COLOMBOS: Access Port for Cross-Platform Bacterial Expression Compendia

Background: Microarrays are the main technology for large-scale transcriptional gene expression profiling, but the large bodies of data available in public databases are not useful due to the large heterogeneity. There are several initiatives that attempt to bundle these data into expression compendia, but such resources for bacterial organisms are scarce and limited to integration of experiments from the same platform or to indirect integration of per experiment analysis results. Methodology/Principal Findings: We have constructed comprehensive organism-specific cross-platform expression compendia for three bacterial model organisms (Escherichia coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium) together with an access portal, dubbed COLOMBOS, that not only provides easy access to the compendia, but also includes a suite of tools for exploring, analyzing, and visualizing the data within these compendia. It is freely available at http://bioi.biw.kuleuven.be/colombos. The compendia are unique in directly combining expression information from different microarray platforms and experiments, and we illustrate the potential benefits of this direct integration with a case study: extending the known regulon of the Fur transcription factor of E. coli. The compendia also incorporate extensive annotations for both genes and experimental conditions; these heterogeneous data are functionally integrated in the COLOMBOS analysis tools to interactively browse and query the compendia not only for specific genes or experiments, but also metabolic pathways, transcriptional regulation mechanisms, experimental conditions, biological processes, etc. Conclusions/Significance: We have created cross-platform expression compendia for several bacterial organisms and developed a complementary access port COLOMBOS, that also serves as a convenient expression analysis tool to extract useful biological information. This work is relevant to a large community of microbiologists by facilitating the use of publicly available microarray experiments to support their research

Characterizing the Physcomitrella patens ecotype Reute

Ecotype collections are used for several plant models to unravel the molecular causes of phenotypic differences and to investigate effects of environmental adaptation and acclimation. For the model moss Physcomitrella patens, collections of accessions are available and have been used e.g. for phylogenetic and taxonomic studies, but few were investigated further for phenotypic differences. My thesis focuses on the accession found in Reute close to Freiburg im Breisgau, Germany. My publication shows that the standard laboratory strain Gransden produces fewer sporophytes than Reute or Villersexel K3, although gametangia develop in the same time course and do not show evident morphological differences. My work provides expression profiling and comparative developmental data for several stages of sporophyte development, as well as information on genetic variation from genomic sequencing. There is variation in the expression profiles of several genes between Gransden and Reute, as well as genome segments that are variation hotspots. With my work I propose that Reute is considered a P. patens ecotype and suggest its use for investigations that involve progression through the life cycle and generational succession. In my experiments I used the P. patens ecotype from Reute in molecular biology experiments introducing fluorescent proteins via chemical protoplast transformation to study codon usage bias and select a strong endogenous promoter. The 35S promoter, which is commonly used in plant systems, is only suitable to a limited extent in Physcomitrella patens due to mediocre and non-constitutive activity. Based on a broad range of Gransden and Reute microarray experiments we selected an LHCS gene that is highly expressed in most tissues and treatments. To measure expression strength I developed a novel fluorescence readout system, utilizing a microplate reader and an internal fluorescence control for normalization. The results from my publications demonstrate that the selected promoter is more active than the double 35S and Actin promoters. Deletion constructs were generated to develop a shorter promoter that retains the high activity of the full-length promoter. In parallel, codon bias within P. patens was analysed and demonstrated that the use of several codons is biased and correlates with expression strength. Two GFP variants were synthesized with different sets of codons and compared, showing that optimized codon usage increases the amount of protein under the control of strong promoters. In summary the findings from my publications characterize the P. patens ecotype from Reute and demonstrate that it will be an useful tool in reverse genetic studies

A calibration method for estimating absolute expression levels from microarray data

Motivation: We describe an approach to normalize spotted microarray data, based on a physically motivated calibration model. This model consists of two major components, describing the hybridization of target transcripts to their corresponding probes on the one hand, and the measurement of fluorescence from the hybridized, labeled target on the other hand. The model parameters and error distributions are estimated from external control spikes. Results: Using a publicly available dataset, we show that our procedure is capable of adequately removing the typical non-linearities of the data, without making any assumptions on the distribution of differences in gene expression from one biological sample to the next. Since our model links target concentration to measured intensity, we show how absolute expression values of target transcripts in the hybridization solution can be estimated up to a certain degree