School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, UK1
Author for correspondence: Andrew P. Morby. Tel: +44 2920 874128. Fax: +44 2920 874116. e-mail: morby{at}cf.ac.uk
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ABSTRACT |
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Keywords: Escherichia coli, metal ion, adaptation, gene array, insA
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INTRODUCTION |
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The recent advent of array technology has allowed the global study of all 4255 genes in Escherichia coli. This work details the investigation of metal-ion tolerance in E. coli adapted to Zn(II), Cd(II), Co(II) and Ni(II), and the results demonstrate that the transcript abundance of a range of genes was altered in tolerant strains. In particular, the transcript level of insA was increased in Zn(II)-, Cd(II)- and Co(II)-adapted strains. The subsequent expression of insA-7 from a heterologous promoter conferred Zn(II) tolerance on E. coli.
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METHODS |
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Adaptation of E. coli TG1 to metal-ion tolerance.
A 10 ml volume of LB medium was inoculated with 150 µl of an overnight culture of E. coli TG1 and grown with shaking for 1·5 h, before 200 µl aliquots were transferred into a 96-well plate containing an increasing range of metal-ion concentrations of Zn(II) (ZnSO4), Cd(II) (CdSO4), Co(II) (CoSO4) and Ni(II) (NiSO4). The plates were incubated at 37 °C for 2448 h with shaking (Camlab Microtherm; 500 r.p.m.) and the OD600 of cell cultures was measured (Molecular Devices Thermo max microplate reader). A 150 µl volume was taken from the well containing the highest concentration of each individual metal ion where the OD600 was 0·100, and this was used to inoculate 10 ml LB medium for further adaptive incubation. This stepwise adaptation generated strains with increased tolerance to Zn(II) (strain EZn), Cd(II) (strain ECd), Co(II) (strain ECo) and Ni(II) (strain ENi). Adaptation was concluded when cultures reached a stable tolerance maximum over two serial culture steps. The resultant cultures were either diluted 50% (v/v) with glycerol for storage or used for the isolation of RNA.
Measurement of metal-ion tolerance (MICs).
A 30 ml volume of LB medium was inoculated with the individual strains of E. coli TG1, EZn, ECd, ECo and ENi, and grown with shaking for 1 h at 37 °C. A 200 µl sample of each culture was transferred into separate wells of a microtitre plate which contained increasing ranges of metal-ion concentrations, and incubated at 37 °C for 24 h with shaking (Camlab Microtherm; 500 r.p.m.). The OD600 of cell cultures was then measured (Molecular Devices Thermo max microplate reader). MICs for each strain were measured for the four metal ions Zn(II), Cd(II), Co(II) and Ni(II). In some cases it was impossible to accurately determine an MIC as the strains grew in medium in which the given metal ion was at the limit of solubility.
Whole-genome transcriptional analysis.
E. coli Panorama arrays were used throughout this work (Sigma-Genosys). Metal-ion-tolerant E. coli strains were grown to stationary phase in LB containing the maximum permissive concentration of the appropriate metal salt. RNA was extracted using a Qiagen RNeasy kit according to the manufacturers instructions. cDNA probes were generated as described by Sigma-Genosys, using primers and reverse transcriptase provided. Hybridized probes were visualized by autoradiography using a phosphorimager (Bio-Rad, Personal FX). Hybridization intensity was quantified and compared using Quantity One (Bio-Rad) and Excel (Microsoft). Transcriptional profiles for each strain of cells were compared to that of the wild-type strain grown to stationary phase in the absence of metal ions. LB medium was used to allow future array experiments involving alternative challenges to be comparable with this data set.
Amplification of the insA-7 coding region and plasmid construction.
All DNA manipulations were carried out according to Sambrook et al. (1989) . The insA-7 coding region was amplified from the Co(II)-tolerant strain ECo using primers A7(V)N-term (5'-CGGAATTCTGCGTGGCTTCCATTTCCATCAGATGTCC-3') and A7C-term (5'-GCTCTAGACGCTGACGTGATTTAGCACCGACG-3') (synthesized by Gibco-BRL). The nucleotide sequence of the amplified insA was determined and was identical to that reported, except for one base change (+150 from start codon: C to A). This change does not affect the primary amino acid sequence. The derivative of pBAD24 (Guzman et al., 1995
) carrying the insA-7 coding region was created by digesting the PCR product with EcoRI and XbaI and ligating together with identically cut pBAD24 to generate pBADinsA-7.
insA-7 expression.
Overnight cultures of TG2(pBAD24) or TG2(pBADinsA-7) were used to inoculate 20 ml fresh LB medium containing arabinose at a final concentration of 0·5% (w/v). A 200 µl sample of each culture was transferred into separate wells of a microtitre plate containing increasing concentration ranges of Zn(II), Cd(II), Co(II) or Ni(II). The cultures were incubated at 37 °C with shaking, and the OD600 was measured (Molecular Devices Thermo max microplate reader) every 1 or 2 h over a 30 h period. Carbenicillin was added to medium to allow selection of pBAD24 and pBADinsA-7 which carry the bla marker gene.
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RESULTS AND DISCUSSION |
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Consistent with tolerance to cations, ompC and ompA, which encode outer-membrane porins (Ried et al., 1990 ), are reduced in most of the adapted strains. b0795, which shows similarity to CzcB (a component of a cation efflux pump; Nies et al., 1989
), is reduced in all four strains. Analysis of the b0795-encoded sequence shows the presence of a signal peptide, suggesting it is periplasmic in location. In addition, the operon which contains b0795 also contains genes whose products are similar to ABC-transporter proteins. It is possible that this operon is involved in cation import and therefore it is consistent for one or more components to be diminished in metal-adapted cells.
Genes which show increased transcriptional levels (Table 2) also have some commonality between strains. Some induced genes have a functional association with metal cations, including nikD [Ni(II) export; Navarro et al., 1993
], yfeC [putative chelated-Fe(II) export; Bearden et al., 1998
], yeaJ (shows sequence similarity to hmsT, a putative regulator of haem storage; Jones et al., 1999
) and bisC (biotin sulfoxide reductase, binds molybdenum; Pierson & Campbell, 1990
). These gene products could be involved in the chelation of excess cytosolic metal ions which may generate tolerance or perhaps represent compensatory alterations which preserve cation [e.g. Mn(II) or Fe(II)] metabolism in the presence of excess Zn(II), Cd(II), Co(II) or Ni(II).
The majority of the characterized genes identified in this experiment encode proteins involved in the transposition of IS1, 2, 3, 5 and 30 (see data at URL http://www.cf.ac.uk/biosi/staff/people/morby.html). It is well documented that IS genes may be induced by cellular stress and it is hypothesized that the movement of IS elements is capable of increasing genetic diversity (Naas et al., 1994 ).
The most consistently induced genes are those for insA (IS1 transposition), for which transcripts are elevated in three out of the four strains. It is impossible to determine from array analyses which of the insA/B sequences were induced, given the close sequence similarity within the gene families, which may have resulted in cross-hybridization during the experiment. The repeated increase in transcript abundance of insA genes led to the investigation of metal tolerance in a wild-type strain expressing insA-7 from a heterologous promoter.
Increase in metal tolerance by overexpression of insA-7
E. coli TG2 carrying pBADinsA-7 was generated in which gene expression was controlled by the level of arabinose in the medium. insA-7 was used since all insA sequences are almost identical and this gene has no apparent associated insB. When grown in medium containing arabinose, but no additional metal ions, TG2(pBADinsA-7) showed a slight growth advantage over TG2(pBAD24) (Fig. 2a). In contrast, when grown in the presence of arabinose plus 0·6 mM Cd(II) and 1·0 mM Zn(II), TG2(pBADinsA-7) shows a slight increase in tolerance to Cd(II) but a marked increase in tolerance to Zn(II) (Fig. 2b
, c
). No increase in tolerance to Co(II) or Ni(II) was observed (data not shown). The insA-dependent increase in tolerance to Zn(II) is consistent with the observation that EZn showed increases in transcript abundance for genes encoded by IS1.
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Concluding remarks
In response to toxic concentrations of Zn(II), Cd(II), Co(II) or Ni(II), E. coli exhibited varying degrees of tolerance (314-fold greater than wild-type) both to the adaptive metal and its congeners. All of the adapted strains showed similar patterns of diminished gene expression, with particular bias towards genes whose products are involved in translation. No single gene whose expression increased was common to the four adapted strains, but surprisingly insA was increased in the Zn(II)-, Cd(II)- and Co(II)-adapted strains. Subsequent expression of insA-7 in E. coli demonstrated that this gene can confer tolerance to Zn(II), suggesting that it may be capable of binding divalent metal ions by virtue of two paired cysteine motifs. This study shows the utility of an array-based approach to functional genomics in E. coli and the capacity for the generation of novel avenues of research using these techniques.
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ACKNOWLEDGEMENTS |
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Received 2 March 2000;
revised 15 May 2000;
accepted 15 June 2000.