Institut für Mikrobiologie der Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, 06099 Halle, Germany1
Author for correspondence: Dietrich H. Nies. Tel: +49 345 55 26352. Fax: +49 345 55 27010. e-mail: d.nies{at}mikrobiologie.uni-halle.de
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ABSTRACT |
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Keywords: heavy metal resistance, cation efflux, silver resistance, RND family
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INTRODUCTION |
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Previously known CBA transport systems for heavy metal cations include the Czc, Cnr and Ncc systems, with Czc being best characterized. Czc provides resistance to Co2+, Zn2+ and Cd2+ 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; J. Goris, P. De Vos, D. Janssens, M. Mergeay & P. Vandamme, unpublished).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 protoncation 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 protein, 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 ylcBCDybdE operon. The ybcZ gene encodes the putative sensor and ylcA the predicted response regulator (Blattner et al., 1997
). This paper describes the initial characterization of this bacterial silver resistance system.
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METHODS |
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Genetic techniques.
Standard molecular genetic techniques were used (Nies et al., 1987 ; Sambrook et al., 1989
). Total RNA of E. coli was isolated as published by Oelmüller et al. (1990)
, van der Lelie et al. (1997)
and Große et al. (1999)
. Amount and quality of total RNA was determined spectrophotometrically at 260 and 280 nm.
Strain constructions.
To prevent any polar effect mediated by deletion of ybdE, the gene was exchanged against a small ORF encoding a polypeptide of 20 amino acids. The first 10 amino acids encoded by this small ORF were identical with the 10 amino-terminal amino acids of YbdE, and the last 8 amino acids were identical with the last, carboxy-terminal amino acids of that protein. Positions 11 and 12 were Glu and Phe, encoded by the hexanucleotide recognition sequence GAATTC of the restriction endonuclease EcoRI. Thus, the 500 bp upstream of ybdE were amplified by PCR (Table 1, primers 1 and 2), and this fragment ended with the 30 bp coding sequence for the first 10 amino acids of the respective gene, followed by an EcoRI hexanucleotide. Secondly, the 500 bp downstream of ybdE were amplified by PCR (Table 1
, primers 3 and 4), and this fragment started with the EcoRI recognition sequence and the last 24 bp of the respective gene. Both fragments were fused by EcoRI restriction and ligation, cloned, verified by DNA sequencing, and finally cloned into pKO3 (Link et al., 1997
). The resulting plasmid was used for mutation in E. coli K38 as described by Link et al. (1997)
, giving strain EC756
ybdE. The mutant genotype was verified by PCR and Southern DNADNA hybridization. The
copA mutation was generated in exactly the same fashion in both strains (primers 58), the K38 wild-type giving EC774
copA and the
ybdE deletion strain EC756 giving the double mutant strain EC773
ybdE
copA.
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For construction of the reporter gene fusion, the ylcBp promoter was amplified by PCR (primers 19 and 20, Table 1) from chromosomal DNA of E. coli strain K38 and cloned upstream of a promoterless lacZ gene (N. Peitzsch & D. H. Nies, unpublished) in plasmid pKO3 (Link et al., 1997
).
Northern DNARNA hybridization.
Northern (RNA) blot analysis was performed as published by Große et al. (1999) by fractionation of RNA samples on agarose/formaldehyde gel (15 g agarose l-1), followed by transfer to a positively charged Qiabrane nylon filter (Qiagen) using a pressure blot (Posi Blot; Stratagene). For quantitative analysis, the same amount of total RNA (40 µg) was loaded into each well of the same gel, and this was verified by ethidium bromide staining after electrophoresis. After prehybridization for 3 h at 55 °C in hybridization buffer (Engler-Blum et al., 1993
), the filters were hybridized for at least 14 h at 55 °C in the same buffer. The filters were probed with PCR fragments representing parts of ybcZylcA (positions 13391813, Fig. 1
) and ybdE (positions 57386339). The DNA fragments were labelled with digoxigenin by random priming using the DIG-DNA labelling kit (Roche). After several washing steps, the filters were developed with the DIG-luminescent detection kit (Roche).
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Primer extension experiments.
Primer extension analysis was performed by a modification of a standard protocol (Sambrook et al., 1989 ; Große et al., 1999
) using fluorescein-labelled oligonucleotides and an automated ALF DNA Sequencer (Pharmacia), as described previously. The fluorescein-labelled 3' antisense primers (Table 1
, primers 9 and 10) were complementary to the corresponding gene regions. The cDNA (obtained by reverse transcription as described above) was vacuum-dried and suspended in 4 µl H2O and 4 µl ALF stop solution (Pharmacia). Following heat denaturation, the sample was loaded onto a 7% polyacrylamide sequencing gel. In parallel, a sequencing reaction was performed with the same fluorescein-labelled primer and a DNA fragment containing the respective gene region. The transcription start site was determined by comparison of the retention time of the primer extension reaction with that of the sequencing reaction.
RT-PCR experiments.
Reverse transcription was carried out with 3' antisense primers (see Fig. 1 for positions, and Table 1
for sequences). PCR amplification was performed with 0·1 vol. of the reverse transcription reaction in a mixture containing 50 mM Tris/HCl (pH 9·0), 20 mM ammonium sulfate, 20 pmol 5' sense primer (see Fig. 1
for positions, and Table 1
for sequences), 0·1 mM of each dNTP, 1·5 mM MgCl2 and 1 U Taq polymerase (Roche) in a final volume of 50 µl. After a hot start (2 min, 96 °C), the amplification profile used was: denaturation at 96 °C for 30 s, annealing at 5058 °C for 20 s, extension at 72 °C for 30 s, final extension at 72 °C for 2 min; 30 cycles were performed. The amplification was performed with a mineral oil overlay in a Trio-Thermoblock (Biometra). Negative controls were: no templates, DNA templates, and PCR reactions with total RNA treated with RNase-free DNase.
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RESULTS |
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The promoters
Using primer extension experiments, the start sites upstream of ylcB and ylcA were determined (Fig. 3). Transcription of both genes started 1020 bp upstream of the predicted RBS. The -10 regions of both promoters, TAAAGT (ylcBp) and TAGAAT (ylcAp), were similar to the consensus motifs of sigma-70 promoters (TATAAT; Rosenberg & Court, 1979
). Conservation of the -35 site with respect to the sigma-70 consensus (Rosenberg & Court, 1979
) was weaker for ylcBp (CGGAAA) than for ylcAp (TTGCCA). Thus, in terms of conservation of the sigma-70 consensus motif, ylcAp should be a stronger promoter than ylcBp. Around position -47 of ylcBp, the inverted repeat 2283-GGCAAAATGACAATTTTGTCATTTTTCTG was identified, which might be an upstream activating sequence used by YlcA. This sequence has similarity to inverted repeats upstream of promoters pcoAp, pcoEp, silCp, silEp, copAp and copHp, genes involved in copper and silver resistance (Nies & Brown, 1998
; Gupta et al., 1999
).
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The ybdE gene is involved in resistance to silver
In the chromosome of E. coli strain K38, the ybdE gene was deleted in-frame giving E. coli strain EC756. In a filter-disk inhibition zone assay, strain EC756 was more sensitive to Ag+, Co2+, Cu+ and Cu2+ than wild-type strain K38 (data not shown); however, the differences were minimal. The strains did not differ in their sensitivity to other metals or organic substances (ethidium bromide, tetracycline, SDS; data not shown).
On solid LB (Sambrook et al., 1989 ) agar plates without NaCl, wild-type strain K38 grew in the presence of 25 µM Ag+, but EC756 did not (data not shown). There was no difference in resistance to Co2+, Cu2+ or Cu+ on solid media (data not shown). In liquid culture, strain EC756
ybdE was more sensitive to Ag+ compared to its wild-type E. coli strain K38 (Fig. 5a
). The deletion could be complemented in trans by the complete ylcBpylcBCDybdE gene region (Fig. 5b
).
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DISCUSSION |
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The CopA P-type copper ATPase was shown to detoxify Cu+ in E. coli (Rensing et al., 2000 ) and increased expression of CopA may therefore compensate for the effect of the missing ybdE gene on copper resistance. Thus,
copA deletions were introduced into wild-type K38 and the
ybdE deletion strain. The resistance phenotypes displayed by the resulting set of single and double mutant strains in comparison with the wild-type (Fig. 5
) ruled out any significant involvement of YbdE in copper detoxification and of CopA in silver detoxification, although both determinants were gratuitously induced by the other monovalent heavy metal cation (Rensing et al., 2000
). Secondly, the Cu2+ cations in the growth medium were indeed reduced to Cu+ in the cytoplasm as expected (Nies, 1999
) or no effect of the
copA deletion on Cu2+ resistance would have been observed.
To study transcriptional regulation of ybdE, the only currently known substrate, Ag+, was used as the inducer. Both operons, ybcZylcA and ylcBCDybdE, are transcribed in different directions from an overlapping promoter/operator region. A possible binding site for YlcA was identified, at position -47 with respect to the ylcBp promoter and -42 with respect to ylcAp. With the membrane-bound histidine kinase sensor YbcZ, these components are sufficient to explain the observed activation of ylcBCDybdE transcription by Ag+ and Cu+ cations.
Since this publication was submitted, another group has characterized the ylc gene region under a different aspect (Munson et al., 2000 ). The data provided by these authors show that the YbcZYlcA two-component system regulates not only the ylcBp promoter, but also the promoter pcoEp of a plasmid-bound copper resistance determinant. However, the main copper resistance gene, pcoA, is only partially regulated by YbcZYlcA. The transcriptional start site of the ylcB gene (Munson et al., 2000
) differs just by 1 base from the start site given here.
The authors speculate (Munson et al., 2000 ) that the genes of the ylc region may be involved in copper resistance in E. coli, but they provide no data for this assumption. In contrast, the data shown in Fig. 5
clearly indicates that the ybdE gene is not involved in copper resistance. If this gene mediates heavy metal resistance at all, it should be silver resistance. The renaming of the ylc genes as cus is therefore highly premature; if these genes are renamed, a name such as agr for silver resistance (agrSR agrCFBA) would be more appropriate.
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ACKNOWLEDGEMENTS |
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Received 24 July 2000;
revised 1 November 2000;
accepted 4 December 2000.