| This research examined how changes in environmental conditions can drive the natural selection of new biological activities, by focusing on the molecular evolution of nitroaromatic degradation pathways in bacteria. Most nitroaromatic compounds are toxic mutagenic chemicals that have been in existence for ∼100 years or less. Yet during this relatively short period of time, bacterial strains that can use nitroaromatic compounds as sole carbon, nitrogen, and energy sources for growth have been isolated from contaminated environments.;Several underlying genetic changes responsible for the evolution of the pathways for nitrobenzene and 2-nitrotoluene catabolism in Comamonas sp. strain JS765 and Acidovorax sp. strain JS42 were successfully identified. In nitrobenzene 1,2-dioxygenase (NBDO), the proper orientation of nitrobenzene and mononitrotoluenes in the active site requires formation of a hydrogen bond between the nitro-group and the asparagine at position 258. Amino acids responsible for the differential specificities of NBDO and 2-nitrotoluene 2,3-dioxygenase (2NTDO) were located near, as well as at positions distal to the active site. Long-term evolution experiments with JS42 resulted in mutant strains that gained the ability to grow on 3-nitrotoluene or 4-nitrotoluene by a new degradation pathway. Mutations at positions 238, 242, and 248 of 2NTDO increased oxidation of 4-nitrotoluene to 4-methylcatechol, and were the only changes required for growth on 4-nitrotoluene. In addition, NBDO and 2NTDO were found to oxidize many other substrates, including chloronitrobenzenes. Engineered strains capable of growth on chloronitrobenzenes by a novel degradation pathway were constructed by incorporating variant NBDO enzymes.;The evolutionary history for nitrotoluene detection by the LysR-type transcriptional regulator NtdR from Acidovorax sp. JS42 was reconstructed through comparison to NagR, a similar regulator differing by five amino acids that does not recognize nitrotoluenes. Mutational analysis revealed that the residues at positions 227 and 232 are key for recognition of nitroaromatic compounds, while position 169 dictates recognition of aromatic acids, and residues at 74 and 189 have minimal contributions to specificity. Residues at positions 169 and 189 also modulated the levels of transcriptional activation. A whole-cell biosensor that emits bioluminescence when exposed to nitroaromatic compounds was constructed using the components of the nitrotoluene regulation system. |
