Institute of Molecular Biology of the Slovak Academy of Sciences, Dúbravská cesta 21, SK-84251 Bratislava, Slovak Republic1
Department of Cell and Molecular Biology, Göteborg University, Medicinaregatan 9C, SE-41390 Göteborg, Sweden2
Author for correspondence: P. Ferianc. Tel: +421 2 5930 7427. Fax: +421 2 5930 7416. e-mail: umikferi{at}savba.sk
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ABSTRACT |
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Keywords: cadmium stress, yodA characterization, YodA protein, RNA polymerase, S1 nuclease mapping
Abbreviations: CDP, cadmium induced protein(s); DIG, digoxigenin; MBP, maltose-binding protein; ppGpp, guanosine 3',5'-bispyrophosphate; RAS, rabbit antiserum; tsp, transcription start point
The GenBank accession number for the E. coli yodA gene and the SWISS-PROT accession number for E. coli YodA protein in this paper are AAC75039 and P76344, respectively.
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INTRODUCTION |
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YodA is a 216 aa residue protein and the product has been identified on two-dimensional polyacrylamide gels (VanBogelen et al., 1996 ; Ferianc et al., 1998
). N-terminal sequencing has demonstrated that the protein is processed and contains a 24 aa signal sequence, suggesting that the mature product is exported from the cytoplasm (Ferianc et al., 1998
). YodA together with two other putative proteins, YrpE of Bacillus subtilis (Sorokin et al., 1997
) and pXO1-130 of Bacillus anthracis (Okinaka et al., 1999
), may constitute a new family of stress proteins based on sequence similarity (44·6% identity; Pu
kárová et al., 2001
) and size (YodA, 216; YrpE, 251; pXO1-130, 237 aa). These three proteins were found to exhibit sequence similarity also with the 200 aa residue C-terminal part of the streptococcal adhesin, AdcA (Pu
kárová et al., 2001
), which is a lipoprotein containing a putative metal-binding site (Dintilhac & Claverys, 1997
). In addition, YodA exhibits weak sequence similarity with the N-terminal part (about 200 aa) of the copper-binding protein amine oxidase, encoded by maoA (Ferianc et al., 1998
), of both E. coli (Azakami et al., 1994
) and Klebsiella aerogenes (Sugino et al., 1992
).
In this work we have identified the yodA promoter and report that induction of the gene during cadmium exposure is dependent on soxS, fur and relA/spoT of the stringent response, but not oxyR. The product of the gene is primarily found in the cytoplasm of non-stressed cells but is exported to the periplasm upon cadmium exposure.
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METHODS |
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Construction of fusion and mutant strains.
The strain carrying PyodAlacZ transcriptional fusion (AL6) was constructed as follows. A 1288 bp SalIBamHI PCR product containing the yodA promoter region was cloned into plasmid pTL61T (Linn & St Pierre, 1990 ). The resulting plasmid (pAL2), in which lacZ is transcribed from the yodA promoter, was transformed into E. coli MC4100 competent cells. The PCR insert was verified by DNA sequencing. Transformants were infected by
phage RS45 (Simons et al., 1987
) and a monolysogenic strain named AL6 for
phage carrying PyodAlacZ was isolated for further studies.
The strain carrying a malEyodA fusion (AL401) was constructed as follows. A 684 bp SmaISalI PCR product containing yodA coding sequence was cloned into plasmid pMal-c cut by the same restriction enzymes downstream of the malE gene, encoding the maltose-binding protein (MBP). The resulting plasmid, pAL401, was used to transform competent cells of strain XL-1 Blue. The PCR insert was verified by DNA sequencing.
Mutations in the oxyR, soxS, fur, rpoS and relA/spoT genes were introduced into AL6 by P1 transduction to generate AL8, AL9, AL10, AL12, AL14, KK223, respectively, selecting for appropriate antibiotic resistance associated with the mutation (Table 1).
Resolution of proteins on two-dimensional polyacrylamide gels.
Cell extracts for two-dimensional polyacrylamide gels were prepared by the methods of OFarrell (1975) with modifications described by VanBogelen & Neidhardt (1990)
.
Measurement of rates of synthesis of individual proteins.
At indicated times, a portion (1 ml) of a culture was removed and placed in a flask containing [3H]leucine (5 mCi mmol-1 [185 MBq mmol-1], 100 µCi ml-1). Incorporation was allowed to proceed for 5 min, after which non-radioactive leucine (2·4 mM) was added for a 3 min chase. To this sample was added a portion of a culture of the same strain grown in [35S]methionine labelling medium (M9 medium; 1·1 mCi mmol-1 [40·7 MBq mmol-1], 11 mCi (35S)methionine ml-1). Radiochemicals were obtained from Amersham Pharmacia Biotech. The mixed sample was analysed by resolution on two-dimensional gels and autoradiograms were prepared to permit visualization of labelled proteins. Protein spots chosen for quantitative assays were sampled from the dried gel with a syringe needle and treated as described by Pedersen et al. (1976) to permit measurement of their 3H and 35S content by scintillation counting. The differential rate of synthesis of a sampled protein was defined as the 3H:35S ratio of the sampled spot divided by the same isotope ratio of unfractionated TCA-precipitated extracts. To confirm these experimental results, another cadmium induced protein, MetK, was monitored in the same manner (not shown).
RNA isolation and Northern blot hybridization.
Total RNA was extracted using the Total RNA Isolation Reagent (Advanced Biotechnologies) according to the manufacturers recommendations. RNA samples were denatured with formamide and formaldehyde, electrophoresed on a 1% (w/v) agarose gel containing formaldehyde and transferred to an NY 12N membrane (0·2 µm) (Schleicher and Schuell) as described by Sambrook et al. (1989) . The blot was then hybridized with the DIG (digoxigenin)-labelled SmaISalI DNA fragment of pAL401 as a probe for the yodA transcript. The DIG DNA Labelling Kit (Boehringer Mannheim) was used for labelling the DNA probe. RNA hybridization was performed by standard procedure essentially as described by Sambrook et al. (1989)
but modified according to the recommendations in the DIG systems users guide for filter hybridization (Boehringer Mannheim). DIG-labelled DNA was detected, after hybridization to target RNA, by using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
S1-nuclease mapping of the transcription start points (tsps).
High-resolution S1-nuclease mapping was performed according to Kormanec (2001) . Samples (40 µg) of RNA (estimated spectrophotometrically) were hybridized to approximately 0·02 pmol DNA probe labelled at one 5' end with [
-32P]ATP (approx. 3x106 c.p.m. pmol-1). The 522 bp probe (Fig. 1a
) was prepared by PCR from E. coli MC4100 using the 5' end-labelled reverse oligonucleotide primer, YodA3r (5'-GCCGTGTGAGTGATGACCATGCG-3') internal to yodA and the forward primer, YodA3f (5'-GTCTATCTTGTCGCGATTCTGGC-3'). The control 444 bp E. coli PrpsM promoter probe was prepared by PCR from E. coli MC4100 using a 5' end-labelled reverse oligonucleotide primer P1 (5'-TGATCAGGAATGTTAATGCCTGCTATACGG-3') and the forward primer P2 (5'-TGCTTATCGTTGTTGTCGTGATTATGGAC-3'). The RNA-protected DNA fragments were analysed on DNA sequencing gels together with G+A and T+C sequencing ladders derived from the end-labelled fragments (Maxam & Gilbert, 1980
).
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ß-Galactosidase assay.
ß-Galactosidase activity was measured as described by Miller (1972) with modifications described by Albertson & Nyström (1994)
. Samples were centrifuged before they were measured spectrophotometrically to determine the A420 (ß-galactosidase). The ß-galactosidase activity is expressed as follows: 1000x(A420/[OD600 culturexreaction timexvolume]). Duplicate measurements within an experiment gave less than 10% variation. Shown in the figures are data from single representative experiments, but all experiments were repeated several times to ensure reproducibility.
Overexpression of yodA.
The yodA gene was overexpressed as a fusion with the malE gene encoding MBP employing the Protein Purification System of New England Biolabs. The E. coli strain containing pAL401 was grown to OD600 0·40·5 and IPTG was added to induce MBPYodA. The incubation was continued for another 24 h at 37 °C. Aliquots (1 ml) were removed at indicated times before and after addition of IPTG. The cells were then harvested by centrifugation. Cell extracts were prepared for SDS-PAGE.
Preparation of a polyclonal antiserum against the MBPYodA fusion protein.
MBPYodA was separated by SDS-PAGE. The gel was stained in 0·1% (w/v) Coomassie brilliant blue R-250 (Serva) for 3 min, washed and the MBPYodA band cut out of the gel. A rabbit was injected subcutaneously at 14 day intervals with a homogenate of about 300 µg fusion protein in crumbled polyacrylamide gel suspended in PBS buffer and Freunds adjuvant. The polyclonal antiserum was prepared from the blood collected two weeks after the third injection (Institute of Virology, Slovak Academy of Science) and was designated RAS-MBPYodA (rabbit antiserum against MBPYodA fusion protein). Using the procedure to absorb antibacterial antibodies (Gruber & Zingales, 1995 ) we removed antibacterial antibodies from the prepared antiserum.
Western immunoblot assays.
Proteins were separated on an 11·5% (w/v) polyacrylamide/SDS gel at 200 V (100 mA). SDS-PAGE analysis was performed as described previously (Laemmli, 1970 ). After electrophoresis, the gels were soaked in transfer buffer (10 mM CAPS, 10% (v/v) methanol, pH 11·0) for 10 min. PVDF Protein Sequencing Membranes (Bio-Rad) were rinsed with 100% methanol and stored in transfer buffer. The gels, sandwiched between sheets of PVDF membrane and several sheets of blotting paper (Whatman), were assembled into a blotting apparatus (Bio-Rad) and electroeluted for 1 h at 50 V (100 mA) in transfer buffer. Immunoblot assay was done by using Immun-Blot Assay Kit (Bio-Rad) according to the protocols provided by the manufacturer. The PVDF membranes were probed with a 1:50000 dilution of RAS-MBPYodA as a primary antibody. Goat anti-rabbit IgG (Bio-Rad) conjugated with alkaline phosphatase (Bio-Rad) was used as a secondary antibody after dilution 1:3000. A colorimetric detection of alkaline phosphatase with 5-bromo-4-chloro-3-indolyl phosphate/4-nitro blue tetrazolium chloride (BCIP/NBT) as a substrate was used according to the manufacturers instructions.
Cell fractionation.
Strain MC4100 was grown to OD600 of 0·3 (exponential-phase) in M9 medium. Before addition of cadmium (273 µM) and 45 min and 150 min after addition, 10 ml culture aliquots were withdrawn to prepare periplasmic fractions and 50 ml culture to obtain cytoplasmic and membrane (outer, inner) fractions. The periplasmic fraction of the cells was isolated using an osmotic shock protocol as described by Rech et al. (1996) . The cytoplasmic and membrane fractions were isolated using differential centrifugation. Cells were harvested by centrifugation and then homogenized in a 0·3 M sucrose, 0·1 M Tris/HCl, pH 7·8 solution. The cell suspension was ultracentrifuged at 150000 g for 30 min (Beckman L8-M). The supernatant was precipitated overnight by the addition of ice-cold acetone at -20 °C and then washed by addition of acetone to a final concentration of 50% (v/v). The precipitated proteins were obtained by centrifugation for 5 min at 12000 g (4 °C). The cell pellet obtained after centrifugation of homogenized cells was resuspended in a 0·1 M Na2CO3 solution. The cell suspension was ultracentrifuged as mentioned above. The supernatant fraction was dialysed overnight against a 0·1 M Tris/HCl, pH 8 solution. The dialysed fraction (outer membrane fraction) was then precipitated by the addition of acetone as described above. The pellet was dissolved in SDS to a final concentration of 4% (w/v). Proteins obtained after precipitation in ice-cold acetone are the proteins of the inner membrane.
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RESULTS |
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To identify the tsp of the yodA promoter, high resolution S1-nuclease mapping was performed using the same RNA as above. The 5'-labelled probe is indicated in Fig. 1(a). As shown in Fig. 3(a)
, lane 1, an RNA-protected fragment with the tsp at a C residue, 29 bp upstream from the proposed translation initiation codon of yodA was identified using RNA from Cd-induced cells (Fig. 3a
, lane 2). No RNA-protected fragment was identified with a control tRNA (Fig. 3a
, lane 3). As an internal RNA control, S1-nuclease mapping was performed with the same RNA samples using a probe fragment specific for the E. coli PrpsM promoter directing expression of the
ribosomal protein operon (Post et al., 1980
). RNA-protected fragments corresponding to the rpsM promoter were identified with all RNA samples (Fig. 3b
). Based on these results we suppose that yodA is transcribed from a single cadmium-induced promoter. The putative -10 region of the yodA promoter (TAACAT) showed similarity to the consensus sequence (TATAAT) of the promoters recognized by the major E. coli sigma factor,
70. The corresponding -35 region of the yodA promoter (TTGCAT) also exhibited similarity to the -35 consensus sequence (TTGACA) (Fig. 1b
). However, an extended spacer region (19 bp) between these two elements was observed (Fig. 1b
).
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DISCUSSION |
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It is possible that this promoter responds to cadmium-induced oxidative stress rather than directly to the presence of the metal since expression of the yodA gene is induced also by hydrogen peroxide. It has been previously shown that the cadmium-stress stimulon shares proteins with the hydrogen peroxide stress stimulon (Lauová et al., 1999
) and many oxyR regulon proteins are induced during the initial hour of cadmium exposure (Ferianc et al., 1998
). It is also known that cadmium exposure results in oxidative damage to macromolecules and many studies have suggested that oxidative stress is a key parameter in the toxicity of this metal (e.g. Faris, 1991
; Manca et al., 1991
). Moreover, the oxidative potential of cadmium is argued to be mediated through generation of the hydroxyl radical (Storz et al., 1990
), which is one of the most reactive oxygen species known. Whether cadmium is involved directly in the production of reactive oxygen species is not clear, but it has been shown that free radical scavengers and antioxidants protect against cadmium toxicity (Stohs & Bagchi, 1994
).
In contrast to hydrogen peroxide, the superoxide generating agent paraquat did not induce yodA expression. Thus, the regulation of yodA as an oxidative stress gene is somewhat atypical since oxyR is dispensable for yodA expression whereas soxS is required for full induction. The increased induction of yodA in the oxyR mutant cells may be explained by the fact that many oxidative stress defence genes, such as katG, require OxyR for their expression, and hydrogen peroxide and other reactive oxygen species are thus likely to accumulate in this mutant. This may cause a superinduction of yodA during cadmium stress in the oxyR mutant.
A mutation in fur drastically attenuated induction of yodA upon cadmium stress. The finding that Fur, originally defined as an iron-response regulator, is a third control system involved in protection against oxidative damage (Niederhoffer et al., 1990 ; Foster & Hall, 1992
; Tardat & Touati, 1991
; Stojiljkovic et al., 1994
) further highlights the link between oxidative stress and yodA expression. However, the fur and soxS mutations appear to affect different pathways affecting yodA expression since the effect of mutating both genes was additive.
Cadmium elicits a stringent response in E. coli when added at high concentrations (600 µM) (VanBogelen et al., 1987 ). The stringent response consists of major adjustments in gene expression and physiological activities, many of which are mediated by ppGpp. An E. coli strain deleted for both the relA and spoT genes fails to accumulate detectable levels of ppGpp (Xiao et al., 1991
). The mutant is defective also in the expression of several stress defence genes, such as rpoS, that are normally induced in stationary phase cells (Nyström, 1994
). A relA spoT double mutant failed to increase yodA expression upon cadmium exposure whereas an rpoS mutation had no effect. Moreover, the relA spoT mutant was more sensitive to cadmium than the rpoS mutant (and wild-type) (not shown) indicating the importance of ppGpp in cadmium accommodation. However, if ppGpp accumulation were the key signal for yodA induction we would expect the gene to be induced in stationary phase. This was not the case, indicating that ppGpp is required but not sufficient for yodA induction.
The presence of a signal sequence confirmed by N-terminal sequencing of YodA protein (Ferianc et al., 1998 ) suggested that the mature YodA protein could be a periplasmic or outer-membrane protein. Because YodA protein has not yet been purified, we used a MBPYodA fusion protein for preparation of polyclonal antibodies to investigate the cellular localization of the YodA protein. The result revealed that the YodA protein was localized in both the cytoplasm and periplasmic space but was only translocated into a periplasmic space upon cadmium stress. The localization of YodA is suggestive with regard to its possible physiological function since YodA is excellently positioned for binding cadmium before it could enter the cytoplasm. Several studies suggest that E. coli is able to accumulate cadmium inside the cell (Mitra et al., 1975
; Morozzi et al., 1986
) but the possible involvement of specific proteins and their identity have not been elucidated. Our theoretical results from searches of sequence databases for YodA homologues (Pu
kárová et al., 2001
) indicate that the proteins YodA, YrpE and pXO1-130 possess common functional and structural features including a potential C-terminal domain that may function as an extracytoplasmic receptor of an ABC-type permease (Dinthilhac et al., 1997
). Perhaps YodA is part of a cadmium-exporting transport protein. Plasmid-encoded transporters for heavy metals have been described in several bacterial species (Silver, 1996
) and recently, a zinc, cadmium and lead translocating P-type ATPase encoded by a chromosomal gene (zntA) has been identified in E. coli (Rensing et al., 1997
, 1999
; Reuven & Ron, 1998
; Binet & Poole, 2000
). Similarly, CopA, a putative copper-translocating P-type ATPase, has been shown to be involved in copper resistance in E. coli (Rensing et al., 2000
). In addition, CadA, similar to cadmium-transporting ATPases known mostly from Gram-positive bacteria and to ZntA from E. coli, and CadR, related to the MerR family of response regulators, have been identified in P. putida 06909, a rhizosphere bacterium (Lee et al., 2001
). Of these metals involved in the expression of the genes encoding metal-translocating P-type ATPases, only zinc and copper are required for growth; there must be a fine balance between uptake and efflux to provide metal ion homeostasis (Rensing et al., 1999
). The presence of Zn- and Cu-translocating P-type ATPases suggested they have roles in either zinc or copper homeostasis in E. coli. On the other hand, it has been suggested that cadmium can displace other metals that are bound to enzymes, such as zinc bound to alkaline phosphatase in E. coli. This may inhibit the enzymic activity and directly damage the cell (Mitra et al., 1975
). Furthermore, if the displaced metals are redox-active, this would lead to oxidative stress (Stohs & Bagchi, 1994
). If YodA is able to bind these displaced redox-active metals, the protein would indirectly protect against the effect of cadmium. However, our results did not indicate the possibility of involvement of YodA in zinc metabolism because there was no confirmation of the dependence of yodA expression on zinc or on other metals such as copper, cobalt and nickel. On the other hand, finding a histidine-rich N-terminal extension of the P. putida CadA sequence, unlike ZntA and other CadA not found in other homologues, and also a histidine-rich C-terminal extension of the P. putida CadR not found in other MerR family response regulators (Lee et al., 2001
), and the occurrence of a histidine-rich N-terminal sequence of the mature YodA (HGHHSH) suggested the possible functional similarity between YodA and CadA or CadR, respectively. Future studies of the phenotype of a yodA deletion mutant and biochemical analysis of purified YodA will reveal the exact function of the YodA cadmium-stress protein.
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ACKNOWLEDGEMENTS |
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Received 10 May 2002;
revised 24 July 2002;
accepted 29 August 2002.
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