Numerous lineage-specific expansions of the transcription factor B (TFB) family in archaea suggests an important role for expanded TFBs in encoding environment-specific gene regulatory programs. Given the characteristics of hypersaline lakes the unusually large numbers of TFBs in halophilic archaea further suggests that they might be especially important in rapid adaptation to the challenges of a dynamically changing environment. Motivated by these observations we have investigated the implications of TFB expansions by correlating sequence variations, regulation, and physical interactions of all seven TFBs in Halobacterium salinarum NRC-1 to their fitness landscapes, functional hierarchies, and genetic interactions across 2,488 experiments covering combinatorial variations in salt, pH, temperature, and Cu-stress. This systems analysis has revealed an elegant scheme in which completely novel fitness landscapes are generated by gene conversion events that introduce subtle changes to the regulation or physical interactions of duplicated TFBs. Based on these insights we have introduced a synthetically redesigned TFB and altered the regulation of existing TFBs to illustrate how archaea can rapidly generate novel phenotypes by simply reprogramming their TFB regulatory network.
The purpose of this gene expression study was to show that reprogrammed synthetic TFB (TFBx) variants are rewired into the gene regulatory network and create global transcriptional changes.
Overall design: The relative changes in transcript levels and transcriptome structure at 37°C and 25°C were determined for wild type, tfbD and tfbE in frame deletion knockouts and all recombinant strains transformed with a plasmid carrying the synthetic TFB constructs. The strains were batch cultured in flasks at either 25°C or 37°C with constant shaking, culture aliquots (~4 ml) were collected over early (OD600: ~0.2), mid (OD600: ~0.4) and late (OD600: ~0.8) phases of growth, centrifuged (16 000 g, 90 s), and flash frozen. Total RNA was prepared from the cell pellets using the mirVANA RNA kit (Ambion, Austin, TX) according to manufacturer's instructions. Whole-genome tiling arrays for H. salinarum NRC-1 were designed with e-Array (Agilent Technologies), using strand-specific 60 mer probes with 24nt spacing between adjacent probes for the main chromosome (NC_002607) and the plasmids pNRC200 (NC_002608) and pNRC100 (NC_001869). Altogether the array contained a total of 60K probes, including manufacturers' controls. The microarrays were printed by Agilent technologies. Labeling with Cyanine 3 (Cy3) and Cyanine5 (Cy5) dyes (Molecular Probes and Kreatech BV), hybridization, and washing were performed as described earlier (Baliga et al, 2004). Arrays were scanned in ScanArray (Perkin Elmer) and spot finding was done using Feature Extraction (Agilent Technologies). Normalization and statistical analysis were performed as described before (Koide et al, 2009).
Less...