense systems against exogenous DNA, cell cycle dynamics, and gene expression, including virulence. Thanks to single-molecule DNA sequencing technologies, genome-wide identification of methylated DNA is becoming feasible on a large scale, providing the possibility to investigate more deeply the presence, variability, and roles of DNA methylation. Here, we present an overview of the multifaceted roles of DNA methylation in prokaryotes and suggest research directions and tools which can enable us to better understand the contribution of DNA methylation to prokaryotic genome evolution and adaptation. In particular, we emphasize the need to understand the presence and role of transgenerational inheritance, as well as the impact of epigenomic signatures on adaptation and genome evolution. Research directions and the importance of novel computational tools are underlined.

Cereals can trap endophytic bacteria with potential beneficial traits when grown ex-situ in harsh soils.

Microbial communities associated with plants growing in harsh conditions, including salinity and water deficiency, have developed adaptive features which permit them to grow and survive under extreme environmental conditions. In the present study, an ex-situ plant trapping method has been applied to collect the culturable microbial diversity associated with the soil from harsh and remote areas. Oryza sativa cv. Baldo and Triticum durum Primadur plants were used as recruiters, while the soil surrounding the roots of Oryza glaberrima plants from remote regions of Mali (West Africa) was used as substrate for their growth. The endophytic communities recruited by the two plant species belonged to Proteobacteria and Firmicutes, and the dominant genera were Bacillus, Kosakonia, and Enterobacter. These endophytes were characterized by analyzing some of the most common plant growth promoting traits. Halotolerant, inorganic phosphate-solubilizing and N-fixing strains were found, and some of them simultaneously showing these three traits. We verified that 'Baldo' recruited mostly halotolerant and P-solubilizers endophytes, while the endophytes selected by 'Primadur' were mainly N-fixers. The applied ex-situ plant trapping method allowed to isolate endophytes with potential beneficial traits that could be applied for the improvement of rice and wheat growth under adverse environmental conditions.

Species-specific gill's microbiome of eight crab species with different breathing adaptations.

Transitions to physically different environments, such as the water-to-land transition, proved to be the main drivers of relevant evolutionary events. Brachyuran crabs evolved remarkable morphological, behavioral, and physiological adaptations to terrestrial life. Terrestrial species evolved new respiratory structures devoted to replace or support the gills, a multifunctional organ devoted to gas exchanges, ion-regulation and nitrogen excretion. It was hypothesized that microorganisms associated with respiratory apparatus could have facilitated the processes of osmoregulation, respiration, and elimination of metabolites along this evolutionary transition. To test if crab species with different breathing adaptations may host similar microbial communities on their gills, we performed a comparative targeted-metagenomic analysis, selecting two marine and six terrestrial crabs belonging to different families and characterised by different breathing adaptations. We analysed anterior and posterior gills separately according to their different and specific roles. Regardless of their terrestrial or marine adaptations, microbial assemblages were strongly species-specific indicating a non-random association between the host and its microbiome. Significant differences were found in only two terrestrial species when considering posterior vs. anterior gills, without any association with species-specific respiratory adaptations. Our results suggest that all the selected species are strongly adapted to the ecological niche and specific micro-habitat they colonise.

Conserved organ-specific microbial assemblages in different populations of a terrestrial crab.

Microorganisms are ubiquitous in the environment and provide genetic and physiological functions to multicellular organisms. Knowledge on the associated microbiota is becoming highly relevant to understand the host's ecology and biology. Among invertebrates, many examples of endosymbiosis have been described, such as those in corals, ants, and termites. At present, however, little is known on the presence, diversity, and putative roles of the microbiota associated to brachyuran crabs in relation to their environment. In this work we investigated the associated microbiota of three populations of the terrestrial brachyuran crab Chiromantes haematocheir to find evidence of a conserved organ-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. Despite the presence of truly marine larval stages and the absence of a gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated with the gut and the gills of crabs from the different populations (with more than 15% of the genera detected specifically enriched only in one organ). These findings suggest the presence of possible functional roles of the organ-specific microbiota.

Ruthenium(II) Polypyridyl Complexes and Metronidazole Derivatives: A Powerful Combination in the Design of Photoresponsive Antibacterial Agents Effective under Hypoxic Conditions.

Ruthenium(II) polypyridyl complexes (RPCs) are gaining momentum in photoactivated chemotherapy (PACT), thanks to the possibility of overcoming the classical reliance on molecular oxygen of photodynamic therapy while preserving the selective drug activation by using light. However, notwithstanding the intriguing perspectives, the translation of such an approach in the development of new antimicrobials has been only barely considered. Herein, MTZH-1 and MTZH-2, two novel analogues of metronidazole (MTZ), a mainstay drug in the treatment of anaerobic bacterial infections, were designed and inserted in the strained ruthenium complexes [Ru(tpy)(dmp)(MTZ-1)]PF6 (Ru2) and [Ru(tpy)(dmp)(MTZ-2)]PF6 (Ru3) (tpy = terpyridine, dmp = 2,9-dimethyl-1,10-phenanthroline) (Chart 1). Analogously to the parental compound [Ru(tpy)(dmp)(5NIM)]PF6 (Ru1) (5-nitroimidazolate), the Ru(II)-imidazolate coordination of MTZ derivatives resulted in promising Ru(II) photocages, capable to easily unleash the bioactive ligands upon light irradiation and increase the antibacterial activity against Bacillus subtilis, which was chosen as a model of Gram-positive bacteria. The photoreleased 5-nitroimidazole-based ligands led to remarkable phototoxicities under hypoxic conditions (<1% O2), with the lead compound Ru3 that exhibited the highest potency across the series, being comparable to the one of the clinical drug MTZ. Besides, the chemical architectures of MTZ derivatives made their interaction with NimAunfavorable, being NimA a model of reductases responsible for bacterial resistance against 5-nitroimidazole-based antibiotics, thus hinting at their possible use to combat antimicrobial resistance. This work may therefore provide fundamental knowledge in the design of novel photoresponsive tools to be used in the fight against infectious diseases. For the first time, the effectiveness of the "photorelease antimicrobial therapy" under therapeutically relevant hypoxic conditions was demonstrated.

Independent origins and evolution of the secondary replicons of the class Gammaproteobacteria.

Multipartite genomes, consisting of more than one replicon, have been found in approximately 10 % of bacteria, many of which belong to the phylum Proteobacteria. Many aspects of their origin and evolution, and the possible advantages related to this type of genome structure, remain to be elucidated. Here, we performed a systematic analysis of the presence and distribution of multipartite genomes in the class Gammaproteobacteria, which includes several genera with diverse lifestyles. Within this class, multipartite genomes are mainly found in the order Alteromonadales (mostly in the genus Pseudoalteromonas) and in the family Vibrionaceae. Our data suggest that the emergence of secondary replicons in Gammaproteobacteria is rare and that they derive from plasmids. Despite their multiple origins, we highlighted the presence of evolutionary trends such as the inverse proportionality of the genome to chromosome size ratio, which appears to be a general feature of bacteria with multipartite genomes irrespective of taxonomic group. We also highlighted some functional trends. The core gene set of the secondary replicons is extremely small, probably limited to essential genes or genes that favour their maintenance in the genome, while the other genes are less conserved. This hypothesis agrees with the idea that the primary advantage of secondary replicons could be to facilitate gene acquisition through horizontal gene transfer, resulting in replicons enriched in genes associated with adaptation to different ecological niches. Indeed, secondary replicons are enriched both in genes that could promote adaptation to harsh environments, such as those involved in antibiotic, biocide and metal resistance, and in functional categories related to the exploitation of environmental resources (e.g. carbohydrates), which can complement chromosomal functions.

Effect of Site and Phenological Status on the Potato Bacterial Rhizomicrobiota.

The potato is the fourth major food crop in the world. Its cultivation can encounter problems, resulting in poor growth and reduced yield. Plant microbiota has shown an ability to increase growth and resistance. However, in the development of effective microbiota manipulation strategies, it is essential to know the effect of environmental variables on microbiota composition and function. Here, we aimed to identify the differential impact of the site of cultivation and plant growth stage on potato rhizosphere microbiota. We performed a 16S rRNA gene amplicon sequencing analysis of rhizospheric soil collected from potato plants grown at four sites in central Italy during two phenological stages. Rhizomicrobiota was mainly composed of members of phyla Acidobacteriota, Actinobacteriota, Chloroflexi, and Proteobacteria and was affected by both the site of cultivation and the plant stages. However, cultivation sites overcome the effect of plant phenological stages. The PiCRUST analysis suggested a high abundance of functions related to the biosynthesis of the siderophore enterobactin. The presence of site-specific taxa and functional profiling of the microbiota could be further exploited in long-term studies to evaluate the possibility of developing biomarkers for traceability of the products and to exploit plant growth-promoting abilities in the native potato microbiota.

Endophytes: Improving Plant Performance.

Endophytes represent microorganisms that reside within plant tissues, without typically causing adverse effects to the plants, for a substantial part of their life cycle, and are primarily known for their beneficial role to their host plant [...].

Microbial Genetics and Evolution.

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 [...].

Synthetic plant microbiota challenges in nonmodel species.

Plant-associated microbiota are becoming central in the development of ways to improve plant productivity and health. However, most research has focussed mainly on a few model plant species. It is essential to translate discoveries to the many nonmodel crops, allowing the design and application of effective synthetic microbiota.

Differential Response of Wheat Rhizosphere Bacterial Community to Plant Variety and Fertilization.

The taxonomic assemblage and functions of the plant bacterial community are strongly influenced by soil and host plant genotype. Crop breeding, especially after the massive use of nitrogen fertilizers which led to varieties responding better to nitrogen fertilization, has implicitly modified the ability of the plant root to recruit an effective bacterial community. Among the priorities for harnessing the plant bacterial community, plant genotype-by-microbiome interactions are stirring attention. Here, we analyzed the effect of plant variety and fertilization on the rhizosphere bacterial community. In particular, we clarified the presence in the bacterial community of a varietal effect of N and P fertilization treatment. 16S rRNA gene amplicon sequence analysis of rhizospheric soil, collected from four wheat varieties grown under four N-P fertilization regimes, and quantification of functional bacterial genes involved in the nitrogen cycle (nifH; amoA; nirK and nosZ) were performed. Results showed that variety played the most important role and that treatments did not affect either bacterial community diversity or bacterial phyla abundance. Variety-specific response of rhizosphere bacterial community was detected, both in relation to taxa (Nitrospira) and metabolic functions. In particular, the changes related to amino acid and aerobic metabolism and abundance of genes involved in the nitrogen cycle (amoA and nosZ), suggested that plant variety may lead to functional changes in the cycling of the plant-assimilable nitrogen.

Scent of a Symbiont: The Personalized Genetic Relationships of Rhizobium-Plant Interaction.

Many molecular signals are exchanged between rhizobia and host legume plants, some of which are crucial for symbiosis to take place, while others are modifiers of the interaction, which have great importance in the competition with the soil microbiota and in the genotype-specific perception of host plants. Here, we review recent findings on strain-specific and host genotype-specific interactions between rhizobia and legumes, discussing the molecular actors (genes, gene products and metabolites) which play a role in the establishment of symbiosis, and highlighting the need for research including the other components of the soil (micro)biota, which could be crucial in developing rational-based strategies for bioinoculants and synthetic communities' assemblage.

A Meta-Analysis Approach to Defining the Culturable Core of Plant Endophytic Bacterial Communities.

Endophytic bacteria are key members of the plant microbiome, which phylogenetic diversity has been widely described through next-generation sequencing technologies in the last decades. On the other side, a synopsis of culturable plant endophytic bacteria is still lacking in the literature. However, culturability is necessary for biotechnology innovations related to sustainable agriculture, such as biofertilizer and biostimulant agents' development. In this review, 148 scientific papers were analyzed to establish a large data set of cultured endophytic bacteria, reported at the genus level, inhabiting different compartments of wild and farmed plants, sampled around the world from different soil types and isolated using various growth media. To the best of our knowledge, this work provides the first overview of the current repertoire of cultured plant endophytic bacteria. Results indicate the presence of a recurrent set of culturable bacterial genera regardless of factors known to influence the plant bacterial community composition and the growth media used for the bacterial isolation. Moreover, a wide variety of bacterial genera that are currently rarely isolated from the plant endosphere was identified, demonstrating that culturomics can catch previously uncultured bacteria from the plant microbiome, widening the panorama of strains exploitable to support plant holobiont health and production.

DNA Methylation in Ensifer Species during Free-Living Growth and during Nitrogen-Fixing Symbiosis with Medicago spp.

Methylation of specific DNA sequences is ubiquitous in bacteria and has known roles in immunity and regulation of cellular processes, such as the cell cycle. Here, we explored DNA methylation in bacteria of the genus Ensifer, including its potential role in regulating terminal differentiation during nitrogen-fixing symbiosis with legumes. Using single-molecule real-time sequencing, six genome-wide methylated motifs were identified across four Ensifer strains, five of which were strain-specific. Only the GANTC motif, recognized by the cell cycle-regulated CcrM methyltransferase, was methylated in all strains. In actively dividing cell cultures, methylation of GANTC motifs increased progressively from the ori to ter regions in each replicon, in agreement with a cell cycle-dependent regulation of CcrM. In contrast, there was near full genome-wide GANTC methylation in the early stage of symbiotic differentiation. This was followed by a moderate decrease in the overall extent of methylation and a progressive decrease in chromosomal GANTC methylation from the ori to ter regions in later stages of differentiation. Based on these observations, we suggest that CcrM activity is dysregulated and constitutive during terminal differentiation, which we hypothesize is a driving factor for endoreduplication of terminally differentiated bacteroids. IMPORTANCE Nitrogen fixation by rhizobia in symbiosis with legumes is economically and ecologically important. The symbiosis can involve a complex bacterial transformation-terminal differentiation-that includes major shifts in the transcriptome and cell cycle. Epigenetic regulation is an important regulatory mechanism in diverse bacteria; however, the roles of DNA methylation in rhizobia and symbiotic nitrogen fixation have been poorly investigated. We show that aside from cell cycle regulation, DNA methyltransferases are unlikely to have conserved roles in the biology of bacteria of the genus Ensifer. However, we present evidence consistent with an interpretation that the cell cycle methyltransferase CcrM is dysregulated during symbiosis, which we hypothesize may be a key factor driving the cell cycle switch in terminal differentiation required for effective symbioses.

Crossing Bacterial Genomic Features and Methylation Patterns with MeStudio: An Epigenomic Analysis Tool.

DNA methylation is one of the most observed epigenetic modifications. It is present in eukaryotes and prokaryotes and is related to several biological phenomena, including gene flow and adaptation to environmental conditions. The widespread use of third-generation sequencing technologies allows direct and easy detection of genome-wide methylation profiles, offering increasing opportunities to understand and exploit the epigenomic landscape of individuals and populations. Here, we present a pipeline named MeStudio, with the aim of analyzing and combining genome-wide methylation profiles with genomic features. Outputs report the presence of DNA methylation in coding sequences (CDSs) and noncoding sequences, including both intergenic sequences and sequences upstream of the CDS. We apply this novel tool, showing the usage and performance of MeStudio, on a set of single-molecule real-time sequencing outputs from strains of the bacterial species Sinorhizobium meliloti.

Nitroimidazole-Based Ruthenium(II) Complexes: Playing with Structural Parameters to Design Photostable and Light-Responsive Antibacterial Agents.

5-Nitroimidazole (5NIMH), chosen as a molecular model of nitroimidazole derivatives, which represent a broad-spectrum class of antimicrobials, was incorporated into the ruthenium complexes [Ru(tpy)(phen)(5NIM)]PF6 (1) and [Ru(tpy)(dmp)(5NIM)]PF6 (2) (tpy = terpyridine, phen = phenanthroline, dmp = 2,9-dimethyl-1,10-phenanthroline). Besides the uncommon metal coordination of 5-nitroimidazole in its imidazolate form (5NIM), the different architectures of the spectator ligands (phen and dmp) were exploited to tune the "mode of action" of the resulting complexes, passing from a photostable compound where the redox properties of 5NIMH are preserved (1) to one suitable for the nitroimidazole phototriggered release (2) and whose antibacterial activity against B. subtilis, chosen as cellular model, is effectively improved upon light exposure. This study may provide a fundamental knowledge on the use of Ru(II)-polypyridyl complexes to incorporate and/or photorelease biologically relevant nitroimidazole derivatives in the design of a novel class of antimicrobials.

Lung and Gut Microbiota Changes Associated with Pseudomonas aeruginosa Infection in Mouse Models of Cystic Fibrosis.

Cystic fibrosis (CF) disease leads to altered lung and gut microbiomes compared to healthy subjects. The magnitude of this dysbiosis is influenced by organ-specific microenvironmental conditions at different stages of the disease. However, how this gut-lung dysbiosis is influenced by Pseudomonas aeruginosa chronic infection is unclear. To test the relationship between CFTR dysfunction and gut-lung microbiome under chronic infection, we established a model of P. aeruginosa infection in wild-type (WT) and gut-corrected CF mice. Using 16S ribosomal RNA gene, we compared lung, stool, and gut microbiota of C57Bl/6 Cftr tm1UNCTgN(FABPCFTR) or WT mice at the naïve state or infected with P. aeruginosa. P. aeruginosa infection influences murine health significantly changing body weight both in CF and WT mice. Both stool and gut microbiota revealed significantly higher values of alpha diversity in WT mice than in CF mice, while lung microbiota showed similar values. Infection with P. aeruginosa did not changed the diversity of the stool and gut microbiota, while a drop of diversity of the lung microbiota was observed compared to non-infected mice. However, the taxonomic composition of gut microbiota was shown to be influenced by P. aeruginosa infection in CF mice but not in WT mice. This finding indicates that P. aeruginosa chronic infection has a major impact on microbiota diversity and composition in the lung. In the gut, CFTR genotype and P. aeruginosa infection affected the overall diversity and taxonomic microbiota composition, respectively. Overall, our results suggest a cross-talk between lung and gut microbiota in relation to P. aeruginosa chronic infection and CFTR mutation.

Endophytes from African Rice (Oryza glaberrima L.) Efficiently Colonize Asian Rice (Oryza sativa L.) Stimulating the Activity of Its Antioxidant Enzymes and Increasing the Content of Nitrogen, Carbon, and Chlorophyll.

Bacterial endophytes support the adaptation of host plants to harsh environments. In this study, culturable bacterial endophytes were isolated from the African rice Oryza glaberrima L., which is well-adapted to grow with poor external inputs in the tropical region of Mali. Among these, six N-fixer strains were used to inoculate O. glaberrima RAM133 and the Asian rice O. sativa L. cv. Baldo, selected for growth in temperate climates. The colonization efficiency and the N-fixing activity were evaluated and compared for the two rice varieties. Oryza sativa-inoculated plants showed a fairly good colonization efficiency and nitrogenase activity. The inoculation of Oryza sativa with the strains Klebsiella pasteurii BDA134-6 and Phytobacter diazotrophicus BDA59-3 led to the highest nitrogenase activity. In addition, the inoculation of 'Baldo' plants with the strain P. diazotrophicus BDA59-3 led to a significant increase in nitrogen, carbon and chlorophyll content. Finally, 'Baldo' plants inoculated with Kl. pasteurii BDA134-6 showed the induction of antioxidant enzymes activity and the maintenance of nitrogen-fixation under salt stress as compared to the unstressed controls. As these endophytes efficiently colonize high-yielding crop varieties grown in cold temperate climates, they become good candidates to promote their growth under unfavorable conditions.

Defining the resilience of the human salivary microbiota by a 520-day longitudinal study in a confined environment: the Mars500 mission.

BACKGROUND: The human microbiota plays several roles in health and disease but is often difficult to determine which part is in intimate relationships with the host vs. the occasional presence. During the Mars500 mission, six crewmembers lived completely isolated from the outer world for 520 days following standardized diet regimes. The mission constitutes the first spaceflight simulation to Mars and was a unique experiment to determine, in a longitudinal study design, the composition and importance of the resident vs. a more variable microbiota-the fraction of the human microbiota that changes in time and according to environmental conditions-in humans. METHODS: Here, we report the characterization of the salivary microbiota from 88 samples taken during and after Mars500 mission for a total of 720 days. Amplicon sequencing of the V3-V4 regions of 16S rRNA gene was performed, and results were analyzed monitoring the diversity of the microbiota while evaluating the effect of the three main variables present in the experimental system: time, diet, and individuality of each subject. RESULTS: Results showed statistically significant effects for either time, diet, and individuality of each subject. The main contribution came from the individuality of each subject, emphasizing salivary microbiota-personalized features, and an individual-based resilience of the microbiota. CONCLUSIONS: The uniqueness of Mars500 mission, allowed to dampen the effect of environmental variables on salivary microbiota, highlighting its pronounced personalization even after sharing the same physical space for more than a year. Video abstract.