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Characterization of the nimR Protein in Rhodococcus sp. I24

Diana Bolton
Department of Biology,Massachusetts Institute of Technology, Cambridge, MA 02139
Work conducted in 7.13 Project Lab

 
   
Non-Scientific Abstract

CRIXIVANÆ is becoming one of the most widely used drugs to treat AIDS; however, there currently exists no efficient way to manufacture this medication. In recent years, the bacterium Rhodococcus has emerged as a vessel for producing large quantities of the drugís necessary chemical intermediates. This bacterium breaks down various aromatic hydrocarbons via several pathways containing mono- and dioxygenases. Although we know that these gene systems exist, scientists do not currently understand many important features of the systems, such as regulation. Once this is understood, the bacteria can be manipulated in such as way as to produce vast quantities of the desired intermediate, thus saving both time and money in production.This study aims to identify the important genetic domains of the nimR regulatory protein and elucidate the regulatory mechanism.

Abstract

Rhodococcus sp. I24 contains a toluene-inducible dioxygenase, a naphthalene-inducible dioxygenase and a naphthalene-inducible monooxygenase, which are all efficient in producing precursors for the HIV drug, CRIXIVANÆ.While the sequence and function of each of these subunits is known, their regulation is not. The objective of these experiments is to characterize nimR, the negative repressor of nimA, which encodoes the naphthalene-inducible monooxygenase. NimR is a member of the GntR family of regulators and contains a helix-turn-helix motif (HTH) in its N-terminus. Point mutations were made in nimR and then analyzed via a reporter enzyme assay. This assay revealed that mutations in the second helix of the HTH motif repressed expression over fifty hours of induction by naphthalene. This finding suggests that the mutation created altered the effector-binding site on nimR.