We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium （named AU 11）, from a water sample collected in Lake Uruguay （King George Island, South Shetlands）. AUI 1 （growth betwe...We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium （named AU 11）, from a water sample collected in Lake Uruguay （King George Island, South Shetlands）. AUI 1 （growth between 4℃ and 30℃） produces a single cold-active extracellular protease （ExPAU11）, differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-ToF MS） as an alkaline metallo-protease （70% coverage with an extracellular protease of Janthinobacterium sp. PI12）, and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene （named JSP8A） showing 60% identity （98% query coverage） to subtilisin peptidases belonging to the $8 family （S8A subfamily） of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from SSA bacterial sequences of other phyla （including its own）. An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer （HGT） from a cyanobacterittrn. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.展开更多
Fildes Peninsula, in King George Island, Antarctica, has a great concentration of international facilities, and it has clearly been affected by human activities. The objective of this 5-year study was to assess the im...Fildes Peninsula, in King George Island, Antarctica, has a great concentration of international facilities, and it has clearly been affected by human activities. The objective of this 5-year study was to assess the impact of anthropogenic activities on the bacterial abundance in water bodies close to Artigas Antarctic Scientific Base （BCAA, in Spanish Base Cientifica Antdrtica Artigas）. Water samples from areas under different human influence （Uruguay Lake, nearby ponds, and meltwater from Collins Glacier） were aseptically collected and refrigerated until processed. The number of heterotrophic bacteria and Pseudomonas spp. was analyzed using a culture-dependent approach. Physico-chemical properties of the water samples （temperature, pH, and conductivity） were also determined. Results showed that water from the highly affected area, Uruguay Lake, where the pump that provides water to the BCAA is located, did not suffer significant fluctuations in heterotrophie bacterial abundance （10^4- 10^5 CFU.mL^-1）; however, Pseudomonas abundance increased until becoming the predominant population. In other water samples, the number of heterotrophie bacteria and Pseudomonas gradually increased during this 5-year study, by 2014 reaching similar values to those observed for Uruguay Lake. The implications of human activities on Antarctic bacterial abundance are discussed.展开更多
Background:Bladder cancer is the tenth most common cancer worldwide.Considering its high prevalence(vulnerability to multiple recurrences and progression despite local therapy),which leads to a substantial health serv...Background:Bladder cancer is the tenth most common cancer worldwide.Considering its high prevalence(vulnerability to multiple recurrences and progression despite local therapy),which leads to a substantial health service burden,it becomes necessary to develop new strategies to increase the effectiveness of bladder tumor therapy.Natural compounds with antiproliferative effect on cancer cells could be a good choice for co-adjuvant chemotherapy.Microorganisms are one of the main sources for natural compounds.Pigments extracted from the cold-adapted microorganisms can contribute to the development of a broader range of applications in biotechnology.Violacein is a purple pigment commonly produced by many bacterial strains.We have previously shown that very low concentrations of violacein extracted from Janthinobacterium sp.produced an antiproliferative effect on HeLa cells.Objective:With the aim to determine if violacein has an antiproliferative activity on bladder cancer cells,as well as to test if it has synergistic effects on cisplatin treated cells in vitro,T24 and 253J cell lines(derived bladder cancer cells from carcinoma in situ and retroperitoneal metastasis,respectively)were exposed to different concentrations of violacein in the presence or absence of cisplatin.Methods:i)Resazurin assay and flow cytometry were performed in two bladder cancer-derived cell lines,namely T24 and 253J,to see if violacein affects cell viability and induce cell death.ii)To find out whether violacein sensitizes bladder cancer cells to cisplatin,the drug interaction among different doses of cisplatin and violacein was analyzed,as well their combination index was determined.iii)The effect of violacein to induce primary genetic damage was determined through the analysis of induced micronuclei frequency and𝛾H2AX foci,as well as performing the comet assay.Results:The half-maximal inhibitory concentration of violacein at 24 h for both cell lines were around 500 nM,and decreased below 400 nM in combination with 10μM of cisplatin,indicating antiproliferative and sensitizing effects of violacein to cisplatin in both cell lines tested.A clear cell cycle delay,as well as an increase in the percentage of cell death was observed by flow cytometry at 300 nM of violacein,either alone or in combination with cisplatin.On the other hand,the analysis of the micronucleus frequency did not evidence an increase in genetic damage.Moreover,in combined treatments with cisplatin there was a slight decrease on micronucleus induction.Besides,the induction of genetic damage was not observed through comet assay when cells were treated with violacein alone,however,when cells were treated with violacein in the presence of cisplatin(10μM).The production of genetic damage was diminished in T24 or 253J cells.By the same token,increase in the frequency of𝛾H2AX foci by violacein was not observed at any tested dose in both cell lines.Conclusion:It was shown that violacein has an in vitro antiproliferative effect in bladder cancer cell lines,sensitizing them to cisplatin.Interestingly,at doses tested,violacein did not induce genotoxicity and reduce the genotoxic effect produced by cisplatin.展开更多
基金supported by PEDECIBA (Programa De Desarrollo de las Ciencias Básicas), Uruguay, and IAU (Instituto Antártico Uruguayo)supported by ANII (Agencia Nacional de Investigación e Innovación)
文摘We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium （named AU 11）, from a water sample collected in Lake Uruguay （King George Island, South Shetlands）. AUI 1 （growth between 4℃ and 30℃） produces a single cold-active extracellular protease （ExPAU11）, differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-ToF MS） as an alkaline metallo-protease （70% coverage with an extracellular protease of Janthinobacterium sp. PI12）, and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene （named JSP8A） showing 60% identity （98% query coverage） to subtilisin peptidases belonging to the $8 family （S8A subfamily） of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from SSA bacterial sequences of other phyla （including its own）. An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer （HGT） from a cyanobacterittrn. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.
基金supported by the Program for the Development of Basic Sciences (Programa de Desarrollo de las Ciencias Básicas, PEDECIBA)provided by the Uruguayan Antarctic Institute (Instituto Antártico Uruguayo, IAU)
文摘Fildes Peninsula, in King George Island, Antarctica, has a great concentration of international facilities, and it has clearly been affected by human activities. The objective of this 5-year study was to assess the impact of anthropogenic activities on the bacterial abundance in water bodies close to Artigas Antarctic Scientific Base （BCAA, in Spanish Base Cientifica Antdrtica Artigas）. Water samples from areas under different human influence （Uruguay Lake, nearby ponds, and meltwater from Collins Glacier） were aseptically collected and refrigerated until processed. The number of heterotrophic bacteria and Pseudomonas spp. was analyzed using a culture-dependent approach. Physico-chemical properties of the water samples （temperature, pH, and conductivity） were also determined. Results showed that water from the highly affected area, Uruguay Lake, where the pump that provides water to the BCAA is located, did not suffer significant fluctuations in heterotrophie bacterial abundance （10^4- 10^5 CFU.mL^-1）; however, Pseudomonas abundance increased until becoming the predominant population. In other water samples, the number of heterotrophie bacteria and Pseudomonas gradually increased during this 5-year study, by 2014 reaching similar values to those observed for Uruguay Lake. The implications of human activities on Antarctic bacterial abundance are discussed.
基金the National Agency for Re-search and Innovation(ANII-Uruguay).
文摘Background:Bladder cancer is the tenth most common cancer worldwide.Considering its high prevalence(vulnerability to multiple recurrences and progression despite local therapy),which leads to a substantial health service burden,it becomes necessary to develop new strategies to increase the effectiveness of bladder tumor therapy.Natural compounds with antiproliferative effect on cancer cells could be a good choice for co-adjuvant chemotherapy.Microorganisms are one of the main sources for natural compounds.Pigments extracted from the cold-adapted microorganisms can contribute to the development of a broader range of applications in biotechnology.Violacein is a purple pigment commonly produced by many bacterial strains.We have previously shown that very low concentrations of violacein extracted from Janthinobacterium sp.produced an antiproliferative effect on HeLa cells.Objective:With the aim to determine if violacein has an antiproliferative activity on bladder cancer cells,as well as to test if it has synergistic effects on cisplatin treated cells in vitro,T24 and 253J cell lines(derived bladder cancer cells from carcinoma in situ and retroperitoneal metastasis,respectively)were exposed to different concentrations of violacein in the presence or absence of cisplatin.Methods:i)Resazurin assay and flow cytometry were performed in two bladder cancer-derived cell lines,namely T24 and 253J,to see if violacein affects cell viability and induce cell death.ii)To find out whether violacein sensitizes bladder cancer cells to cisplatin,the drug interaction among different doses of cisplatin and violacein was analyzed,as well their combination index was determined.iii)The effect of violacein to induce primary genetic damage was determined through the analysis of induced micronuclei frequency and𝛾H2AX foci,as well as performing the comet assay.Results:The half-maximal inhibitory concentration of violacein at 24 h for both cell lines were around 500 nM,and decreased below 400 nM in combination with 10μM of cisplatin,indicating antiproliferative and sensitizing effects of violacein to cisplatin in both cell lines tested.A clear cell cycle delay,as well as an increase in the percentage of cell death was observed by flow cytometry at 300 nM of violacein,either alone or in combination with cisplatin.On the other hand,the analysis of the micronucleus frequency did not evidence an increase in genetic damage.Moreover,in combined treatments with cisplatin there was a slight decrease on micronucleus induction.Besides,the induction of genetic damage was not observed through comet assay when cells were treated with violacein alone,however,when cells were treated with violacein in the presence of cisplatin(10μM).The production of genetic damage was diminished in T24 or 253J cells.By the same token,increase in the frequency of𝛾H2AX foci by violacein was not observed at any tested dose in both cell lines.Conclusion:It was shown that violacein has an in vitro antiproliferative effect in bladder cancer cell lines,sensitizing them to cisplatin.Interestingly,at doses tested,violacein did not induce genotoxicity and reduce the genotoxic effect produced by cisplatin.
