RESUMO
Shewanella sp. G5, a psychrotolerant marine bacterium, has a cold-shock protein (CspA) and three ß-glucosidases, two of which were classified in the glycosyl hydrolase families 1 and 3 and are encoded by bgl-A and bgl genes, respectively. Shewanella sp. G5 was cultured on Luria-Bertani (LB) and Mineral Medium Brunner (MMB) media with glucose and cellobiose at various temperatures and pH 6 and 8. Relative quantification of the expression levels of all three genes was studied by real-time PCR with the comparative Ct method (2(-ΔΔCt)) using the gyrB housekeeping gene as a normalizer. Results showed that the genes had remarkably different genetic expression levels under the conditions evaluated, with increased expression of all genes obtained on MMB with cellobiose at 30 °C. Specific growth rate and specific ß-glucosidase activity were also determined for all the culture conditions. Shewanella sp. G5 was able to grow on both media at 4 °C, showing the maximum specific growth rate on LB with cellobiose at 37 °C. The specific ß-glucosidase activity obtained on MMB with cellobiose at 30 °C was 25 to 50 % higher than for all other conditions. At pH 8, relative activity was 34, 60, and 63 % higher at 30 °C than at 10 °C, with three peaks at 10, 25, and 37 °C on both media. Enzyme activity increased by 61 and 47 % in the presence of Ca(2+) and by 24 and 31 % in the presence of Mg(2+) on LB and MMB at 30 °C, respectively, but it was totally inhibited by Hg(2+), Cu(2+), and EDTA. Moreover, this activity was slightly decreased by SDS, Zn(2+), and DTT, all at 5 mM. Ethanol (14 % v/v) and glucose (100 mM) also reduced the activity by 63 and 60 %, respectively.
Assuntos
Proteínas de Bactérias/genética , Celobiose/metabolismo , Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial , Shewanella/genética , beta-Glucosidase/genética , Adaptação Fisiológica/genética , Organismos Aquáticos , Proteínas de Bactérias/metabolismo , Cátions Bivalentes , Celobiose/farmacologia , DNA Girase/genética , DNA Girase/metabolismo , Fermentação , Genes Essenciais , Glucose/metabolismo , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Magnésio/metabolismo , Salinidade , Shewanella/efeitos dos fármacos , Shewanella/enzimologia , Shewanella/crescimento & desenvolvimento , Temperatura , beta-Glucosidase/metabolismoRESUMO
Surface-active compounds such as synthetic emulsifiers have been used for several decades, both for the degradation of hydrocarbons and increasing desorption of soil-bound metals. However, due to their high toxicity, low degradability, and production costs unaffordable for use in larger ecosystems, synthetic emulsifiers have been gradually replaced by those derived from natural sources such as plants or microbes. In previous studies, the bacterium Streptomyces sp. MC1 has shown the ability to reduce and/or accumulate Cr(VI), a highly promising advance in the development of methods for environmental clean-up of sites contaminated with chromium. Here, new studies on the production of emulsifier from this strain are presented. The cultivation factors that have a significant influence on emulsifier biosynthesis, as well as the interactions among them, were studied by factorial design. Based upon optimization studies, maximum bioemulsifier production was detected in the culture medium having an initial pH of 8 with phosphate 2.0 g L(-1) and Ca(+2) 1.0 g L(-1) added, with an emulsification index about 3.5 times greater compared to the basal value. Interestingly, in the presence of 5.0 g L(-1) Cr(VI), Streptomyces sp. MC1 retained about 65% of its emulsifier production ability. Partially purified emulsifier presented high thermo-stability and partial water solubility. These findings could have promising future prospects for the remediation of organic- and metal-contaminated sites.
Assuntos
Produtos Biológicos/metabolismo , Cromo/toxicidade , Emulsificantes/metabolismo , Poluentes Ambientais/toxicidade , Streptomyces/metabolismo , Actinobacteria/metabolismo , Adaptação Fisiológica , Biodegradação Ambiental , Streptomyces/efeitos dos fármacosRESUMO
Heavy metal pollution is widespread causing serious ecological problems in many parts of the world; especially in developing countries where a budget for remediation technology is not affordable. Therefore, screening for microbes with high accumulation capacities and studying their stable resistance characteristics is advisable to define cost-effective any remediation strategies. Herein, the copper-resistome of the novel copper-resistant strain Amycolatopsis tucumanensis was studied using several approaches. Two dimensional gel electrophoresis revealed that proteins of the central metabolism, energy production, transcriptional regulators, two-component system, antioxidants and protective metabolites increased their abundance upon copper-stress conditions. Transcriptome analysis revealed that in presence of copper, superoxide dismutase, alkyl hydroperoxide reductase and mycothiol reductase genes were markedly induced in expression. The oxidative damage of protein and lipid from A. tucumanensis was negligible compared with that observed in the copper-sensitive strain Amycolatopsis eurytherma. Thus, we provide evidence that A. tucumamensis shows a high adaptation towards copper, the sum of which is proposed as the copper-resistome. This adaptation allows the strain to accumulate copper and survive this stress; besides, it constitutes the first report in which the copper-resistome of a strain of the genus Amycolatopsis with bioremediation potential has been evaluated.
Assuntos
Actinomycetales/efeitos dos fármacos , Actinomycetales/genética , Cobre/toxicidade , Poluentes Ambientais/toxicidade , Actinomycetales/isolamento & purificação , Actinomycetales/metabolismo , Antioxidantes/metabolismo , Argentina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Cobre/farmacocinética , Farmacorresistência Bacteriana/genética , Poluentes Ambientais/farmacocinética , Genoma Bacteriano , Peroxidação de Lipídeos/efeitos dos fármacos , Oxirredução , TranscriptomaRESUMO
Copper is a redox-active metal, which acts as a catalyst in the formation of Reactive Oxygen Species (ROS) encouraging oxidative stress. Protection against oxidants is intrinsic to every living cell; however, in stress conditions, cells are forced to increase and expand their antioxidative network. In this work, the novel copper-resistant strain Amycolatopsis tucumanensis and the copper-sensitive Amycolatopsis eurytherma were grown under copper increasing concentrations in order to elucidate the dissimilar effects of the metal on the strains viability, mainly on morphology and antioxidant capacity. Although biosorbed copper encouraged ROS production in a dose-dependent manner in both strains, the increase in ROS production from the basal level to the stress conditions in A. tucumanensis is lesser than in the copper-sensitive strain; likewise, in presence of copper A. eurytherma suffered inexorable morphological alteration while A. tucumanensis was not affected. The levels of antioxidant enzymes and metallothioneins (MT) were all greater in A. tucumanensis than in A. eurytherma; in addition MT levels as well as superoxide dismutase and thioredoxin reductase activities in A. tucumanensis, were higher as higher the concentration of copper in the culture medium. This work has given evidence that an efficient antioxidant defense system might aid microorganisms to survive in copper-stress conditions; besides it constitutes the first report of oxidative stress study in the genus Amycolatopsis and contributes to enlarge the knowledge on the copper-resistance mechanisms of A. tucumanensis.
Assuntos
Actinomycetales/efeitos dos fármacos , Antioxidantes/metabolismo , Cobre/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Actinomycetales/enzimologia , Actinomycetales/crescimento & desenvolvimento , Actinomycetales/ultraestrutura , Catalase/metabolismo , Metalotioneína/metabolismo , Viabilidade Microbiana , Microscopia Eletrônica de Varredura , Oxirredução , Superóxido Dismutase/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
From seawater and the intestines of benthonic organisms collected from the Beagle Channel, Argentina, 230 marine bacteria were isolated. Cultivable bacteria were characterized and classified as psychrotolerant, whereas few isolates were psychrophiles. These isolates were capable of producing proteases at 4 and 15 °C under neutral (pH 7.0), alkaline (pH 10.0) and acidic (pH 4.5) conditions on different media, revealing 62, 33 and 22% producers at cold and 84, 47 and 33% producers at low temperatures, respectively. More protease-producing strains (67%) were detected when isolated from benthic invertebrates as compared to seawater (33%), with protease production under neutral conditions resulting in milk protein hydrolysis halos between 27 and 30 ± 2 mm in diameter. Using sterile 0.22 µm membrane filters, 29 isolates exhibiting extracellular protease activity were detected. These were grouped into six operational taxonomic units by restriction analysis and identified based on 16S rDNA as γ-proteobacteria of the genera Pseudoalteromonas, Pseudomonas, Shewanella, Alteromonas, Aeromonas, and Serratia. Plasmids were found to be harbored by eight strains, mainly within the isolates from benthonic organisms.
Assuntos
Bactérias/classificação , Bactérias/metabolismo , Biodiversidade , Trato Gastrointestinal/microbiologia , Peptídeo Hidrolases/metabolismo , Água do Mar/microbiologia , Animais , Regiões Antárticas , Argentina , Bactérias/genética , Bactérias/isolamento & purificação , Análise por Conglomerados , Temperatura Baixa , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Invertebrados , Dados de Sequência Molecular , Filogenia , Plasmídeos , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Amycolatopsis tucumanensis DSM 45259, the strain of a recently recognized novel species of the genus Amycolatopsis with remarkable copper resistance, was used to bioaugment soil microcosms experimentally polluted with copper and for studying the ability of this strain to effectively diminish phytoavailable copper from soils. Our results demonstrated that A. tucumanensis was capable of profusely colonizing both, copper polluted and non-polluted soil. Copper bioimmobilization ability of A. tucumanensis on soil was assessed measuring the bioavailable copper in the soil solution extracted from polluted soil by using chemical and physical methods and, in this way, 31% lower amounts of the metal were found in soil solution as compared to non-bioaugmented soil. The results obtained when using Zea mays as bioindicator correlated well with the values obtained by the chemical and physical procedures: 20% and 17% lower tissue contents of copper were measured in roots and leaves, respectively. These data confirmed the efficiency of the bioremediation process using A. tucumanensis and at the same time proved that chemical, physical and biological methods for assessing copper bioavailability in soils were correlated. These results suggest a potential use of this strain at large scale in copper soil bioremediation strategies. To our knowledge, this work is the first to apply and to probe the colonization ability of an Amycolatopsis strain in soil microcosms and constitutes the first application of an Amycolatopsis strain on bioremediation of polluted soils.
Assuntos
Actinobacteria/metabolismo , Cobre/metabolismo , Poluentes do Solo/metabolismo , Zea mays/metabolismo , Actinobacteria/crescimento & desenvolvimento , Biodegradação Ambiental , Cobre/análise , Solo/análise , Microbiologia do Solo , Poluentes do Solo/análiseRESUMO
A novel actinomycete strain, ABO(T), isolated from copper-polluted sediments showed remarkable copper resistance as well as high bioaccumulation abilities. Classical taxonomic methods, including chemotaxonomy and molecular techniques, were used to characterize the isolate. Strain ABO(T) developed a honey-yellow substrate mycelium on all ISP media tested. Abundant, white, aerial mycelium was only formed on ISP 2, 5 and 7 and MM agar. Both types of hyphae fragmented into squarish rod-shaped elements. The aerial mycelium displayed spore-like structures with smooth surfaces in long, straight to flexuous chains. The organism has a type-IV cell wall lacking mycolic acids and type-A whole-cell sugar pattern (meso-diaminopimelic acid, arabinose and galactose) in addition to a phospholipid type-II profile. 16S rRNA gene sequence studies indicated that this organism is a member of the family Pseudonocardiaceae and that it forms a monophyletic clade with Amycolatopsis eurytherma NT202(T). The DNA-DNA relatedness of strain ABO(T) to A. eurytherma DSM 44348(T) was 39.5 %. It is evident from these genotypic and phenotypic data that strain ABO(T) represents a novel species in the genus Amycolatopsis, for which the name proposed is Amycolatopsis tucumanensis sp. nov. The type strain is ABO(T) (=DSM 45259(T) =LMG 24814(T)).
Assuntos
Actinomycetales/classificação , Cobre/farmacologia , Microbiologia da Água , Poluição Química da Água , Actinomycetales/efeitos dos fármacos , Actinomycetales/genética , Sequência de Bases , Farmacorresistência Bacteriana , Dados de Sequência Molecular , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Homologia de Sequência do Ácido NucleicoRESUMO
46 actinomycetes were isolated from two polluted sites and one unpolluted site. One strain, F4, was selected through primary qualitative screening assays because of its cadmium resistance, and physiologically and taxonomically characterized. F4 was able to grow at 7.5% NaCl and 100 microg/ml lysozyme and at a pH between 6 and 10. 16S rDNA sequence analysis showed that F4 was closely related to Streptomyces tendae. Growth of Streptomyces sp. F4 on culture medium with 8 mg/l Cd(2+) for 8 days showed 80% inhibition. Maximum specific biosorption was 41.7 mg Cd(2+)/g dry weight after 7 days of growth and highest Cd(2+ )concentration was found in the cell wall (41.2%). The exopolysaccharide layer only contained 7.4%, whereas 39.4% of Cd(2+) was found in the cytosolic fraction. Twelve % was found in the ribosomes and membrane fraction. This was verified with TEM, showing Streptomyces sp. F4 cytoplasm with dark granulate appearance. This study could present the potential capacity of Streptomyces sp. F4 for Cd(2+) bioremediation.
Assuntos
Cádmio/metabolismo , Microbiologia do Solo , Poluentes do Solo/metabolismo , Streptomyces/metabolismo , Absorção , Biodegradação Ambiental , Parede Celular/química , Meios de Cultura , DNA Bacteriano/genética , Sedimentos Geológicos/microbiologia , Concentração de Íons de Hidrogênio , RNA Ribossômico 16S/genética , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , Streptomyces/isolamento & purificaçãoRESUMO
Morphological, physiological and molecular characterization of three copper-resistant actinobacterial strains (AB2A, AB3 and AB5A) isolated from copper-polluted sediments of a drainage channel showed that they belonged to the genus Streptomyces. These characteristics plus their distinctive copper resistance phenotypes revealed considerable divergence among the isolates. Highly dissimilar growth patterns and copper removal efficiency were observed for the selected Streptomyces strains grown on minimal medium (MM) added with 0.5 mM of copper sulfate (MM(Cu)). Strain AB2A showed an early mechanism of copper uptake/retention (80% until day 3), followed by a drastic metal efflux process (days 5-7). In contrast, Streptomyces sp. AB3 and AB5A showed only copper retention phenotypes under the same culture conditions. Particularly, Streptomyces sp. AB5A showed a better efficiency in copper removal (94%), although a longer lag phase was observed for this microorganism grown for 7 days in MM(Cu). Cupric reductase activity was detected in both copper-adapted cells and nonadapted cells of all three strains but this activity was up to 100-fold higher in preadapted cells of Streptomyces sp. AB2A. To our knowledge, this is the first time that cupric reductase activity was demonstrated in Streptomyces strains.
Assuntos
Cobre/metabolismo , Oxirredutases/metabolismo , Streptomyces/enzimologia , Streptomyces/crescimento & desenvolvimento , Biodegradação Ambiental , Cobre/farmacologia , Meios de Cultura , Farmacorresistência Bacteriana , Genótipo , Dados de Sequência Molecular , Oxirredutases/genética , Fenótipo , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Microbiologia do Solo , Poluentes do Solo/metabolismo , Poluentes do Solo/farmacologia , Streptomyces/classificação , Streptomyces/isolamento & purificaçãoRESUMO
Amycolatopsis sp. AB0, a copper resistant actinobacterium isolated from polluted sediments, has shown high copper specific biopsortion ability (25 mg g(-1)). Two approaches were used to confirm metal accumulation in growing cells of Amycolatopsis sp. AB0; we performed subcellular fractioning assays which showed that the retained copper was associated with the extra-cellular fraction (exopolymer, 40%), but mainly within the cells. Intracellular distribution of copper was: 86% in the cytosolic fraction, 11% at the cell wall and 3% associated with the ribosome/membrane fraction. Its copper bioaccumulation ability was corroborated by using silver enhanced staining of copper with the Timm's reagent technique, which has not been used to detect metal deposits in bacteria before. In addition, we constructed specific oligonucleotides for targeting genes coding for copper P-Type ATPases that could be involved in the copper uptake ability of this strain. A 607 bp DNA fragment was amplified and sequenced from Amycolatopsis sp AB0. BLAST search analysis showed 71% protein homology of the deduced sequence with a putative cation-transporting ATPase of Nocardia farcinica and 65% with a copper translocating ATPase of Mycobacterium flavescens. To our knowledge this is the first report of the presence of copper P-type ATPase genes in the Amycolotopsis genus.
Assuntos
Actinomycetales/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Cobre/metabolismo , Actinomycetales/genética , Actinomycetales/ultraestrutura , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , ATPases Transportadoras de Cobre , DNA Bacteriano/genética , Marcação de Genes , Microscopia Eletrônica , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
beta -Glucosidase is a highly desired glycosidase, especially for hydrolysis of glycoconjugated precursors in musts and wines for the release of active aromatic compounds. A Shewanella sp. G5 strain was isolated from the intestinal content of benthonic organism (Munida subrrugosa) from different coastal areas of the Beagle Channel, Tierra del Fuego (Argentina). This marine bacterium was able to grow at a temperature range between 4 to 20 degrees C using different beta-glycoside substrates, such as cellobiose, as carbon source. In this work, the Shewanella sp. G5 strain exhibited high beta-glucosidase activity on plate at low temperature (4 and 20 degrees C). Two genes encoding different cold-active beta-glucosidases were amplified and sequenced and the nucleotide sequences were submitted to the GenBank. 16S rDNA and gyrB gene sequences were used for the molecular characterization of Shewanella sp. G5.