Integrating the Molecular Machines of Mercury Detoxification into Host Cell Biology

Abstract

Integrating the Molecular Machines of Mercury Detoxification into Host Cell Biology The bacterial mercury resistance (mer) operon, one of the most evolutionarily successful genetic loci in any defined organism, detoxifies organic and inorganic mercury compounds. Several major biotic processes in the global Hg(II) cycle are carried out by bacteria with this highly mobile detoxification locus that occurs in Gram negative and high and low GC Gram positive bacteria. The functions of many individual mer operon components are well described, so we aim to dissect the higher order interactions of the enzymes, transporters, and regulators of this paradigm metal metabolizing system with each other and with the larger metabolism of the host cell. Understanding how this ubiquitous detoxification system fits into the biology and ecology of its bacterial host is essential to guide interventions that support and enhance Hg remediation. Specifically, we will test the hypotheses that: (a) the organomercurial lyase, MerB, and the mercuric reductase, MerA, act synergistically together and with the membrane-bound Hg(II) transporters, MerT and MerC, to detoxify mercurials; (b) the interaction of the metalloregulator MerR with RNA polymerase (RNAP) and with its DNA binding site, MerO, modulates its metal response, and interaction with its antagonist, MerD, prevents RNA polymerase from binding to the structural gene promoter, P merT and (c) exposure of cells to Hg(II) makes specific demands on cellular resources and expression of the mer operon modulates those demands and is, in turn, modulated by them. To test these hypotheses we propose to: (a) use enzymology, NMR, fluorescence anisotropy, protein-crosslinking, crystallography, and calorimetry in vitro along with in vivo measurements of Hg(II) volatilization and HgR phenotyping to detect and define interactions between the mer enzymes, MerA and MerB, and the transporters, MerT and MerC,and their functional fragments and specific mutant variants; (b) use NMR, fluorescence anisotropy, protein-crosslinking, crystallography, affinity pull-downs, and calorimetry in vitro along with in vivo measurements of transcript synthesis and HgR phenotyping to detect and define interactions between mer regulatory proteins (MerR and MerD), DNA sites (MerO, PT and PR), and RNA polymerase and their functional fragments and specific mutant variants; and (c) use 2D microarrays to define the Hg-inducible transcriptome of the model bacterium E. coli and of radionuclide-remediation model microorganisms, Shewanella oneidensis and Desulfovibrio vulgaris,with and without the mer operon. The information and insights obtained from this work will benefit the DOE-NABIR program by providing (a) a model for an evolutionarily successful metal detoxification system and (b) guidance for manipulations of field conditions so as to optimize the functioning of the cells which carry this detoxification system. The work will also contribute to the fundamental understanding of (a) the evolution of modular architecture in multi-domain proteins and (b) the integration of horizontally transferred genetic elements into pre-existing networks of cellular functions.