Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis

Directed evolution combined with rational design increases activity of GpdQ toward a non-physiological substrate and alters the oligomeric structure of the enzyme

Directed evolution was used to enhance the activity of the glycerophosphodiesterase enzyme from Enterobacter aerogenes, GpdQ, toward bis(para-nitrophenol) phosphate (BpNPP), a substrate that is frequently used to assay phosphodiesterases. Native GpdQ has a low level of activity toward BpNPP while the evolved enzymes exhibited k(cat) values that were well over 100 times better while improvements in k(cat)/K(m) of around 500 times were observed along with improved activity we observed a change in the oligomeric structure in the evolved enzymes. The native enzyme is a hexamer with tightly associated dimers related by a 3-fold axis. The stability of the dimer was attributed in part to the cap domain that forms a disulfide bond with its 2-fold-related subunit and in part due to the fact that dimerization results in burying 23.6% of the monomer's accessible surface area. The cap domain also forms the top of the active site and contributes an essential part of the interface between 3-fold-related molecules. The evolved proteins quickly lost one of the cysteine residues that formed the disulfide bond and other mutations that might stabilize the cap domain. The likely effect of these mutations was to open up the active site for the new substrate and to favor the formation of dimeric molecules. The breakdown of the oligomeric structure was accompanied by a reduction in the thermal stability of the protein-as monitored by the residual activity of the native and mutant proteins following pre-incubation at elevated temperatures. A discussion on the evolutionary implications of these studies is presented