Laboratoire de Microbiologie et Génétique Moléculaire, UMR 5100 CNRS Université Toulouse III, 118 Route de Narbonne, F-31062, Toulouse Cedex, France1
Author for correspondence: Annie Conter. Tel: +33 561 33 58 95. Fax: +33 561 33 58 86. e-mail: aconter{at}ibcg.biotoul.fr
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ABSTRACT |
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Keywords: transcriptional regulation, osmoregulation, bacterial promoters, NhaR
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INTRODUCTION |
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METHODS |
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Genetic procedures.
Bacterial strains carrying nhaR::Kan or hns205::Tn10 mutations were constructed by P1vir transduction, as described by Silhavy et al. (1984) , using strains OR100 and GM229 (Table 1
) as donors, and selecting for resistance to kanamycin (40 µg ml-1) or tetracycline (10 µg ml-1), respectively. The NhaR phenotype was tested by growth on minimal medium A plates (Miller, 1992
) containing melibiose as a carbon source and 100 mM LiCl. Mutants exhibit very poor growth in comparison with that of an isogenic wild-type strain (Rahav-Manor et al., 1992
). Strain CLG723 carries a
(malPlacZ) transcriptional fusion in which an intact lac operon is fused to the first gene of the malPQ operon (Debarbouille et al., 1978
). Strains carrying osmCplac fusions were constructed as follows. DNA fragments harbouring various portions of the osmCp region were PCR-amplified with oligonucleotides introducing EcoRI sites at both ends (sense: OsmC1E, OsmC9E, OsmC10E and OsmC13E; antisense: OsmC3E; Table 2
). To generate fusions with the wild-type promoter region (osmCp1+ osmCp2+), the template was plasmid pCG321 (Table 1
). To generate fusions with only one functional promoter, derivatives of pCG321 carrying the same insert with mutations osmCp11 (osmCp1- osmCp2+) or osmCp21 (osmCp1+ osmCp2-) were used as templates (Bouvier et al., 1998
; Fig. 1
). The DNA fragments were cloned in the unique EcoRI site of the vector pOM41 (Vidal-Ingigliardi & Raibaud, 1985
). After transformation of CLG723 with the resulting plasmids, the osmCp region was inserted in front of the
(malPlacZ) fusion, in place of the malP promoter, by homologous recombination, as described previously (Gutierrez & Devedjian, 1991
).
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Methods used with nucleic acids.
Isolation of plasmid DNA, digestion with restriction enzymes, ligation with T4 DNA ligase, and transformation were carried out by using standard methods (Silhavy et al., 1984 ; Sambrook et al., 1989
). Amplification of DNA fragments with Hot Tub DNA polymerase (Amersham) was performed according to the manufacturers protocol. DNA sequencing was done with the thermo-sequenase sequencing kit (USB), using oligonucleotide 209H or 209E 5'-end-labelled with [
-32P]ATP.
Construction of plasmids.
During the screening for osmC activators, we isolated an nhaR+ clone carrying nhaR on a 3968 bp Sau3A chromosomal DNA fragment ligated to the vector pJPB209. An internal deletion between two unique restriction sites (BspEI in the chromosomal insert and XmaI in the linker of pJPB209) yielded the plasmid pNHAR, carrying 1647 bp of chromosomal DNA with only one intact ORF (nhaR, transcribed from its own promoter). Plasmid pAPTnhaR was obtained by PCR amplification of nhaR, using NHAR1 and NHAR2 oligonucleotides (Table 2) and pNHAR as the template, digestion of the resulting DNA fragment with AseI and BamHI, and ligation with the 7007 bp BamHINdeI fragment of the vector pAPT156 (Table 1
). nhaR was then placed under the control of the lacUV5 promoter.
Preparation of E. coli crude extracts.
Bacterial cells were grown in LB170 medium to an OD600 of 0·6 and induced with 500 µM IPTG for 2 h. Cells were harvested by centrifugation and then washed in Tris-NaCl buffer (10 mM Tris/HCl, pH 8·0, 100 mM NaCl). They were then washed in buffer B (20 mM HEPES, pH 8·0, 1 mM EDTA, 150 mM NaCl, 7 mM ß-mercaptoethanol, 10%, v/v, glycerol) and lysed by sonication. Lysates were centrifuged for 1 h at 12000 g at 4 °C, and each supernatant was mixed with an equal volume of saturated ammonium sulphate and incubated for 30 min at 4 °C. After centrifugation at 12000 g at 4 °C, pellets were resuspended in buffer B and adjusted to 1 µg protein µl-1 after assay of the total protein content (protein assay kit; Bio-Rad).
Electrophoretic mobility shift experiments.
The DNA probes carrying the osmC promoter region were obtained by PCR amplification using pCG302 (Table 1) as a template and primers OsmC7 plus OsmC3, OsmC1 plus OsmC3 or OsmC13 plus OsmC3 (Table 2
). The probe carrying nhaAp was amplified from the DNA of bacteriophage
6H3 from Koharas collection (Kohara et al., 1987
) and the primers NhaA1 and NhaA2 (Table 2
). PCR amplifications were performed in the presence of 20 µCi [
-32P]ATP. A 10 ng sample of the labelled DNA fragment was incubated for 10 min at room temperature with crude extract (0·62 µg protein) in buffer B and 1 µg poly(dI-dC)/poly(dI-dC) (Pharmacia) competitor DNA. The binding mix was loaded onto 5% polyacrylamide gels in TBE at a voltage of 6 V cm-1, and run at a voltage of 12 V cm-1.
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RESULTS |
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Analysis of the new regulators of the osmCp1 promoter
The next step of our genetic screening was to determine which of the two osmC promoters was sensitive to the presence of the plasmids. Strains CLG685 and CLG686 carry osmClac transcriptional fusions expressed under the control of the osmCp2 promoter or the osmCp1 promoter, respectively (Table 1). After transformation of these two strains with each candidate plasmid and plating on MacConkey-lactose agar, we observed that seven plasmids were able to stimulate expression of osmCp1 but had no apparent effect on osmCp2. Previous work had established that (in standard laboratory conditions) osmCp2 was 10-fold more active than osmCp1, suggesting that osmCp1 plays a minor role in osmC expression (Gordia & Gutierrez, 1996
; Bouvier et al., 1998
). Therefore, we decided to focus our attention on these putative activators of osmCp1. By making use of oligonucleotides hybridizing with either side of the pJPB209 multi-site linker (209E and 209H, Table 2
), we then sequenced the two ends of the inserts on each of the seven candidate plasmids and compared the sequences with the E. coli genome, using the Colibri genebank (http://genolist.pasteur.fr/Colibri/index.html). Two of these clones carried the gene rcsB, encoding the response regulator of the two-component system RcsB/RcsC, and the analysis of these clones will be reported elsewhere. The five other clones carried different but overlapping DNA fragments from the same chromosomal region. All had in common the gene nhaR, already identified as a transcriptional activator of the Na+/H+ antiporter NhaA (Rahav-Manor et al., 1992
).
In addition, the sequence data showed that the single plasmid giving pink colonies (and thus able to repress expression of the osmClac fusion) carried the gene hns, which is already known to have a negative effect on the expression of the osmC promoters (Gutierrez & Devedjian, 1991 ; Bouvier et al., 1998
).
The gene nhaR is sufficient to activate osmCp1 when present in multicopy
The five nhaR+ clones carried inserts ranging in size from 3968 bp to 4490 bp. To demonstrate that nhaR was responsible for the stimulation of osmCp1, we made an internal deletion on the nhaR+ plasmid carrying the smaller insert (see Methods), yielding a derivative (pNHAR; Table 1) with nhaR as the sole intact ORF. Strains CLG685 (osmCp1- osmCp2+) and CLG686 (osmCp1+ osmCp2-) were transformed with pNHAR or with the empty vector pJPB209. Overnight cultures of the four resulting strains were diluted 1000-fold in LB170 medium and grown for 300 min (to an OD600 of approximately 2) before assay of ß-galactosidase. Derivatives of CL685 exhibited activities of 160 and 166 Miller units, in the presence of pJBP209 and pNHAR, respectively, demonstrating that overexpression of nhaR has no effect on transcription from osmCp2. In contrast, the presence of pNHAR resulted in a ninefold activation of osmCp1 (257 Miller units for CLG686/pNHAR versus 27 Miller units for CLG686/pJPB209).
Expression of osmCp1 and osmCp2 in nhaR mutants
We then checked the effect of the chromosomal copy of nhaR on expression of the osmC promoters. Strains CLG685 and CLG686 and their respective nhaR derivatives (CLG688 and CLG689) were grown in LB0 and LB400. Measurement of ß-galactosidase activity in samples of these cultures indicated that, in the absence of NhaR, osmCp2 expression was unchanged (Fig. 2a) whereas osmCp1 expression was reduced in both media (Fig. 2b
).
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Overexpression of NhaR produces an osmC promoter region binding activity in E. coli crude extracts
Crude extracts were prepared from strain CLG686 containing either the vector pAPT110 or the plasmid pAPTnhaR and used for electrophoretic mobility shift experiments with DNA fragments carrying either the nhaA or the osmC promoter region (see Methods). In agreement with previous observations (Rahav-Manor et al., 1992) , incubation with a crude extract containing overexpressed NhaR retarded the migration of a DNA probe carrying the nhaA promoter region (Fig. 5
, compare lanes 2 and 3). The migration of a 91 bp osmCp DNA probe that was sufficient to produce an effect of NhaR in vivo (Table 3
) was also retarded after incubation with a crude extract enriched in NhaR (Fig. 5
, compare lanes 59 with lanes 10 and 11). No retardation was observed after incubation with a control crude extract that did not contain overexpressed NhaR, demonstrating that the retardation was not a non-specific effect of some protein present in the crude extract. A longer DNA probe extending further upstream of the osmCp1 promoter exhibited the same behaviour as the 91 bp DNA probe (Fig. 5
, lanes 1216). In contrast, a smaller DNA probe that did not carry enough sequence to be responsive to NhaR in vivo (Table 3
) showed no retardation of its migration after incubation with the crude extract containing overexpressed NhaR (Fig. 5
, lanes 1719).
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DISCUSSION |
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Until now, the only gene that was known to be regulated by NhaR was nhaA, a gene encoding a Na+/H+ antiporter of E. coli (Rahav-Manor et al., 1992 ; Dover et al., 1996
). This gene is necessary for adaptation to high salinity and alkaline pH in the presence of Na+, but is not essential (Goldberg et al., 1987
; Karpel et al., 1988
). Like nhaA, osmC is not essential in E. coli (Gutierrez & Devedjian, 1991
). To date, the biochemical function of the OsmC protein remains unknown. However, we have shown that inactivation of osmC results in higher sensitivity to organic peroxides and faster decay in viable cell counts of bacterial cultures during long-term stationary phase (Conter et al., 2001
). Therefore, it is clear that the physiological role of OsmC is to participate in the response of the cells to adverse conditions. We have shown previously that transcription of osmC is stimulated by elevated osmolarity, through the stimulation of its two promoters. It has been shown that osmotic shocks result in an accumulation of the
s sigma factor and that this can account for the osmotic induction of some
s-dependent genes (Hengge-Aronis et al., 1993
; Hengge-Aronis, 1996a
). Since the transcription of osmCp2 is
s-dependent (Gordia & Gutierrez, 1996
), the osmotic stimulation of this promoter may be mediated by
s. In contrast, transcription of osmCp1 is probably assured by the
70 sigma factor, and the mechanism of its osmotic induction was not clear. The data presented here indicate that NhaR is responsible for the stimulation of osmCp1 by NaCl, LiCl, and, to a lesser extent, the non-ionic solute sucrose (Fig. 4
). Therefore, NhaR is responsible for the osmotic induction of osmCp1.
It has been demonstrated recently that nhaA is also transcribed from two promoters (Dover & Padan, 2001 ). A proximal, Na+- and NhaR-dependent promoter (P1), is the main promoter during exponential growth. A distal, Na+- and NhaR-independent, but
s-dependent, promoter (P2) becomes the major promoter upon entry into stationary phase. Therefore, it appears that nhaA and osmC exhibit similar complex regulatory patterns. Overall, having two different promoters inducible by different mechanisms provides a broader spectrum of conditions for the induction of nhaA and osmC. In early exponential phase, when the amount of
s in the cells is very low, the osmotic induction of nhaA and osmC is due to the NhaR-dependent activation of P1 and osmCp1. At a later stage of growth, even in the absence of osmotic stress, the two genes are expressed under the control of
s, preparing the cells for eventually encountering stress during stationary phase. It must be noted that similar dual-promoter organization has been described for other stress-responsive genes. For instance, proP, a gene encoding a transporter for the osmoprotectant compound glycine betaine, is induced by both osmotic stress, through stimulation of a
s-independent promoter, and upon entry into stationary phase, through stimulation of a second,
s-dependent, promoter (Mellies et al., 1995
).
The molecular mechanism of activation of nhaAp by NhaR has been studied extensively (Karpel et al., 1991 ; Rahav-Manor et al., 1992
; Carmel et al., 1997
; Dover & Padan, 2001
). It involves a Na+-dependent interaction between the activator and several binding sites near the promoter (Rahav-Manor et al., 1992
; Carmel et al., 1997
). The activation of osmCp1 by NhaR might also be direct, involving binding of NhaR near osmCp1. Alternatively, it might be indirect, involving an unknown mechanism. Our band-shift experiments show that overexpression of NhaR results in the appearance of a promoter-binding activity in the crude extracts (Fig. 5
). This binding is sequence specific, since it is only observed with a DNA probe carrying enough sequence upstream from the promoter to confer an NhaR-dependent stimulation of transcription from osmCp1. However, we do not know if NhaR is directly responsible for this binding. Comparison of the sequences of the NhaR-binding sites near nhaAp shows that the recognition sequence is quite variable (Carmel et al., 1997
), and it is not possible to derive a clear consensus with which to scan the sequence near osmCp1. We note also that the pattern observed in band-shift experiments with the osmCp1 DNA fragments is quite different from that obtained with the nhaAp DNA (Fig. 5
). The retarded bands migrate at almost the same rate as the naked osmCp1 DNA fragments, suggesting that the overall structure of the osmCp1NhaR complex is different from that of the nhaApNhaR complex. In addition, we constructed and purified a variant of NhaR, carrying a six-histidine tag at its amino-terminal end. Although we could reproduce with this modified protein the binding to nhaAp reported previously (Carmel et al., 1997
), this variant of NhaR was unable to bind the 91 bp osmCp1 DNA fragment (data not shown). In view of the weak binding of native NhaR, it is possible that minor structural alterations induced by the His6 tag render binding by the tagged protein too weak to be observed in band-shift experiments. Additional experiments will be needed to resolve this question.
After leucine-responsive protein, H-NS and s, NhaR and RcsB are the fourth and fifth regulators of osmC transcription to have been identified The participation of so many factors in the regulation of osmC illustrates the notion of cooperation of global regulators in the fine-tuning of stress-inducible genes (Bouvier et al., 1998
; Hengge-Aronis, 1999
). Work on the relationships between these multiple factors is in progress in our laboratory.
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ACKNOWLEDGEMENTS |
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Received 19 April 2001;
revised 13 June 2001;
accepted 18 June 2001.