Replication origin location might contribute to genetic variability in Trypanosoma cruzi

Background: DNA replication in trypanosomatids operates in a uniquely challenging environment, since most of their genomes are constitutively transcribed. Trypanosoma cruzi, the etiological agent of Chagas disease, presents high variability in both chromosomes size and copy number among strains, though the underlying mechanisms are unknown. Results: Here we have mapped sites of DNA replication initiation across the T. cruzi genome using Marker Frequency Analysis, which has previously only been deployed in two related trypanosomatids. The putative origins identified in T. cruzi show a notable enrichment of GC content, a preferential position at subtelomeric regions, coinciding with genes transcribed towards the telomeres, and a pronounced enrichment within coding DNA sequences, most notably in genes from the Dispersed Gene Family 1 (DGF-1). Conclusions: These findings suggest a scenario where collisions between DNA replication and transcription are frequent, leading to increased genetic variability, as seen by the increase SNP levels at chromosome subtelomeres and in DGF-1 genes containing putative origins

Insights Into Limnothrix sp. Metabolism Based on Comparative Genomics

Currently only four genome sequences for Limnothrix spp. are publicly available, and information on the genetic properties of cyanobacteria belonging to this genus is limited. In this study, we report the draft genome of Limnothrix sp. CACIAM 69d, isolated from the reservoir of a hydroelectric dam located in the Amazon ecosystem, from where cyanobacterial genomic data are still scarce. Comparative genomic analysis of Limnothrix revealed the presence of key enzymes in the cyanobacterial central carbon metabolism and how it is well equipped for environmental sulfur and nitrogen acquisition. Additionally, this work covered the analysis of Limnothrix CRISPR-Cas systems, pathways related to biosynthesis of secondary metabolites and assembly of extracellular polymeric substances and their exportation. A trans-AT PKS gene cluster was identified in two strains, possibly related to the novel toxin Limnothrixin biosynthesis. Overall, the draft genome of Limnothrix sp. CACIAM 69d adds new data to the small Limnothrix genome library and contributes to a growing representativeness of cyanobacterial genomes from the Amazon region. The comparative genomic analysis of Limnothrix made it possible to highlight unique genes for each strain and understand the overall features of their metabolism

In silico analysis of the cyanobacterial lectin scytovirin : new insights into binding properties

We acknowledge Fundação Amazônia de Amparo a Estudos e Pesquisas do Pará (FAPESPA) for financially supporting (ICAAF 099/2014) our project. Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) also supported individual authors through Grant 311686/2015-0 (ECG).Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Exatas e Naturais. Laboratórios de Investigação Sistemática em Biotecnologia e Biodiversidade Molecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Exatas e Naturais. Laboratórios de Investigação Sistemática em Biotecnologia e Biodiversidade Molecular. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Scytovirin is a lectin isolated from the cyanobacterium Scytonema varium that has shown activity against HIV, SARS coronavirus and Zaire Ebola virus. Its 95 amino acids are divided into two structural domains (SD), the first spanning amino acids 1–48 (SD1) and the second 49–95 (SD2). Interestingly, the domains are nearly identical but differ in their affinities for carbohydrates. With the aim of enhancing understanding of the binding properties of scytovirin, we performed molecular dynamics (MD) simulations of scytovirin complexed with Man4. We set up three systems: (i) Man4 bound to both domains (SD1+SD2) using the full-length protein; (ii) Man4 bound to an incomplete protein, containing only SD1 and (iii) Man4 bound to an incomplete protein containing only SD2. Contrary to other reports, binding free energy results suggest that Man4 can bind simultaneously to SD1 and SD2 binding regions, but SD1 individually has the best values of energy and the best affinity for Man4. Decomposition of the binding free energy showed that the residues that interact with Man4 were different in the three systems, suggesting that the binding mechanism of Man4 varies between full-length protein, SD1 and SD2. The results presented here may help to formulate strategies to use scytovirin and promote mutagenesis studies to improve the antiviral activity of scytovirin

Genomic screening of new putative antiviral lectins from Amazonian cyanobacteria based on a bioinformatics approach

Fundação Amazônia Paraense de Amparo à Pesquisa. Grant Number: ICAAF 099/2014; Conselho Nacional de Desenvolvimento Científico e Tecnológico. Grant Number: 311686/2015‐0Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Naturais. Laboratórios de Investigação Sistemática em Biotecnologia e Biodiversidade Molecular. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Lectins are proteins of nonimmune origin, which are capable of recognizing and binding to glycoconjugate moieties. Some of them can block the interaction of viral glycoproteins to the host cell receptors acting as antiviral agents. Although cyanobacterial lectins have presented broad biotechnological potential, little research has been directed to Amazonian Cyanobacterial diversity. In order to identify new antiviral lectins, we performed genomic analysis in seven cyanobacterial strains from Coleção Amazônica de Cianobactérias e Microalgas (CACIAM). We found 75 unique CDS presenting one or more lectin domains. Since almost all were annotated as hypothetical proteins, we used homology modeling and molecular dynamics simulations to evaluate the structural and functional properties of three CDS that were more similar to known antiviral lectins. Nostoc sp. CACIAM 19 as well as Tolypothrix sp. CACIAM 22 strains presented cyanovirin‐N homologues whose function was confirmed by binding free energy calculations. Asn, Glu, Thr, Lys, Leu, and Gly, which were described as binding residues for cyanovirin, were also observed on those structures. As for other known cyanovirins, those residues in both our models also made favorable interactions with dimannose. Finally, Alkalinema sp. CACIAM 70d presented one CDS, which was identified as a seven‐bladed beta‐propeller structure with binding sites predicted for sialic acid and N‐acetylglucosamine. Despite its singular structure, our analysis suggested this molecule as a new putative antiviral lectin. Overall, the identification and the characterization of new lectins and their homologues are a promising area in antiviral research, and Amazonian cyanobacteria present biotechnological potential to be explored in this regard

Anti-dengue virus activity of scytovirin and evaluation of point mutation effects by molecular dynamics and binding free energy calculations

Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Centro de Inovações Tecnológicas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Naturais. Laboratórios de Investigação Sistemática em Biotecnologia e Biodiversidade Molecular. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Centro de Inovações Tecnológicas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.The absence of a specific treatment against DENV has led to intensive research into developing strategies for curing the infection. One lectin with high antiviral activity is scytovirin, which was isolated from the cyanobacterium Scytonema varium and has proven activity against HIV and Zaire Ebola Virus. To achieve the results presented here, we tested the affinity of full-length scytovirin, SD1 and SD2 separately, and six SD1 mutants for DENV glycoprotein E carbohydrate by Molecular Dynamics (MD) simulations and binding free energy calculations. It was possible to identify the key residues for protein-ligand interaction such as Glu10, Ala11, Pro17, Ans18, Arg30, Thr41, Ser42 and Arg43, which also has importance action against HIV. All binding free energy calculations showed negative values to ΔGbind of protein-DENV carbohydrate complexation. Additionally, these results are similar to the values of scytovirin and HIV gp120 carbohydrate complexation (-32.20 kcal/mol). Furthermore, we found that SD1 individually has more affinity to the carbohydrate and the Asn9, Glu10, Asn18, Arg30 and Arg43 demonstrated an important role in this matter. We also found that mutant G48R has better affinity (-34.10 kcal/mol) for the DENV carbohydrate than the wild type protein (-27.15 kcal/mol)

Comparative modeling and molecular dynamics suggest high carboxylase activity of the Cyanobium sp. CACIAM14 RbcL protein

Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brasil.Universidade Federal Rural da Amazônia. Belém, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brasil.Rubisco catalyzes the first step reaction in the carbon fixation pathway, bonding atmospheric CO2/O2 to ribulose 1,5-bisphosphate; it is therefore considered one of the most important enzymes in the biosphere. Genetic modifications to increase the carboxylase activity of rubisco are a subject of great interest to agronomy and biotechnology, since this could increase the productivity of biomass in plants, algae and cyanobacteria and give better yields in crops and biofuel production. Thus, the aim of this study was to characterize in silico the catalytic domain of the rubisco large subunit (rbcL gene) of Cyanobium sp. CACIAM14, and identify target sites to improve enzyme affinity for ribulose 1,5-bisphosphate. A three-dimensional model was built using MODELLER 9.14, molecular dynamics was used to generate a 100 ns trajectory by AMBER12, and the binding free energy was calculated using MM-PBSA, MM-GBSA and SIE methods with alanine scanning. The model obtained showed characteristics of form-I rubisco, with 15 beta sheets and 19 alpha helices, and maintained the highly conserved catalytic site encompassing residues Lys175, Lys177, Lys201, Asp203, and Glu204. The binding free energy of the enzyme substrate complexation of Cyanobium sp. CACIAM14 showed values around −10 kcal mol−1 using the SIE method. The most important residues for the interaction with ribulose 1,5-bisphosphate were Arg295 followed by Lys334. The generated model was successfully validated, remaining stable during the whole simulation, and demonstrated characteristics of enzymes with high carboxylase activity. The binding analysis revealed candidates for directed mutagenesis sites to improve rubisco s affinity

Draft genome sequence of Alkalinema sp. Strain CACIAM 70d, a cyanobacterium isolated from an Amazonian freshwater environment

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grants 554321/2010-6, 140218/2016-5, and 311686/2015-0) and Fundação Amazônia de Amparo an Estudos e Pesquisa (FAPESPA; grant ICAAF 099/2014)Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Maranhão. Programa de Pós-Graduação em Saúde e Ambiente. Laboratório de Farmacognosia. São Luís, MA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal do Maranhão - Campus de Bacabal. Bacabal, MA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, BrazilIn order to increase the genomic data of cyanobacterial strains isolated in Brazil, we hereby present the draft genome sequence of the Alkalinema sp. strain CACIAM 70d, isolated from an Amazonian freshwater environment. This report describes the first genome available for this genus

In silico improvement of the cyanobacterial lectin microvirin and mannose interaction

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) ICAAF 099/2014; National Council for Scientific and Technological Development (CNPq) 311686/2015-0.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Lectins that bind to HIV envelope glycoprotein can inhibit virus-cell fusion and be used for rational drug design. This paper presents the results of anin silicoapproach to improve affinity interaction between the cyanobacterial lectin microvirin and its ligand Man alpha(1-2)Man. Comparative modeling and molecular dynamics tools were used. Additionally, the alanine scanning webserver was used to study the importance of protein residues in the binding site and to guide mutant production. The model obtained presented two homologous domains designated as domains A and B, each consisting of a single strand with triple and antiparallel beta-sheets of (beta 1-beta 3 and beta 6-beta 8). Disulfide bonds between the cysteines (Cys60-Cys80, Cys63-Cys78 and Cys8-Cys24) were also found. The highly conserved binding site, including residues Asn44, Ile45, Asp46, Gln54, Asn55, Glu58, Thr59, Gln81, Thr82 and Met83. The RMSD values of the di-mannose and the interaction site were very stable during the molecular dynamics. Calculations of the occupation time of the hydrogen bonds were made for the residues that showed interaction in the complex lectin and ligand. The residue that contributed most to the interaction with Man alpha(1-2)Man was Asn55. After validation, the model generated remained stable during the entire simulation. Despite its structural similarity with the template we used, our mutant (Thr82Arg) showed a higher affinity interaction with Man alpha(1-2)Man

Draft genome sequence of Flavihumibacter sp. Strain CACIAM 22H1, a heterotrophic bacterium associated with cyanobacteria

Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil / Universidade Federal do Maranhão - Campus de Bacabal. São Luis, MA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Núcleo de Medicina Tropical. Laboratório de Patologia Clínica e Doenças Tropicais. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Here, we present a draft genome and annotation of Flavihumibacter sp. CACIAM 22H1, isolated from Bolonha Lake, Brazil, which will provide further insight into the production of substances of biotechnological interest

Draft genome sequence of Limnobacter sp. Strain CACIAM 66H1, a heterotrophic bacterium associated with cyanobacteria

Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil / Universidade Federal do Maranhão - Campus de Bacabal. São Luis, MA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, Brazil.Universidade Federal do Pará.Núcleo de Medicina Tropical. Laboratório de Patologia Clínica e Doenças Tropicais. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Tecnologia Biomolecular. Belém, PA, BrazilEcological interactions between cyanobacteria and heterotrophic prokaryotes are poorly known. To improve the genomic studies of heterotrophic bacterium-cyanobacterium associations, the draft genome sequence (3.2 Mbp) of Limnobacter sp. strain CACIAM 66H1, found in a nonaxenic culture of Synechococcus sp. (cyanobacteria), is presented here