Functional participation of a nifH–arsA2 chimeric fusion gene in arsenic reduction by Escherichia coli Surobhi Lahiri, Lakshmi Pulakat, Nara Gavini * Department of Biological Sciences, Mississippi State University, P.O. Box GY, Mississippi State, MS 39762, USA Received 11 January 2008 Available online 28 January 2008 Abstract The NifH (dimer) and ArsA proteins are structural homologs and share common motifs like nucleotide-binding domains, signal transduction domains and also possible similar metal center ligands. Given the similarity between two proteins, we investigated if the NifH protein from Azotobacter vinelandii could functionally substitute for the ArsA1 half of the ArsA protein of Escherichia coli. The chimeric NifH–ArsA2 protein was expressed and detected in the E. coli strain by Western blotting. Growth comparisons of E. coli strains containing plasmids encoding for complete ArsA, partial ArsA (ArsA2) or chimeric ArsA (NifH–ArsA2) in media with increasing sodium arsenite concentrations (0–5 mM) showed that the chimeric NifH–ArsA2 could substitute for the ArsA. This func- tional complementation demonstrated the strong conservation of essential domains that have been maintained in NifH and ArsA even after their divergence to perform varied functions. Ó 2008 Published by Elsevier Inc. Keywords: nifH; arsA; Arsenite reduction; Chimeric gene; Complementation Nitrogen fixation and arsenic resistance are both pro- cesses that had developed very early in the evolution of life [1,2]. Nitrogen fixation is driven by the nitrogenase enzyme and its major structural components are the NifH, D, and K proteins [2–4]. The NifH is the obligate electron donor to the NifDK protein for the nitrogen fixation reaction. The biosynthesis and assembly of the [4Fe–4S] cluster found in NifH and the FeMoco and P-Cluster found in NifDK is carried out by a host of nif accessory factors that mainly include the NifE, N, M, B, Q, V, and X proteins ([5–8], for reviews, see [9,10]). In case of arsenic resistance, the most well studied mechanism of arsenic resistance is the ArsAB pump that is encoded by the ars operon of conjugative R- factor R773 in Escherichia coli and is part of the arsR- DABC operon [11]. The arsA gene encodes for a 583-amino acid catalytic subunit (63 kDa) with two ABC domains, whereas the arsB gene encodes a 429-amino acid (45.5 kDa) inner membrane protein that serves as both the anion channel and an anchor for the ArsA protein [12,13]. It has been speculated that nitrogen fixation and arsenite detoxification evolved from an inter-related process in the ancient past since the NifH protein of the nitrogenase com- plex and the ArsA protein of the arsenite detoxification pump share many structurally homologous features as revealed by the crystallized structure of the ArsA protein [14,15]. ArsA has homologous N-terminal (A1) and C-ter- minal (A2) halves, ‘‘indicating an evolutionary gene dupli- cation and fusion” [16]. This ATPase contains two consensus nucleotide-binding sites (NBSs), one each in the A1 and A2 halves. The A1 and A2 halves are held together by a 25-residue linker peptide [17]. ArsA is twice the size of NifH, yet since it consists of two similar domains connected by a short linker, each ArsA monomer could actually be considered to be a pseudodimer [15]. The percentage protein sequence similarity between NifH and ArsA1 is 53.7% and between NifH and ArsA2 is 48.7% (alignment shown in Fig. 1A). Further informa- tion obtained on ArsA (PDB ID: 1F48) and NifH (PDB 0006-291X/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.bbrc.2008.01.086 * Corresponding author. Fax: +1 662 325 7939. E-mail address: gavini@biology.msstate.edu (N. Gavini). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 368 (2008) 311–317