RESUMO
The cyanobacterium Synechococcus elongatus PCC 7942 holds significant potential as a biofactory for recombinant protein (RP) production due to its capacity to harness light energy and utilize CO2. This study aimed to enhance RP production by integration of native promoters and magnetic field application (MF) in S. elongatus PCC 7942. The psbA2 promoter, which responds to stress conditions, was chosen for the integration of the ZsGreen1 gene. Results indicated successful gene integration, affirming prior studies that showed no growth alterations in transgenic strains. Interestingly, exposure to 30 mT (MF30) demonstrated a increase in ZsGreen1 transcription under the psbA2 promoter, revealing the influence of MF on cyanobacterial photosynthetic machinery. This enhancement is likely attributed to stress-induced shifts in gene expression and enzyme activity. MF30 positively impacted photosystem II (PSII) without disrupting the electron transport chain, aligning with the "quantum-mechanical mechanism" theory. Notably, fluorescence levels and gene expression with application of 30 mT were significantly different from control conditions. This study showcases the efficacy of utilizing native promoters and MF for enhancing RP production in S. elongatus PCC 7942. Native promoters eliminate the need for costly exogenous inducers and potential cell stress. Moreover, the study expands the scope of optimizing RP production in photoautotrophic microorganisms, providing valuable insights for biotechnological applications.
Assuntos
Synechococcus , Regiões Promotoras Genéticas , Synechococcus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
This study aimed to analyze the effect of magnetic field (MF) application on the metabolism of Synechococcus elongatus PCC 7942. Concentrations of biomass, carbohydrate, protein, lipid, and photosynthetic pigments (chlorophyll-a, C-phycocyanin, allophycocyanin and phycoerythrin) were determined. In cultures with MF application (30 mT for 24 h d-1), there were increases of 47.5% in total protein content, 87.4% in C-phycocyanin, and 332.8% in allophycocyanin contents, by comparison with the control. Allophycocyanin is the most affected pigment by MF application. Therefore, its biosynthetic route was investigated, and four genes related to its synthesis were found. However, the analysis of the gene expression showed no statistical differences from the control culture, which suggests that induction of such genes may occur soon after MF application with consequent stabilization over time. MF application may be a cost-effective alternative to increase production of compounds of commercial interest by cyanobacteria.
Assuntos
Ficocianina , Synechococcus , Ficocianina/genética , Ficocianina/metabolismo , Ficobiliproteínas/metabolismo , Ficobiliproteínas/farmacologia , Synechococcus/genética , Campos MagnéticosRESUMO
Cyanobacteria of the genus Synechococcus are major contributors to global primary productivity and are found in a wide range of aquatic ecosystems. This Synechococcus collective (SC) is metabolically diverse, with some lineages thriving in polar and nutrient-rich locations and others in tropical or riverine waters. Although many studies have discussed the ecology and evolution of the SC, there is a paucity of knowledge on its taxonomic structure. Thus, we present a new taxonomic classification framework for the SC based on recent advances in microbial genomic taxonomy. Phylogenomic analyses of 1085 cyanobacterial genomes demonstrate that organisms classified as Synechococcus are polyphyletic at the order rank. The SC is classified into 15 genera, which are placed into five distinct orders within the phylum Cyanobacteria: (i) Synechococcales (Cyanobium, Inmanicoccus, Lacustricoccus gen. Nov., Parasynechococcus, Pseudosynechococcus, Regnicoccus, Synechospongium gen. nov., Synechococcus and Vulcanococcus); (ii) Cyanobacteriales (Limnothrix); (iii) Leptococcales (Brevicoccus and Leptococcus); (iv) Thermosynechococcales (Stenotopis and Thermosynechococcus) and (v) Neosynechococcales (Neosynechococcus). The newly proposed classification is consistent with habitat distribution patterns (seawater, freshwater, brackish and thermal environments) and reflects the ecological and evolutionary relationships of the SC.
Assuntos
Genoma Bacteriano/genética , Synechococcus/classificação , Synechococcus/genética , Ecossistema , Água Doce/microbiologia , Genômica , Ferro/metabolismo , Filogenia , Águas Salinas , Água do Mar/microbiologia , Synechococcus/metabolismoRESUMO
Understanding how ecological traits have changed over evolutionary time is a fundamental question in biology. Specifically, the extent to which more closely related organisms share similar ecological preferences due to phylogenetic conservation - or if they are forced apart by competition - is still debated. Here, we explored the co-occurrence patterns of freshwater cyanobacteria at the sub-genus level to investigate whether more closely related taxa share more similar niches and to what extent these niches were defined by abiotic or biotic variables. We used deep 16S rRNA gene amplicon sequencing and measured several abiotic environmental parameters (nutrients, temperature, etc.) in water samples collected over time and space in Furnas Reservoir, Brazil. We found that relatively more closely related Synechococcus (in the continuous range of 93%-100% nucleotide identity in 16S) had an increased tendency to co-occur with one another (i.e. had similar realized niches). This tendency could not be easily explained by shared preferences for measured abiotic niche dimensions. Thus, commonly measured abiotic parameters might not be sufficient to characterize, nor to predict community assembly or dynamics. Rather, co-occurrence between Synechococcus and the surrounding community (whether or not they represent true biological interactions) may be a more sensitive measure of realized niches. Overall, our results suggest that realized niches are phylogenetically conserved, at least at the sub-genus level and at the resolution of the 16S marker. Determining how these results generalize to other genera and at finer genetic resolution merits further investigation.
Assuntos
Evolução Biológica , Synechococcus/fisiologia , Brasil , Ecossistema , Água Doce/microbiologia , Filogenia , RNA Ribossômico 16S , Synechococcus/classificação , Synechococcus/genéticaRESUMO
Ultraviolet radiation from sunlight causes DNA damage in skin cells by formation of photoproducts, mainly cyclobutane pyrimidine dimers (CPD), which are reverted by exogenous CPD-photolyase, preventing photoaging and skin cancer. High performance liquid chromatography tandem mass spectrometry method for quantification of CPD-photolyase activity was developed to search new enzymes sources for dermatology or clinical studies. The method was based in the enzymatic conversion of a 15mer oligonucleotide, containing a center cyclobutane thymidine dimer, to the restored 15mer oligonucleotide. Three ion pair reagent were evaluated by response surface methodology to increase mass intensities. Additionally, chromatographic separation of oligonucleotides was performed. The selected mobile phase was 15â¯mM diisopropylethylamine/20â¯mM hexafluoroisopropanol in methanol. The method allowed total separation between the oligonucleotides studied (resolution of 2.3) by using the core shell technology, which reduce the diffusion time of the analyte into the column, increasing the efficiency and minimizing the analysis time at 7â¯min. The mass spectrometry detection allowed a high selectivity and sensitivity. This is the first time where MRM modality has been employed with this specific purpose. Oligonucleotides recovery from reaction mixture was â¼ 94% and the limit of quantification was 13.4â¯nM for 15mer. The method was evaluated with a recombinant CPD-photolyase from Synechococcus leopoliensis using purified and crude protein extract. CPD-photolyase could be measured in terms of activity for enzymatic kinetics studies, for evaluation of UV-R effects in (micro)organisms and to identify new enzymes.
Assuntos
Proteínas de Bactérias/química , Cromatografia Líquida/métodos , Desoxirribodipirimidina Fotoliase/química , Ensaios Enzimáticos/métodos , Oligonucleotídeos/análise , Synechococcus/enzimologia , Espectrometria de Massas em Tandem/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Cinética , Dímeros de Pirimidina/química , Synechococcus/química , Synechococcus/genéticaRESUMO
High-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and, so far, they have predominantly been targeted using universal bacterial primers, providing a limited resolution of the picocyanobacterial community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here, we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high-throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for the genera Synechococcus, Prochlorococcus, and Cyanobium An in silico analysis showed that the V5, V6, and V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zero-radius operational taxonomic units [zOTUs]), which allowed us to detect higher genetic diversity and infer ecologically relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door to future studies investigating picocyanobacterial diversity in aquatic systems.IMPORTANCE The molecular diversity of the aquatic picocyanobacterial community cannot be accurately described using only the available universal 16S rRNA gene primers that target the whole bacterial and archaeal community. We show that the hypervariable regions V5, V6, and V7 of the 16S rRNA gene are better suited to study the diversity, community structure, and dynamics of picocyanobacterial communities at a fine scale using Illumina MiSeq sequencing. Due to its variability, it allows reconstructing phylogenies featuring topologies comparable to those generated when using the complete 16S rRNA gene sequence. Further, we successfully designed a new set of primers flanking the V5 to V7 region whose specificity for picocyanobacterial genera was tested in silico and validated in several freshwater and marine aquatic communities. This work represents a step forward for understanding the diversity and ecology of aquatic picocyanobacteria and sets the path for future studies on picocyanobacterial diversity.
Assuntos
Cianobactérias/classificação , Cianobactérias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Microbiota , Filogenia , Argentina , Simulação por Computador , Cianobactérias/isolamento & purificação , Primers do DNA/genética , Primers do DNA/isolamento & purificação , Ecologia , Água Doce/microbiologia , Variação Genética , Prochlorococcus/classificação , Prochlorococcus/genética , Prochlorococcus/isolamento & purificação , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Água do Mar/microbiologia , Análise de Sequência de DNA , Synechococcus/classificação , Synechococcus/genética , Synechococcus/isolamento & purificaçãoRESUMO
The development of new heterologous hosts for polyketides production represents an excellent opportunity to expand the genomic, physiological, and biochemical backgrounds that better fit the sustainable production of these valuable molecules. Cyanobacteria are particularly attractive for the production of natural compounds because they have minimal nutritional demands and several strains have well established genetic tools. Using the model strain Synechococcus elongatus, a generic platform was developed for the heterologous production of polyketide synthase (PKS)-derived compounds. The versatility of this system is based on interchangeable modules harboring promiscuous enzymes for PKS activation and the production of PKS extender units, as well as inducible circuits for a regulated expression of the PKS biosynthetic gene cluster. To assess the capability of this platform, we expressed the mycobacterial PKS-based mycocerosic biosynthetic pathway to produce multimethyl-branched esters (MBE). This work is a foundational step forward for the production of high value polyketides in a photosynthetic microorganism.
Assuntos
Engenharia Metabólica , Microrganismos Geneticamente Modificados , Policetídeos/metabolismo , Synechococcus , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Synechococcus/genética , Synechococcus/metabolismoRESUMO
Bacteria are highly important for the cycling of organic and inorganic matter in freshwater environments; however, little is known about the diversity of bacterioplankton in tropical systems. Studies on carbon and nutrient cycling in tropical lakes suggest a very different seasonality from that of temperate climates. Here, we used 16S rRNA gene next-generation sequencing (NGS) to investigate seasonal changes in bacterioplankton communities of two tropical lakes, which differed in trophic status and mixing regime. Our findings revealed seasonally and depth-wise highly dynamic bacterioplankton communities. Differences in richness and structure appeared strongly related to the physicochemical characteristics of the water column, especially phosphate, pH and oxygen. Bacterioplankton communities were dominated by common taxonomic groups, such as Synechococcus and Actinobacteria acI, as well as rare and poorly characterized taxa such as 'Candidatus Methylacidiphilum' (Verrucomicrobia). Stratification and oxygen depletion during the rainy season promoted the occurrence of anoxygenic phototrophic and methanotrophic bacteria important for carbon and nutrient cycling. Differences in lake mixing regime were associated with seasonal beta diversity. Our study is the first attempt to use NGS for cataloging the diversity of bacterioplankton communities in Brazilian lakes and thus contributes to the ongoing worldwide endeavor to characterize freshwater lake bacterioplankton signatures.
Assuntos
Actinobacteria/isolamento & purificação , Bactérias/classificação , Bactérias/isolamento & purificação , Lagos/microbiologia , Synechococcus/isolamento & purificação , Actinobacteria/genética , Bactérias/genética , Brasil , Florestas , Lagos/química , Filogenia , Plâncton/classificação , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Synechococcus/genéticaRESUMO
Unicellular cyanobacteria are ubiquitous photoautotrophic microbes that contribute substantially to global primary production. Picocyanobacteria such as Synechococcus and Prochlorococcus depend on chlorophyll a-binding protein complexes to capture light energy. In addition, Synechococcus has accessory pigments organized into phycobilisomes, and Prochlorococcus contains chlorophyll b. Across a surface water transect spanning the sparsely studied tropical Indian Ocean, we examined Synechococcus and Prochlorococcus occurrence, taxonomy and habitat preference in an evolutionary context. Shotgun sequencing of size fractionated microbial communities from 0.1 µm to 20 µm and subsequent phylogenetic analysis indicated that cyanobacteria account for up to 15% of annotated reads, with the genera Prochlorococcus and Synechococcus comprising 90% of the cyanobacterial reads, even in the largest size fraction (3.0-20 mm). Phylogenetic analyses of cyanobacterial light-harvesting genes (chl-binding pcb/isiA, allophycocyanin (apcAB), phycocyanin (cpcAB) and phycoerythin (cpeAB)) mostly identified picocyanobacteria clades comprised of overlapping sequences obtained from Indian Ocean, Atlantic and/or Pacific Oceans samples. Habitat reconstructions coupled with phylogenetic analysis of the Indian Ocean samples suggested that large Synechococcus-like ancestors in coastal waters expanded their ecological niche towards open oligotrophic waters in the Indian Ocean through lineage diversification and associated streamlining of genomes (e.g. loss of phycobilisomes and acquisition of Chl b); resulting in contemporary small celled Prochlorococcus. Comparative metagenomic analysis with picocyanobacteria populations in other oceans suggests that this evolutionary scenario may be globally important.
Assuntos
Cianobactérias/genética , Metagenoma , Prochlorococcus/genética , Synechococcus/genética , Evolução Biológica , Clorofila/química , Clorofila A , Ecossistema , Genoma Bacteriano , Oceano Índico , Funções Verossimilhança , Metagenômica , Ficobilissomas/genética , Filogenia , Água do Mar/microbiologia , TemperaturaRESUMO
Synechococcus sp PCC 7336 represents a newly sequenced strain, and its genome is obviously different from that of other Synechococcus strains. In this analysis, local alignment and annotation databases were constructed and combined with various bioinformatic tools to carry out gene annotation and functional analysis of this strain. From this analysis, we identified 5096 protein-coding genes and 47 RNA genes. Of these, 116 genes that were classified into 9 categories were associated with photosynthesis, and type V polymerase proteins that were identified are unique for this strain. An additional 107 genes were closely related to signal transduction pathways, which primarily comprised parts of two-component regulatory systems. Gene ontogeny analysis showed that 2377 genes were annotated with a total number of 9791 functional categories, and specifically that 41 genes distributed in 4 protein complexes were involved in oxidative phosphorylation. Clusters of orthologous groups classification showed that there were 1463 homologous proteins associated with 17 specific metabolic pathways, and that most of the proteins participated in primary metabolic processes such as binding and catalysis. The phylogenetic tree based on 16S rRNA sequences indicated that Synechococcus PCC 7336 is highly likely to represent a new branch.