©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Activation of the Escherichia coli Adaptive Response Gene aidB Is Mediated by Binding of Methylated Ada Protein
EVIDENCE FOR A NEW CONSENSUS SEQUENCE FOR Ada-BINDING SITES (*)

(Received for publication, December 12, 1994; and in revised form, February 7, 1995)

Paolo Landini Michael R. Volkert (§)

From the Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Escherichia coli aidB gene is part of the adaptive response to DNA methylation damage. Genes belonging to the adaptive response are positively regulated by the ada gene; the Ada protein acts as a transcriptional activator when methylated in one of its cysteine residues at position 69. Through DNaseI protection assays, we show that methylated Ada (Ada) is able to bind a DNA sequence between 40 and 60 base pairs upstream of the aidB transcriptional startpoint. Binding of Ada is necessary to activate transcription of the adaptive response genes; accordingly, in vitro transcription of aidB is dependent on the presence of Ada. Unmethylated Ada protein shows no protection against DNaseI digestion in the aidB promoter region nor does it promote aidBin vitro transcription. The aidB Ada-binding site shows only weak homology to the proposed consensus sequences for Ada-binding sites in E. coli (AAANNAA and AAAGCGCA) but shares a higher degree of similarity with the Ada-binding regions from other bacterial species, such as Salmonella typhimurium and Bacillus subtilis. Based on the comparison of five different Ada-dependent promoter regions, we suggest that a possible recognition sequence for Ada might be AATnnnnnnGCAA. Higher concentrations of Ada are required for the binding of aidB than for the ada promoter, suggesting lower affinity of the protein for the aidB Ada-binding site. Common features in the Ada-binding regions of ada and aidB are a high A/T content, the presence of an inverted repeat structure, and their position relative to the transcriptional start site. We propose that these elements, in addition to the proposed recognition sequence, are important for binding of the Ada protein.


INTRODUCTION

In Escherichia coli, the adaptive response to DNA methylation damage is activated by sublethal concentrations of methylating agents such as methyl methanesulfonate or N-methyl-N`-nitro-N-nitrosoguanidine (MNNG)(^1)(1, 2) . Upon activation, cells are protected from the toxic and mutagenic effects of these agents. The Ada protein is the key enzyme of the adaptive response; this protein is a methyltransferase able to remove methyl groups from damaged DNA, transferring them to two of its own cysteine residues. Ada protein methylated at Cys-69 (Ada) can bind to specific DNA sequences in the promoter region of several genes; binding of Ada makes the promoters accessible to RNA polymerase and therefore activates their transcription(3, 4) . Moreover, a direct protein-protein interaction takes place, since RNA polymerase mutants resulting from deletions in the C-terminal domain of the alpha subunit are unable to support transcription from the ada promoter, even when Ada is present(5) . Unmethylated Ada protein, on the contrary, seems to be involved in negative regulation of the ada promoter(6) . Methylated Ada activates transcription of its own gene, ada, and of at least two other genes, alkA and aidB. The ada promoter is also responsible for the transcription of another gene, alkB, whose function is unknown(7) . The product of the alkA gene is a glycosylase that recognizes and excises methylated DNA bases(8) . The aidB gene encodes a protein homologous to mammalian isovaleryl-coenzyme A dehydrogenase. Overexpression of the AidB protein is able to counteract MNNG-mediated mutagenesis in vivo(9) , and this effect seems to be specific for MNNG. (^2)It seems possible that the AidB protein is involved in a detoxification pathway for MNNG, although it is still unclear if this function is related to its isovaleryl-coenzyme A dehydrogenase activity.

For both the ada/alkB and the alkA promoters, the Ada-binding site has been determined by DNaseI protection experiments; two short sequences common to both ada and alkA promoter regions (AAANNAA and AAAGCGCA) have been proposed as the Ada-binding sequence (Ada box) by different papers ( (10) and (11) , respectively). Mutations in the AAAGCGCA sequence have been shown to abolish transcriptional activation of the ada gene by Ada(11) , although the same is true for mutations outside of this proposed consensus sequence(11, 12) .

Counterparts of the ada genes have been found in many other bacteria, as well as in eukaryotic and mammalian cells(13, 14) . In Salmonella typhimurium and Bacillus subtilis, Ada-binding sites have been identified and sequenced(15, 16) ; in both of these bacterial species, no precise consensus sequence has been assigned. However, it has been shown that E. coli Ada protein is able to activate transcription from the S. typhimurium ada gene(15) .

The aidB gene is ada regulated(17) , but unlike other adaptive response genes, aidB can also be induced by oxygen-limiting conditions, or by the addition of sodium acetate to the growth medium, at a final pH of 6.0-6.5(18, 19) . This second mechanism of induction is ada independent and requires a functional rpoS gene. rpoS encodes ^s, an alternative factor for RNA polymerase that is required for expression of stationary phase-specific genes(20) . In this report, we will consider only the mechanism of the ada-dependent activation of aidB. It has already been shown that methylated Ada protein is able to bind to the aidB promoter region in vitro(9) , although aidB lacks both the AAANNAA and the AAAGCGCA sequences common to ada and alkA. This suggests that both of these sequences might be non-essential features of the Ada-binding site. In this paper, we describe the aidB Ada-binding site and discuss its importance as a general model for gene regulation by the Ada protein.


MATERIALS AND METHODS

Chemicals

Restriction endonucleases were purchased from Boehringer Mannheim. [alpha-P]dATP, [alpha-P]dCTP, and [alpha-P]CTP were from Amersham Corp. E. coli RNA polymerase and RNase-free DNaseI were from Pharmacia Biotech Inc.

DNA

For gel retardation assays, DNA fragments were labeled with [alpha-P]dATP by end-filling with DNA polymerase I (Klenow fragment). The following fragments were used: for ada, a 176-bp HindIII-EcoRI fragment from plasmid pYN3066(3) ; for aidB, a 106-bp BamHI-StyI fragment from pMV120 ( (9) and Fig. 1). For DNaseI protection assays, a 246-bp BamHI-MluI aidB fragment from plasmid pMV120 was labeled with [alpha-P]dATP at the BamHI site (template strand); for the coding strand, a BamHI-BstXI fragment (Fig. 1) was subcloned into the BamHI-MluI sites of pSport1 (Life Technologies, Inc.) using a BstXI-MluI linker oligonucleotide (Operon Technologies, Alameda, CA) and producing plasmid pMV442. The EcoRI-MluI fragment from pMV442 (165 bp) was labeled at the MluI site with [alpha-P]dCTP by the Klenow end-filling reaction. For in vitro transcription, the HindIII-EcoRI fragment from ada, the BamHI-MluI fragment from aidB, and the lacUV5 promoter (a 205-bp EcoRI fragment from plasmid pYN3072(3) ) were used. All DNA fragments were isolated by electrophoresis and purified from agarose using a Geneclean kit (Bio 101, La Jolla, CA).


Figure 1: aidB promoter and regulatory region. Sequence numbering is relative to the first transcriptional start site (+1). The nucleotides protected from DNaseI by Ada are shown in boldfacetype. The twoarrows running in opposite directions indicate an imperfect palindromic sequence. The possible -35 and -10 promoter elements are boxed. The two transcriptional start sites identified in vivo(8) are numbered+1 and +3, and the smallarrowabove indicates direction of transcription. The two possible translation starts for the AidB protein are outlined, and the dottedunderline shows the Shine-Dalgarno sequence. Restriction sites used in the in vitro experiments are underlined, and the different enzymes are indicated.



Gel Retardation Assays

Methylation of the Ada protein was produced by incubating the protein (120 pmol) with 1 µg of methylated calf thymus DNA for 15 min at 37 °C in 20 µl of 70 mM Na-Hepes, pH 7.8, 1 mM EDTA, and 1 mM dithiothreitol. For unmethylated Ada, the protein was incubated in the same conditions with 1 µg of unmethylated DNA. 10,000 cpm of labeled DNA, corresponding to approximately 10 fmol, were used in each reaction. The binding reaction was performed at 22 °C for 45 min in 10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5% glycerol, 0.05% Nonidet P-40, 0.1 µg of salmon sperm DNA, and 10 µg/ml bovine serum albumin in a final volume of 20 µl. The samples were loaded on a 6% native polyacrylamide gel and run in 0.25 times TBE at 12 V/cm.

DNaseI Protection Assays

Samples were treated as described for the gel retardation assay. After 45 min at 22 °C, 6 mM CaCl(2) and 200 ng of DNaseI were added, and samples were incubated for 30 s at 22 °C. DNaseI digestion was stopped with an equal volume (25 µl) of DNaseI stop solution (600 mM NaCl, 100 mM EDTA, 30 µg/ml of salmon sperm DNA), and samples were precipitated with ethanol and resuspended in loading buffer. Samples were heated for 3 min at 100 °C and loaded onto a denaturing polyacrylamide/urea gel.

In Vitro Transcription

0.5 pmol of the promoter regions from lac, ada, and aidB were pre-incubated as described for the gel retardation experiment, with the addition of 4 pmol of RNA polymerase. Transcription reactions were started by the addition of 6 mM MgCl(2), 0.2 mM ATP, 0.2 mM UTP, 0.2 mM GTP, 120 µg/ml heparin, and 1 µCi of [alpha-P]CTP. Incubation was for 6 min at 30 °C. After addition of 1000-fold unlabeled CTP, the reaction was continued for 5 min and blocked with an equal volume of DNaseI stop solution (see above). Samples were processed as described for DNaseI protection experiments and analyzed on a 6% polyacrylamide/urea gel.


RESULTS

DNaseI Protection Experiments

To identify the Ada-binding site in the aidB promoter region, DNaseI protection experiments were performed. Results are shown in Fig. 2A for the coding strand and in Fig. 2B for the template strand. For the coding strand, clear protection by 12 pmol of Ada can be observed between nucleotides -62 to -38 relative to the transcriptional start site. This area is marked by the solidline in Fig. 2A. Two DNaseI-hypersensitive sites, at positions -35 and -28, could also be detected. The region between these two hypersensitive sites also shows a weak protection by Ada.


Figure 2: DNaseI protection of aidB promoter region. A, coding strand. Lane1, no Ada protein; lane2, 12 pmol of unmethylated Ada; lane3, 12 pmol of methylated Ada. B, template strand. Lanenumbering is the same as A. The sequencing ladder of a known DNA fragment (GATC) is shown as a molecular weight standard. The solidlines indicate protection by Ada; arrows show DNaseI-hypersensitive sites.



Although the borders of the protected region appear to be less clear on the template strand, the pattern emerging from DNaseI protection experiments is very similar; the bands corresponding to positions -62 to -38 are protected by Ada (marked by a solidline in Fig. 2B). Two hypersensitive sites are also present at nucleotides -68 and -75 from the transcription start. It is noteworthy that the position of the hypersensitive sites is downstream of the protected region in the coding strand and upstream of it in the template strand. This might suggest that binding of Ada induces a symmetrical distortion of the DNA helix in the bordering regions. No DNaseI-protected or -hypersensitive site was detectable with 12 pmol of unmethylated Ada in either of the two strands (Fig. 2, A and B).

Fig. 3compares the aidB Ada-binding site to those from the E. coli ada and alkA genes(3) , from the S. typhimurium ada gene(15) , and from the B. subtilis alkA gene(16) . The Ada-binding site from aidB displays a higher similarity with the Ada boxes from the B. subtilis alkA gene (14 identical nucleotides out of 18, 78% identity) and for the S. typhimurium ada gene (67% identity) than for both the ada and alkA genes from E. coli (56% identity).


Figure 3: Comparison between Ada-binding sites in the regulatory regions of different genes. The regions with higher similarity are indicated by largeletters. The nucleotides conserved in each sequence are underlined. Numbers give the position of the different Ada-binding regions relative to the transcriptional start sites. Abbreviations are as follows: st, S. typhimurium; bs, B. subtilis; ec, E. coli.



Gel Retardation Assays

Since aidB does not share a very high homology with the Ada-binding site from the ada promoter, it was possible that the methylated Ada protein could display a different affinity for the two sites. To answer this question, we performed a gel retardation assay with both the ada and the aidB Ada-binding regions in the presence of different concentrations of unmethylated and methylated Ada protein. 100% retardation is obtained at a concentration of Ada as low as 0.3 pmol for ada (Fig. 4B), while it takes an approximately 10-fold higher concentration for aidB (Fig. 4A). Binding was also observed with unmethylated Ada, although 100% retardation was not obtained for aidB at the concentrations tested and was obtained for ada only at 10 pmol. At high concentrations of Ada, new bands with a slower electrophoretic mobility began to appear, particularly for the ada promoter. Although we cannot rule out that more than one molecule of Ada might be able to bind specifically at the same time, it seems likely that the slower bands result from nonspecific interaction between DNA and Ada and/or residual unmethylated protein.


Figure 4: Gel retardation experiments. A, aidB regulatory region (BamHI-StyI fragment, 106 bp). Lanes1 and 6, free DNA; lanes2-5, 0.3, 1, 3.3, and 10 pmol of unmethylated Ada protein; lanes7-10, same concentrations of Ada. B, ada promoter region (HindIII-EcoRI, 176 bp). Lanes1 and 6, free DNA; lanes2-5: 0.3, 1, 3.3, and 10 pmol of unmethylated Ada protein; lanes7-10, same concentrations of Ada.



In Vitro Transcription

In the reaction conditions described by Teo et al.(4) , in which Ada is able to stimulate transcription from the ada promoter, we could not detect any transcription from the aidB promoter region. However, in a cell-free transcription/translation system, Ada was required for transcription of the aidB gene from the supercoiled plasmid pMV426(9) , suggesting that binding of Ada is necessary for aidB transcription. Fig. 5shows in vitro transcription assays with purified RNA polymerase performed using the reaction buffer from the footprint experiments instead of the transcription buffer previously used(4) . In these conditions, transcription from the aidB promoter produces an mRNA of approximately 120 nucleotides in size, determined using a known DNA sequencing ladder as a molecular weight marker (Fig. 5B, lanes2-4). This value is in good agreement with the position of the transcriptional start sites identified in vivo ( (9) and Fig. 1). This transcript was the only one to be affected by the presence of Ada (Fig. 5B, lane4). It is likely that the other transcripts originate from nonspecific transcription from either promoter-like elements or the ends of the DNA fragment. Fig. 5A shows transcription from the ada promoter using the same experimental conditions; the size of the transcripts (92-98 nucleotides) and the presence of multiple start sites are in agreement with the results of Teo et al.(4) .


Figure 5: In vitro transcription experiments. DNA fragments used were HindII-EcoRI (176 bp) for ada and BamHI-MluI (246 bp) for aidB. A lacUV5 promoter fragment (205 bp) was always added as an internal standard. The expected RNA transcripts were 92-98 nucleotides for ada, 118-120 nucleotides for aidB, and 72 nucleotides for lacUV5. The sequencing ladder of a known DNA fragment was used to confirm the size of the transcripts. A: lane1, p lacUV5 alone; lane2, p lac and p ada, no Ada protein; lane3, 10 pmol of unmethylated Ada protein added; lane4, 10 pmol of methylated Ada added. B: lane1, p lacUV5 alone; lane2, p lac and p aidB no Ada protein; lane3, 10 pmol of unmethylated Ada protein added; lane4, 10 pmol of methylated Ada added.




DISCUSSION

The aidB gene is ada regulated in vivo(17) , and the Ada protein, in its methylated form (Ada), is able to interact with the aidB promoter region and stimulate its transcription(9) . However, neither of the two consensus sequences so far proposed for Ada-binding sites (10, 11) has been found in aidB. With DNaseI protection experiments, it has been possible to identify the specific binding site for Ada; this area spans approximately 24 nucleotides, slightly more than the number of nucleotides protected in both the ada and the alkA promoter regions(4) . Ada-binding sites are centered at different positions relative to the promoter elements and the transcriptional start site in ada and alkA, but they are always located in the proximity of the -35 region; in the ada gene, the protein binds immediately upstream, protecting the residues from -40 to -60, while in alkA the binding site overlaps the -35 region, spanning from -32 to -52(4, 21) . The location of the Ada-binding site of aidB is the same as that of ada; Ada protects from DNaseI digestion an area extending from -38 to -62.

The location of the Ada-binding site in aidB may provide information on the -35 and -10 promoter elements and define the areas to target in future site-specific mutagenesis analysis. The aidB promoter, like many other positively controlled promoters, lacks -10 and -35 consensus sequences; based on the position of the transcriptional start site, the most likely candidate for a -10 sequence is CATACT (Fig. 1). This hexamer maintains the sixth (T) and the second (A) nucleotide residues that are strongly conserved in positively controlled promoters (22) and is similar to CAGCCT, which is the -10 sequence for the ada promoter (11) . Assuming that CATACT is the -10 for aidB, a possible -35 box might be CTGTCA, which is located 18 base pairs upstream and is similar to TTGACA, the consensus sequence for the -35 region.

In Fig. 3, we have compared the Ada-binding site of aidB with the E. coli ada and alkA genes, as well as to the ada gene from S. typhimurium(15) and the alkA gene from B. subtilis(16) . It is noteworthy that when the ada gene from S. typhimurium is cloned into E. coli, it functions as part of the adaptive response, i.e. its Ada-binding site is recognized by the E. coli methylated Ada protein(15) . Common elements among these binding sites are the presence of an AGCAA-like sequence (AGCAA in aidB and in alkA from both E. coli and B. subtilis, ACGCAA in ada from S. typhimurium, and AGCGCAA in the ada gene from E. coli) preceded by an A/T-rich region. Another common sequence is a short AAT that is always present five nucleotides upstream of the AGCAA-like element. These observations suggest that a possible recognition sequence for Ada might be AATnnnnnnGCAA. However, it is likely that elements other than the simple nucleotide sequence play a role in the recognition of the binding site by Ada. The A/T-rich sequence is likely to determine local DNA structural modification such as bending(23) . Another common feature is the location of the Ada-binding site, immediately upstream of the -35 box. This position is important for protein-protein interaction between Ada and RNA polymerase, interaction known to be necessary for Ada-mediated transcriptional activation(5) . In the aidB and ada genes from E. coli, the AGCAA (aidB) and the AGCGCAA (ada) sequences are at the center of an imperfect palindrome ( Fig. 1and (4) ); although this structure might be involved in the regulation of the two genes, it does not appear to be a general feature for Ada-binding sites.

The affinity of Ada for its binding site in ada is higher than for aidB, as determined using gel retardation assays. Fig. 4shows that a 100% bandshift is observed at a concentration of 0.3 pmol of Ada for the ada promoter, while the same effect is obtained at concentrations of Ada at least 10-fold higher for the aidB regulatory region. This suggests that additional determinants, such as local modification of DNA structure, may be necessary for optimal interaction between Ada and its binding site. This view is supported by the fact that, in the ada promoter, mutations affecting local DNA bending have been shown to abolish transcriptional activation by Ada (12) , although they fall outside any consensus sequence so far proposed for the Ada-binding site.

The lower affinity of Ada for aidB is likely to play a role in the lower transcriptional levels displayed by aidB both in vitro and in vivo. In the in vitro experiments, the levels of induced transcription for aidB are much lower than for the ada promoter (Fig. 5). In experiments performed in vivo using aidB::lacZ chromosomal fusions, aidB transcription requires higher concentrations of methylating agents for induction compared with the ada or the alkA promoters(24) . aidB induction, however, is neither indirect nor accidental, since aidB has been shown to be effective in counteracting MNNG-mediated mutagenesis(9) . It is likely that induction of adaptive response genes may take place at different levels of methylation damage, and this ``fine tuning'' may be determined by different affinities of Ada for different Ada-binding sites. According to this hypothesis, aidB might be regarded as an ``emergency gene,'' activated when ada and alkA induction are no longer enough to counteract high levels of methylation damage.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM37052. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. Tel.: 508-856-2314; Fax: 508-856-5920; mvolkert{at}umassmed.ummed.edu.

(^1)
The abbreviations used are: MNNG, N-methyl-N`-nitro-N-nitrosoguanidine; bp, base pair(s); Ada, methylated Ada.

(^2)
M. R. Volkert, unpublished data.


ACKNOWLEDGEMENTS

We thank Graham Walker, Bradford Saget, and Robert Pohlman for the gift of purified Ada protein and for useful discussion and Laurel Hajec for critical reading of the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.