Microarray analysis in the archaeon Halobacterium salinarum strain R1

Abstract

Background: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea.

Methodology/principal findings: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis.

Conclusion/significance: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.

Figure 1. Box plots of background sample (upper diagram) and reference fluorescence (lower diagram) values around probe spots.

The values are grouped according to the spotting pin. The horizontal line within the boxes shows the median. The borders of the boxes show the 75th and 25th percentiles. The dotted lines outside the boxes indicate upper and lower limit values. Open circles represent individual outlier spots.

Figure 2. Scatter plots of local background values of probe spots in reference and sample fluorescence channel.

The contour lines (green) were calculated using the minimum volume ellipsoid method for all spots printed by an individual pin (red).

Figure 3. MA plot of raw fluorescence data within a single microarray.

The data are colored pin-wise (fluorescence intensities obtained from probes spotted by a certain pin are colored individually).

Figure 4. Box plots of sample (upper diagram) and reference fluorescence (lower diagram) values of probe spots.

See legend of Figure 1 for a detailed description.

Figure 5. Distribution of all genes versus distribution of significantly regulated genes on the genome.

Chr: chromosome; pHs1-4: plasmids.

Figure 6. Graphical overview of the functional classification of the significantly regulated genes.