Bacteria that assimilate man-made nitroarene compounds provide useful models for the study of evolution,as they demonstrate that populations of microorganisms can rapidly adapt when challenged with new potential carbon and nitrogen sources.These strains represent unique evolutionary systems,as their metabolic pathways are in the process of changing and are undergoing optimization for the consumption of toxic,synthetic industrial chemicals.We have been using Acidovorax sp.strain JS42,which is capable of growth on the synthetic compounds nitrobenzene and 2-nitrotoluene,in experiments to examine how a nitroarene degradation pathway evolves when its host strain is challenged with direct selective pressure to assimilate non-native substrates.Although the same dioxygenase enzyme that allows for growth on nitrobenzene and 2-nitrotoluene also oxidizes 3-and 4-nitrotoluene to methylcatechols,which are growth substrates for JS42,the strain is incapable of growth on 3-or 4-nitrotoluene.Using laboratory evolution experiments,we obtained mutants of JS42 that gained the ability grow on either 3-or 4-nitrotoluene.The underlying basis for this new activity resulted from the accumulation of specific mutations in the gene encoding the initial dioxygenase that catalyzes the oxidation of both nitrobenzene and 2-nitrotoluene.Amino acid substitutions that allowed growth on 3-nitrotoluene were near the active site iron,while those that allowed growth on 4-nitrotoluene were located at positions distal to the active site in a region previously unknown to affect activity in this or related enzymes.Further evolution experiments were carried out to select for strains that grow on all three mononitrotoluene isomers;these strains had additional changes near the active site of the dioxygenase as well as additional and as yet unidentified mutations elsewhere in the genome.