Bismuth–dithiol inhibition of the Escherichia coli rho transcription termination factor Andrew P. Brogan a , Jacob Verghese b , William R. Widger b, * , Harold Kohn a, * a Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA b Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001, USA Received 10 September 2004; received in revised form 23 December 2004; accepted 24 December 2004 Available online 22 January 2005 Abstract Bismuth–dithiol mixtures are proven antimicrobial agents with unknown mechanism(s) of action. We show that select bismuth– dithiol solutions inhibit the Escherichia coli rho transcription termination factor. Rho is an essential enzyme in most Gram-negative prokaryotes and without rho function the cells are not viable. Bismuth complexes with 2,3-dimercapto-1-propanol (BiBAL) (3:1 solutions) functioned as a noncompetitive inhibitor with respect to ATP in the rho poly(C)-dependent ATPase assay (I 50 = 60 lM) and as a competitive inhibitor with respect to ribo(C) 10 in the poly(dC)-ribo(C) 10 -dependent ATPase assay. The min- imum inhibitory concentration (MIC) of bacterial growth for BiBAL (3:1) in the liquid culture assay using E. coli W3350 was 16 lM. Using the tnaA/lacZ fusion reporter assay we showed that sublethal amounts (3 lM) of BiBAL (3:1 solution) led to a small increase (37%) in in vivo b-glactosidase activity in E. coli SVS1144, which corresponds to antitermination of the tna operon as a result of rho inhibition. We concluded that BiBAL was a potent in vitro rho inhibitor but its effect on in vivo rho processes was modest indicating that other mechanisms contributed to the antibacterial activity of BiBAL. Our study suggests that structural changes in the dithiol unit that provide greater bismuth binding may improve rho specificity, a macromolecular target not previously recognized for bismuth therapy. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Rho transcription termination factor; Bismuth–dithiols; Inhibitors; Antibacterial activity 1. Introduction Rho is an essential cellular protein for controlling the expression of specific genes in many Gram-negative organisms, and without rho, cell death results [1]. Func- tional rho consists of six identical 47 kDa subunits (419 amino acids) arranged in a toroidal shape with two dis- tinct types of RNA binding sites [1,2]. Mechanistic and structural investigations support either a ‘‘tethered- tracking’’ or ‘‘simple’’ mechanism for rho translocation [1,3,4]. The primary RNA binding sites sit like a cap on top of the rho hexamer, bind specific nucleotide se- quences that contain high cytosine content, and will bind both single-stranded RNA and DNA [1,3]. The sec- ondary RNA binding sites are located within the central hole, are specific for RNA and trigger ATP hydrolysis [5] that translocates the protein (5 0 ! 3 0 ) along the nas- cent RNA. Transcript termination occurs when rho encounters the stalled RNA polymerase. The only known selective inhibitor of rho is the com- mercial antibiotic bicyclomycin (BCM) (Chart 1) [6]. In 2003, we reported that 2+ metal (zinc, cadmium, nickel) –dithiol complexes were potent inhibitors of the Esche- richia coli rho transcription termination factor and that 0162-0134/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2004.12.019 * Corresponding authors. Tel.: +1 713 743 8368; fax: +1 713 743 8351 (W. Widger); Tel.: +1 919 966 2680; fax: +1 919 843 9835 (H. Kohn). E-mail addresses: widger@uh.edu (W.R. Widger), harold_kohn @unc.edu (H. Kohn). www.elsevier.com/locate/jinorgbio Journal of Inorganic Biochemistry 99 (2005) 841–851 JOURNAL OF Inorganic Biochemistry