Institute for Genetics, University of Cologne, Zülpicherstr. 47, 50674 Cologne, Germany
Correspondence
Karin Schnetz
schnetz{at}uni-koeln.de
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ABSTRACT |
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INTRODUCTION |
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The E. coli bgl operon is one example of a system whose repression by H-NS is exceptionally specific. The bgl operon encodes the gene products for the fermentation of aryl -D-glucosides, including a
-glucoside-specific permease EIIBgl (or BglF) and a phospho-
-D-glucosidase BglB (Fig. 1
b). Also encoded within the operon is the positive regulator and anti-terminator protein BglG, whose activity is regulated by phosphorylation and which, under inducing conditions, allows transcription elongation beyond two terminators (t1 and t2) within the operon (Amster-Choder & Wright, 1993
; Görke, 2003
).
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In this work, we screened for suppressors of the more effective downstream repression of bgl by H-NS in a lon mutant. This transposon-mutagenesis screen yielded mutations in loci encoding products known to affect bgl operon expression, including leuO, yjjQbglJ, rpoS and crl. These mutations increase the bgl promoter activity, as shown here for BglJ and LeuO and as known for RpoS and Crl, and thus compensate indirectly for the more efficient downstream repression in the lon mutant. In addition, we isolated a suppressor mutant that carries a transposon insertion upstream of the dnaK open reading frame, which expresses decreased levels of DnaK. This mutant, which is novel in the context of bgl, was found to specifically affect downstream repression by H-NS.
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METHODS |
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-Galactosidase assays.
For enzyme assays, cells were grown in M9 medium containing 1 % (w/v) glycerol, 0·66 % (w/v) Casamino acids (Difco) and 1 µg vitamin B1 ml1, or in NB medium (Difco), as indicated. Cultures were inoculated to an OD600 of 0·10·15 from fresh overnight cultures grown in the same medium and grown at 37 or 30 °C as indicated. IPTG was added to this fresh culture, where indicated. Cells were harvested at an OD600 of 0·5. The -galactosidase assays were performed as described previously (Miller, 1992
; Dole et al., 2002
). The enzyme activities were determined at least three times from at least two independent transformants or integration derivatives. SD values were <10 %.
DnaK Western analysis.
Cultures were grown in LB at 30 °C to an OD600 of 0·5. IPTG (1 mM) was added where indicated. Cultures were stopped on ice. Then, cells were harvested by centrifugation and resuspended in SDS-PAGE sample buffer (Laemmli, 1970) at an OD600 of 0·05 per 10 µl sample buffer. A 5 µl (0·025 OD600) aliquot was separated by SDS-PAGE (12 % gel) using an SE600 16 cm gel-electrophoresis unit (GE Healthcare). The gel was blotted onto a 0·45 µm pore size PVDF transfer membrane by using a TE70 semi-dry blotting apparatus (GE Healthcare). The blot was handled using a standard Western blotting protocol (Coligan et al., 2005
). Monoclonal mouse antisera directed against DnaK (Stressgen Bioreagents) were used as the primary antibody at 1 µg ml1; Alexa Fluor 680 rabbit anti-mouse IgG(H+L) (Molecular Probes) was used as the secondary antibody at a concentration of 0·5 µg ml1. Visualization and quantification were done by using the Odyssey Imaging System (Li-Cor Biosciences) according to the instructions of the manufacturer.
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RESULTS |
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Mutagenesis of the lon mutant strain S764 carrying the complete bgl regulatory region fused to the lac operon yielded in total 25 transposon mutants with a clear double-phenotypic change to Bgl+ and Lac+. Of these mutants, which were characterized by sequencing of the miniTn10 insertion site, 16 mapped in leuO, four mapped in yjjQbglJ, three in crl, one in rpoS and one in dnaK (Fig. 2a). Interestingly, the second mutagenesis screen for suppressors using the lon mutant strain S2103, which carries the lacUV5bglGorflacZ reporter specific for downstream repression (Fig. 1c
), yielded three insertion mutations with a clear phenotype change to Bgl+ and Lac+, all of which mapped at the dnaKJ locus (Fig. 2b
). This result suggests that DnaK (and DnaJ) may affect downstream repression by H-NS, whilst the mutations mapping in leuO, bglJ, rpoS and crl may affect the promoter. In agreement with this is the fact that RpoS (together with Crl) is known to repress the bgl promoter (Schnetz, 2002
). Thus, the miniTn10 insertion mutations mapping in crl and rpoS are likely to compensate indirectly for the more efficient, H-NS-mediated downstream repression in the lon mutant by increasing the promoter activity. The rpoS and crl mutants were not further analysed. The analysis of the other mutants is presented below. Another interesting result is that no hns mutant was isolated, although the bgl operon and the bgllacZ reporter constructs direct a Bgl- and Lac-positive phenotype in a lon hns double mutant (Dole et al., 2004a
). A similar change in the spectrum of mutations that affect bgl was detected before in an rpoS background (Moorthy & Mahadevan, 2002
).
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The insertion mutations isolated at the bglJ and leuO loci are similar to mutations isolated before. It has also been shown before that constitutive expression of BglJ and LeuO, respectively, relieves silencing of bgl (Giel et al., 1996; Ueguchi et al., 1998
). Here, we analysed whether the leuO and yjjQbglJ mutations relieve silencing of the bgl promoter, downstream silencing by H-NS or both levels of silencing. To this end, we used chromosomally encoded bgllacZ reporter constructs specific for promoter and downstream repression, respectively (Figs 3
and 4
). The expression directed by these bgllacZ reporter constructs was tested in the wild-type, as well as in yjjQbglJ and leuO mutants, grown to the exponential phase (OD600=0·5) in minimal M9 glycerol medium.
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The bgllacZ downstream reporter construct (which carries the bglGorf downstream regulatory region inserted between the lacUV5 promoter and lacZ as described above) was also analysed in the wild-type and the bglJ-Y6 and leuO-Y1 mutants. The -galactosidase level directed by the downstream reporter (Fig. 4a
) did not vary significantly between the wild-type (62 units), the yjjQbglJ-Y6 mutant (69 units) and the leuO-Y1 mutant (75 units), demonstrating that LeuO and BglJ have no impact on downstream repression by H-NS. Thus, suppression of the more effective H-NS-mediated downstream repression in the lon mutant by expression of bglJ and leuO is indirect and compensated for by an increased promoter activity.
In the dnaKJ : : miniTn10 mutant, downstream repression by H-NS is specifically suppressed
In both screening strategies (Fig. 1), insertion mutations mapping at the dnaKJ locus were isolated (Fig. 2
). The single dnaKJ : : miniTn10 mutant isolated in the first screen is identical to two of the three mutants isolated in the second screen, which was performed in the strain carrying the bgllacZ downstream reporter. These mutants carry a miniTn10 insertion that disrupts the dnaK promoter and that maps 63 bp upstream of the dnaK ATG start codon. In the other insertion mutation, miniTn10 also maps upstream of the dnaK open reading frame (26 bp upstream of the ATG) but, in addition, the mutant carries a small deletion of 29 bp. The phenotype of this latter mutant is weaker and this mutant was not further characterized.
The possible role of DnaKJ in bgl promoter and bgl downstream repression by H-NS and the modulation of the latter by Lon were addressed by using the same approach as described above for the leuO and bglJ mutants, i.e. by using bgl promoter and bgl downstream lacZ reporter constructs. The dnaKJ : : miniTn10 mutants were grown in NB medium, because the mutants were also analysed in combination with lon, and lon mutants grow poorly in minimal medium.
First, we analysed whether the dnaKJ : : miniTn10 mutant (allele dnaKJ-M2) affects the bgl promoter activity by using the bgl promoterlacZ reporter described above (Fig. 3c). In the wild-type, this reporter directs the expression of 140 units of
-galactosidase activity when cells are grown in NB. In the dnaKJ : : miniTn10 mutant, 165 units were detected, i.e. the promoter activity was not changed significantly. Likewise, the promoter activity remained unaffected in a lon mutant (Dole et al., 2004a
) and a lon dnaKJ : : miniTn10 double mutant (Fig. 3c
). The bgl promoter allele (
Pbgl) that lacks the upstream silencer sequence necessary for repression by H-NS was likewise not affected by the mutation of dnaKJ (Fig. 3d
). Thus DnaK (and DnaJ) have no impact on the activity of the bgl promoter or its repression by H-NS.
In contrast, the expression level of -galactosidase directed by the bgllacZ downstream reporter, which decreases from 95 units in the wild-type to 40 units in the lon mutant (Fig. 4b
) (Dole et al., 2004a
), was affected by DnaKJ. In the dnaKJ : : miniTn10 mutant, the expression level increased to 220 units, i.e. approximately twofold compared with the wild-type, and in the lon dnaKJ : : miniTn10 double mutant, the expression level increased to 155 units, i.e. approximately fourfold compared with the lon single mutant (40 units). These data show that DnaK (and possibly DnaJ) specifically affect the downstream repression of bgl by H-NS and its modulation by Lon.
DnaK is required for efficient downstream silencing by H-NS
In the dnaKJ : : miniTn10 mutant, the miniTn10 insertion maps upstream of the dnaK open reading frame. Therefore, these insertions may direct constitutive expression of the dnaKJ operon by the promoter of the chloramphenicol-resistance gene located within the miniTn10 transposon. To analyse whether an increase or a decrease in the cellular DnaK level affects downstream repression by H-NS, an additional dnaKJ mutant was used, in which the dnaKJ promoter is replaced by a lacI PA1/lacO cassette (Tomoyasu et al., 1998). Thus, dnaKJ expression requires IPTG and DnaKJ levels are very low when cells are grown without IPTG. Downstream repression of bgl by H-NS was tested in the PA1/lacO dnaKJ strain using the downstream reporter (lacUV5bglGorflacZ) as described above. As these cultures had to be grown in LB at 30 °C when grown without IPTG, the
-galactosidase assay for the wild-type and the dnaKJ : : miniTn10 mutant was repeated at these conditions (Fig. 4c
). In the wild-type, 156 units of
-galactosidase activity were detected and the activity increased to 335 units in the dnaKJ : : miniTn10 mutant. In the PA1/lacO dnaKJ strain, 76 units of
-galactosidase activity were detected when IPTG for induction of the pA1/lacO1 promoter was present, i.e. high DnaKJ levels support downstream repression. In contrast, the expression level increased to 415 units when IPTG for induction of dnaKJ was omitted. This effect was specific for downstream repression. The activity of the PbgllacZ promoter construct did not vary in the PA1/lacO dnaKJ strain when grown with or without IPTG (Fig. 3e
). These data indicate that expression of dnaKJ is required for downstream repression by H-NS. To further strengthen this argument, the DnaK levels were analysed in a Western blot by using DnaK-specific antibodies (Fig. 5
). The quantitative Western analysis demonstrated that DnaK levels are reduced by approximately twofold in the dnaKJ : : miniTn10 mutant compared with the wild-type. In the PA1/lacO dnaKJ strain, DnaK levels were very low (fivefold lower than the wild-type) when cells were grown without IPTG, whilst the DnaK protein level was much higher upon induction with IPTG than in the wild-type strain (17-fold increase) (Fig. 5
). Thus, the cellular DnaK protein levels correlate well with the differences in downstream repression of bgl by H-NS (Fig. 4c
), i.e. downstream repression is more effective when DnaKJ is present.
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DISCUSSION |
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Mutations characterized so far that relieve silencing (or repression) of the bgl operon by H-NS affect only one level of repression, the repression of the bgl promoter. Here, we have shown that the two transcription factors LeuO and BglJ also counteract the H-NS-mediated repression of the promoter. As, under laboratory growth conditions, the bgl operon is always repressed, the expression level of leuO and bglJ may be too low for activation of bgl. The regulation of bglJ has not been studied to date. Preliminary data from our lab indicate that it is repressed by H-NS. The expression of leuO is likewise repressed by H-NS, and not detectable during exponential growth (Klauck et al., 1997; Chen et al., 2004
). However, leuO expression is induced in a ppGpp-dependent manner by starvation for branched-chain amino acids, and LeuO is necessary for resuming growth after such a starvation (Fang et al., 2000
; Majumder et al., 2001
). It is not known whether such a transient induction of leuO is sufficient to relieve silencing of bgl. The miniTn10 insertion mutants isolated in this work are similar to Tn10 mutants isolated before in the context of bgl (Giel et al., 1996
; Ueguchi et al., 1998
) and apparently direct constitutive expression of bglJ and leuO, respectively. Constitutive expression of leuO and bglJ results in activation of the bgl promoter. This explains the dominance of the bglJ and leuO mutants over the wild-type copy of the bglJ and leuO gene, respectively, also present in the strains, as they are repressed by H-NS. Similar leuO : : Tn10 transposon mutants to those isolated here were isolated in different contexts and shown to enhance expression of cadA, encoding an acid-inducible lysine decarboxylase, as well as to reduce RpoS levels (Shi & Bennett, 1994
; Klauck et al., 1997
). The regulation of RpoS levels by LeuO is indirect and mediated through the small regulatory RNA DsrA, which, at low temperature, enhances translation of the rpoS mRNA and represses translation of the hns mRNA (Klauck et al., 1997
; Lease & Belfort, 2000
; Repoila & Gottesman, 2003
). Thus, LeuO belongs to a regulatory network that involves RpoS, H-NS, Hfq and DsrA (Klauck et al., 1997
; Repoila & Gottesman, 2003
), and the bgl operon is clearly a system that is controlled by this network.
The second, downstream level of bgl operon repression by H-NS is not affected by LeuO, BglJ or RpoS/Crl. However, this level of repression is modulated by Lon (Dole et al., 2004a). In Lon-deficient mutants, the repression is more effective. This effect is independent of the H-NS homologue StpA (Dole et al., 2004a
), a Lon target protein (Johansson & Uhlin, 1999
; Johansson et al., 2001
). In addition, as shown here, downstream repression is modulated by the heat shock-induced DnaKJ chaperone system. The dnaKJ mutants carry a miniTn10 insertion upstream of the dnaK open reading frame. Western analysis revealed that this mutant expresses lower levels of DnaK than the wild-type. Likewise, downstream repression is inefficient in a PA1/lacO dnaKJ strain when expression of dnaKJ is not induced with IPTG. These data imply that DnaKJ is required for efficient downstream repression, which indicates a modulation of H-NS activity by chaperones. Interestingly, the mutation of an HSP66 protein and DnaK homologue encoded by hscA was also found to have some effects on silencing by H-NS (Kawula & Lelivelt, 1994
). Presently, it is not known whether Lon and the DnaKJ chaperone system affect downstream repression of bgl by H-NS directly or indirectly. One speculative possibility is that Lon and DnaKJ modulate the H-NS repressing complex or change the level of hns expression. However, Lon and DnaKJ specifically modulate downstream repression by H-NS, but not bgl promoter repression. In addition, we found no role for DnaKJ in silencing of proU by H-NS (data not shown). Therefore, Lon and DnaKJ may affect an additional process that is specific for downstream repression, e.g. modulation of the elongating RNA polymerase complex and its translocation along the DNA.
Results presented here are in agreement with and extend the finding that repression of the bgl operon is linked tightly to the regulatory network controlling the stress response in E. coli. Strictly controlled repression of bgl supports the idea that the utilization of aryl -D-glucosides is deleterious under certain conditions (Reynolds et al., 1981
), whilst conservation of silencing of the bgl operon that is present in a majority of E. coli strains (G. Neelakanta and K. Schnetz, unpublished data) suggests an important role for bgl under specific conditions in nature.
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ACKNOWLEDGEMENTS |
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Received 31 March 2005;
revised 20 July 2005;
accepted 21 July 2005.
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