34 research outputs found
Genômica e metabolômica secundária de cianobactérias de lagoas salino-alcalinas do Pantanal (Brasil)
No full textSecondary metabolites are molecules usually described as non-essential for the basal metabolism of an organism, however, they are highly specialized, with a wide spectrum of activities that allow competitive gain for the organism. Due to this high bioactive diversity, many of these molecules have already been described as natural products, which are compounds used in several industries and have great economic and pharmacological interest. In the last decade, there has been a growing interest for cyanobacterial natural products, due to the high adaptative capacity of these microorganisms and the methodological developments in the fields of genomic and chemical studies, which allow accessing new information about these understudied compounds. In this study, genomes of isolated cyanobacteria and the metagenomes from the extreme environment of the Brazilian Pantanal soda lakes were analyzed, in order to determine the potential for the synthesis of natural products of these organisms, using the combination of large-scale genomic sequencing and chromatography-coupled mass-spectrometry. The genomic analyzes allowed the robust classification of the cyanobacterias using phylogenomic methodologies, with the submission of the first genome of an Anabaenopsis species, and the prediction of gene clusters of secondary metabolites, which identified potential genes associated with the routes for bacteriocin synthesis, NRPS/PKS, cyanobactins, terpenoids, and others, which among these include the grouping of the cyanotoxin saxitoxin identified in the metagenome. Molecular networking analyzes based on mass spectrometry data identified a diverse potential in the six strains isolates for the synthesis of different types of peptides and carotenoids of pharmacological interest. These results demonstrate the potential richness for the exploration of natural products of cyanobacterias from underexplored extreme environments, and also provide possibilities for new studies, which can analyze extracts of these cyanobacterias in order to test different bioactivities.Metabólitos secundários são moléculas normalmente descritas como não essenciais para metabolismo basal de um organismo, porém são altamente especializadas, com um amplo espectro de atividades que permitem ganho competitivo do organismo. Devido a essa alta diversidade bioativa, muitas dessas moléculas já foram descritas como produtos naturais, que são compostos empregados em diversas indústrias e possuem grande interesse econômico e farmacológico. Na última década, houve um crescente interesse por produtos naturais cianobacterianos, devido à alta capacidade adaptativa desses microrganismos e às evoluções metodológicas nos campos de estudo genômicos e químicos, que permitem acessar novas informações acerca desses compostos pouco conhecidos. Neste estudo, genomas de cianobactérias isoladas e do metagenoma do ambiente extremo das lagoas salino-alcalinas do Pantanal brasileiro foram analisadas para conhecer o potencial de síntese de produtos naturais desses organismos, utilizando a combinação de sequenciamento genômicos de larga escala e cromatografia acoplada a espectrometria de massas. As análises genômicas permitiram a robusta classificação das cianobactérias utilizando metodologias filogenômicas, com a submissão do primeiro genoma de uma espécie do Anabaenopsis, e a predição de agrupamentos gênicos de metabólitos secundários, que identificou potenciais genes associados as rotas para síntese de bacteriocinas, NRPS/PKS, cianobactinas, terpenoides, além de outros, que dentre estes, inclui o agrupamento da cianotoxina saxitoxina identificado no metagenoma. As análises de molecular networking com base nos dados de espectrometria de massas identificou potencial diverso nas seis linhagens isoladas para a síntese de diferentes tipos de peptídeos e carotenóides de interesse farmacológico. Esses resultados demonstram a potencial riqueza para a exploração de produtos naturais de cianobactérias de ambientes extremos ainda pouco explorados, e também fornecem possibilidades para novos estudos, que possam analisar extratos dessas cianobactérias com a finalidade testar diferentes biatividades
Host species, microhabitat and time of the year modulate the community structure of diazotrophic microorganisms in the Amazon rainforest
No full textÉ crescente o reconhecimento da importância de micro-organismos diazotróficos assimbióticos em florestas tropicais. Nos últimos anos, tem sido demostrado que suas contribuições para o ganho de nitrogênio em ecossistemas naturais podem ser até maiores do que as contribuições dos sistemas simbióticos. No entanto, a diversidade e os mecanismos que determinam a estruturação das comunidades de diazotróficos assimbióticos são pouco conhecidos. Para compreender melhor a diversidade e a estruturação de suas comunidades na Floresta Amazônica, os diazotróficos assimbióticos da filosfera, serapilheira e rizosfera de nove espécies arbóreas em três épocas distintas foram avaliados utilizando-se sequenciamento em larga escala dos genes rRNA 16S e nifH. Os resultados mostraram que cada micro-habitat seleciona comunidades diazotróficas distintas, as quais variam também com a época do ano. As comunidades de diazotróficos também mostraram ser dependentes dos táxons das árvores, com o maior efeito seletivo na filosfera. Para todas as condições estudadas, as comunidades diazotróficas apresentaram altos índices de diversidade e riqueza, com os maiores índices de Shannon e Chao1 na Serapilheira (9,57 e 5222, respectivamente), em relação a filosfera (7,4 e 3145, respectivamente) e a rizosfera (8,65 e 2868, respectivamente). A maior proporção de bactérias diazotróficas em relação a comunidade total de bactérias, com base na abundância de sequências do gene rRNA 16S, foi observada na filosfera (23%), em relação a serapilheira (20%) e rizosfera (18%). De uma maneira geral, essa proporção relativa não foi afetada pela época de amostragem, muito embora as estruturas das comunidades tenham variado, sugerindo existência de redundância funcional para FBN. Os três micro-habitats mostraram dominância do filo Proteobacteria, com frequências aproximadas a 81% na filosfera, 93% na rizosfera de 82% na serapilheira. O filo Cyanobacteria apresentou frequências relevantes na filosfera e serapilheira, com aproximadamente 17% e 5%, respectivamente. Firmicutes mostrou frequências, na serapilheira e rizosfera, de aproximadamente 12% e 5%, respectivamente. Estes resultados mostram que os micro-organismos diazotróficos assimbóticos associados com as árvores podem ter um importante papel na entrada do nitrogênio em florestas tropicais e que a estrutura de tais comunidades é determinada pelo seu micro-habitat, hospedeiro e época do ano.There is increasing recognition of the importance of assimbiotic diazotrophic microorganisms in tropical forests. In recent years, it has been shown that their contributions to the nitrogen inputs can be even higher than the contributions of symbiotic systems in natural ecosystems. However, the diversity and the mechanisms driving the assembling of the communities of assimbiotic diazotrophs in tropical forests are poorly understood. To better understand the diversity and assembling of the diazothrophs communities in the Amazon forest, the assimbiotic diazotrophs in the phyllosphere, litter and rhizosphere from nine tree species in three distinct times of year were evaluated using large-scale sequencing of 16S rRNA and nifH genes. The results showed that each microhabitat selected distinct diazotrophic communities, which were also affected by the time of year. The structure of the communities of putative diazotrophs were also dependent on the tree taxa, with greater selection effect on the phyllosphere. For all conditions studied, the communities of putiative diazothrophs showed high diversity indexes and OTU richness, with the highest Shannon and Chao1 indexes detected for the litter (9.57 and 5222, respectively), as compared to phyllosphere (7.4 and 3145, respectively) and rhizosphere (8.65 and 2868, respectively). The highest proportion of diazotrophic bacteria in relation to the total bacteria, based on the abundance of sequences of 16S rRNA genes, was observed in the phyllosphere (23%), as compared to litter (20%) and rhizosphere (18%). Overall, the relative abundance of dizothrophic bacteria in the bacterial community was not affected by the sampling time, even though the community structures varied, suggesting the occurrence of functional redundancy for BNF. In the three microhabitats, dominance of the phylum Proteobacteria, with frequencies of approximately 81% in the phyllosphere, 93% in the rhizosphere and 82% in the litter, was observed. Cyanobacteria showed significantly higher frequencies in the phyllosphere and litter, approximately 17% and 5%, respectively, as compared to rhizosphere. Firmicutes also showed high frequencies in the litter and rhizosphere, approximately 12 % and 5%, respectively. These results showed that the assimbiotic putative diazotrophic microorganisms associated with trees may play an important role in the input of nitrogen in tropical forests and that the structure of their communities is determined by the microhabitat, host taxon and time of year
Mucilaginibacter sp. Strain Metal(loid) and Antibiotic Resistance Isolated from Estuarine Soil Contaminated Mine Tailing from the Fundao Dam
No full textIdentification of a homoarginine biosynthetic gene from a microcystin biosynthetic pathway in Fischerella sp. PCC 9339
No full textMicrocystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein phosphatases 1 and 2A and are responsible for the toxicosis and death of wild and domestic animals around the world. Microcystins are synthesized on large enzyme complexes comprised of peptide synthetases, polyketide synthases, and additional modifying enzymes. Bioinformatic analysis identified the presence of an additional uncharacterized enzyme in the microcystin (mcy) biosynthetic gene cluster in Fischerella sp. PCC 9339, which we named McyK, that lacked a clearly defined role in the biosynthesis of microcystin. Further bioinformatic analysis suggested that McyK belongs to the inosamine-phosphate amidinotransferase family and could be involved in synthesizing homo amino acids. Quadrupole time-of-flight tandem mass spectrometry (Q-TOFMS/MS) analysis confirmed that Fischerella sp. PCC 9339 produces MC-Leucine2-Homoarginine4(MC-LHar) and [Aspartic acid3]MC-Leucine2-Homoarginine4 ([Asp3]MC-LHar) as the dominant chemical variants. We hypothesized that the McyK enzyme might be involved in the production of microcystin variants containing homoarginine (Har) in the strain. Heterologous expression of a codon-optimized mcyK gene in Escherichia coli confirmed that McyK is responsible for the synthesis of l-Har. These results confirm the production of MC-LHar, a novel microcystin chemical variant [Asp3]MC-LHar, and a new microcystin biosynthetic enzyme involved in supply of the rare homo-amino acid Har to the microcystin biosynthetic pathway in Fischerella sp. PCC 9339. This study provides new insights into the logic underpinning the biosynthesis of microcystin chemical variants and broadens our knowledge of structural diversity of the microcystin family of toxins.Peer reviewe
Metadata for: Environmental adaptations by the intertidal Antarctic cyanobacterium Halotia branconii CENA392 as revealed using long-read genome sequencing
No full textAntarctica poses numerous challenges to life such as cold shock, low nutrient concentrations and periodic desiccation over a wide range of extreme temperatures. Cyanobacteria survive this harsh environment having evolved adaptive metabolic plasticity to become the dominant primary producers. The type strain cyanobacterium Halotia branconii CENA392 was isolated from an Antarctic intertidal seashore. The complete circular genome of this strain is presented herein, which was assembled using long sequence reads. The genome encoded some stress-related genes associated with low-temperature adaptation and biosynthesis of mycosporine-like amino acid (MAA) photoprotective compounds. Empirical experimentation demonstrated constitutive production of the MAA porphyra-334 and total carotenoids without exposure to low temperatures or ultraviolet radiation stress. Phylogenetic analysis provided insights on the taxonomic placement and the evolutionary history of some annotated genes. These data exemplify the importance of generating complete quality genome sequences of microorganisms isolated from extreme intertidal environments, facilitating in-depth evaluation of ecological and taxonomic inferences.The data are available in .sh and .xlsx files. The first can be opened with any basic text reader (such as Notepad or File Reader) while the second is a spreadsheet from Office Excel.Funding provided by: Fundação de Amparo à Pesquisa do Estado de São PauloCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001807Award Number: 2013/07914-8Funding provided by: Fundação de Amparo à Pesquisa do Estado de São PauloCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001807Award Number: 2021/00149-0Funding provided by: Fundação de Amparo à Pesquisa do Estado de São PauloCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001807Award Number: 2016/14227-5Funding provided by: Universidade de São PauloCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100005639Award Number: 13.1.1502.9.8Funding provided by: Conselho Nacional de Desenvolvimento Científico e TecnológicoCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100003593Award Number: 439065/2018-6Funding provided by: Conselho Nacional de Desenvolvimento Científico e TecnológicoCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100003593Award Number: 433166/2018-5Obtaining the genome:
Bacteria growing over the surface of H. branconii CENA392 were removed by serial washing following a procedure reported by Delbaje et al. (2021). Total gDNA was extracted from washed cyanobacterial cells using an All-Prep DNA/RNA Mini kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. DNA stable Plus (Biomatrica, San Diego, USA) was added at a final volume of 25 %(v/v) to preserve integrity of the gDNA during lyophilization. The lyophilized gDNA was sent for whole-genome sequencing to the Joint Genome Institute (Berkley, CA, USA; JGI Project ID: 1338759). A PacBio SMRTbell library was prepared for circular consensus sequencing using a PacBio RS platform. The reads were filtered using BBMap v38.90 (Bushnell et al. 2017) with the icecreamfinder.sh program using default parameters. De novo genome assembly was performed with default parameters using Flye v2.9 (Kolmogorov et al. 2019). The assembled scaffolds were classified with Kaiju v1.7.2 (Menzel et al., 2016) to obtain only cyanobacterial sequences. Circularity of the assembled genome was confirmed with Bandage v0.8.1 (Wick et al., 2015). Completeness and quality assessments were measured using CheckM v1.0.13 (Parks et al., 2015) and Quast v5.0.2 (Gurevich et al., 2013). The genome file was uploaded to National Center for Biotechnology Information (NCBI) and will be publicaly available once it receives an accession number.
The bash script used for the assembly is shown here:
#!/bin/bash
# -q all.q
# -V
# -cwd
# -pe smp 8
module load Flye/2.9
flye --pacbio-hifi /Storage/data/JGI_data/CENA392/ccs.filter.fasta.gz -o flye_out392 -t 8 -i 4
The script starts with a shebang code (#!/bin/bash) that identifies the file as a bash script. Afterwards, the next 4 lines are resource requests identified by #$
-q all.q recognizes the location in which the work will be performed;
-V and -cwd permit that the output files generated are stored within the same location as the script;
-pe smp reserves the indicated cores in the computational module for running the script.
module load Flye2.9 loads the necessary program from the server.
flye --pacbio-hifi indicates the type of input data.
/Storage/data/JGI_data/CENA392/ccs.filter.fasta.gz shows the path in which the reads were stored in the server.
-o flye_out392 names the output file that will contain the results of the assembly.
-t exits after the script is done, setting the allocated cores free.
-i indicates that the script is interactive and X number of reserved cores are the minimum amount needed for completing the script.
Mycosporine-like amino acid quantification:
Identification and quantification of the MAAs palythine, shinorine and porphyra-334 used previously published procedures (Geraldes et al. 2019; 2020) from biomass that had been grown for 45 days in Z8 medium (Kotai 1972) under fluorescent light (45 µmol photons·μm-2·s-1) in a 14:10h light/dark cycle and temperature of 22 ± 1 ºC. Cells were not exposed to additional UV irradiance. After 45 days of growth, the biomass generated in each replicate (n =3) was centrifuged and lyophilized at -80°C. Later, the biomass was submitted to the following extraction process:
Weighing 5.00 mg of lyophilized biomass in an Eppendorf;
Add 2.00 mL of acid solvent (0.1% (v/v) formic acid + 0.2 mM ammonium formate, pH ~2.55) and vortex for 5s;
Sample sonication with ultrasound probe for 1 min (amplitude 30%, impulse 10) and vortex again for 5s;
Extract at room temperature (~22°C) for 1 hour;
Centrifuge samples (10,000 rpm for 10 min at 5°C) and pipette the supernatant in a syringe attached to a 0.45 µm filter;
Filtration to a HPLC 1.0 mL vial for sample reading.
The HPLC/MS-MS readings followed the parameters described in Geraldes et al. 2019. Blanks containing only growth media were used in between readings and standard patterns for shinorine, palythine and porphyra-334 were used. The standards were donated by Prof. Ernani Pinto. HPLC reading values are provided in an EXCEL file attached to this submission.
Chlorophyll a and carotenoids quantification:
On the first and last days of the experiment, 1.0 mL samples were taken from each replicate to evaluate photosynthetic-related pigments chlorophyll a and total carotenoids. These aliquots were filtered in glass membranes with 47 mm diameter in a setting coupled to a vacuum pump. The resulting filters were stored in amber Eppendorf's in an ultra-freezer (-80°C) until extraction, performed as described here:
Addition of 1.1 mL of cold 90% acetone in each Eppendorf containing the frozen filters;
Sample vortexed for 30s and extraction in dark and refrigerated storage for 24h;
Centrifuge samples (3,000 rpm for 30 min at 5°C) and pipette the supernatant in a new Eppendorf for spectrometer readings.
The data concerning specific wavelengths were taken to be used in specific equations that allow the calculation of chlorophyll a and total carotenoids concentrations (Kirk and Allen, 1965). The readings are provided in an EXCEL file attached to this submission.
Chlorophyll a (μg mL-1) = (12,7 x A663) – (2,69 x A645)
Carotenoids (μg mL-1) = A480 + (0,114 x A663) – (0,638 x A645
Plasmid-mediated microcystin production in Fischerella CENA161
No full textThe strain Fischerella sp. CENA161 is a microcystin producing cyanobacterium isolated from spring water in Brazil. The complete biosynthetic mcy gene cluster and potential microcystin congeners were published in 2018. In this work, bioinformatic analysis of whole-genome sequence and hybrid assembled data from Fischerella sp. CENA161 was performed. For detection of putative plasmids in this strain, the plasmid assembly mode in SPAdes was used on reads sequenced with the MiSeq and PacBio plataforms, followed by annotation of resulting contigs using PlasmidFinder. The complete genome size of CENA161 is 5.9 Mb and three plasmids were annotated with sizes of 728, 242 and 192 kb. The complete mcy gene cluster was found in the larger plasmid. Genome mining of available microcystin-producing cyanobacteria was also carried out to detect the partitioning of microcystin biosynthetic pathways in chromosomes and plasmids, and will be presented. Plasmid-mediated microcystin production is a significant public health concern given its potential to easily spread among aquatic cyanobacteria
Exploring the Relationship between Biosynthetic Gene Clusters and Constitutive Production of Mycosporine-like Amino Acids in Brazilian Cyanobacteria
No full textCyanobacteria are oxygenic phototrophic prokaryotes that have evolved to produce ultraviolet-screening mycosporine-like amino acids (MAAs) to lessen harmful effects from obligatory exposure to solar UV radiation. The cyanobacterial MAA biosynthetic cluster is formed by a gene encoding 2-epi-5-epi-valiolone synthase (EVS) located immediately upstream from an O-methyltransferase (OMT) encoding gene, which together biosynthesize the expected MAA precursor 4-deoxygadusol. Accordingly, these genes are typically absent in non-producers. In this study, the relationship between gene cluster architecture and constitutive production of MAAs was evaluated in cyanobacteria isolated from various Brazilian biomes. Constitutive production of MAAs was only detected in strains where genes formed a co-linear cluster. Expectedly, this production was enhanced upon exposure of the strains to UV irradiance and by using distinct culture media. Constitutive production of MAAs was not detected in all other strains and, unexpectedly, production could not be induced by exposure to UV irradiation or changing growth media. Other photoprotection strategies which might be employed by these MAA non-producing strains are discussed. The evolutionary and ecological significance of gene order conservation warrants closer experimentation, which may provide a first insight into regulatory interactions of genes encoding enzymes for MAA biosynthesis
A refactored biosynthetic pathway for the production of glycosylated microbial sunscreens
No full textMycosporine-like amino acids (MAAs) are a family of water-soluble and colorless secondary metabolites, with high extinction coefficients, that function as microbial sunscreens. MAAs share a cyclohexinimine chromophore that is diversified through amino acid substitutions and attachment of sugar moieties. The genetic and enzymatic bases for the chemical diversity of MAAs remain largely unexplored. Here we report a series of structurally distinct MAAs and evidence for an unusual branched biosynthetic pathway from a cyanobacterium isolated from lake sediment. We used a combination of high-resolution liquid chromatography-mass spectrometry (HR-LCMS) analysis and nuclear magnetic resonance (NMR) spectroscopy to identify diglycosylated-palythine-Ser (C22H36N2O15) as the dominant chemical variant in a series of MAAs from Nostoc sp. UHCC 0302 that contained either Ser or Thr. We obtained a complete 9.9 Mb genome sequence to gain insights into the genetic basis for the biosynthesis of these structurally distinct MAAs. We identified MAA biosynthetic genes encoded at two locations on the circular chromosome. Surprisingly, direct pathway cloning and heterologous expression of the complete mysABCJ1D1G1H biosynthetic gene cluster in Escherichia coli (E. coli) led to the production of 450 Da monoglycosylated-palythine-Thr (C18H30N2O11). We reconstructed combinations of the two distant biosynthetic gene clusters in refactored synthetic pathways and expressed them in the heterologous host. These results demonstrated that the MysD1 and MysD2 enzymes displayed a preference for Thr and Ser, respectively. Furthermore, one of the four glycosyltransferases identified, MysG1, was active in E. coli and catalysed the attachment of a hexose moiety to the palythine-Thr intermediate. Together these results provide the first insights into the enzymatic basis for glycosylation of MAAs and demonstrates how paralogous copies of the MysD enzymes allow the simultaneous biosynthesis of specific chemical variants to increase the structural variation in this family of microbial sunscreens.peerReviewe
Aspergillus fumigatus mitogenomes and their influence on azole-resistant and -susceptible populations
No full textAbstract The role of the fungal mitochondria goes far beyond energy metabolism. The genomes of 318 Aspergillus fumigatus clinical and environmental isolates from different geographic origins were analyzed aiming to study the mitochondrial sequences from populations sensitive and resistant to azoles. Our results show that A. fumigatus mitogenomic sequences are very conserved and only show variation in small intergenic regions and one intronic sequence in the cox3 gene. Furthermore, a genome-wide association analysis of accessory mitochondrial genes revealed potential mitochondria-based genotypes that may interact synergistically with the ergosterol biosynthesis pathway to confer the resistant phenotype. This includes a mutation in the AMID-like mitochondrial oxidoreductase (aifA, AFUA_3G01290) and the absence of the mitochondrial carrier protein (pet8, AFUA_8G01400). Deletion of these genes did not change the azole-susceptibility but increased the azole-persistence, suggesting mitochondrial genes could be involved in azole-persistence. Our work opens new hypotheses for the involvement of mitochondria in A. fumigatus azole-resistance
