Characterization of the Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus: Effects of Thioredoxin Fusion Tag and Triton X-100

In this work, the lipase from Pyrococcus furiosus encoded by ORF PF2001 was expressed with a fusion protein (thioredoxin) in Escherichia coli. The purified enzymes with the thioredoxin tag (TRX−PF2001Δ60) and without the thioredoxin tag (PF2001Δ60) were characterized, and various influences of Triton X-100 were determined. The optimal temperature for both enzymes was 80°C. Although the thioredoxin presence did not influence the optimum temperature, the TRX−PF2001Δ60 presented specific activity twice lower than the enzyme PF2001Δ60. The enzyme PF2001Δ60 was assayed using MUF-acetate, MUF-heptanoate, and MUF-palmitate. MUF-heptanoate was the preferred substrate of this enzyme. The chelators EDTA and EGTA increased the enzyme activity by 97 and 70%, respectively. The surfactant Triton X-100 reduced the enzyme activity by 50% and lowered the optimum temperature to 60°C. However, the thermostability of the enzyme PF2001Δ60 was enhanced with Triton X-100

Transfer RNA-dependent asparagine biosynthesis in Gluconacetobacter diazotrophicus and its influence on biological nitrogen fixation

8 p. : il.Background and aims Gluconacetobacter diazotrophicus is a nitrogen-fixing endophytic bacterium isolated from sugarcane, rice, elephant grass, sweet potato, coffee, and pineapple. These plants have high level of asparagine, which promotes microbial growth and inhibits nitrogenase activity. The regulation of intracellular concentrations of this amino acid is essential for growth and biological nitrogen fixation (BNF) in this diazotroph; however its asparagine metabolic pathway has not yet been clearly established. Methods The work reported here is the first to demonstrate the use of an alternative route for asparaginyl-tRNA (Asn-tRNA) and asparagine formation in an endophytic nitrogen-fixing bacterium by using in silico and in vitro analysis. Results The indirect route involves transamidation of incorrectly charged tRNA via GatCAB transamidase. Nitrogenase activity was completely inhibited by 20 mM Asn in LGI-P medium, which in contrast promotes protein synthesis and microbial growth. Conclusions The analysis carried out in this work shows that intracellular levels of asparagine regulate the expression of nitrogenase nifD gene (GDI0437), suggesting that the presence of an alternative route to produce asparagine might give the G. diazotrophicus a tighter control over cell growth and BNF, and may be of importance in the regulation of the endophytic plant-microbe interaction

Archaea: potencial biotecnológico

Submitted by Alexandre Sousa ([email protected]) on 2015-11-09T16:49:10Z No. of bitstreams: 1 Biotec Cienc Desenv_30.pdf: 256940 bytes, checksum: 79b715b41243787328bc6b1c614a3e4a (MD5)Approved for entry into archive by Alexandre Sousa ([email protected]) on 2015-11-09T17:06:54Z (GMT) No. of bitstreams: 1 Biotec Cienc Desenv_30.pdf: 256940 bytes, checksum: 79b715b41243787328bc6b1c614a3e4a (MD5)Made available in DSpace on 2015-11-09T17:06:54Z (GMT). No. of bitstreams: 1 Biotec Cienc Desenv_30.pdf: 256940 bytes, checksum: 79b715b41243787328bc6b1c614a3e4a (MD5) Previous issue date: 2003Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas. Departamento de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas. Departamento de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de Biologia. Departamento de Biologia Marinha. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, COPPE, Programa de Engenharia Química. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas. Departamento de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas. Departamento de Bioquímica Médica. Rio de Janeiro, RJ, Brasil

Explorando as aplicações biotecnológicas do domínio Archaea

Submitted by Alexandre Sousa ([email protected]) on 2015-12-16T10:28:15Z No. of bitstreams: 1 Braz_J_Microbiol_38_398-405.pdf: 488726 bytes, checksum: b81824bef49afc9dfa9158af9528d14d (MD5)Approved for entry into archive by Alexandre Sousa ([email protected]) on 2015-12-16T10:39:55Z (GMT) No. of bitstreams: 1 Braz_J_Microbiol_38_398-405.pdf: 488726 bytes, checksum: b81824bef49afc9dfa9158af9528d14d (MD5)Made available in DSpace on 2015-12-16T10:39:55Z (GMT). No. of bitstreams: 1 Braz_J_Microbiol_38_398-405.pdf: 488726 bytes, checksum: b81824bef49afc9dfa9158af9528d14d (MD5) Previous issue date: 2007Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Química. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.As arqueas representam uma considerável fração dos procariotos nos ecossistemas marinhos e terrestes, indicando que estes organismos devem possuir um grande impacto nos ciclos energéticos. A natureza extremofílica de muitas arqueas tem estimulado intensos esforços para compreender sua adaptação fisiológica a ambientes extremos. Suas propriedades incomus as tornam uma fonte valiosa no desenvolvimento de novos processos biotecnológicos e aplicações industriais como novos fármacos, cosméticos, suplementos nutricionais, sondas moleculares, enzimas e reagentes. Na presente mini-revisão, mostramos e discutimos algumas de suas características exclusivas correlacionando-as com seu potencial biotecnológico e aplicação industrial. Os tópicos são: características das arqueas, divisão filogenética, aplicações biotecnológicas, isolamento e cultivo de novos microrganismos, genoma e metagenoma.Archaea represent a considerable fraction of the prokaryotic world in marine and terrestrial ecosystems, indicating that organisms from this domain might have a large impact on global energy cycles. The extremophilic nature of many archaea has stimulated intense efforts to understand the physiological adaptations for living in extreme environments. Their unusual properties make them a potentially valuable resource in the development of novel biotechnological processes and industrial applications as new pharmaceuticals, cosmetics, nutritional supplements, molecular probes, enzymes, and fine chemicals. In the present mini-review, we show and discuss some exclusive characteristics of Archaea domain and the current knowledge about the biotechnological uses of the archaeal enzymes. The topics are: archaeal characteristics, phylogenetic division, biotechnological applications, isolation and cultivation of new microbes, achievements in genomics, and metagenomic

Microrganismos associados a poríferos: potencial biotecnológico da microbiota associada às esponjas marinhas

Submitted by Alexandre Sousa ([email protected]) on 2015-11-18T15:30:57Z No. of bitstreams: 1 Turque_2007.pdf: 485860 bytes, checksum: cb1c21ca51eca2a6e04e5e66a5d6657a (MD5)Approved for entry into archive by Alexandre Sousa ([email protected]) on 2015-11-18T16:02:58Z (GMT) No. of bitstreams: 1 Turque_2007.pdf: 485860 bytes, checksum: cb1c21ca51eca2a6e04e5e66a5d6657a (MD5)Made available in DSpace on 2015-11-18T16:02:58Z (GMT). No. of bitstreams: 1 Turque_2007.pdf: 485860 bytes, checksum: cb1c21ca51eca2a6e04e5e66a5d6657a (MD5) Previous issue date: 2007Universidade Federal do Rio de Janeiro. Instituto de Bioquimica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal Fluminense. Biologia Marinha. Niterói, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquimica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Museu Nacional. Rio de Janeiro, RJ, Brasil.Fundação Centro Universitário Estadual da Zona Oeste. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquimica Médica. Rio de Janeiro, RJ, Brasil.Os Poríferos, também conhecidos como esponjas, são invertebrados que filtram grandes quantidades de água e adquirem seus nutrientes por fagocitose dos micróbios capturados durante a filtração (Fig.1). Habitam os oceanos tropicais, temperados e polares, com algumas espécies encontradas em água doce (Taylor et al., 2007). As esponjas formam uma das mais antigas radiações dos metazoários, cuja origem data do Período Pré-Cambriano há cerca de 500 milhões de anos (Hentschel, 2004). São organismos sésseis (fixos no substrato) que apresentam uma alta biodiversidade, com uma estimativa de aproximadamente 15.000 espécies representadas em três classes: Demospongiae, Calcarea e Hexactinellida (Fig.2), sendo a maioria pertencente ao primeiro grupo (Hooper e Van Soest, 2002)

Potencial biotecnológico da microbiota associada às esponjas marinhas

6 p. : il.Os Poríferos, também conhecidos como esponjas, são invertebrados que filtram grandes quantidades de água e adquirem seus nutrientes por fagocitose dos micróbios capturados durante a filtração (Fig.1). Habitam os oceanos tropicais, temperados e polares, com algumas espécies encontradas em água doce (Taylor et al., 2007). As esponjas formam uma das mais antigas radiações dos metazoários, cuja origem data do Período Pré-Cambriano há cerca de 500 milhões de anos (Hentschel, 2004). São organismos sésseis (fixos no substrato) que apresentam uma alta biodiversidade, com uma estimativa de aproximadamente 15.000 espécies representadas em três classes: Demospongiae, Calcarea e Hexactinellida (Fig.2), sendo a maioria pertencente ao primeiro grupo (Hooper e Van Soest, 2002)

Archaea, bacteria, and algal plastids associated with the reef-building corals siderastrea stellata and mussismilia hispida from Búzios, South Atlantic Ocean, Brazil

10 p. : il.Reef-building corals may be seen as holobiont organisms, presenting diverse associated microbial communities. Best known is the symbiotic relationship with zooxanthellae, but Archaea, Bacteria, fungi, viruses, and algal plastids are also abundant. Until now, there is little information concerning microbial communities associated with Brazilian corals. The present study aims to describe the diversity of Archaea, Bacteria, and eukaryotic algal plastid communities associated with two sympatric species, Siderastrea stellata and Mussismilia hispida, from Southeastern Brazil, using 16S rRNA gene libraries. Since corals present a high number of other associated invertebrates, coral barcoding (COI) was performed to confirm the exclusive occurrence of coral DNA in our samples. Our analysis yielded 354 distinct microbial OTUs, represented mainly by novel phylotypes. Richness (Chao1 and ACE) and diversity (H') estimations of the microbial communities associated with both species were high and comparable to other studies. Rarefaction analyses showed that microbial diversity of S. stellata is higher than that of M. hispida. Libshuff comparative analyses showed that the highest microbial community similarity between the two coral species occurred in the bacterial libraries, while archaeal and plastidial communities were significantly different. Crenarchaeota dominated archaeal communities, while Proteobacteria was the most abundant bacterial phylum, dominated by alpha-Proteobacteria. Plastids were also represented by novel phylotypes and did not match with any 16S rRNA sequences of Cyanobacteria and zooxanthellae from GenBank. Our data improves the pool of available information on Brazilian coral microbes and shows corals as sources of diverse prokaryotic and picoeukaryotic communities

Relationships between bacterial diversity and environmental variables in a tropical marine environment, Rio de Janeiro

11 p. : il.This study is the first to apply a comparative analysis of environmental chemistry, microbiological parameters and bacterioplankton 16S rRNA clone libraries from different areas of a 50 km transect along a trophic gradient in the tropical Guanabara Bay ecosystem. Higher bacterial diversity was found in the coastal area, whereas lower richness was observed in the more polluted inner bay water. The significance of differences between clone libraries was examined with LIBSHUFF statistics. Paired reciprocal comparisons indicated that each of the libraries differs significantly from the others, and this is in agreement with direct interpretation of the phylogenetic tree. Furthermore, correspondence analyses showed that some taxa are related to specific abiotic, trophic and microbiological parameters in Guanabara Bay estuarine system

Diversity of bacterial communities related to the nitrogen cycle in a coastal tropical bay

7 p. : il.A culture-independent molecular phylogenetic analysis was carried out to study for the first time the diversity of bacterial ammonia monooxygenase subunit A (amoA) and nitrogenase reductase subunit H (nifH) genes from Urca inlet at Guanabara Bay in Rio de Janeiro, Brazil. Most bacterial amoA and nifH sequences exhibited identities of less than 95% to those in the GenBank database revealing that novel ammonia-oxidizing bacteria and nitrogen-fixing microorganisms may exist in this tropical marine environment. The observation of a large number of clones related to uncultured bacteria also indicates the necessity to describe these microorganisms and to develop new cultivation methodologies

Bacterial communities of the marine sponges Hymeniacidon heliophila and Polymastia janeirensis and their environment in Rio de Janeiro, Brazil

12 p. : il.In this study we performed a survey of the bacterial communities associated with the Western Atlantic demosponges Hymeniacidon heliophila and Polymastia janeirensis, based on 16S rRNA sequencing and transmission electron microscopy (TEM). We compared diversity and composition of the sponge-associated bacteria to those of environmental bacteria, represented by free-living bacterioplankton and by bacteria attached to organic particulate matter in superWcial sediments. Partial bacterial 16S rRNA sequences from seawater, sediment, and sponges were retrieved by PCR, cloning, and sequencing. Sequences were subjected to rarefaction analyses, phylogenetic tree construction, and LIBSHUFF quantitative statistics to verify coverage and similarity between libraries. Community structure of the free-living bacterioplankton was phylogenetically different from that of the sponge-associated bacterial assemblages. On the other hand, some sediment-attached bacteria were also found in the sponge bacterial community, indicating that sponges may incorporate bacteria together with sediment particles. Rare and few prokaryotic morphotypes were found in TEM analyses of sponge mesohyl matrix of both species. Molecular data indicate that bacterial richness and diversity decreases from bacterioplankton, to particulate organic sediment, and to H. heliophila and P. janeirensis. Sponges from Rio de Janeiro harbor a pool of novel and exclusive sponge-associated bacterial taxa. Sponge-associated bacterial communities are composed of both taxons shared by many sponge groups and by species-speciWc bacteria