RESUMO
Arica and Parinacota Region is located at the extreme north of the Atacama Desert, where the high levels of salts and boron, lack of rain, high UV radiation, among other conditions, make this zone an extreme environment. Despite these characteristics, in the transversal valleys, different types of crops are cultivated in this region, which are associated to beneficial microorganisms with specific traits that allow plants surviving and developing under extreme conditions. However, there is incomplete information related to these microorganisms. In this work, bacteria associated with ancestral crops were isolated from oregano, alfalfa, maize, potato, and grapevine samples from Belén, Codpa, Molinos, Poconchile and Socoroma localities, representing the first report of these microorganisms in those sites. Bacteria were identified, being γ-Proteobacteria, the most frequent class (~ 74.4%), with members of Pseudomonas genus the most common isolated genus. All bacteria were functionally characterized for plant growth-promoting activities, including siderophores and auxins production, phosphate solubilization, and nitrogen fixation, revealing an extraordinary potential from these microorganisms for agricultural applications under arid and semiarid conditions.
Assuntos
Bactérias , Microbiologia do Solo , Bactérias/genética , Produtos Agrícolas , Clima Desértico , Fixação de Nitrogênio , ProteobactériasRESUMO
BACKGROUND: Antifreeze proteins (AFPs) production is a survival strategy of psychrophiles in ice. These proteins have potential in frozen food industry avoiding the damage in the structure of animal or vegetal foods. Moreover, there is not much information regarding the interaction of Antarctic bacterial AFPs with ice, and new determinations are needed to understand the behaviour of these proteins at the water/ice interface. RESULTS: Different Antarctic places were screened for antifreeze activity and microorganisms were selected for the presence of thermal hysteresis in their crude extracts. Isolates GU1.7.1, GU3.1.1, and AFP5.1 showed higher thermal hysteresis and were characterized using a polyphasic approach. Studies using cucumber and zucchini samples showed cellular protection when samples were treated with partially purified AFPs or a commercial AFP as was determined using toluidine blue O and neutral red staining. Additionally, genome analysis of these isolates revealed the presence of genes that encode for putative AFPs. Deduced amino acids sequences from GU3.1.1 (gu3A and gu3B) and AFP5.1 (afp5A) showed high similarity to reported AFPs which crystal structures are solved, allowing then generating homology models. Modelled proteins showed a triangular prism form similar to ß-helix AFPs with a linear distribution of threonine residues at one side of the prism that could correspond to the putative ice binding side. The statistically best models were used to build a protein-water system. Molecular dynamics simulations were then performed to compare the antifreezing behaviour of these AFPs at the ice/water interface. Docking and molecular dynamics simulations revealed that gu3B could have the most efficient antifreezing behavior, but gu3A could have a higher affinity for ice. CONCLUSIONS: AFPs from Antarctic microorganisms GU1.7.1, GU3.1.1 and AFP5.1 protect cellular structures of frozen food showing a potential for frozen food industry. Modeled proteins possess a ß-helix structure, and molecular docking analysis revealed the AFP gu3B could be the most efficient AFPs in order to avoid the formation of ice crystals, even when gu3A has a higher affinity for ice. By determining the interaction of AFPs at the ice/water interface, it will be possible to understand the process of adaptation of psychrophilic bacteria to Antarctic ice.
Assuntos
Proteínas Anticongelantes/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Aminoácidos , Regiões Antárticas , Proteínas Anticongelantes/química , Proteínas Anticongelantes/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cucurbita/metabolismo , Cucurbitaceae/metabolismo , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Sequenciamento Completo do GenomaRESUMO
Four lipases were purified from ID17, a thermophilic bacterium belonging to Geobacillus genus isolated from Deception Island, Antarctica. Lipase activity was detected by opacity test and p-nitrophenyl laurate methods. Lipase production was better in a medium containing tryptone as the carbon and nitrogen source, without non-ionic detergents and pH 7.5. Proteins were ultrafiltered from supernatant and separated using anion exchange and size exclusion chromatography resulting in four distinct fractions with lipase activity (called Lip1-4). Purified lipases showed an optimal pH at 9.0, 9.5, 10.0 and 8.0 and temperature at 65, 70, 75 and 80 °C for Lip1-4, respectively. Lip1 and Lip2 showed higher activity using p-nitrophenol decanoate as substrate, whereas Lip3 and Lip4 prefer p-nitrophenol laurate. Based on their molecular weight Lip1 and Lip2 are trimeric and pentameric proteins, respectively, whereas Lip3 and Lip4 are monomeric proteins. Lip1 was exceptionally thermostable maintaining 70 % of its activity after incubating it at 70 °C for 8 h. Based on their characteristics, the four lipases obtained from ID17 are good candidates to understand the mechanisms of lipase stability and to be used in different types of industrial applications.