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Concentration of bacterial species indicative of fecal contamination in the gut of mangrove oysters (Crassostrea gasar) is a major concern for public health and food surveillance. Our work aimed to determine the occurrence, antibiotic-resistance, phylogenetic profile and virulence of Escherichia coli strains isolated from C. gasar farmed in four estuaries of Amazonia. Santo Antônio de Urindeua was the sampling point with the highest number of E. coli cells in oyster samples (104 per 100 g of sample). Twenty-four isolates (52.2%) showed resistance to cephalotin and 18 to amoxicillin (39.1%). Eighteen clonal populations were determined by rep-PCR and were mainly affiliated to the pathogenic and commensal phylo-groups B1 and D. The presence of elt genes suggests that 10 of these clones belong to the Enterotoxigenic Escherichia coli pathotype. Plasmids, mostly of the F incompatibility group, were detected in the majority of the strains. All isolates were susceptible to last-resort antibiotics.
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Crassostrea , Escherichia coli , Animais , Antibacterianos , Brasil , Estuários , Filogenia , VirulênciaRESUMO
Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2â¡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments.
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Bacillales/genética , Bacillales/metabolismo , Biologia Computacional/métodos , Ácidos Graxos/biossíntese , Ácidos Graxos/metabolismo , Redes e Vias Metabólicas/genética , Bases de Dados Genéticas , Genoma BacterianoRESUMO
BACKGROUND: FapR protein from the psychrotrophic species Exiguobacterium antarcticum B7 was expressed and purified, and subsequently evaluated for its capacity to bind to the promoter regions of the fabH1-fabF and fapR-plsX-fabD-fabG operons, using electrophoretic mobility shift assay. The genes that compose these operons encode for enzymes involved in the de novo synthesis of fatty acids molecules. In Bacillus subtilis, FapR regulates the expression of these operons, and consequently has influence in the synthesis of long or short-chain fatty acids. To analyze the bacterial cold adaptation, this is an important metabolic pathway because psychrotrophic microrganisms tend to synthesize short and branched-chain unsaturated fatty acids at cold to maintain cell membrane fluidity. RESULTS: In this work, it was observed that recombinant protein was able to bind to the promoter of the fully amplified fabH1-fabF and fapR-plsX-fabD-fabG operons. However, FapR was unable to bind to the promoter of fapR-plsX-fabD-fabG operon when synthesized only up to the protein-binding palindrome 5'-TTAGTACCAGATACTAA-3', thus showing the importance of the entire promoter sequence for the correct protein-DNA interaction. CONCLUSIONS: Through this observation, we demonstrate that the FapR protein possibly regulates the same operons as described for other species, which emphasizes its importance to cold adaptation process of E. antarcticum B7, a psychrotrophic bacterium isolated at Antarctica.
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Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Ácidos Graxos/biossíntese , Regiões Antárticas , Bacillus/isolamento & purificação , Sequência de Bases , Ensaio de Desvio de Mobilidade EletroforéticaRESUMO
Corynebacterium pseudotuberculosis causes significant loss to goat and sheep farmers because it is the causal agent of the infectious disease caseous lymphadenitis, which may lead to outcomes ranging from skin injury to animal death (Ruiz et al., 2011) [1]. This bacterium was grown under osmotic (2 M), acid (pH) and heat (50 °C) stress and under control (Normal-BHI brain heart infusion) conditions, which simulate the conditions faced by the bacteria during the infectious process. Subsequently, cDNA of each condition was sequenced by the SOLiD3 Plus platform using the RNA-Seq technique [2], [3], [4]. The data produced was processed to evaluate the differential gene expression, which is helpful to understand the adaptation mechanisms during the infection in the host. The sequencing data of all conditions are available in the European Bioinformatics Institute (EBI) repository under accession number E-MTAB-2017.
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Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field.
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The genome of Corynebacterium pseudotuberculosis MB20 bv. equi was sequenced using the Ion Personal Genome Machine (PGM) platform, and showed a size of 2,363,089 bp, with 2,365 coding sequences and a GC content of 52.1%. These results will serve as a basis for further studies on the pathogenicity of C. pseudotuberculosis bv. equi.
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Corynebacterium pseudotuberculosis equi is a Gram-positive pathogenic bacterium which affects a variety of hosts. Besides the great economic losses it causes to horse-breeders, this organism is also known to be an important infectious agent to cattle and buffaloes. As an outcome of the efforts in characterizing the molecular basis of its virulence, several complete genome sequences were made available in recent years, enabling the large-scale assessment of genes throughout distinct isolates. Meanwhile, the RNA-seq stood out as the technology of choice for comprehensive transcriptome studies, which may bring valuable information regarding active genomic regions, despite of the still impeditive associated costs. In an attempt to increase the use of generated reads per instrument run, by effectively eliminating unwanted rRNAs from total RNA samples without relying on any commercially available kits, we applied denaturing high-performance liquid chromatography (DHPLC) as an alternative method to assess the transcriptional profile of C. pseudotuberculosis. We have found that the DHPLC depletion method, allied to Ion Torrent sequencing, allows mapping of transcripts in a comprehensive way and identifying novel transcripts when a de novo approach is used. These data encourage us to use DHPLC in future transcriptional evaluations in C. pseudotuberculosis.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Corynebacterium pseudotuberculosis/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Desnaturação de Ácido Nucleico/genética , RNA Ribossômico/química , Transcriptoma , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/classificação , Corynebacterium pseudotuberculosis/genética , Doenças dos Cavalos/microbiologia , Cavalos/microbiologia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Especificidade da EspécieRESUMO
Corynebacterium pseudotuberculosis is a facultative intracellular pathogen and the causative agent of several infectious and contagious chronic diseases, including caseous lymphadenitis, ulcerative lymphangitis, mastitis, and edematous skin disease, in a broad spectrum of hosts. In addition, Corynebacterium pseudotuberculosis infections pose a rising worldwide economic problem in ruminants. The complete genome sequences of 15 C. pseudotuberculosis strains isolated from different hosts and countries were comparatively analyzed using a pan-genomic strategy. Phylogenomic, pan-genomic, core genomic, and singleton analyses revealed close relationships among pathogenic corynebacteria, the clonal-like behavior of C. pseudotuberculosis and slow increases in the sizes of pan-genomes. According to extrapolations based on the pan-genomes, core genomes and singletons, the C. pseudotuberculosis biovar ovis shows a more clonal-like behavior than the C. pseudotuberculosis biovar equi. Most of the variable genes of the biovar ovis strains were acquired in a block through horizontal gene transfer and are highly conserved, whereas the biovar equi strains contain great variability, both intra- and inter-biovar, in the 16 detected pathogenicity islands (PAIs). With respect to the gene content of the PAIs, the most interesting finding is the high similarity of the pilus genes in the biovar ovis strains compared with the great variability of these genes in the biovar equi strains. Concluding, the polymerization of complete pilus structures in biovar ovis could be responsible for a remarkable ability of these strains to spread throughout host tissues and penetrate cells to live intracellularly, in contrast with the biovar equi, which rarely attacks visceral organs. Intracellularly, the biovar ovis strains are expected to have less contact with other organisms than the biovar equi strains, thereby explaining the significant clonal-like behavior of the biovar ovis strains.
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Corynebacterium/genética , Genoma Bacteriano/genética , Animais , Deleção de Genes , Genes Bacterianos/genética , Variação Genética , Ilhas Genômicas/genética , Família Multigênica/genética , Especificidade da Espécie , Fatores de Virulência/genéticaRESUMO
Corynebacterium pseudotuberculosis is a bacterium which causes diseases such as caseous lymphadenitis in small ruminants, resulting in large-scale economic losses for agribusiness worldwide. Consequently, this bacterium including its transcriptional profile analysis has been the focus of various studies. Identification of the transcripts that appear under conditions that simulate the environment encountered by this bacterial species in the host is of great importance in discovering new targets for the production of more efficient vaccines. We sequenced the cDNA of Corynebacterium pseudotuberculosis strain 1002, using the SOLiD V3 system, under the following conditions: osmotic stress (2 M), acidity (pH), heat shock (50 °C) and control condition (N). To identify the transcripts shared among the stimulons and integrate this information with the results from BLAST and BLAST2GO, we developed the software CoreStImulon (CSI) which allows the user to individually distinguish the genes in terms of their participation in biological processes, their function and cellular location. In the biosynthetic processes, eleven genes represented in the core stimulon and twenty genes in the control were observed. This validates the hypothesis that the organisms strategy for surviving in a hostile environment is through growth reduction. The oxidation reduction process, response to stress process, and cell adhesion are controlled by genes that contribute to bacterial cell maintenance under stress conditions; these could be involved in their pathogenicity. The methodology for identification of transcripts obtained by ab initio assembly and shared among the stimulons permitted candidates selection for vaccine studies. CSI is available at https://sourceforge.net/projects/corestimulon/.
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Biologia Computacional/métodos , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/metabolismo , Regulação Bacteriana da Expressão Gênica , Aderência Bacteriana , DNA/metabolismo , DNA Complementar/metabolismo , Perfilação da Expressão Gênica , Concentração de Íons de Hidrogênio , Osmose , Oxirredução , Linguagens de Programação , RNA Mensageiro/metabolismo , Software , Temperatura , Transcrição GênicaRESUMO
Metalloendopeptidases are zinc-dependent hydrolases enzymes with many different roles in biological systems, ranging from remodeling conjunctive tissue to removing signaling sequences from nascent proteins. Here, we describe the three-dimensional structure of the metalloendopeptidase from Corynebacterium pseudotuberculosis generated by homology modeling and molecular dynamics. Analysis of key distances shows that His-132, Asp-136, His-211, Leu-212 and one molecule of water play an important role in the protein-Zn(2+) ion interaction. The model obtained may provide structural insights into this enzyme and can be useful for the design of new caseous lymphadenitis vaccines based on genetic attenuation from key point mutation.
Assuntos
Corynebacterium pseudotuberculosis/enzimologia , Metaloendopeptidases/química , Sequência de Aminoácidos , Metaloendopeptidases/isolamento & purificação , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Chromobacterium violaceum is a gram-negative betaproteobacterium that has been isolated from various Brazilian ecosystems. Its genome contains the cyn operon, which gives it the ability to metabolize highly toxic cyanate into ammonium and carbon dioxide. We used a proteomics approach to investigate the effects of cyanate on the metabolism of this bacterium. The proteome of cells grown with and without cyanate was compared on 2-D gels. Differential spots were digested and identified by mass spectrometry. The bacterium was able to grow at concentrations of up to 1 mM cyanate. Eighteen spots were differentially expressed in the presence of cyanate, of which 16 were downregulated and only two were upregulated. An additional 12 spots were detected only in extracts of cells unexposed to cyanate, and one was expressed only by the exposed cells. Fourteen spots were identified, corresponding to 13 different proteins. We conclude that cyanate promotes expression of enzymes that combat oxidative stress and represses enzymes of the citric acid cycle, strongly affecting the energetic metabolism of the cell. Other proteins that were under-expressed in bacteria exposed to cyanate are involved in amino-acid metabolism or are hypothetical proteins, demonstrating that cyanate also affects expression of genes that are not part of the cyn operon.
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In this study, 15 microsatellite DNA loci used in comparative tests by the International Society for Animal Genetics were applied to the evaluation of genetic diversity and management, and the efficiency of paternity testing in Marajoara horses and Puruca ponies from the Marajó Archipelago. Based on the genotyping of 93 animals, mean allelic diversity was estimated as 9.14 and 7.00 for the Marajoara and Puruca breeds, respectively. While these values are similar to those recorded in most European breeds, mean levels of heterozygosity were much lower (Marajoara 49 percent, Puruca 40 percent), probably as a result of high levels of inbreeding in the Marajó populations. The mean informative polymorphic content of this 15-marker system was over 50 percent in both breeds, and was slightly higher in the Marajoara horses. The discriminative power and exclusion probabilities derived from this system were over 99 percent for both populations, emphasizing the efficacy of these markers for paternity testing and genetic management in the two breeds.
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Animais , Cavalos/genética , DNA Satélite/genética , Variação Genética , Brasil , Repetições de Microssatélites , Paternidade , LinhagemRESUMO
Chromobacterium violaceum is a Gram-negative bacterium found in a wide variety of tropical and subtropical ecosystems. The complete genome sequence of C. violaceum ATCC 12472 is now available, and it has considerable biotechnological potential for various applications, such as environmental detoxification, as well as medical and agricultural use. We examined the biotechnological potential of C. violaceum for environmental detoxification. Three operons, comprising the ars operon, involved in arsenic resistance, the cyn operon, involved in cyanate detoxification, and the hcn operon, encoding a cyanase, responsible for biogenic production of cyanide, as well as an open reading frame, encoding an acid dehalogenase, were analyzed in detail. Probable catalytic mechanisms for the enzymes were determined, based on amino acid sequence comparisons and on published structural information for these types of proteins.
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Proteínas de Bactérias/genética , Biotecnologia , Chromobacterium/genética , Arsênio/metabolismo , Arsênio/farmacologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biodegradação Ambiental , Chromobacterium/metabolismo , Cianetos/metabolismo , Farmacorresistência Bacteriana/genética , Hidrolases/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Óperon/genéticaRESUMO
An electrophoretic survey of 15 protein systems (22 loci) was employed to determine the genetic relationships among 9 populations (441 individuals) of South American squirrel monkeys (Saimiri sciureus sciureus, S. sciureus boliviensis, and S. sciureus ustus). Genetic markers capable of differentiating the second from the two other taxa were observed mainly in the ADA and GPI systems. Heterogeneity for ADA and CA2 between populations from opposite banks of the Jamari river was verified in S. sciureus ustus. The average heterozygosities ranged from 3% to 5%, the lowest being in S. sciureus sciureus and the highest in S. sciureus boliviensis. Low genetic distances (D = 0.001-0.057) were observed between populations within taxa or between S. sciureus sciureus and S. sciureus ustus. But both differed to a larger extent from S. sciureus boliviensis (D = 0.11 in both comparisons). There is a positive correlation between the genetic and geographic distance matrices. The three taxa are more clearly separated (D = 0.76-0.77) from the outgroup used for comparison (Cebus apella). Our data suggest that there is only one large, polytypic species of squirrel monkeys in South America, S. sciureus, forming a contiguous ring of geographical races or subspecies. Two of the most differentiated forms meet at the Peruvian Amazonia where natural hybrids and secondary intergradation have been reported. © 1993 Wiley-Liss, Inc.
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Four esterase D (ESD) phenotypes, presumably resulting from the segregation of three alleles with moderate polymorphic frequencies, were observed in a sample of the black handed tamarin, Saguinus midas niger, from the northern Amazonian region (Brazil). Previous surveys in the non-human Anthropoidea indicate that the ESD locus is polymorphic in at least 4 of the 19 taxonomic entities listed.
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Um total de 201 indivíduos, descendentes de escravos africanos que fugiram e formaram um comunidade independente às margens dos rios Trombetas e Cuminá, foi estudada com relaçäo a 16 sistemas genéticos (dois grupos sangüíneos, hemoglobina, oito sistemas eritrocitários e cinco proteínas séricas). Dezesseis (59 por cento) dos 27 marcadores genéticos estudados apresentaram freqüências que säo típicas de populaçöes com ancestralidade africana e alguma mistura. A presença de HB*S, CA2*2, ACP*R, GD*A, GD*A- e HP*2M também indicou um conjunto gênico em grande parte africano. A estimativa quantitativa da mistura, usando 11 dos sistemas que apresentaram menos heterogeneidade, forneceu as seguintes percentagens, quanto aos componentes raciais: 62 por cento de negro, 11 por cento de indígena e 27 por cento de branco. Estimativas prévias de miscigenaçäo africana em populaçöes da regiäo amazônica resultaram em números muito menores (13 por cento a 24 por cento). Conclui-se que esta comunidade ainda mantém muito das suas características ancestrais, apesar do processo de mistura racial que está ocorrendo lá e praticamente em todo Brasil