Independent regulation of H-NS-mediated silencing of the bgl operon at two levels: upstream by BglJ and LeuO and downstream by DnaKJ

S. Madhusudan, Andreas Paukner, Yvonne Klingen and Karin Schnetz

Institute for Genetics, University of Cologne, Zülpicherstr. 47, 50674 Cologne, Germany

Correspondence
Karin Schnetz
schnetz{at}uni-koeln.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Silencing of the Escherichia coli bgl operon by the histone-like nucleoid-structuring protein H-NS occurs at two levels. Binding of H-NS upstream of the promoter represses transcription initiation, whilst binding within the coding region is also proposed to repress transcription elongation. The latter, downstream level of repression is counteracted by the protease Lon and, thus, silencing of the bgl operon is more effective in lon mutants. Transposon-mutagenesis screens for suppression of this lon phenotype on bgl were performed and insertion mutations disrupting rpoS and crl were obtained, as well as mutations mapping upstream of the open reading frames of bglJ, leuO and dnaK. In rpoS and crl mutants, bgl promoter activity is known to be higher. Likewise, as shown here, bgl promoter activity is increased in the bglJ and leuO mutants, which express BglJ and LeuO constitutively. However, BglJ and LeuO have no impact on downstream repression. A dnaKJ mutant was isolated for the first time in the context of the bgl operon. The mutant expresses lower levels of DnaK than the wild-type. Interestingly, in this dnaKJ : : miniTn10 mutant, downstream repression of bgl by H-NS is less effective, whilst upstream repression by H-NS remains unaffected. Together, the data show that the two levels of bgl silencing by H-NS are regulated independently.


Abbreviations: H-NS, histone-like nucleoid-structuring protein


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The abundant histone-like nucleoid-structuring protein H-NS is a key regulator in the adaptation of Escherichia coli to its environment (Ussery et al., 1994; Schröder & Wagner, 2002) and it affects the expression of ~5 % of the genes in E. coli K-12 (Hommais et al., 2001). The current view of the mechanism of H-NS-mediated repression is that H-NS binds as a dimer to AT-rich and planar-bent DNA sequences and then forms an extended oligomeric complex on the DNA, which represses transcription initiation when located next to a promoter. Efficient repression by H-NS often involves binding of H-NS to two close binding (or nucleation) sites and probably the formation of a DNA loop, by ‘zipping’ the two sites together (Dame et al., 2002; Dorman, 2004). In several H-NS-repressed systems analysed so far, the H-NS-mediated repression is relieved by specific mechanisms that disturb the formation of the repressing complex, for example by binding of a specific transcription factor or by a temperature-dependent change in the DNA bend (Dorman, 2004; Prosseda et al., 2004). To date, little is known about whether and how the activity of the H-NS protein itself and its capacity to form repressing complexes are modulated. One possibility is that the formation of heterodimers and hetero-oligomers between H-NS and its homologue StpA affects its activity (Williams et al., 1996; Johansson & Uhlin, 1999; Johansson et al., 2001). Interaction of H-NS with Hha and Hfq has also been reported (Kajitani & Ishihama, 1991; Nieto et al., 2000). Furthermore, it was found that hscA, encoding an HSP66 protein and DnaK homologue, modulates the H-NS-mediated repression of pilA and the bgl operon in an hns mutant with reduced levels of H-NS protein (Kawula & Lelivelt, 1994). H-NS may also be modulated by a post-translational modification with short-chain poly(R)-3-hydroxybutyrate (Reusch et al., 2002).

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 {beta}-D-glucosides, including a {beta}-glucoside-specific permease EIIBgl (or BglF) and a phospho-{beta}-D-glucosidase BglB (Fig. 1b). 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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Fig. 1. Mutagenesis screen for suppressors of efficient bgl operon silencing in lon mutants. (a) Plasmid pKESK18 was used to perform a transposon-mutagenesis screen to isolate suppressors of the more effective repression of bgl in lon mutants. Plasmid pKESK18 is a replication temperature-sensitive (repts) pSC101 derivative, which carries a mini-Tn10 transposon (miniTn10) with a chloramphenicol-resistance gene (cam), the Tn10 transposase gene under control of the lambda PR promoter and the lambda cI857 gene (see Methods). Transposition was induced by a temperature shift from 28 to 42 °C and single miniTn10-Camr transposon mutants were selected at 42 °C on chloramphenicol plates and screened for Bgl- and Lac-positive mutants. Strains S764 (b) and S2103 (c) are lon mutants that carry an activated bgl operon and a fusion of the bgl regulatory region to the lac operon. The bgl operon is activated by a point mutation that improves the CRP-binding site (allele bgl-CRP+). (b) In strain S764, the bgllacZ fusion carries the same activated bgl promoter allele, bgl-CRP+, as is present at the bgl operon. Expression of this bgllacZ fusion is independent of BglG-mediated anti-termination, due to a deletion of the terminator bgl-t1 located in the leader of the operon. In addition, the bglG allele bglGorf is present, carrying a mutated translation start codon (Dole et al., 2004a). (c) In strain S2103, the expression of the bglGorflacZ fusion, which carries the downstream-regulatory site of bgl, is directed by the constitutive lacUV5 promoter. Both of these lon mutants are Bgl- and Lac-negative, due to more efficient bgl operon silencing by H-NS, whilst the phenotype is Bgl- and Lac-positive in the corresponding wild-type strains [(b), strain S594; (c), strain S2101 (Dole et al., 2004a) and this study].

 
Silencing of the bgl operon by H-NS occurs under all laboratory growth conditions tested (Prasad & Schaefler, 1974; Reynolds et al., 1981; Lopilato & Wright, 1990). Silencing involves two sites (Dole et al., 2004b): H-NS represses transcription initiation at the cAMP receptor protein (CRP)-dependent promoter by binding to an AT-rich and presumably bent upstream silencer sequence (Schnetz, 1995; Singh et al., 1995; Schnetz & Wang, 1996; Mukerji & Mahadevan, 1997). In addition, H-NS binds to a site located 600–700 bp downstream of the transcription-initiation site within the coding region of the first gene, bglG. Binding to this downstream silencer is likely to hinder transcription elongation (Schnetz, 1995; Dole et al., 2004b). Silencing of bgl can be relieved by mutations that disrupt the upstream H-NS-binding site or that improve the CRP-binding site (Reynolds et al., 1986; Schnetz & Rak, 1988; Singh et al., 1995). Recently, we found that the silencing by H-NS mediated via the downstream site (‘downstream repression’) is more efficient in lon mutants (Dole et al., 2004a), suggesting that it is modulated by the ATP-dependent Lon protease (Gottesman, 1996). Therefore, a bgl allele (bgl-CRP+) that escapes silencing in the wild-type due to a mutation that improves the CRP binding of the bgl promoter remains repressed in a lon mutant (Dole et al., 2004a).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, plasmids and media.
The genotypes of the E. coli strains used in this study are listed in Table 1. All experiments were performed using isogenic strains derived from E. coli K-12 strain S541 carrying a deletion of the bgl operon and the lacZ gene (Dole et al., 2002). Mutations were transduced by using phage T4GT7 (Wilson et al., 1979). Integration of bgllacZ reporter-gene fusions into the chromosomal phage lambda attachment site attB was performed as described previously (Diederich et al., 1992; Dole et al., 2002). Plasmids were constructed according to standard techniques (Sambrook & Russell, 2001). Plasmid pKESK18 (see Fig. 1a) is a temperature-sensitive derivative of pSC101 (Hashimotoh-Gotoh et al., 1981) with a kanamycin-resistance gene. The plasmid carries the phage lambda cI-857 allele encoding the temperature-sensitive lambda repressor, the Tn10 transposase gene under control of the phage lambda PR promoter and a miniTn10 transposon (miniTn10-Camr) with a chloramphenicol-resistance gene derived from plasmid pNK2884 (Kleckner et al., 1991). Plasmid pKEAP4 is a pKK223-3 derivative (Brosius & Holy, 1984) that carries a lacIq tacOP cassette followed by a truncated phage T7 gene 10 Shine–Dalgarno sequence [GenBank accession no. NC_001604 (phage T7), positions 22949–22966] to which the bglJ open reading frame (GenBank no. AE000507, positions 7109–7786) is fused for high expression of BglJ. Plasmid pKEAP10 also carries a lacIq tacOP cassette followed by the leuO gene with its native translation start (GenBank no. AE000118: positions 625–1786) cloned into a backbone carrying the pBR322 origin of replication and the bla gene (Bolivar et al., 1977). Bacteria were grown in LB (Difco), NB (Difco) or minimal M9 (Miller, 1992) media, as indicated. Antibiotics were added to final concentrations of 12 µg tetracycline ml–1, 25 µg kanamycin ml–1, 50 µg ampicillin ml–1, 15 µg chloramphenicol ml–1 and 50 µg spectinomycin ml–1 where necessary.


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Table 1. E. coli K-12 strains

 
Transposon mutagenesis.
Transposon-mutagenesis screens were performed by using pKESK18 (Fig. 1a) carrying a miniTn10-Camr transposon. In this plasmid, replication is temperature-sensitive and, also, expression of the transposase is repressed at 28 °C and induced at 42 °C. Thus, at 28 °C, the plasmid replicates, whilst the transposase is not expressed. Upon a temperature shift, expression of the transposase gene and thus transposition are induced, whilst replication of the plasmid stops, allowing the selection of transposon mutants on chloramphenicol plates at 42 °C. With this system, transposition takes place in approximately 1–5 % of the cells and all of the mutants that we characterized carried single miniTn10-Camr transposon insertions. Transformants of strains S594 and S2103 with plasmid pKESK18 were grown at 28 °C in LB medium containing kanamycin and chloramphenicol. To select for transposon mutants, dilutions were plated on MacConkey lactose (Difco)/chloramphenicol plates and incubated at 42 °C. Mutants with a change in the lactose phenotype were restreaked and their Bgl phenotype was tested on bromothymol blue/salicin indicator plates (Dole et al., 2002). In addition, the phenotypes were re-evaluated on M9 minimal plates containing 0·4 % glycerol, 0·1 % tryptone, 0·1 % yeast extract (Difco), 0·2 % Casamino acids and 20 µg 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside or 5-bromo-4-chloro-3-indolyl-{beta}-D-glucopyranoside ml–1. Of mutants with a double phenotype change, the insertion position of the miniTn10 transposon was determined by sequencing of chromosomal DNA using the miniTn10-specific primer S156 of the sequence 5'-GATGATAAAAGGCACCTTTGGTCA-3' or by a semi-random, two-step PCR protocol as described by Chun et al. (1997). Briefly, in a first, semi-specific PCR, a ‘random primer’ (S360, 5'-GGCCACGCGTCGACTAGTACNNNNNNNNNNGATC-3') and a miniTn10-specific primer (S357, 5'-GGCAGGGTCGTTAAATAGCCGCTTATGT-3', or S358, 5'-CGGTATCAACAGGGACACCAGGATTTATTTATTCT-3') were used. The amplification products of this first PCR were reamplified in a second PCR using a primer (S361, 5'-GCTCTAGAGGCCACGCGTCGACTAGTAC-3') that matches to the ‘random primer’ S360 and a nested miniTn10-specific primer (S359, 5'-GCTCTAGAGATCATATGACAAGATGTGTATCCACCTTAACT-3'). The PCR products were gel-purified and sequenced with primer S359.

{beta}-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 ml–1, or in NB medium (Difco), as indicated. Cultures were inoculated to an OD600 of 0·1–0·15 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 {beta}-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 ml–1; Alexa Fluor 680 rabbit anti-mouse IgG(H+L) (Molecular Probes) was used as the secondary antibody at a concentration of 0·5 µg ml–1. Visualization and quantification were done by using the Odyssey Imaging System (Li-Cor Biosciences) according to the instructions of the manufacturer.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mutagenesis screen for suppression of more effective downstream repression of bgl by H-NS in lon mutants
In order to identify factors that suppress the more effective H-NS-mediated downstream repression of bgl in a lon mutant, we performed a transposon-mutagenesis screen using a modified miniTn10 system (Kleckner et al., 1991) present on the low-copy and replication temperature-sensitive plasmid pKESK18 (Fig. 1a; see Methods for details). To avoid mutations that map in cis to the operon, a double-phenotype screening strategy was used (Fig. 1b and c) (Dole et al., 2004a). Both of the lon mutant strains S764 and S2103 (containing allele lon-107 : : miniTn10-Tcr; Table 1), which were screened for suppressor mutations, carry a bgl operon allele (bgl-CRP+) whose promoter is activated by a point mutation improving the CRP-binding site. This bgl allele confers a Bgl-positive phenotype in the wild-type, but a Bgl-negative phenotype in the lon mutants (Fig. 1b and c) (Dole et al., 2004a). As a second reporter, the lon mutants carry bgllacZ fusions. In one strain (Fig. 1b, S764) the bgl promoter, including upstream- and downstream-regulatory elements, was fused to the chromosomal lac operon genes (Dole et al., 2004a). In this bgllac fusion, the same bgl promoter allele, Pbgl-CRP+, is present as at the bgl locus. In addition, the terminator t1 within the leader is deleted ({Delta}t1) and translation of bglG is excluded by mutation of the translation start codon to avoid the necessity for anti-termination by BglG and crosstalk with the bgl operon. The second lon mutant strain (S2103) carries a bgllacZ fusion, which is specific for downstream repression by H-NS (Dole et al., 2004b). In this fusion, the bgl downstream-regulatory fragment, encompassing the bglG allele bglGorf (which cannot be translated), is inserted between the constitutive lacUV5 promoter and the lacZ gene (Fig. 1c). Both bgllac fusions direct a Lac-positive phenotype in the wild-type strains (S594 and S2101), but a Lac-negative phenotype in the lon mutant strains S764 and S2103 (Fig. 1b and 1c).

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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Fig. 2. Mutants that suppress efficient silencing of bgl in lon mutants. The insertion sites of miniTn10-Camr mutations causing suppression of more efficient silencing of bgl in the lon mutant are indicated by arrowheads. (a) Suppressor mutants that increase expression of bgl and the bgllacZ fusion in strain S764 (Fig. 1b) map in leuO (16 mutants), yjjQ–bglJ (four mutants), crl (three mutants), rpoS (one mutant) and dnaK (one mutant). In the leuO, bglJ and dnaK mutants, the miniTn10-Camr insertion maps 5' to the coding region and is thus likely to direct constitutive expression of these genes. In contrast, the miniTn10-Camr insertions mapping in crl and at the 3' end of rpoS disrupt the coding region of these genes. (b) Three suppressor mutants were isolated that increase expression of the bgl and the lacUV5bglGorflacZ fusion (strain S2103, Fig. 1c). All three mutants map 5' to the dnaK coding region (alleles M1, M2 and M3). One of them (allele dnaK-M3) is associated with a 29 bp deletion (indicated by an open arrowhead). (a, b) The sequence position of the miniTn10-Camr insertion sites of all mutants is given in Table 1: leuO mutants include strain S1729 and its identical isolates S1730, S1735, S1741, S1747–S1750, S1752, S1755–S1756 and S1760–S1763, as well as strain S1739; yjjQbglJ mutants include the identical strains S1733 and S1742, as well as strains S1734 and S1754; the crl mutants are strains S1740, S1743 and S1753; the rpoS mutant is strain S1751; and the dnaK mutants are strain S1738, as well as strains S2141, S2142 and S2143, which were isolated in the second screen (Fig. 1c). All leuO and yjjQbglJ miniTn10 insertion mutants retain a copy of the wild-type gene in addition to the mutant allele. This is not the case in the rpoS, crl or dnaK miniTn10 mutants.

 
Constitutive expression of BglJ and LeuO enhances the activity of the bgl promoter
All leuO : : miniTn10 insertion mutants map upstream of the leuO open reading frame. In 15 of 16 mutants, the insertion site maps 26 bp upstream of an ATG that is equivalent to the translation start characterized for the Salmonella typhimurium leuO gene (Chen et al., 2004). In one mutant (leuO-Y18, strain S1739), the insertion site maps 139 bp upstream of leuO. Four mutants map within the putative yjjQbglJ operon at the 3' end of the yjjQ open reading frame and upstream of bglJ. In allele yjjQbglJ-Y5 (which was isolated twice), the insertion maps 181 bp upstream of the putative translation start codon of bglJ, in allele Y6 it maps 37 bp upstream and in allele Y33 it maps 176 bp upstream. In all of these mutants, the orientation of the miniTn10 transposon may allow constitutive expression of leuO and bglJ, respectively, by the promoter of the chloramphenicol-resistance gene present within miniTn10-Camr. Furthermore, PCR analyses revealed that, in all of these mutants, a copy of the wild-type leuO gene and yjjQbglJ operon is retained (this is not the case in the rpoS, crl or dnaK insertion mutants) (see Discussion).

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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Fig. 3. The activity of the bgl promoter increases upon overexpression of BglJ or LeuO, but not in dnaKJ mutants. The wild-type bgl promoter (encompassing positions –450 to +25) (a, c) and a bgl promoter lacking the upstream silencer ({Delta}Pbgl, encompassing positions –76 to +25) (b, d) were fused to lacZ, and these reporter constructs were integrated into the chromosomal attB site. {beta}-Galactosidase activities shown were determined for cultures grown to OD600=0·5 in minimal M9 glycerol medium (a, b) or in NB medium (c, d). (a) In the wild-type (strain S1213), the bgl promoter directs 74 units of {beta}-galactosidase activity. In the yjjQbglJ mutants, the promoter directs 225 units (allele bglJ-Y5, strain S1787) and 675 units (allele bglJ-Y6, strain S1799) of {beta}-galactosidase activity. In the leuO-Y1 mutant (strain S1776), 215 units of {beta}-galactosidase activity were detected. Upon overexpression of BglJ (+BglJ) and LeuO (+LeuO), the promoter activity increased to 570 and 735 units, respectively. Overexpression of BglJ and LeuO was accomplished by using plasmids pKEAP4 (BglJ) and pKEAP10 (LeuO), which carry the bglJ and leuO genes, respectively, under control of the inducible tac promoter. For induction of bglJ and leuO expression, 1 mM IPTG was added to the exponentially growing cultures. (b) Activity of the bgl promoter lacking the upstream silencer varied only slightly, from 300 units in the wild-type (strain S2111) to 320 units in the bglJ-Y6 mutant (strain S1801) and to 425 units in the leuO-Y1 mutant (strain S1777). (c, d) The activity of the promoter constructs did not differ significantly in the dnaK : : miniTn10-Camr mutant. (c) When cells were grown in NB medium, the wild-type promoter directed 140 units in the wild-type strain (S1213), 165 units in the dnaK mutant (S2674), 135 units in the lon mutant (S1556) and 145 units in the lon dnaK double mutant (S2710). (d) The promoter lacking the upstream silencer directed 470 units in the wild-type background (S1211), 525 units in the dnaK mutant (S2676), 520 in the lon mutant (S1554) and 450 units in the lon dnaK double mutant (S2708). (e) The promoter activity was not affected in a PA1/lacO1 dnaKJ strain grown without or with 1 mM IPTG in LB medium at 30 °C. The promoter directed 80 units in the wild-type (S1213), 335 units in the dnaKJ : : miniTn10 mutant (S2674), 90 units in the PA1/lacO1 dnaKJ strain (S2904) when grown without IPTG (–) and 75 units when grown with 1 mM IPTG (+).

 


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Fig. 4. In dnaK mutants, downstream repression of bgl by H-NS is specifically reduced, whilst BglJ and LeuO have no impact. The role of LeuO and BglJ (a), as well as DnaK and Lon, on downstream repression of bgl was tested by using a bgllacZ downstream reporter construct that carries the bglG coding region, bglGorf, inserted between the lacUV5 promoter and the lacZ gene integrated into the chromosomal attB site. {beta}-Galactosidase activities shown were determined for cultures grown to OD600=0·5 in minimal M9 glycerol medium at 37 °C (a), NB medium at 37 °C (b) or LB medium at 30 °C (c). (a) In the wild-type (strain S1195), 62 units of {beta}-galactosidase activity were measured, which was not changed significantly in the bglJ-Y6 mutant (strain S1797) and the leuO-Y1 mutant (strain S1773). (b) Wild-type cells (strain S1195) grown in NB medium expressed 95 units of {beta}-galactosidase activity. In the dnaKJ mutant (strain S2712), the expression level increased by approximately twofold to 220 units. In the lon mutant (strain S1564), 40 units were detected, whilst in the dnaKJ lon double mutant (strain S2670), the expression level increased by approximately fourfold to 155 units. (c) When cells were grown at 30 °C in LB medium, 155 units were detected in the wild-type (S1195) and 335 units in the dnaKJ : : miniTn10 mutant (S2712). In the PA1/lacO dnaKJ strain (S2888), the expression level was increased to 415 units when IPTG was omitted, and decreased to 76 units when dnaKJ expression was induced with IPTG.

 
The expression level of {beta}-galactosidase directed by the bgl promoter–lacZ reporter (which carries the promoter fused at position +25 to lacZ) increased from 74 units in the wild-type to 225 units in the bglJ-Y5 mutant and to 675 units in the bglJ-Y6 mutant, demonstrating that, presumably, constitutive expression of bglJ activates the bgl promoter. The effect observed with allele yjjQbglJ-Y6 is bigger, possibly because the insertion element maps closer to bglJ (37 bp upstream). Likewise, in the leuO-Y1 mutant, the bgl promoter activity increased to 215 units, which suggests that LeuO also activates the bgl promoter (Fig. 3a). In addition, the bglJ and leuO genes were cloned under the control of the IPTG-inducible tac promoter in the pBR-derived plasmids pKEAP4 and pKEAP10 (see Methods for details). These plasmids were used for transformation of the wild-type strain carrying the bgl promoter–lacZ fusion. Induction of bglJ expression with IPTG resulted in an increase of {beta}-galactosidase expression directed by the bgl promoter to 570 units, whilst induction of leuO expression caused an increase to 735 units (Fig. 3a), confirming that BglJ and LeuO are activators of the bgl promoter and that the miniTn10 insertions cause constitutive expression of these genes. A bgl promoter allele ({Delta}Pbgl) that lacks the upstream silencer sequence necessary for H-NS-mediated repression was also tested. The activity of this promoter ({Delta}Pbgl) is approximately fourfold higher than that of the wild-type promoter (Dole et al., 2004b) (also compare Fig. 3a and b, 74 versus 300 units). The bglJ-Y6 mutation had no effect on the {Delta}Pbgl promoter (320 units compared with 300 units in the wild-type), whilst the leuO-Y1 mutations caused a rather moderate 1·4-fold increase (to 425 units). These data suggest that constitutive expression of BglJ and LeuO activates the bgl promoter by counteracting the H-NS-mediated repression of the promoter.

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 {beta}-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 bgl–lacZ 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 promoter–lacZ reporter described above (Fig. 3c). In the wild-type, this reporter directs the expression of 140 units of {beta}-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 ({Delta}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 {beta}-galactosidase directed by the bgl–lacZ 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 {beta}-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 {beta}-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 {beta}-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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Fig. 5. The DnaK level is lower in the dnaKJ : : miniTn10-Camr mutant. A quantitative Western blot analysis of DnaK protein levels expressed in the wild-type (S1195), the dnaKJ : : miniTn10 mutant (allele dnaKJ-M2; strain S2712) and the PA1/lacO dnaKJ strain (S2888) grown without IPTG (–IPTG) and with IPTG (+IPTG) at 30 °C in LB medium to OD600=0·5 was performed. The Western blot and the result of the quantitative analysis given as peak intensity, as well as relative protein levels compared with the wild-type (wt; set as 100 arbitrary units), are shown.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Silencing of bgl by H-NS occurs through binding of H-NS to two sites, located upstream and downstream of the promoter. The latter level of repression by H-NS also affects transcription elongation (Dole et al., 2004b). Here, we have shown that this downstream repression is modulated by DnaK, an HSP70 family chaperone (Dougan et al., 2002), and possibly DnaJ, the DnaK co-chaperone. Lon protease also affects the downstream repression (Dole et al., 2004a). DnaKJ (as Lon) has no impact on silencing of the promoter. Furthermore, we have shown that constitutive expression of transcription factors LeuO and BglJ relieves silencing of the bgl operon by counteracting repression of the promoter by H-NS through the upstream site, whilst BglJ and LeuO do not affect downstream repression. These data demonstrate that upstream and downstream repression of bgl by H-NS is regulated independently by LeuO and BglJ, as well as DnaKJ. Although upstream and downstream repression can be separated, silencing of bgl is significantly more effective when both silencers are present, indicating cooperativity between the two sites, as shown previously (Dole et al., 2004b).

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 {beta}-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.


   ACKNOWLEDGEMENTS
 
We thank Sandra Malcher for excellent technical assistance and Sudhanshu Dole for suggestions. The work was funded by the Deutsche Forschungsgemeinschaft through grant Schn 371/8 and the International Graduate School of Genetics and Functional Genomics at the University of Cologne.


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Received 31 March 2005; revised 20 July 2005; accepted 21 July 2005.



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