Background: The common agricultural pesticide and nerve agent byproduct p-nitrophenol is a potentially carcinogenic nerve agent. Bacteria in the environment have been observed to degrade p-nitrophenol by a number of pathways. Our laboratory has been using a pathway from a Pseudomonas sp ENV2030 through the plasmid pSB337, but has not examined the specifics of the native operons cloned from a cosmid library. Methods: One way of examining how a system works is trying to reconstruct the system heterologously in a different host. A set of novel plasmids were constructed to degrade p-nitrophenol using genes from pSB337. The pCM62 (Marx and Lidstrom 2001) vector backbone was utilized and re-engineered to allow insertion of standard biological parts. Gene number, order and inducible promoter strengths were varied. We assayed for growth in flasks, agar, and 96-well plates and analyzed metabolic intermediates using CE-MS and LC-MS. Results: The engineered strain can efficiently mineralize p-nitrophenol within 24 hours up to the minimum inhibitory concentration of the host bacterium E. coli. A minimum complement of four genes from the original plasmid was necessary for mineralization of p-nitrophenol, and a total of seven genes from the host organism (including three from the chromosome) were necessary for growth on the contaminant. Conclusions: Use of this system in situ has the potential to reduce worldwide nitrophenol contamination problems. The alternative method of redesigning a catabolic pathway from putative gene sequences holds great promise for the study of novel endogenous catabolic pathways.