The Feather Moss Hylocomium splendens Affects the Transcriptional Profile of a Symbiotic Cyanobacterium in Relation to Acquisition and Turnover of Key Nutrients.

Moss-cyanobacteria symbioses were proposed to be based on nutrient exchange, with hosts providing C and S while bacteria provide N, but we still lack understanding of the underlying molecular mechanisms of their interactions. We investigated how contact between the ubiquitous moss Hylocomium splendens and its cyanobiont affects nutrient-related gene expression of both partners. We isolated a cyanobacterium from H. splendens and co-incubated it with washed H. splendens shoots. Cyanobacterium and moss were also incubated separately. After 1 week, we performed acetylene reduction assays to estimate N2 fixation and RNAseq to evaluate metatranscriptomes. Genes related to N2 fixation and the biosynthesis of several amino acids were up-regulated in the cyanobiont when hosted by the moss. However, S-uptake and the biosynthesis of the S-containing amino acids methionine and cysteine were down-regulated in the cyanobiont while the degradation of selenocysteine was up-regulated. In contrast, the number of differentially expressed genes in the moss was much lower, and almost no transcripts related to nutrient metabolism were affected. It is possible that, at least during the early stage of this symbiosis, the cyanobiont receives few if any nutrients from the host in return for N, suggesting that moss-cyanobacteria symbioses encompass relationships that are more plastic than a constant mutualist flow of nutrients.

Chemical Stimulation of Heterocyte Differentiation by the Feather Moss Hylocomium splendens: a Potential New Step in Plant-Cyanobacteria Symbioses.

Cyanobacteria associated with mosses play a key role in the nitrogen (N) cycle in unpolluted ecosystems. Mosses have been found to release molecules that induce morphophysiological changes in epiphytic cyanobionts. Nevertheless, the extent of moss influence on these microorganisms remains unknown. To evaluate how mosses or their metabolites influence N2 fixation rates by cyanobacteria, we assessed the nitrogenase activity, heterocyte frequency and biomass of a cyanobacterial strain isolated from the feather moss Hylocomium splendens and a non-symbiotic strain when they were either growing by themselves, together with H. splendens or exposed to H. splendens water, acetone, ethanol, or isopropanol extracts. The same cyanobacterial strains were added to another moss (Taxiphyllum barbieri) and a liverwort (Monosolenium tenerum) to assess if these bryophytes affect N2 fixation differently. Although no significant increases in nitrogenase activity by the cyanobacteria were observed when in contact with H. splendens shoots, both the symbiotic and non-symbiotic cyanobacteria increased nitrogenase activity as well as heterocyte frequency significantly upon exposure to H. splendens ethanol extracts. Contact with T. barbieri shoots, on the other hand, did lead to increases in nitrogenase activity, indicating low host-specificity to cyanobacterial activity. These findings suggest that H. splendens produces heterocyte-differentiating factors (HDFs) that are capable of stimulating cyanobacterial N2 fixation regardless of symbiotic competency. Based on previous knowledge about the chemical ecology and dynamics of moss-cyanobacteria interactions, we speculate that HDF expression by the host takes place in a hypothetical new step occurring after plant colonization and the repression of hormogonia.

Mining of Cyanobacterial Genomes Indicates Natural Product Biosynthetic Gene Clusters Located in Conjugative Plasmids.

Microbial natural products are compounds with unique chemical structures and diverse biological activities. Cyanobacteria commonly possess a wide range of biosynthetic gene clusters (BGCs) to produce natural products. Although natural product BGCs have been found in almost all cyanobacterial genomes, little attention has been given in cyanobacterial research to the partitioning of these biosynthetic pathways in chromosomes and plasmids. Cyanobacterial plasmids are believed to disperse several natural product BGCs, such as toxins, by plasmids through horizontal gene transfer. Therefore, plasmids may confer the ability to produce toxins and may play a role in the evolution of diverse natural product BGCs from cyanobacteria. Here, we performed an analysis of the distribution of natural product BGCs in 185 genomes and mapped the presence of genes involved in the conjugation in plasmids. The 185 analyzed genomes revealed 1817 natural products BGCs. Individual genomes contained 1-42 biosynthetic pathways (mean 8), 95% of which were present in chromosomes and the remaining 5% in plasmids. Of the 424 analyzed cyanobacterial plasmids, 12% contained homologs of genes involved in conjugation and natural product biosynthetic pathways. Among the biosynthetic pathways in plasmids, manual curation identified those to produce aeruginosin, anabaenopeptin, ambiguine, cryptophycin, hassallidin, geosmin, and microcystin. These compounds are known toxins, protease inhibitors, odorous compounds, antimicrobials, and antitumorals. The present study provides in silico evidence using genome mining that plasmids may be involved in the distribution of natural product BGCs in cyanobacteria. Consequently, cyanobacterial plasmids have importance in the context of biotechnology, water management, and public health risk assessment. Future research should explore in vivo conjugation and the end products of natural product BGCs in plasmids via chemical analyses.

TnCentral: a Prokaryotic Transposable Element Database and Web Portal for Transposon Analysis.

We describe here the structure and organization of TnCentral (https://tncentral.proteininformationresource.org/ [or the mirror link at https://tncentral.ncc.unesp.br/]), a web resource for prokaryotic transposable elements (TE). TnCentral currently contains ∼400 carefully annotated TE, including transposons from the Tn3, Tn7, Tn402, and Tn554 families; compound transposons; integrons; and associated insertion sequences (IS). These TE carry passenger genes, including genes conferring resistance to over 25 classes of antibiotics and nine types of heavy metal, as well as genes responsible for pathogenesis in plants, toxin/antitoxin gene pairs, transcription factors, and genes involved in metabolism. Each TE has its own entry page, providing details about its transposition genes, passenger genes, and other sequence features required for transposition, as well as a graphical map of all features. TnCentral content can be browsed and queried through text- and sequence-based searches with a graphic output. We describe three use cases, which illustrate how the search interface, results tables, and entry pages can be used to explore and compare TE. TnCentral also includes downloadable software to facilitate user-driven identification, with manual annotation, of certain types of TE in genomic sequences. Through the TnCentral homepage, users can also access TnPedia, which provides comprehensive reviews of the major TE families, including an extensive general section and specialized sections with descriptions of insertion sequence and transposon families. TnCentral and TnPedia are intuitive resources that can be used by clinicians and scientists to assess TE diversity in clinical, veterinary, and environmental samples. IMPORTANCE The ability of bacteria to undergo rapid evolution and adapt to changing environmental circumstances drives the public health crisis of multiple antibiotic resistance, as well as outbreaks of disease in economically important agricultural crops and animal husbandry. Prokaryotic transposable elements (TE) play a critical role in this. Many carry "passenger genes" (not required for the transposition process) conferring resistance to antibiotics or heavy metals or causing disease in plants and animals. Passenger genes are spread by normal TE transposition activities and by insertion into plasmids, which then spread via conjugation within and across bacterial populations. Thus, an understanding of TE composition and transposition mechanisms is key to developing strategies to combat bacterial pathogenesis. Toward this end, we have developed TnCentral, a bioinformatics resource dedicated to describing and exploring the structural and functional features of prokaryotic TE whose use is intuitive and accessible to users with or without bioinformatics expertise.

Residual biomass from surfactin production is a source of arginase and adsorbed surfactin that is useful for environmental remediation.

Lipopeptides are important secondary metabolites produced by microbes. They find applications in environmental decontamination and in the chemical, pharmaceutical and food industries. However, their production is expensive. In the present work we propose three strategies to lower the production costs of surfactin. First, the coproduction of surfactin and arginase in a single growth. Second, extract the fraction of surfactin that adsorbs to the biomass and is removed from the growth medium through centrifugation. Third, use microbial biomass for the remediation of organic and inorganic contaminants. The coproduction of surfactin and arginase was evaluated by factorial design experiments using the LB medium supplemented with arginine. The best conditions for surfactin production were 22 h of growth at 37 °C using LB supplemented with arginine 7.3 g/L. Almost similar conditions were found to produce highest levels of arginase, 24 h and 6.45 g/L arginine. Decontamination of phenol and copper from artificial samples was attained by treatment with residues from lipopeptide production. Thus, cell suspensions and wash-waters used to extract surfactin from the biomass. Cell suspensions were used to successfully remove hydroquinone. Cell suspensions and wash-waters containing surfactin were successfully used to recover copper from solution. Specific monitoring methods were used for phenol and metal solutions, respectively a biosensor based on tyrosinase and either atomic absorption flame ionization spectrometry or absorbance coupled to the Arduino™ platform. Therefore, we report three alternative strategies to lower the production costs in lipopeptide production, which include the effective recovery of copper and phenol from contaminated waters using residues from surfactin production. Sustainable and profitable production of surfactin can be achieved by a coproduction strategy of lipopeptides and enzymes. Lipopeptides are collected in the supernatant and enzymes in the biomass. In addition, lipopeptides that coprecipitate with biomass can be recovered by washing. Lipopeptide wash-waters find applications in remediation and cells can also be used for environmental decontamination.

Unraveling a Lignocellulose-Decomposing Bacterial Consortium from Soil Associated with Dry Sugarcane Straw by Genomic-Centered Metagenomics.

Second-generation biofuel production is in high demand, but lignocellulosic biomass' complexity impairs its use due to the vast diversity of enzymes necessary to execute the complete saccharification. In nature, lignocellulose can be rapidly deconstructed due to the division of biochemical labor effectuated in bacterial communities. Here, we analyzed the lignocellulolytic potential of a bacterial consortium obtained from soil and dry straw leftover from a sugarcane milling plant. This consortium was cultivated for 20 weeks in aerobic conditions using sugarcane bagasse as a sole carbon source. Scanning electron microscopy and chemical analyses registered modification of the sugarcane fiber's appearance and biochemical composition, indicating that this consortium can deconstruct cellulose and hemicellulose but no lignin. A total of 52 metagenome-assembled genomes from eight bacterial classes (Actinobacteria, Alphaproteobacteria, Bacilli, Bacteroidia, Cytophagia, Gammaproteobacteria, Oligoflexia, and Thermoleophilia) were recovered from the consortium, in which ~46% of species showed no relevant modification in their abundance during the 20 weeks of cultivation, suggesting a mostly stable consortium. Their CAZymes repertoire indicated that many of the most abundant species are known to deconstruct lignin (e.g., Chryseobacterium) and carry sequences related to hemicellulose and cellulose deconstruction (e.g., Chitinophaga, Niastella, Niabella, and Siphonobacter). Taken together, our results unraveled the bacterial diversity, enzymatic potential, and effectiveness of this lignocellulose-decomposing bacterial consortium.

Amazonocrinis nigriterrae gen. nov., sp. nov., Atlanticothrix silvestris gen. nov., sp. nov. and Dendronalium phyllosphericum gen. nov., sp. nov., nostocacean cyanobacteria from Brazilian environments.

The cyanobacterial genus Nostoc is an important contributor to carbon and nitrogen bioavailability in terrestrial ecosystems and a frequent partner in symbiotic relationships with non-diazotrophic organisms. However, since this currently is a polyphyletic genus, the diversity of Nostoc-like cyanobacteria is considerably underestimated at this moment. While reviewing the phylogenetic placement of previously isolated Nostoc-like cyanobacteria originating from Brazilian Amazon, Caatinga and Atlantic forest samples, we detected 17 strains isolated from soil, freshwater, rock and tree surfaces presenting patterns that diverged significantly from related strains when ecological, morphological, molecular and genomic traits were also considered. These observations led to the identification of the evaluated strains as representative of three novel nostocacean genera and species: Amazonocrinis nigriterrae gen. nov., sp. nov.; Atlanticothrix silvestris gen. nov., sp. nov.; and Dendronalium phyllosphericum gen. nov., sp. nov., which are herein described according to the rules of the International Code of Nomenclature for algae, fungi and plants. This finding highlights the great importance of tropical and equatorial South American ecosystems for harbouring an unknown microbial diversity in the face of the anthropogenic threats with which they increasingly struggle.

Kryptousia macronema gen. nov., sp. nov. and Kryptousia microlepis sp. nov., nostocalean cyanobacteria isolated from phyllospheres.

Tropical ecosystems worldwide host very diverse microbial communities, but are increasingly threatened by deforestation and climate change. Thus, characterization of biodiversity in these environments, and especially of microbial communities that show unique adaptations to their habitats, is a very urgent matter. Information about representatives of the phylum Cyanobacteria in tropical environments is scarce, even though they are fundamental primary producers that help other microbes to thrive in nutrient-depleted habitats, including phyllospheres. In order to increase our knowledge of cyanobacterial diversity, a study was conducted to characterize isolates from Avicennia schaueriana and Merostachys neesii leaves collected at a mangrove and an Atlantic forest reserve located at the littoral of São Paulo state, south-east Brazil. The morphological, ultrastructural, phylogenetic, molecular and ecological features of the strains led to the recognition of the new genus Kryptousia, comprising two new species, Kryptousiamacronema gen. nov., sp. nov. and Kryptousiamicrolepis sp. nov., described here according to the International Code of Nomenclature for algae, fungi and plants. The new genus and species were classified in the nostocalean family Tolypotrichaceae. This finding advances knowledge on the microbial diversity of South American ecosystems and sheds further light on the systematics of cyanobacteria.

Evaluating methods for purifying cyanobacterial cultures by qPCR and high-throughput Illumina sequencing.

Cyanobacteria are commonly found in association with other microorganisms, which constitutes a great challenge during the isolation of cyanobacterial strains. Although several methods have been published for obtaining axenic cyanobacterial cultures, their efficiency is usually evaluated by observing the growth of non-cyanobacteria in culture media. In order to verify whether uncultured bacteria should be a concern during cyanobacterial isolation, this work aimed to detect by molecular methods sequences from cyanobacteria and other bacteria present before and after a technique for obtaining axenic cultures from plating and exposure of Fischerella sp. CENA161 akinetes to the Extran detergent and sodium hypochlorite. Solutions containing 0.5, 1, and 2% sodium hypochlorite were able to remove contaminant bacterial CFUs from the culture. However, qPCR pointed that the quantity of sequences amplified with universal bacteria primers was higher than the number of cyanobacteria-specific sequences before and after treatments. The presence of uncultured bacteria in post-hypochlorite cultures was confirmed by high-throughput Illumina sequencing. These results suggest that culturing may overlook the presence of uncultured bacteria associated to cyanobacterial strains and is not sufficient for monitoring the success of cyanobacterial isolation by itself. Molecular methods such as qPCR could be employed as an additional measure for evaluating axenity in cyanobacterial strains.

Aliterella atlantica gen. nov., sp. nov., and Aliterella antarctica sp. nov., novel members of coccoid Cyanobacteria.

Two Cyanobacteria isolated from South Atlantic Ocean continental shelf deep water and from a marine green algae inhabiting the Admiralty Bay, King George Island, Antarctica were investigated based on morphological and ultrastructural traits, phylogeny of 16S rRNA gene sequences, secondary structure of the 16S-23S internal transcribed spacer regions and phylogenomic analyses. The majority of these evaluations demonstrated that both strains differ from the genera of cyanobacteria with validly published names and, therefore, supported the description of the novel genus as Aliterella gen. nov. The identity and phylogeny of 16S rRNA gene sequences, together with the secondary structure of D1D1' and BoxB intergenic regions, further supported the two strains representing distinct species: Aliterella atlantica gen. nov., sp. nov. (type SP469036, strain CENA595T) and Aliterella antarctica sp. nov. (type SP469035, strain CENA408T). The phylogenomic analysis of A. atlantica sp. nov. CENA595T, based on 21 protein sequences, revealed that this genus belongs to the cyanobacterial order Chroococcidiopsidales. The isolation and cultivation of two geographically distant unicellular members of a novel cyanobacterial genus and the sequenced genome of the type strain bring new insights into the current classification of the coccoid group, and into the reconstruction of their evolutionary history.

Cyanobacterial diversity in the phyllosphere of a mangrove forest.

The cyanobacterial community colonizing phyllosphere in a well-preserved Brazilian mangrove ecosystem was assessed using cultivation-independent molecular approaches. Leaves of trees that occupy this environment (Rhizophora mangle,Avicennia schaueriana and Laguncularia racemosa) were collected along a transect beginning at the margin of the bay and extending upland. The results demonstrated that the phyllosphere of R. mangle and L. racemosa harbor similar assemblages of cyanobacteria at each point along the transect. A. schaueriana, found only in the coastal portions of the transect, was colonized by assemblages with lower richness than the other trees. However, the results indicated that spatial location was a stronger driver of cyanobacterial community composition than plant species. Distinct cyanobacterial communities were observed at each location along the coast-to-upland transect. Clone library analysis allowed identification of 19 genera of cyanobacteria and demonstrated the presence of several uncultivated taxa. A predominance of sequences affiliated with the orders Nostocales and Oscillatoriales was observed, with a remarkable number of sequences similar to genera Symphyonemopsis/Brasilonema (order Nostocales). The results demonstrated that phyllosphere cyanobacteria in this mangrove forest ecosystem are influenced by environmental conditions as the primary driver at the ecosystem scale, with tree species exerting some effect on community structure at the local scale.