Process of Environmental Microbiology and Molecular Ecotoxicology, Swiss Federal Institute for Environmental Science and Technology (EAWAG), Postbox 611, CH-8600 Dübendorf, Switzerland1
Author for correspondence: Jan Roelof van der Meer. Tel: +41 1 823 5438. Fax: +41 1 823 5547. e-mail: vdmeer{at}eawag.ch
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ABSTRACT |
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Keywords: NtrC-type transcriptional activator, regulation, evolution
Abbreviations: 2-HBP, 2-hydroxybiphenyl; UAS, upstream activating sequence; RNAP, RNA polymerase; IHF, integration host factor
a Present address: Department of Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands.
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INTRODUCTION |
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Transcription activation of the hbpCA and hbpD genes by the HbpR protein is mediated from two different 54-dependent promoters, designated PhbpC and PhbpD (Jaspers et al., 2001
) (Fig. 1
). Also for the HbpR system, IHF was needed as a co-regulator, in order to obtain full activation of the PhbpC and PhbpD promoters (Jaspers et al., 2001
). Solely on the basis of the DNA sequence, regions could be identified with homology to the UASs of the XylR/DmpR type and to IHF-binding sites. It is interesting to study the HbpR-mediated regulatory system, since HbpR is the only member of the XylR/DmpR subclass described so far which is activated by biaromatic structures, such as 2-HBP and 2,2'-diHBP, and not by the classical effectors xylene and o-cresol (Jaspers et al., 2000
). Furthermore, the organization of the hbp genes pointed to recent evolutionary changes, which allows us to study the processes leading to the optimization of regulatory systems (Jaspers et al., 2001
). Here we report on the unusual location of two pairs of UASs within the hbpRhbpC intergenic region. We addressed the questions whether both pairs of UASs are functional and necessary for transcriptional activation of the hbpC promoter, and whether they have any importance for transcription of the hbpR gene itself. These questions were studied by analysing expression from promoter/operator deletions fused to the luciferase gene and by determining the transcription start site for the hbpR gene.
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METHODS |
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Recombinant DNA techniques, DNA sequencing and Southern analysis.
Plasmid DNA isolations, ligations, transformations and other DNA manipulations were carried out according to well-established procedures (Sambrook et al., 1989 ). Restriction endonucleases and other DNA-modifying enzymes were obtained from Amersham, Boehringer Mannheim and New England Biolabs, and used according to the specifications of the manufacturer. DNA fragments were isolated from agarose gels using QIAquick spin columns (QIAGEN). Double-stranded template sequencing was performed on plasmids using the dideoxy chain-termination method (Sanger et al., 1977
) with primers that were labelled with the fluorescent dye IRD-800 at the 5'-end, as described elsewhere (Ravatn et al., 1998
).
Construction of luxAB based promoter-probe plasmids.
Plasmid pHYBP103 contains a 704 bp fragment with the hbpRhbpC intergenic region (Jaspers et al., 2000 ) transcriptionally fused to the luxAB genes (Fig. 1
). To obtain a promoter-probe construct for the hbpR promoter (PhbpR) the hbpRhbpC intergenic region was retrieved from plasmid pHYBP100 (Jaspers et al., 2000
) as a 0·7 kb SphI fragment, making use of a second SphI site in the multi-cloning site, and cloned into pJAMA8 digested with SphI. The plasmid in which luxAB expression was driven from PhbpR was designated pHYBP113.
Deletion derivatives of pHYBP103 (hbpC'::luxAB) were constructed to test the contribution of the different pairs of UASs on transcriptional activation from PhbpC. Plasmids pHYBP134 and pHYBP135 were obtained by deletion of the 0·13 kb SphIBamHI or the 0·28 kb NcoIBamHI DNA fragments within the hbpRhbpC intergenic region of plasmid pHYBP103, respectively (Fig. 1). In plasmid pHYBP136 the 0·41 kb SphINcoI fragment of pHYBP103 was deleted (Fig. 1
). To vary the relative position of the distal pair of UASs (i.e. UASs C-3/C-4) on the DNA-helix with respect to the
54 promoter and the IHF-binding site, small insertions (4 and 8 bp) were introduced into plasmid pHYBP135. To do this, the plasmid was cut with NcoI, protruding ends filled in with Klenow DNA polymerase and religated. This resulted in a 4 bp insertion (plasmid pHYBP137) and a new NsiI site (Fig. 1
). Subsequently, an additional 4 bp were inserted by cutting pHYBP137 with BamHI (adjacent to the NsiI site), filling in with Klenow and religating (plasmid pHYBP138) (Fig. 1
).
Similarly, deletion derivatives of plasmid pHYBP113, containing an hbpR'::luxAB fusion, were constructed by deleting the 0·13 kb SphIBamHI fragment (plasmid pHYBP139) or the 0·28 kb NcoIBamHI fragment (plasmid pHYBP140). Plasmid pHYBP141 was derived by deleting the 0·41 kb SmaINcoI fragment from pHYBP113.
Testing hbp::lux promoter-probe constructs in E. coli.
All the different hbp::lux promoter-probe plasmids were cotransformed in E. coli with a compatible plasmid expressing either the hbpR gene (plasmid pHYBP124) or a dysfunctional hbpR gene (hbpR), which carries a frameshift mutation (plasmid pHYBP125) (Jaspers et al., 2001
).
Single chromosomal insertion of an hbpR'::luxAB fusion in P. azelaica.
By using the unique NotI sites at the flanks, the hbpR'::luxAB fusion in plasmid pHYBP113 was recovered and exchanged with the 3·2 kb NotI fragment present in the Tn5 delivery vector PCK218 (Kristensen et al., 1995 ) to produce plasmid pHYBP117 (Table 1
). By using mini-Tn5 delivery, the hbpR'::luxAB promoter-probe fusion of plasmid pHYBP117 was inserted into the chromosome of P. azelaica HBP1, in a triparental mating procedure, as described previously (Jaspers et al., 2000
). Selection for P. azelaica exconjugants was done on MM plates with Km and 2·9 mM 2-HBP. Proper insertion of the constructs was verified by Southern hybridization of the P. azelaica exconjugants (data not shown). The resulting strain is referred to as HBP117.
Luciferase assays.
Induction experiments with luxAB-harbouring E. coli and P. azelaica strains were performed in mineral medium at 30 °C as described elsewhere (Jaspers et al., 2000 ). Expression of luciferase was analysed by measuring bioluminescence on whole cells at a final n-decanal concentration of 2 mM in a MicroLumat LB 96 P luminometer (Berthold) as described previously (Sticher et al., 1997
).
RNA isolation and primer extension analysis.
Total RNA was isolated from a carbon-limited continuous culture of P. azelaica HBP1, 30 min after induction with 2-HBP, as described elsewhere (Jaspers et al., 2001 ). Primer extension analysis was performed with primer PE_HbpR2 (5'-GATTTCATGGCGATGGTTCAGG-3'; 5 bp downstream of the hbpR ATG start codon; Fig. 2a
), which was labelled with the fluorescent dye IRD-800 at the 5'-end as described previously (Jaspers et al., 2001
).
Synthetic oligonucleotides and chemicals.
Primers labelled with the fluorescent dye IRD-800 at the 5'-end were purchased from MWG-BIOTECH, all other primers from Microsynth. Ultrapure agarose, APS, TEMED, Tris and urea were purchased from Gibco-BRL Life Technologies, and Rapid Gel-XL-40% acrylamide gel solution was obtained from Amersham. IPTG and X-Gal were obtained from Biosynth and n-decanal from Sigma. Nutrient broth, yeast extract and tryptic casein were purchased from Biolife and ultrapure agar from Merck. Antibiotics, inorganic salts, silicon antifoam and all other organic chemicals were obtained from Fluka Chemie.
Nucleotide sequence accession number.
The nucleotide sequence of the hbpRC intergenic region can be retrieved from the GenBank database under accession no. U73900.
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RESULTS |
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Functional analysis of the two pairs of UASs in mediating expression from the hbpC promoter
To establish the in vivo functionality of each pair of putative UASs in transcription activation from the hbpC promoter, we applied a heterologous E. coli-based reporter system that had previously been used to study expression from this promoter (Jaspers et al., 2001 ). Plasmids were constructed that contained transcriptional fusions between different portions of the DNA region upstream of the hbpC promoter and the luxAB genes of Vibrio harveyi. These plasmids were then cotransformed into E. coli with a plasmid expressing either a functional HbpR (pHYBP124) or a truncated HbpR
(pHYBP125). Plasmid pHYBP103 contained the complete hbpRC intergenic region. In the presence of a functional HbpR and after induction with 2-HBP for 3 h E. coli strains carrying pHYBP103 gave rise to a 23-fold increase in bioluminescence as compared to uninduced conditions (with no 2-HBP added) (Fig. 3a
). E. coli containing plasmid pHYBP134, in which the distal pair of UASs C-3/C-4 was removed, gave rise to comparable levels of both luciferase activity and induction ratio after induction with 2-HBP as the native configuration (Fig. 3b
). In contrast, deletion of the proximal pair of UASs C-1/C-2 (E. coli with plasmid pHYBP135) resulted in a 23-fold decrease of luciferase activity. However, expression from the hbpC promoter was still significantly higher under induced conditions than uninduced (2·6-fold) (Fig. 3c
). Deletion of both pairs of UASs (as in pHYBP136) abolished inducible transcription activation from the hbpC promoter completely (Fig. 3d
). In all cases, no inducible response of the hbpC promoter was seen in the absence of a functional HbpR. These results indicated clearly that only the proximal pair of UASs C-1/C-2 is needed to obtain full (and wild-type) expression levels from the hbpC promoter (Fig. 3a
, b
). However, the distal UASs (C-3/C-4) seemed to be suitable in principle for HbpR-mediated activation. Their relatively poor performance in plasmid pHYBP135 might have been caused by an unsuitable relative geometry or spacing between the UASs C-3/C-4 pair compared to the -12/-24 promoter.
To determine if the geometry could have an influence on the suitability of the distal UASs to act as sites for HbpR-mediated transcription activation, we constructed derivatives of plasmid pHYBP135 which contained four (pHYBP137) and eight (pHYBP138) additional basepairs between the UASs and the other more downstream positioned promoter elements (IHF-binding site, -12/-24 motifs) (Fig. 1, 3e
, f
). E. coli carrying pHYBP137 with the 4 bp insertion gave a luciferase activity and induction ratio that were 1·5- and 2·3-fold higher, respectively, than those observed for pHYBP135 (Fig. 3e
). Plasmid pHYBP138 with the 8 bp insertion resulted in a further increase of luciferase activity (2·8-fold) and induction ratio (3·7-fold) compared to pHYBP135 (Fig. 3f
). These results demonstrated that the C-3/C-4 pair of UASs are indeed capable of mediating transcription activation from the hbpC-promoter upon induction with 2-HBP and in the presence of HbpR. The absolute luciferase response from the C-3/C-4 pair of UASs under these geometrical conditions, however, was still 9·3-fold lower than found for the UASs C-1/C-2 activated promoter (compare induced levels in Fig. 3b
and 3f
).
In vivo expression of the hbpR regulatory gene
We then determined which of the elements in the hbpRC intergenic region were important for expression of HbpR itself. To study the expression of the hbpR gene we used plasmid pHYBP113, which contained a luxAB-based transcriptional fusion with the complete hbpRhbpC intergenic region in the direction of hbpR. Induction studies were carried out in E. coli DH5 in a similar way to before. In the absence of 2-HBP, the expression from the hbpR promoter was 4·3-fold higher than the basal expression observed previously from the hbpC promoter (Figs 3a
and 4a
). However, in the presence of 40 µM 2-HBP, activation from the hbpR promoter did not increase, whereas that from the hbpC promoter increased 23-fold (Figs 3a
and 4a
). Based upon these results in E. coli, we concluded that the hbpR gene was transcribed constitutively. The expression studies were then repeated in P. azelaica. A P. azelaica strain was constructed that contained one copy of a chromosomally integrated hbpR'::luxAB transcriptional fusion (strain HBP117). Similarly as in E. coli, luciferase expression levels obtained under uninduced conditions from the hbpR promoter in strain HBP117 were about fourfold higher than those from the hbpC promoter (Fig. 5
). Transcription from the hbpR promoter was again independent of addition of 2-HBP, whereas the response from an hbpC'::luxAB fusion in P. azelaica increased 40-fold over a range of 1 µM to 0·4 mM 2-HBP (Fig. 5b
). At higher 2-HBP concentrations, however, luciferase activity decreased similarly for both strains. This was probably not due to down-regulating of the hbpR and hbpC promoters per se, but more likely reflected the inhibitory effects of 2-HBP on the cellular metabolism and/or on the luciferase activity (Folkert, 1996
; Oelze & Kamen, 1975
).
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DISCUSSION |
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Induction experiments in E. coli with different deletions of the hbpRhbpC intergenic region fused to luxAB showed that most if not all of the transcriptional output from the hbpC promoter is mediated from the proximal pair of UASs (i.e. UASs C-1/C-2). We concluded this from the observations that deleting UASs C-3/C-4 did not result in a decrease of luciferase activity or of induction ratio as compared to the native promoter (Fig. 3). However, our data also indicated that the distal pair of UASs (C-3/C-4) is principally functional for HbpR-mediated activation, since placing it at a similar position (-225 to -271) relative to the hbpC transcription start site as UASs C-1/C-2 (-180 to -227) resulted in transcription activation in the presence of 2-HBP. More recently, we also observed binding of an HbpR-fusion protein to DNA fragments containing either the proximal or distal pair of UASs, which indicated that these DNA sequences indeed act as HbpR-binding sites in vitro (D. Tropel & J. R. van der Meer, unpublished).
By aligning the UASs of the hbp system with those of other known XylR/DmpR-type regulators (Fig. 7), we can conclude that the distance between the centres of the binding motifs is not dramatically different among them, except for the UASs of the hbpD promoter. However, this larger distance between the UAS motifs in the hbpD promoter does not result in a much smaller promoter output compared to the hbpC promoter (Jaspers et al., 2001
). Interestingly, the UASs of the hbp system all have a larger positive twist between their centres (Fig. 7
), but it is not clear whether this positive turn is a specific binding determinant for HbpR. Compared to the location of the UASs in the Po, Pu, Ps and PtoMO promoters, the UASs of the hbpC and hbpD operators are also further away from the -24/-12 sequences (Fig. 7
). Based upon an alignment including the UAS-sequences in the hbp genes, we propose a refined consensus sequence for the distal and proximal region (Fig. 7b
, c
).
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Is there any advantage in keeping the unusual organization of the hbpRhbpC intergenic region? Keeping two pairs of UASs does not seem to increase transcription from the hbpC promoter since the distal pair of UASs can be deleted without affecting expression of the hbpC promoter. On the other hand, any recombinatorial deletion between the homologous regions would result in loss of the hbpR transcription start site and place the UASs at a position rather close to the start of hbpR (46 bp, whereas the distances between the distal UAS and the start of dmpR or xylR are 218 and 120 bp, respectively). Therefore, there seems to be a selective disadvantage for recombined structures in this region. We speculate that the unusual organization of pairs of UASs in the hbpRC intergenic region was the result of a DNA duplication. The original hbpR configuration would have had the C-3/C-4 pair of UASs relatively close to the HbpR translation start, which resulted in poor expression, since no proper 70 promoter sequence was available. A short duplication now placed the UASs further away (the present C-1/C-2 pair) and provided a promoter within the duplicated region. Interestingly, this scenario is not so far-fetched. A similar 5' duplication, although larger (0·9 kb) in size, was isolated in E. coli carrying a plasmid with the phlR system for phenol degradation of P. putida strain H (Burchhardt et al., 1997
). This spontaneous promoter-up mutation in phlR was isolated upon selection for better growth on phenol and led to an increase in the expression of phlR and the phenol pathway enzymes. The duplication had created a slightly different -35 box and -35/-10 spacing for the phlR promoter at the 3' end of the duplicated fragment. Maybe the regions upstream of hbpR, phlR and other homologous systems are prone to duplication, which might help to overcome limiting amounts of enzymes on new potential substrates that are inefficient effectors.
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ACKNOWLEDGEMENTS |
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Received 12 February 2001;
revised 17 April 2001;
accepted 8 May 2001.