Unité de Génétique des Génomes Bactériens, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France1
Author for correspondence: Evelyne Krin. Tel: +33 01 40 61 35 56. Fax: +33 01 45 68 89 48. e-mail: ekrin{at}pasteur.fr
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ABSTRACT |
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Keywords: catabolite repression, cya, regulatory network
Abbreviations: CAP, catabolite activator protein; H-NS, histone-like nucleoid-structuring; PTS, phosphoenolpyruvate:carbohydrate phosphotransferase system
a Present address: Genopole, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
b Present address: Laboratory of Microbiology, Radioactive Waste & Clean-up Division, SCK/CEN, Boeretang 200, 2400 Mol, Belgium.
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INTRODUCTION |
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It has been demonstrated that the H-NS protein interacts with other regulatory systems. For example, the leucine-responsive regulatory protein Lrp regulates the transcription of a number of genes by binding DNA at a specific site located upstream from the transcription start site of the genes (Calvo & Matthews, 1994 ). Some of these target genes and the Lrp structural gene itself are regulated by the H-NS protein (Levinthal et al., 1994
; Oshima et al., 1995
). The H-NS protein also possesses common targets with the cAMP-catabolite activator protein (CAP) complex: nmpC and malT genes (Coll et al., 1994
; Johansson et al., 1998
) and pap, bgl, mccABCDE and flhDC operons (Forsman et al., 1992
; Gonzalez-Pastor et al., 1995
; Schnetz & Wang, 1996
; Soutourina et al., 1999
). In contrast to its role in the control of flhDC expression (Bertin et al., 1994
; Soutourina et al., 1999
), the cAMP-CAP complex acts as an H-NS protein antirepressor in the regulation of pap and bgl operons (Forsman et al., 1992
; Schnetz & Wang, 1996
).
The cAMP-CAP complex, which controls the expression of a multitude of genes or operons, has been characterized for its role in catabolite repression. Indeed, high glucose levels reduce Enzyme IIAGlc phosphorylation, which decreases adenylate cyclase activity and cAMP concentration. This results in a repressed synthesis of the enzymes needed for the catabolism of alternative carbon sources. It is now known that CAP is also involved in the expression of genes needed for adaptation to changes in growth conditions. Moreover, CAP regulates the synthesis of some membrane components, numerous proteins involved in various stresses and some regulator-encoding genes. Finally, it is worth mentioning that many CAP-regulated genes are also controlled by other transcription factors (for a review see Busby & Kolb, 1996 ).
It has been suggested that the cellular CAP concentration might be somewhat reduced in hns strains (Johansson et al., 1998 ). As it has been demonstrated that the activity of CAP is mainly modulated by the intracellular level of cAMP (Roy et al., 1983
; Kolb et al., 1993
), we wanted to know whether the H-NS protein could be involved in the control of the intracellular cyclic AMP (cAMP) concentration. In the present paper, we show that the H-NS protein plays a role in this process by acting on crr gene expression. The effect on the crr-specific P2 promoter was mainly observable in the absence of CAP and, although the H-NS protein has been considered as a transcriptional repressor, our results constitute a new example of a positive effect of this regulatory protein on bacterial physiology. Moreover, we show for the first time that crr expression is regulated and that a twofold reduction in the global amount of Enzyme IIAGlc is sufficient to significantly decrease adenylate cyclase activity and the cAMP level.
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METHODS |
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In vitro adenylate cyclase assay.
Exponentially growing cell cultures were centrifuged at 9000 g for 10 min at 20 °C. The pellet, corresponding to 0·5 g bacteria, was resuspended in 3 ml 25 mM Tris/HCl, 10 mM MgCl2, pH 8·3, (Tris-Mg buffer) to obtain a 20 mg ml-1 protein concentration. Bacteria were then broken with the FastPrep System and FastProtein Blue (Bio-101) and centrifuged at 13000 g at 4 °C. Protein (0·40·8 mg) of the bacterial supernatant was added to 1·2 ml of assay mixture (125 mM Tris/HCl, pH 8·3, 50 mM MgCl2, 5 mM dithiothreitol, 5 mM ATP) and 4·68 ml Tris-Mg buffer. During incubation at 28 °C, several samples of 1 ml were taken between 0 and 60 min and heated for 5 min at 100 °C, according to the method of Joseph et al. (1982) . The cAMP synthesized was quantified with the cAMP[125I]-RIA kit (NEN).
ß-Galactosidase assay.
ß-Galactosidase activity was determined by the method of Miller (1992) on exponentially growing cells. The assay was performed on more than three samples from at least two independent cultures.
Determination of the phosphorylation state of Enzyme IIAGlc.
The phosphorylation state of Enzyme IIAGlc was determined with a Western blotting experiment with anti-IIAGlc antibodies (kindly provided by P. Postma, University of Amsterdam, The Netherlands) on 20 µl protein extracts from strains grown to an OD600 of precisely 0·400 (Takahashi et al., 1998 ). Immunoblots were scanned with a JX-330 Sharp scanner and quantified using PDI software, PDQuest, based on a SUN computer system.
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RESULTS |
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Adenylate cyclase activity was measured with an in vitro assay on exponentially growing cells. A more than 30-fold increase in enzyme activity was measured in the crp strain TP2139 [12600 pmol cAMP (mg protein)-1 min-1] compared to the wild-type TP2101 [400 pmol cAMP (mg protein)-1 min-1], in accordance with the results of others (Rephaeli & Saier, 1976 ; Joseph et al., 1982
). In contrast, an eightfold decrease in activity was measured in the crp hns double mutant BE1420 [1600 pmol cAMP (mg protein)-1 min-1] compared to the crp strain. These results are in agreement with the total cAMP level measured in bacterial cultures (Table 2
).
To investigate the role of the H-NS protein on the transcription of the adenylate cyclase encoding gene, we measured the activity of a cyaA-lacZ operon fusion from plasmid pDIA1973 (Roy et al., 1988 ). Compared to the wild-type TP2101 [6320 Miller units (mg protein)-1], a moderate increase in ß-galactosidase activity was observed in the crp strain TP2139 (Table 3
), in agreement with the data of others (Kawamukai et al., 1985
). In contrast, in the crp hns double mutant BE1421 (Table 3
), a more than twofold decrease in ß-galactosidase activity was observed compared to that in the crp strain.
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DISCUSSION |
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Our results demonstrate, for the first time, that crr is regulated at the transcriptional level. This regulation only affects the P2-specific promoter and has almost no effect on the two other promoters of the PTS operon. The H-NS protein controls the expression of numerous genes involved in bacterial adaptation to environmental changes (Hommais et al., 2001 ) and it is generally considered as a transcriptional repressor. Although the mechanism, which could be indirect, remains to be determined, our results constitute a new example of the positive effect of this regulatory protein on bacterial physiology. Recently, the existence of a complex between HhA and the H-NS protein has been shown to be involved in the regulation of the haemolysin operon in E. coli (Nieto et al., 2000
). Moreover, an hha mutation results in a fivefold decrease in Enzyme IIAGlc in rich medium under high osmolarity conditions (Balsalobre et al., 1999
). Taken together, these obervations may suggest an interaction of both proteins in the regulation of crr expression. However, in contrast to the H-NS protein (Table 3
and Fig. 2
), the effect of HhA on Enzyme IIAGlc was only observed in conditions of high osmolarity, suggesting that the two regulatory proteins affect crr expression by a different mechanism. Finally, the repression by the cAMP-CAP complex is predominant with regard to the activation by the H-NS protein. Indeed, in the presence of the cAMP-CAP complex, only a small fraction of Enzyme IIAGlc was phosphorylated (Fig. 2
). This suggests that adenylate cyclase has its lowest activity in the wild-type strain, which could explain that a twofold alteration in the amount of Enzyme IIAGlc in hns strains has no major effect on adenylate cyclase activation.
We also showed that, despite a large excess of Enzyme IIAGlc in the cell [about 15000 Enzyme IIAGlc phosphorylated molecules in comparison with 1550 adenylate cyclase molecules (Yang & Epstein, 1983 ; Mitchell et al., 1987
)], a twofold variation in its accumulation level resulted in an eightfold decrease in adenylate cyclase activity. In a glucose-rich medium, the phosphorylated form of Enzyme IIAGlc interacts preferentially with the glucose permease (Enzyme IIBCGlc) to allow the entry of glucose into the cell. This suggests that the affinity of phosphorylated Enzyme IIAGlc is much lower for adenylate cyclase, or for the putative intermediary that could activate it (Saier et al., 1996
), than for glucose permease. Depending on environmental conditions, adenylate cyclase may be regulated either by the amount or by the phosphorylation of Enzyme IIAGlc.
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ACKNOWLEDGEMENTS |
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Received 27 November 2001;
revised 21 January 2002;
accepted 23 January 2002.
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