Integrative “Omics”-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses
Bacterial cultures are subjected to stress (e.g., singlet oxygen, 1O2) and relative changes monitored for mRNAs in total RNA isolates (transcriptome), mRNAs in polysomes (translatome), and for proteins (proteome). For the proteome approach, a culture has to be fully labeled with the heavy amino acid 13C6-lysine (Lys6; heavy standard). Protein samples from a reference (no stress) or stressed cells are separately mixed in a 1∶1 ratio with heavy standard protein and subsequently applied to protein digest followed by MS analysis. SILAC ratios are calculated by comparing intensities of heavy (red) to light (green) peaks of individual peptides (m, mass and z, charge). Direct ratios, reflecting relative changes, are determined thereof. The translatome is assessed by microarray analysis of RNA from polysome fractions. Polysomes are enriched by sucrose density gradient centrifugation of cells that have been treated with chloramphenicol. For transcriptome data, total RNA is isolated and applied to microarray analysis.