Unité de Génétique des Génomes Bactériens1 and Génopole-Plateau Protéomique2, Institut Pasteur, Paris, France
Author for correspondence: Philippe N. Bertin. Tel: +32 14 33 34 36. Fax: +32 14 32 03 13. e-mail: pbertin{at}sckcen.be
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ABSTRACT |
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Keywords: acidic pH, osmolarity, DNA supercoiling, DNA array
Abbreviations: CAT, chloramphenicol acetyltransferase
a Present address: Laboratoire de Biochimie, UMR 7654, CNRS-Ecole Polytechnique, 91128 Palaiseau Cedex, France.
b Present address: Laboratory of Microbiology, Boeretang 200, B-2400 Mol, Belgium.
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INTRODUCTION |
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Bacterial flagellum biosynthesis is under the control of the flhDC master operon, which governs motility and chemotaxis, as well as differentiation into swarming cells in enterobacteria. Moreover, this operon ensures global communication between flagellar genes and external factors, as well as cell division (Aizawa & Kubori, 1998 ). The complex motility and chemotaxis system in Escherichia coli includes nearly 50 genes organized in an ordered cascade in which the expression of a gene located at a given level requires the transcription of another one at a higher level (Macnab, 1996
). This system is subject to a complex regulation by multiple environmental factors and regulatory proteins. For example, flagellum biosynthesis is sensitive to catabolite repression (Adler & Templeton, 1967
; Silverman & Simon, 1974
; Yokota & Gots, 1970
) and is inhibited by stressful environmental conditions, such as increased temperature and high osmolarity (Adler & Templeton, 1967
; Li et al., 1993
). Moreover, numerous mutations in stress-related genes, such as those encoding heat-shock proteins, membrane components or DNA replication initiation factors, are known to affect motility (Farr et al., 1989
; Kitamura et al., 1994
; Mizushima et al., 1995
, 1997
; Shi et al., 1992
, 1993a
) by repressing transcription of the flhDC master operon (Mizushima et al., 1995
, 1997
; Shi et al., 1993b
). Unlike flhDC transcriptional control by cAMPCAP complex (Soutourina et al., 1999
), the mechanism by which stress-related conditions affect master operon expression remains still largely unknown.
H-NS is a nucleoid-associated protein known to be involved in the control of motility in E. coli (Bertin et al., 1994 ) and in Salmonella typhimurium (Hinton et al., 1992
). This protein positively controls the master flagellar operon but the mechanism of this regulation remains unclear (Kutsukake, 1997
; Soutourina et al., 1999
). In the present study we demonstrated that flagellar gene expression is inhibited under low-pH conditions and that this regulation of the flhDC master operon may be dependent on the H-NS protein. For the first time, we provide evidence that the 5' end of mRNA plays a crucial role in flhDC expression in response to environmental factors.
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METHODS |
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Resistance to low pH.
Strains were grown to stationary phase overnight in M9 medium, pH 5·5, supplemented with glucose and Casamino acids. Acidic stress was analysed in M9 medium at pH 2·5 supplemented with 0·012% glutamate as previously described (Hommais et al., 2001 ).
Two-dimensional gel electrophoresis.
Strains were grown in M9 medium supplemented with Casamino acids and glycerol at pH 7·0 or 4·6 to an OD600 of 0·7. Total protein extracts and two-dimensional gel electrophoresis were carried out as previously described (Hommais et al., 2001 ; Laurent-Winter et al., 1997
).
Expression profiling.
Bacterial cells were grown in M63 minimal medium supplemented with glucose (Miller, 1992 ) to an OD600 of 0·6. Handling of RNA, cDNA synthesis from 10 µg RNA, hybridization on DNA arrays (Panorama E. coli gene arrays from Sigma-GenoSys Biotechnologies) and data analysis were performed as previously described (Hommais et al., 2001
). Briefly, hybridization probes were generated from 10 µg RNA following standard cDNA synthesis using [
-33P]dCTP (7·4x10131·1x1014 Bq mmol-1, New England Nuclear), AMV reverse transcriptase (Roche) and E. coli labelling primers (Sigma-Genosys). The prehybridization and hybridization were carried out according to the manufacturers recommendations with some modifications (Hommais et al., 2001
). Blots were exposed to PhosphorImager screen (Molecular Dynamics) and were then scanned on a 445SI PhosphorImager. The intensity of each dot was measured with the XDOTSREADER software (Cose) and analysed using an Excel spreadsheet.
In vitro transcription assays.
In vitro transcription experiments were performed with pDIA546 containing the entire flhDC regulatory region as previously described (Soutourina et al., 1999 ). Plasmid pDIA546 was restricted by EcoRI for 2 h at 37 °C and used as linearized template for in vitro transcription.
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RESULTS |
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To investigate the effect of the suppressor mutations on the physiology of E. coli, we tested various H-NS-related phenotypes in strains BE2120 and BE2121. Like the original hns mutant, both strains remained mucoid and able to use salicin as a carbon source (Table 2). In contrast, the mutations reversed, as expected, the loss of motility on semi-solid medium. Surprisingly, the suppressor mutants also showed a strong susceptibility to low pH, similar to that of the wild-type strain (Table 2
).
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Regulation of motility under acidic pH conditions
In E. coli, the optimum pH for motility and chemotaxis is close to that for growth (Adler, 1973 ; Adler & Templeton, 1967
). However, the control of bacterial motility by acidic pH, which can reflect the growth conditions frequently encountered by enterobacteria inside their host (Mahan et al., 1996
), has not yet been well documented. To investigate the direct effect of low pH on motility, we tested the swarming behaviour of wild-type E. coli on semi-solid plates at neutral and acidic pH. As seen in Fig. 1(A)
, a loss of motility was observed under low pH conditions. To determine whether this alteration in swarming behaviour resulted from a lack of flagella, we analysed the flagellin content by two-dimensional gel electrophoresis. As seen in Fig. 1(B)
, the spot corresponding to the FliC protein on our E. coli two-dimensional protein map (Hommais et al., 2001
) was undetectable in the protein extract of bacteria grown at low pH by comparison with those grown at neutral pH. It has been proposed that disintegration of flagella into subunits occurs at acidic pH (Stocker & Campbell, 1959
; Weibull, 1948
). However, to prevent unnecessary energy consumption the cost to cell of flagellar synthesis is about 2% of total biosynthetic energy expenditure (Macnab, 1996
) it can be assumed that detrimental conditions such as acidic pH repress expression of flagellar genes. To test this hypothesis, we measured the expression level of the flagellin-encoding gene using a fliCcat transcriptional fusion under neutral and acidic pH conditions. A sevenfold decrease in fliC transcription was observed at acidic as compared to neutral pH: CAT activities of 530±30 and 68±8 units were measured at pH 7·0 and pH 4·6, respectively (1 unit corresponds to 1 µmol chloramphenicol acetylated per min per µg protein).
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One possibility that could explain the role of H-NS in the control of gene expression is its implication in DNA topology (Higgins et al., 1988 ; Dorman et al., 2001
). Moreover, some environmental factors or drugs that are known to inhibit bacterial motility also affect DNA supercoiling, e.g. high salt concentration or novobiocin (Anderson & Bauer, 1978
; Goldstein & Drlica, 1984
; Higgins et al., 1988
; Shi et al., 1993b
). The overexpression of DNA gyrase subunit GyrB resulted in a partial restoration of motility in an hns mutant (data not shown). Furthermore, in the presence of novobiocin, a DNA gyrase inhibitor decreasing DNA supercoiling, we observed a more than twofold decrease in flhDC activity from the flhDC transcriptional fusion containing the entire regulatory region, similar to that obtained in the presence of an hns mutation or at low pH (Fig. 2
). Finally, as compared to the wild-type strain, which was non-motile in the presence of novobiocin (28 and 4 mm swarming ring diameter in the absence and presence of 200 µM novobiocin, respectively), a significant restoration of motility was observed in the presence of plasmid pPM61 overexpressing the flhDC operon (10 mm swarming ring diameter).
To further investigate the effect of DNA topology on flhDC expression, we performed in vitro transcription experiments with either supercoiled or linearized plasmid pDIA546 carrying the entire flhDC regulatory region. A severe reduction in the flhDC transcription level was observed when linearized plasmid was used as a template (Fig. 3). This suggests that variations in DNA topology involving the 5' end of the flhDC operon may play an important role in the transcriptional control of the flagellar master operon by H-NS in response to low pH.
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DISCUSSION |
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Despite the similarities between E. coli and S. typhimurium, some differences have been reported in the regulation of motility in these two organisms. These include the autogenous and global control by H-NS on flhDC master operon expression (Kutsukake, 1997 ) and the initiation of flhDC transcription (Soutourina et al., 1999
; Yanagihara et al., 1999
). On the other hand, some specificities may also exist in the response of these bacteria to acidic pH (Lin et al., 1995
), including the PhoPQ-mediated acid tolerance response (ATR) (Bearson et al., 1998
). As the synthesis of H-NS protein has been shown to be unaffected by low pH (Adams et al., 2001
), it has been recently suggested that H-NS is not involved in the control of flagellar biosynthesis in S. typhimurium under the acidic conditions tested. Similarly, in E. coli, we observed no alteration in hns expression at acidic pH. However, flhDC transcription measurement (Fig. 2
) and characterization of suppressive mutants (Table 3
) demonstrated the role of H-NS protein in motility control under low-pH conditions in E. coli. Nevertheless, these observations do not exclude the possibility that other regulators may participate in this process.
The presence of a 5' untranslated mRNA region has been usually associated with post-transcriptional regulation mechanisms. For example, such regions are responsible for either transcriptional anti-termination and RNA processing or translational control of threonyl-tRNA synthetase genes in E. coli and Bacillus subtilis (Condon et al., 1997 ; Putzer et al., 1995
; Sacerdot et al., 1998
). As compared to the wild-type strain, an up to threefold decrease in flhDC activity was observed in an hns strain from both transcriptional and translational fusions (Soutourina et al., 1999
). Moreover, a similar reduction in the level of flhDC mRNA was measured in the hns mutant as compared to the wild-type in RT-PCR experiments and this reduction did not result from an effect of the H-NS protein on the flhDC mRNA stability (Soutourina, 2001
). Finally, in vivo activity measurements from a transcriptional fusion containing the extended flhDC regulatory region (Fig. 2
) demonstrated that H-NS, acidic pH and DNA supercoiling affect the flhDC expression at the level of transcription initiation.
Despite numerous studies on the control of bacterial motility by environmental factors, the molecular basis of this process remains largely unknown. In E. coli, the H-NS protein affects the expression of many genes involved in the cellular response to environmental changes, including those required for acidic pH resistance (Hommais et al., 2001 ). Although the mechanism by which H-NS controls gene expression remains the subject of debate (Williams & Rimsky, 1997
), an alteration of plasmid and chromosomal DNA supercoiling has been demonstrated in vivo in an hns mutant (Mojica & Higgins, 1997
). Moreover, the involvement of DNA supercoiling has been proposed, for example, to explain the regulation by H-NS of osmotically regulated genes (Higgins et al., 1988
), stringently controlled bacterial promoters (Johansson et al., 2000
) or virulence gene expression in Shigella flexneri (Dorman et al., 2001
). On the other hand, various environmental conditions are also known to affect the level of DNA supercoiling, even though a cause-and-effect relationship has not yet been established (Higgins et al., 1988
; Tse-Dinh et al., 1997
). It has been proposed that a direct effect of environmental signals on promoter architecture, and then transcription, through influencing the interaction of architectural proteins with DNA, might be an important concept in understanding the environmental regulation of gene expression in bacteria (Jordi et al., 1997
). Similarly, different environmental cues might influence the action of H-NS by changing the structure of regulatory regions, the ability of H-NS to bind to DNA target and/or the conformation or the oligomerization state of H-NS. We did not observe any alteration in the level of hns gene expression or in the isoform composition of the H-NS protein under low pH or in the presence of DNA gyrase inhibitor (data not shown). In contrast, flhDC expression could be modulated by local alteration of DNA topology, resulting from interactions between H-NS and the regulatory region. Several observations argue in favour of this hypothesis. First, the alteration of swarming properties in presence of novobiocin, a DNA gyrase inhibitor, or in strains overproducing DNA gyrase or mutated in its structural gene suggests the existence of a critical DNA supercoiling level for normal motility in E. coli (see Results) (Shi et al., 1993b
; O.S., unpublished). Second, we observed a severe reduction in flhDC expression when linearized rather than supercoiled plasmid was used in in vitro transcription assay (Fig. 3
). Third, the involvement of the extended flhDC regulatory region in the control of the master operon by H-NS, acidic pH and novobiocin suggests a strong correlation between these regulatory processes (Fig. 2
). Finally, further support is provided by the partial restoration of motility in the hns mutant by overexpression of DNA gyrase subunit gene gyrB (data not shown), and the alteration of topoisomer distribution of plasmids carrying the entire flhDC regulatory region in the presence of chloroquine phosphate as an intercalating agent (Soutourina, 2001
). Taken together, these data suggest that the H-NS-mediated effect on motility may be at least in part explained by an alteration in the level of DNA topology of the flhDC regulatory region. They are consistent with the recent demonstration that a 339 bp DNA fragment having a bent structure can strongly affect the level of plasmid DNA supercoiling (Rohde et al., 1999
) and suggest that the regulatory region of the flagellar master operon may play a crucial role for an adequate control by the H-NS protein and environmental factors.
The control of bacterial motility via the flhDC operon includes several participants at multiple regulatory levels, e.g. transcription initiation control by cAMPCAP complex and H-NS (Soutourina et al., 1999 ), mRNA stability control by CsrA (Wei et al., 2001
) in E. coli, or FlhDC protein degradation by Lon protease in Proteus mirabilis (Claret & Hughes, 2000
). Our results extend the knowledge of the regulation of the flagellar system and represent an important step toward the understanding of complex mechanisms governing bacterial motility in response to environmental challenges.
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ACKNOWLEDGEMENTS |
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Financial support came from the Institut Pasteur and the Centre National de la Recherche Scientifique (URA 2171). O.S. was supported by a French Government fellowship.
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Received 1 November 2001;
revised 9 January 2002;
accepted 17 January 2002.