Analysis of nifH DNA and RNA reveals a disproportionate contribution to nitrogenase activities by rare plankton-associated diazotrophs.

BACKGROUND: Holobionts comprising nitrogen-fixing diazotrophs and phytoplankton or zooplankton are ubiquitous in the pelagic sea. However, neither the community structure of plankton-associated diazotrophs (PADs) nor their nitrogenase transcriptional activity are well-understood. In this study, we used nifH gene Illumina sequencing and quantitative PCR to characterize the community composition and nifH expression profile of PADs with > 100 µm size fraction in the euphotic zone of the northern South China Sea. RESULTS: The results of DNA- and RNA-derived nifH gene revealed a higher alpha-diversity in the active than in the total community. Moreover, the compositional resemblance among different sites was less for active than for total communities of PADs. We characterized the 20 most abundant OTUs by ranking the sum of sequence reads across 9 sampling stations for individual OTUs in both nifH DNA and RNA libraries, and then assessed their phylogenetic relatedness. Eight of the 20 abundant OTUs were phylogenetically affiliated with Trichodesmium and occurred in approximately equal proportion in both the DNA and RNA libraries. The analysis of nifH gene expression level showed uneven attribute of the abundance and nitrogenase activities by the remaining 12 OTUs. Taxa belonging to cluster III and Betaproteobacteria were present at moderate abundance but exhibited negligible nitrogenase transcription activity. Whereas, the abundances of Richelia, Deltaproteobacteria and Gammaproteobacteria were low but the contribution of these groups to nitrogenase transcription was disproportionately high. CONCLUSIONS: The substantial variation in community structure among active dizatrophic fractions compared to the total communities suggests that the former are better indicators of biological response to environmental changes. Altogether, our study highlights the importance of rare PADs groups in nitrogen fixation in plankton holobionts, evidenced by their high level of nitrogenase transcription.

[Protective effects of exogenous H2S to the recovery of hypoxia post-conditioning on aged H9C2 cells and the underling mechanisms].

OBJECTIVE: To investigate the recovery of protective effects of exogenous hydrogen sulfide (H2S) on hypoxia post-conditioning in aged H9C2 cells and its mechanism. METHODS: H9C2 cells (cardiomyocytes line) were treated with 30 µmol/L hydrogen peroxide (H2O2) for 2 hours, then cultured for 3 days in order to induce cellular aging. Aged H9C2 cells were randomly divided into 5 groups (n=8):Control group (Control), hypoxia/reoxygenation group (H/R), H/R + NaHS group, hypoxia post-conditioning (PC) group, PC+NaHS group. H/R model:the cells were exposed to hypoxic culture medium (serum and sugar free medium, pH=6.8) for 3 hours and then cultured at normal condition for 6 hours. PC model:at the end of hypoxia for 3 hours, the cells were exposed to normoxic culture solution for 5 minutes, then the cells were placed in hypoxic solution for 5 minutes, the cycle above-mentioned was repeated 3 times and followed by reoxygenation for 6 hours. Advanced glycation end products (AGEs) content and caspase-3 activity were detected by ELISA. The cell viability was observed by cell counting kit-8 (CCK-8). The reactive oxygen species (ROS) levels were analyzed using 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The apoptotic rate was determined through Hoechst 33342 staining. The mRNA levels of relative gene expression were detected by real-time PCR. RESULTS: Thirty µmol/L H2O2 induced H9C2 cell senescence while did not lead to apoptosis. Compared with control group, cell viability was decreased, the apoptotic rate、levels of ROS and the mRNA of caspase-3, caspase-9 and Bcl-2 were increased in H/R and PC groups (P<0.01). There were no differences in the above indexes between PC group and H/R group. Supplementation of NaHS increased cell viability and decreased apoptotic rate and oxidative stress. The effects of PC + NaHS on the above indexes were better than those of H/R+NaHS group. CONCLUSIONS: Exogenous H2S can restore the protective effect of PC on the aged H9C2 cells, and its mechanism is related to the inhibition of oxidative stress and apoptosis.

Responses of bacterial communities in seagrass sediments to polycyclic aromatic hydrocarbon-induced stress.

The seagrass meadows represent one of the highest productive marine ecosystems, and have the great ecological and economic values. Bacteria play important roles in energy flow, nutrient biogeochemical cycle and organic matter turnover in marine ecosystems. The seagrass meadows are experiencing a world-wide decline, and the pollution is one of the main reasons. Polycyclic aromatic hydrocarbons (PAHs) are thought be the most common. Bacterial communities in the seagrass Enhalus acoroides sediments were analyzed for their responses to PAHs induced stress. Dynamics of the composition and abundance of bacterial communities during the incubation period were explored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR assay, respectively. Both the incubation time and the PAHs concentration played significant roles in determining the microbial diversity, as reflected by the detected DGGE bands. Analysis of sequencing results showed that the Gammaproteobacteria were dominant in the seagrass sediments, accounting for 61.29 % of all sequenced bands. As PAHs could be used as carbon source for microbes, the species and diversity of the PAH-added groups (group 1 and 2) presented higher Shannon Wiener index than the group CK, with the group 1 showing the highest values almost through the same incubation stage. Patterns of changes in abundance of the three groups over the experiment time were quite different. The bacterial abundance of the group CK and group 2 decreased sharply from 4.15 × 10(11) and 6.37 × 10(11) to 1.17 × 10(10) and 1.07 × 10(10) copies/g from day 2 to 35, respectively while bacterial abundance of group 1 increased significantly from 1.59 × 10(11) copies/g at day 2 to 8.80 × 10(11) copies/g at day 7, and then dropped from day 14 till the end of the incubation. Statistical analysis (UMPGA and PCA) results suggested that the bacterial community were more likely to be affected by the incubation time than the concentration of the PAHs. This study provided the important information about dynamics of bacterial community under the PAHs stress and revealed the high bacterial diversity in sediments of E. acoroides. Investigation results also indicated that microbial community structure in the seagrass sediment were sensible to the PAHs induced stress, and may be used as potential indicators for the PAHs contamination.

Illumina-based analysis the microbial diversity associated with Thalassia hemprichii in Xincun Bay, South China Sea.

In order to increase our understanding of the microbial diversity associated with seagrass Thalassia hemprichii in Xincun Bay, South China Sea, 16S rRNA gene was identified by highthrough sequencing method. Bacteria associated with seagrass T. hemprichii belonged to 37 phyla, 99 classes. The diversity of bacteria associated with seagrass was similar among the geographically linked coastal locations of Xincun Bay. Proteobacteria was the dominant bacteria and the α-proteobacteria had adapted to the seagrass ecological niche. As well, α-proteobacteria and Pseudomonadales were associated microflora in seagrass meadows, but the interaction between the bacteria and plant is needed to further research. Burkholderiales and Verrucomicrobiae indicated the influence of the bay from anthropogenic activities. Further, Cyanobacteria could imply the difference of the nutrient conditions in the sites. γ-proteobacteria, Desulfobacterales and Pirellulales played a role in the cycle of sulfur, organic mineralization and meadow ecosystem, respectively. In addition, the less abundance bacteria species have key functions in the seagrass meadows, but there is lack knowledge of the interaction of the seagrass and less abundance bacteria species. Microbial communities can response to surroundings and play key functions in the biochemical cycle.

The diversity of coral associated bacteria and the environmental factors affect their community variation.

Coral associated bacterial community potentially has functions relating to coral health, nutrition and disease. Culture-free, 16S rRNA based techniques were used to compare the bacterial community of coral tissue, mucus and seawater around coral, and to investigate the relationship between the coral-associated bacterial communities and environmental variables. The diversity of coral associated bacterial communities was very high, and their composition different from seawater. Coral tissue and mucus had a coral associated bacterial community with higher abundances of Gammaproteobacteria. However, bacterial community in seawater had a higher abundance of Cyanobacteria. Different populations were also found in mucus and tissue from the same coral fragment, and the abundant bacterial species associated with coral tissue was very different from those found in coral mucus. The microbial diversity and OTUs of coral tissue were much higher than those of coral mucus. Bacterial communities of corals from more human activities site have higher diversity and evenness; and the structure of bacterial communities were significantly different from the corals collected from other sites. The composition of bacterial communities associated with same coral species varied with season's changes, geographic differences, and coastal pollution. Unique bacterial groups found in the coral samples from more human activities location were significant positively correlated to chemical oxygen demand. These coral specific bacteria lead to coral disease or adjust to form new function structure for the adaption of different surrounding needs further research.