Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 15 Mar 2019 08:21:16 Microbiology (2001), 147, 965–972 Printed in Great Britain The product of the ybdE gene of the Escherichia coli chromosome is involved in detoxification of silver ions Sylvia Franke, Gregor Grass and Dietrich H. Nies Author for correspondence : Dietrich H. Nies. Tel : 49 345 55 26352. Fax: 49 345 55 27010. e-mail : d.niesmikrobiologie.uni-halle.de Institut fu  r Mikrobiologie der Martin-Luther- Universita  t Halle- Wittenberg, Kurt-Mothes- Str. 3, 06099 Halle, Germany Transcription of the ybcZ–ylcA ylcBCD–ybdE region of the Escherichia coli K38 chromosome was analysed by Northern RNA–DNA hybridization, RT-PCR and primer extension. Transcription of a dicistronic ybcZ–ylcA mRNA and a tetracistronic ylcBCD–ybdE mRNA was induced by silver and was initiated from the sigma-70 promoters ylcAp and ylcBp. Expression of β-galactosidase activity from a Φ (ylcBp–lacZ) operon fusion was also induced by Ag M and Cu 2M , but not by Zn 2M . In-frame deletion of ybdE from the chromosome yielded a silver- sensitive E. coli mutant strain which did not differ in its copper resistance from its wild-type strain. On the other hand, deletion of the copA gene for the copper-exporting P-type ATPase CopA resulted in copper sensitivity, but not in silver sensitivity. A ΔybdE ΔcopA double mutant strain behaved towards copper as the ΔcopA strain and towards silver as the ΔybdE strain. Thus, in E. coli, the YlcBCD–YbdE system may be involved in silver- but not in copper resistance, and CopA may be involved in copper- but not in silver resistance. Keywords : heavy metal resistance, cation efflux, silver resistance, RND family INTRODUCTION Silver (Ag + ) combines a high toxicity with relatively frequent occurrence in natural ecosystems and a long tradition of medical use (Nies, 1999). The toxic effects of silver on bacteria have been investigated for more than 60 years and many silver-resistant bacteria have been isolated (Yudkins, 1937 ; Nies, 1999). However, the molecular background of silver resistance has only recently become clear. In Gram-positive bacteria, Ag + resistance may be the result of efflux catalysed by a P- type ATPase which is also responsible for the export of Cu + (Solioz & Odermatt, 1995). In Gram-negative bacteria, a plasmid-encoded silver resistance determi- nant mediates efflux of Ag + by the RND-driven SilCBA transport system (Gupta et al., 1998, 1999), and physio- logical evidence for a chromosomally encoded silver efflux system in Escherichia coli has also been reported (Li et al., 1997). Previously known CBA transport systems for heavy metal cations include the Czc, Cnr and Ncc systems, with Czc being best characterized. Czc provides re- sistance to Co+ , Zn+ and Cd+ in the Gram-negative bacterium ‘ Ralstonia metallidurans ’ CH34 (previously Alcaligenes eutrophus ; Brim et al., 1999 ; Mergeay, 2000; J. Goris, P. De Vos, D. Janssens, M. Mergeay & P. Vandamme, unpublished). The CzcCBA efflux pump (Nies et al., 1989 ; Rensing et al., 1997) is composed of three subunits. CzcA transports the cations across the cytoplasmic membrane. The protein has been purified (Goldberg et al., 1999) and shown to be an inner- membrane proton–cation antiporter. CzcA belongs to the RND protein superfamily (TC 2.A.6.1.1 ; Saier, 2000) of proton-driven sym- and antiporters (Tseng et al., 1999). CzcB, a membrane fusion protein, and CzcC, an outer-membrane-associated protein, may transport the cations across the periplasmic space and the outer membrane to the outside (Rensing et al., 1997). The total E. coli genome contains seven genes encoding RND proteins. We have demonstrated that one of them, ybdE (gb AE000162.1), is involved in chromosomal silver resistance. The YbdE protein is similar to CzcA from ‘ R. metallidurans ’ CH34 (Nies et al., 1989) and SilA from Salmonella typhimurium (Gupta et al., 1999). Like the czcA or silA genes, ybdE is preceded by a gene encoding a membrane fusion protein, ylcD, and a gene encoding a putative outer-membrane-associated pro- tein, ylcB. Between ylcB and ylcD is located the small ORF ylcC, which is homologous to a small ORF in the sil operon at the same respective location. Finally, the genes for a two-component regulatory system are located adjacent to the putative ylcBCD–ybdE operon. 0002-4414  2001 SGM 965