Avaliação do processo de morte celular em bactérias aquáticas em dois modelos de ecossistemas aquáticos tropicais

Abstract

The bacterioplankton is an important component of the food web structure in aquatic ecosystems and it is regulated by many factors such as nutrient supply, temperature, predation and virus infection. Bacterial death is characterized by functional and morphological loss of the bacterial cell with roles in the control process of abundance and bacterial production of aquatic ecosystems and functional meaning in the carbon and nutrient cycles. However, the cell death process in aquatic bacteria remains to be defined. Programmed cell death (PCD) is a regulated process largely known in eukaryotic organisms. There are not studies dealing with PCD occurrence in aquatic bacteria. This study aimed to investigate the occurrence of PCD in free-living aquatic bacteria in two models of aquatic ecosystems: Batata lake and Manacás lake. Batata Lake is located on floodplain Trombetas river in northern of Brazilian Amazon. This ecosystem has been impacted for bauxite tailings over teen years and it is presently divided in impacted and natural stations. Manacás Lake is an artificial system located in Minas Gerais. Three groups of bacteria from Manacás Lake, Batata lake – impacted area - and Batata lake – natural area -were studied. Water samples were collected from the subsurface of these lakes and processed for analyses of bacterial viability by fluorescence microscopy and DNA fragmentation by flow citometry. In parallel, samples from Batata lake – impacted area - were processed for transmission electron microscopy (TEM) for characterization of the bacterial community ultrastructural diversity and investigation of bacteria alterations. Our cell viability results using specific markers for membrane integrity (LIVE/DEAD BacLight) showed directly the presence of live/viable and dead/ not viable bacteria. Batata lake presented a higher proportion of bacterial death (36,20%) compared to Manacás lake (19,66%). DNA fragmentation analyses (TUNEL assay) showed that PCD is a phenomenon occurring in all aquatic ecosystems investigated, with higher frequency in the Batata lakeimpacted area compared to the other ecosystems. Our TEM analyses revealed a great bacterial ultrastructural diversity represented by morphological differences in the cellular envelope, capsule, mesosomes, membrane vesicles, attached particles and thylacoids. Moreover, around 34,28% of the bacteria showed virus in their cytoplasm. The ultrastructural diversity may represent the large metabolic and adaptative diversity of aquatic bacteria while the presence of virus may be related to bacterial death. Our ultrastructural integrity analyses showed that (i) the higher proportion of bacteria was in death process or dead (damaged and empty), (ii) the capsule frequency is lower in empty bacteria, (iii) the higher bacteria-particle association was found in altered bacteria, (iv) the mean number of viruses per cell section was higher in altered compared to intact bacteria. TEM also showed the presence occurrence of typical ultrastructural changes indicative of MPC, such as cell retraction and condensation. Altogether, our data demonstrate that PCD occur in aquatic bacteria, and that this event may be a survival mechanism for bacterial communities in these ecosystems.