Institut für Genetik, Universität zu Köln, Weyertal 121, 50931 Köln, Germany1
Tel: +49 221 4703815. Fax: +49 221 4705975. e-mail: schnetz{at}uni-koeln.de
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ABSTRACT |
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Keywords: ß-glucoside, pleiotropic regulation, H-NS, lac operon region
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INTRODUCTION |
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The expression level of the activated bgl operon derivatives and the effect of mutations that partially disrupt the hns gene can vary with the strain background (Higgins et al., 1988 ; Lopilato & Wright, 1990
; Tsui et al., 1994
; Free et al., 2001
). In addition to H-NS and CRP, the bgl operon is affected by other pleiotropic regulators including the DNA-bending protein FIS, which competes with CRP for binding (Caramel & Schnetz, 2000
), and the transcriptional-regulator-like proteins LeuO and BglJ (Giel et al., 1996
; Ueguchi et al., 1998
). RpoS, the key regulator in the stress response of E. coli and the H-NS homologue StpA are necessary for silencing of the wild-type bgl operon by a truncated H-NS protein lacking its C-terminal DNA-binding domain (Free et al., 1998
, 2001
; Ohta et al., 1999
). Furthermore, RpoS downregulates the expression of activated bgl operon alleles (Dole et al., 2002
). Interestingly, the up to 50-fold repression of the bgl operon by RpoS is based on the amplification of a moderate (two- to threefold) repression of the transcription rate by RpoS via a second, post-transcriptional level of regulation involving the specific antiterminator protein BglG. BglG is encoded by the first gene of the operon. At low transcription rates BglG is limiting and transcription halts at rho-independent transcriptional terminators t1 in the leader and t2 within the operon. If the transcription rate increases above a threshold the basal synthesis of BglG is sufficient for antitermination and, as a result, bglG and the operon are expressed at high levels (Dole et al., 2002
).
RpoS activity is controlled by multiple signals and at various levels. Predominant is the post-transcriptional control, for example its translation is regulated by the RNA-binding protein Hfq and small regulatory RNAs and its proteolysis is controlled by the response regulator RssB and the ClpXP protease (Hengge-Aronis, 2000 ). In addition, RpoS activity is affected by Crl, a 15 kDa protein that is conserved among the Enterobacteriaceae (for example, it is present in the complete genome sequences of Salmonella enterica, Salmonella typhimurium LT2, Yersinia pestis and Vibrio cholerae) but shares no homology with other proteins. Crl was discovered because it is required for the RpoS-dependent expression of the csgBA operon encoding the fibronectin-binding curli fimbria (Crl=curli) (Olsen et al., 1989
; Arnquist et al., 1992
). Genetic analyses revealed that Crl stimulates to various extents all RpoS-dependent transcription processes and that Crl acts either in concert with RpoS or upstream of it in the same pathway (Pratt & Silhavy, 1998
).
Here the expression level of activated bgl operon derivatives and bgllacZ reporter constructs were tested in strain CSH50 which carries a large deletion in the lac region, (gptlac), and derivatives that carry the smaller
(argFlac) deletion or the wild-type gptprolac (
lacZ) region. In strain CSH50, activated bgl operon alleles are expressed at high levels. However, their expression is low in the other two deletion derivatives. In these strains the expression is increased up to 50-fold when a rpoS mutation is introduced (see also Dole et al., 2002
). RpoS has no effect in CSH50. A complementation experiment shows that Crl, which is encoded in the gptargF region, is required for the RpoS-dependent repression of the bgl operon and responsible for these strain-dependent variations in the expression level of the bgl operon.
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METHODS |
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RESULTS AND DISCUSSION |
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Crl is required for the RpoS-mediated repression of the bgl operon
The crl gene maps in the gptargF region and thus was an obvious candidate for the observed difference. To test whether Crl complements the (gptlac) strain, plasmid pKESK19 carrying the crl gene under control of the constitutive antisense promoter of the tetracycline resistance gene (Stüber & Bujard, 1981
) was constructed. In transformants of the
(gptlac) strain the repression of the activated bgl operon alleles by RpoS was restored (Fig. 2
; +Crl). As a control, in the RpoS-negative
(gptlac) strain providing Crl in trans caused a less than twofold decrease [Fig. 2
;
(gptcrllac) rpoS +Crl]. This demonstrates that Crl is required for the RpoS-mediated repression.
Crl is required for the RpoS-mediated repression of bgllacZ reporter constructs
Next a chromosomal bgllacZ reporter system was used in which expression is independent of the ß-glucoside specific regulation by BglG-mediated antitermination due to a mutation (t1-L) of terminator t1 (Fig. 3) (Dole et al., 2002
). In this system the changes in the transcription rate are not amplified by BglG-mediated antitermination and thus correlate with changes in the levels of expressed ß-galactosidase (see Introduction). RpoS has an approximately threefold effect on the expression level of the bgllacZ reporter gene system (Dole et al., 2002
). Analysed were bgllacZ fusions that carry the wild-type bgl promoter (Fig. 3a
) or activated alleles, including the mutants that carry the improved CRP-binding site (Fig. 3b
) or the integration of IS1 (Fig. 3c
). In addition, mutants carrying an integration of IS5 (Fig. 3d
) and a deletion of the upstream silencer were tested (Fig. 3e
) (Dole et al., 2002
). Expression levels are given in units of ß-galactosidase activity and thus the values are higher than in the phospho-ß-glucosidase assay shown in Fig. 2
. Similar to the results obtained using the bgl operon, the bgllacZ constructs were repressed by RpoS when Crl was present (
lacZ) but not when Crl was missing (
gptcrllac) (Fig. 3
). Repression of the activated alleles by RpoS is approximately two- to threefold (Fig. 3
). Again, providing Crl in trans restored repression by RpoS in the
gptcrllac strain, but had no effect in the rpoS mutant of this strain (Fig. 3
). As a control, the expression of a lacOPlacZ fragment integrated into attB varied only moderately in the various strain backgrounds (Fig. 3
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 25 March 2002;
revised 3 May 2002;
accepted 7 May 2002.