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Heterotrophic activity and Ecology of abundant versus RAre marine bacterial phylotypes

Final Report Summary - HERA (Heterotrophic activity and Ecology of abundant versus RAre marine bacterial phylotypes)

The project aimed to study the diversity and activity of rare versus abundant heterotrophic marine bacterial phylotypes by using methodological innovations and conventional approaches based on cloning and sequencing of PCR amplification products. The rare biosphere consists of a plethora of low abundance phylotypes, which apparently escape conventional sequencing approaches, but the fractionation of complex natural prokaryotic communities results in greatly enhanced detection of low abundant phylotypes by increasing the efficiency of the PCR. We focused on the fractionation done by high-throughput cell sorting using a FACSAria flow cytometer and on the confinement of bacterial community, which allowed us the identification of specific groups of bacteria as well as monitoring changes in bacterial diversity.

The three main objectives of the project were:
1. Determine the number of phylotypes recovered from complex (unfractionated) versus fractionated bacterial communities.
2. Determine the abundance and phylogeny of rare versus abundant phylotypes.
3. Determine the metabolic activity (leucine incorporation) of selected rare versus abundant bacteria.

In order to identify members of the rare biosphere, the low abundance of cells collected by cell sorting needed specific molecular approaches. Real-time monitoring in multiple displacement amplification (MDA) following phi29 polymerase amplification of the genome of the sorted cells allows phylogenetic affiliation on almost a single cell level. In our work (Months 1-7), we assayed different amplification conditions of phi29 polymerase to establish an accurate protocol for single-cell MDA. This aim was possible for cultured cells but we were not able to amplify one single cell from marine bacterial assemblages.

An important part of the project was the participation in a seasonal sampling in the coastal Adriatic Sea and in a cruise to the Vema Fracture Zone, Central Atlantic Ocean. The work during the coastal sampling (Months 8-17) additionally allowed us to explore the effects of incubation time and concentration of erythromycin on bacterial activity. Specific work with metabolic inhibitors was originally not planned, but previous works of researchers in Herndl's group (Taichi et al, in press) revealed that erythromycin is selectively inhibiting Bacteria, but not Archaea. The sampling in Adriatic coastal waters also allowed us to estimate growth rates of relevant marine bacterial groups. Microbial degradation of dissolved organic matter (DOM) in planktonic ecosystems is carried out by diverse prokaryotic communities, whose growth rates and patterns of DOM utilisation modulate carbon and nutrient biogeochemical cycles at local and global scales. Our results suggest that different bacterial groups exhibit characteristic growth rates, and, consequently, distinct competitive abilities to succeed under contrasting environmental conditions.

The participation in the cruise to the Vema Fracture Zone of the Atlantic and the subsequent processing of the samples (Months 18-24) aimed to monitor changes in the structure of the bacterioplankton communities of surface and mesopelagic waters subjected to confinement. We analyzed the composition of bacterial assemblages at the beginning and the end of the incubations by terminal restriction fragment length polymorphism (T-RFLP). In two selected experiments, we analyzed by cloned and sequenced the 16S rDNA genes of the bacterial community. Sequence analysis confirmed the T-RFLP data revealing a significant decrease in the diversity of bacterial communities during the incubations and a clear depth-related pattern after incubation. Additionally, T-RFLP of selected clones allowed us to link the OTUs obtained by T-RFLP to specific phylogenetic groups, enabling to track the occurrence of specific phylotypes in the T-RFLP fingerprints throughout the different experiments. Hence, further coupling between T-RFLP profiles and 16S rDNA clone libraries could become an effective and powerful tool to obtain specific taxonomic information from a fingerprinting technique. During the participation in the cruise to the Vema Fracture Zone, we also had the opportunity of taking samples for cryopreservation for further single cell DNA analyses in collaboration with Stepanauskas's lab (Bigelow Laboratory for Ocean Sciences, USA).

Taken together, all these results will improve our current understanding of the ecological role of the rare biosphere as part of the heterotrophic bacterial community driving the biogeochemical cycles. Due to the effects of global ocean warming on the biomass and activity of bacterial assemblages, understanding the key biogeochemical players and regulating mechanisms are urgent issues for the scientific community, and society in general.

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