TP2

Project TP2: (Meta)transcriptomic of marine model bacteria and model habitats

Responsible: Frank Oliver Glöckner and Hanno Teeling, Jacobs

 

Transcriptomics and metatranscriptomics explore the active fraction of the genes in an organism or a microbial community in situ and are an essential element of a comprehensive “omics” analysis. 

 

The importance of this approach can be imagined by a macroscopic analogy: a caterpillar and a butterfly have the same genome, but show a completely different phenotype and fulfil different functions in the ecosystem. This metamorphosis is regulated by the specific activation and deactivation of genes. In general, all organisms react to environmental changes or trigger morphological changes by adapting their gene expression. 

 

Transcriptomics and metatranscriptomics target the messenger RNA (mRNA) level and hence provide a snapshot of the expressed fraction of genes. Therefore, the method shows how bacteria respond to changes like temperature or nutrient availability. This should lead to predictions how the bacterial community will react to future changes like global warming or ocean acidification. Gene expression data in correlation with contextual (meta)data also paves the way to unravel the function of so far unknown genes, see www.metafunctions.org. 

 

The subproject 2 (TP2) of MIMAS implies gene expression analysis (transcriptomics) of model organisms as well as metatranscriptomics of bacterial communities in the North Sea and Baltic Sea.

 

In order to learn more about the behaviour of bacteria and their gene functions, the model organisms Rhodopirellula baltica SH 1T (Glöckner et al. 2003) and Gramella forsetii KT0803 (Bauer et al. 2006) will be cultivated under various, well defined conditions. The gene expression will be analyzed using whole genome microarrays (Wecker et al. 2009) and pyrosequencing. The results of the two model organisms will provide new insights into the complex life-style and adaptation mechanisms of environmentally relevant marine bacteria. It will further set the ground for the validation of the metatranscriptome approach.

 

Rhodopirellula-baltica

 

Rhodopirellula baltica

 

The aim of the metatranscriptome analysis in the North Sea and Baltic Sea is to gain insights about the interaction of microbes and their environment as well as their reaction to seasonal changes. By comparison of the data sets from different sampling time points and by taking a rich set of contextual data into consideration, key genes might be identified and promising new gene functions detected. This will provide the cornerstones for a future ecosystems biology approach at the two long term ecological research sides Helgoland Roads and Gotland Deep.

 

Especially the metatranscriptomic part is challenging. A pipeline for sampling, the processing of the samples for pyrosequencing and data analysis is currently developed in cooperation with all MIMAS partners. Low biomass concentrations, force the need to filter more than 100 L of water in short time. Additionally, the need to find and validate appropriate methods for mRNA enrichment and double stranded cDNA synthesis, are among the issues to be dealt with. 

 

Sampling for Metatranscriptomics

Bacteria can react within minutes to changes of their surrounding with a change of their gene expression. Hence, the conditions during sample processing should be similar to the natural environment and the biomass enrichment should not take longer than 20 minutes. On the other hand, the mRNA content per cell and the biomass concentration in the water column are low. Therefore, the biomass from a large amount of water needs to be extracted in the given time frame. Another issue is the enzymatic degradation of RNA by RNAses, making RNAse-free handling imperative.

 

 

 

The sampling boat Diker and its lager version Sorcerer II at Helgoland

 

 

Considering these requirements, a sampling pipeline with two prefiltration steps, one capture filtration with two filtration units running in parallel for each step, has been set up. The filter material of the capture filter has been optimized for fast flow-through and downstream processing.

 

Cut up filter with biomass

 

Cut up filter with biomass

 

 

Downstream processing

Once the biomass is fixed on the filter the RNA extraction starts. Appropriate methods for cracking tough cell walls of marine Bacteria and Archaea and suitable solutions for sample clean-up have been evaluated. The complex and mostly unknown composition of the samples, possible contaminations from sediments, which can hamper further processing, are challenging.

 

 

Img.1 Cell breakup
Img.2 Measuring RNA quality

Img.3 Checking mRNA quality with Bioanalyzer 

 

The major part of RNA consists of ribosomal RNA (rRNA). In classical microarray based expression analysis this is not problematic, because no corresponding capture probes are available on the microarray slide. If the expression analysis is carried out via sequencing, however, the rRNA will occupy the majority of the sequencing capacity. For metatranscriptome analysis, this part of the RNA is not vital and should be reduced. Several methods have been tested to find the best solution for this task and finally a method to enrich the amount of mRNA has been successfully tested.

Subsequently, the enriched mRNA is transcribed to double stranded cDNA and sequenced via pyrosequencing. 

 

Data analysis

A major challenge will be the processing, integration and interpretation of the data. Diversity as well as genomic (link zu TP3), transcriptomic and proteomic (link zu TP1) data will be integrated with respect to on site and long term contextual data by the technological platform of the MIMAS project (link zu TP5). The metatranscriptome will be mapped on the metagenome to determine which parts of the genomes are in use under the respective conditions. The data will be screened for seasonal patterns and correlations between gene expression and environmental parameters.

Additionally, the results will be checked for key genes and potential candidates for biotechnology or medical use.

 

References:

Bauer M, Kube M, Teeling H, Richter M, Lombardot T, Allers E, et al. (2006). Whole genome analysis of the marine Bacteriodetes 'Gramella forsetii' reveals adaptations to degradation of polymeric organic matter. Environ Microbiol 8: 2201-2213.

Glöckner FO, Kube M, Bauer M, Teeling H, Lombardot T, Ludwig W, et al. (2003). Complete genome sequence of the marine planctomycete Pirellula sp. strain 1. Proc Natl Acad Sci U S A 100: 8298-8303

Wecker P, Klockow C, Ellrott A, Quast C, Langhammer P, Harder J and Glöckner FO, Transcriptional response of the model planctomycete Rhodopirellula baltica SH1T to changing environmental conditions, under revision