JOURNAL OF BACTERIOLOGY, June 1996, p. 3331–3334 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 178, No. 11
NOTES Cyclic AMP Receptor Protein Positively Controls gyrA Transcription and Alters DNA Topology after Nutritional Upshift in Escherichia coli JOSE MARIA GOMEZ-GOMEZ, FERNANDO BAQUERO,
AND
JESUS BLAZQUEZ*
Servicio de Microbiologı´a, Hospital Ramo ´n y Cajal, Madrid 28034, Spain Received 15 August 1995/Accepted 29 March 1996
The expression of a transcriptional gyrA-lacZ gene fusion throughout the Escherichia coli growth cycle and the effect that mutation Dcrp39 had on this expression were studied. The data obtained indicate that the expression of gyrA is growth phase dependent and under the positive control of the cyclic AMP receptor protein (CRP). Complementation analysis of gyrA-lacZ expression with wild-type CRP or variant CRPpc (with a T-to-A mutation at position 158) in a CRP-deficient background suggests that this CRP action is mediated by a class I or class II CRP-dependent promoter(s). Our results also indicate that CRP may be involved in the modulation of DNA topology in the transition from the lag period to the exponential phase of growth. suggested to play an important role in cell adaptability to stress conditions (1, 10, 15, 16). Negative DNA supercoiling is under homeostatic control by the opposite actions of DNA topoisomerases, which are responsible for the regulation of DNA topology. Topoisomerase I is coded for by the topA gene, and DNA gyrase is coded for by the gyrA and gyrB genes. While DNA gyrase introduces negative supercoils (in an ATP-dependent reaction), DNA topoisomerase I is capable of removing negative superhelical turns (11, 22, 23). A possible link between these two global regulatory processes (CRP and DNA supercoiling) might be that the cAMPCRP complex regulates the expression of genes encoding the DNA topoisomerases. To test this hypothesis, we constructed a gyrA-lacZ operon fusion and measured its expression in a CRP2 background. We used plasmid pSLS447 (27), which contained the wild-type gyrA gene from E. coli on a 7-kb BamHI fragment inserted in the BamHI site of pBR322 (5). The BamHI fragment was cloned in the unique BamHI site of plasmid pACYC184 (8) to give pGYA11 (Fig. 1). A derivative of pGYA11, pGYA12, was constructed by producing an internal deletion of the shortest PstI fragment (Fig. 1). The lacZKmr gene fusion cassette from pKOK6 (17) was inserted into the unique PstI site of plasmid pGYA12 to give pGYL12 (data not shown). The appropriate orientation of lacZ relative to the gyrA gene and its promoters was tested by digestion with EcoRI and HindIII enzymes. The BamHI fragment carrying the gyrAlacZ gene fusion (Fig. 1) was ligated to a BglII (3.5-kb) fragment harboring the replication origin of monocopy plasmid pMccB17 (20) to give plasmid pMGL25. Monocopy plasmid pMGL25 was introduced into strain MC4100 [F2 araD139 D(argF-lacU169) rpsL flbB5801 DfruA25 deoC1 relAl ptsF rbsR] by transformation (7). The b-galactosidase activity from MC4100(pMGL25) was determined throughout the bacterial cycle. As can be seen from Fig. 2, the b-galactosidase activity of the gyrA-lacZ fusion fluctuated with the bacterial cycle. The b-galactosidase activity of the gyrA-lacZ fusion began to increase (by fourfold) at the mid-exponential phase and reached its peak at the late exponential growth phase. During the transition from the late exponential phase to the early
Regulation of gene expression is a pivotal process in the adaptability of bacterial cells to stress conditions. Gene expression is regulated by several mechanisms in bacterial cells. A common way to regulate this expression is the modulation of the activity of proteins involved in the transcriptional activation of diverse promoters. In Escherichia coli, cyclic AMP (cAMP) receptor protein (CRP, also referred to as CAP) is a global transcriptional regulator. CRP modulates positively or negatively the expression of a large number of genes and operons in response to glucose depletion (9, 18). Under conditions in which cAMP levels are high (e.g., when cells are growing in glycerol minimal medium or in Luria-Bertani [LB] medium) CRP activates transcription of many promoters, including those required for the utilization of such alternative carbohydrate carbon sources as lactose, galactose, arabinose, and maltose (29). The active cAMP-CRP complex binds to specific DNA sites located at or upstream of CRP-dependent promoters. Binding of CRP to these DNA sites modulates transcription initiation by RNA polymerase. Two classes of CRP-dependent promoters have been described: (i) simple CRPdependent promoters (classes I and II) and (ii) complex promoters that require a regulon-specific activator in addition to CRP (class III) for their modulation (12). The activation of class I and class II promoters requires an activating region in the CRP molecule that comprises amino acids 156 to 162 (32, 33). Additionally, the cAMP-CRP complex influences the transcription of different groups of genes and operons not related to the catabolite (6). As an example, expression of the cts group, which is induced in the stationary phase or under carbon starvation conditions, is dependent on the cAMP-CRP complex (19). On the other hand, changes in DNA supercoiling represent a second global transcriptional regulatory mechanism (14, 30). Environmentally induced changes in DNA topology have been
* Corresponding author. Mailing address: Servicio de Microbiologı´a, Hospital Ramo ´n y Cajal, Carretera de Colmenar Km 9.100, Madrid 28034, Spain. Phone: (34)-1-3368330. Fax: (34)-1-336 88 09 or (34)-1336 90 16. Electronic mail address:
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FIG. 1. Genetic and physical map of the BamHI fragment carrying the gyrAlacZ operon fusion in monocopy plasmid pMGL25. The arrows indicate the directions of transcription for the corresponding genes. The zigzag line indicates transcription from the gyrA promoter. Restriction enzyme abbreviations: B, BamHI; H, HindIII; K, KpnI; Sn, SnaBI; St, StuI.
stationary phase, the activity decreased to a level similar to that during mid-exponential growth. This result indicates that expression of the gyrA gene of E. coli is growth phase dependent. It is well known that the growth-dependent expression of some genes is under the control of the cAMP-CRP complex (19, 25, 26, 31). To test the hypothesis that the growth-dependent expression of the gyrA gene could be mediated by the cAMP-CRP complex, the activity of a gyrA-lacZ fusion was tested in E. coli SBS688 (MC4100 Dcrp39) harboring monocopy plasmid pMGL25. The b-galactosidase activity was examined throughout the growth cycle and compared with that obtained with strain MC4100 harboring the same plasmid, pMGL25 (Fig. 3). The increased b-galactosidase activity during the exponential growth phase with the crp1 background
FIG. 2. Growth-phase-dependent expression of a gyrA-lacZ gene fusion and the effect of glucose. An overnight culture of MC4100 harboring a plasmid-borne transcriptional fusion of the gyrA promoter with lacZ (pMGL25) was grown with kanamycin (40 mg/ml). Cells were subcultured (1/200) in fresh LB medium with and without glucose (0.2%) and incubated at 378C with agitation. At the indicated time intervals, aliquots were removed and cell density and b-galactosidase activity were measured as described by Miller (24). Open symbols indicate OD600 and closed symbols indicate b-galactosidase activity. E, no glucose; h, 0.2% glucose.
FIG. 3. Effect of the Dcrp39 mutation on growth-phase-dependent gyrA-lacZ expression. An overnight culture of Dcrp39 strain SBS688 harboring plasmid pMGL25 was grown with kanamycin (40 mg/ml) in LB medium. The procedure was the same as that described in the legend to Fig. 2. The b-galactosidase activity obtained with strain Dcrp39 was compared with that of the wild type (crp1). b-Galactosidase activity is plotted versus growth (OD600). F, crp1; ■, Dcrp39.
was not observed with the crp mutant. This result indicates that CRP positively regulates the transcription of gyrA. In addition, we examined the putative inhibitory effect of glucose on the gyrA-lacZ fusion. As was expected, when glucose was present in the medium, the b-galactosidase activity was lower than that obtained in glucose-free medium (Fig. 2). Overall, these results suggest that gyrA transcription is subject to catabolite control. Several potential hypotheses might account for the CRP action on gyrA transcription. First, CRP action may be mediated by CRP binding to specific sequences located at the promoter region of the gyrA gene. A CRP dimer has been shown to recognize two 5-bp sequences (consensus, TGTGA and TCACA) that are related by a twofold rotational symmetry and that have a 6- or 8-bp spacer region in between (2–4, 12, 13). A visual examination of the sequence containing the gyrA promoter (27) did not reveal any potentially appropriate CRPbinding site. However, we identified the sequence 59-ggaTGT GAataaagcgTATAGg-39 (297 to 276 bp from the translation start point of GyrA) in the promoter region of gyrA. Therefore, we cannot discard the possibility that the mentioned sequence (with an A1T-rich N8 spacer) might be the target for CRP in the regulation of gyrA transcription. Second, CRP could indirectly regulate gyrA expression by means of a positive regulator directly controlled by CRP. This control is expected to be mediated by CRP-dependent promoters. The positive regulator could interact with the gyrA promoter to increase the transcription levels. Finally, CRP could regulate the expression of other topoisomerase genes. This regulatory process might alter DNA topology and gyrA transcription in turn. In support of this hypothesis, we have detected three putative CRP-binding sites in the promoter region of the topA gene: cacTGTGAcgctt tCGTCAatc (positions 1078 to 1100, according to reference 28), accTGTTAactcagTCACCtga (1064 to 1085), and tttCGT GAacagagTCACGaca (1187 to 1210). The last site has previously been proposed to be a potential recognition site for a regulatory factor (28). We suggest that this factor could be CRP. These sequences have high identity with the proposed CRP-binding consensus sequence. In addition, CRP could regulate gyrA transcription through the regulation of topA expression. On the basis of our experimental results, no preference for any of these three hypotheses can be given. The observed effect of the crp mutation on gyrA expression could simply be the consequence of the slow growth of strain SBS688, which carries the Dcrp39 mutation, at 378C in LB
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FIG. 6. Effect of the Dcrp39 mutation on plasmid supercoiling during the transition from the lag period to the exponential growth phase. An overnight culture of strains MC4100 and SBS688 harboring plasmid pACYC184 was grown with chloramphenicol (40 mg/ml) in LB medium. Cells were subcultured (1/200) in fresh LB medium and incubated at 378C with agitation. At the indicated time intervals, aliquots were removed. Cell density (OD600) was measured, and the cells were collected by centrifugation. Plasmid DNA was extracted (with the Wizard miniprep kit [Promega Corp.]), and topoisomers were separated by 0.8% agarose gel electrophoresis in Tris phosphate-EDTA buffer at 3 V/cm for 20 h in the presence of 12 mg of chloroquine per ml. Under these conditions, the more negatively supercoiled topoisomers migrate faster. The gel was washed for 3 h in distilled water before it was stained with ethidium bromide and photographed under UV light (the negative is shown). Lanes 1 to 7, crp1 at 15, 30, 45, 60, 90, 150, and 180 min after nutritional upshift, respectively. The OD600s were 0.04, 0.05, 0.07, 0.1, 0.2, 0.8, and 1.4, respectively. Lanes 8 to 13, Dcrp39 after 15, 30, 60, 150, 330, and 420 min, respectively, corresponding to OD600s of 0.04, 0.05, 0.08, 0.3, 0.8, and 1.1, respectively. FIG. 4. Growth of control strain MC4100 (pMGL25) and strain SBS688 (pMGL25) carrying plasmids pBR322 and pYZCRP. E, crp1; h, Dcrp39; Ç, Dcrp39 (wild-type CRP); É, Dcrp39 (CRP [T158A]).
medium. To rule out this possibility, we examined gyrA-lacZ expression in SBS688 variant strain JMG158, which harbors plasmid pYZCRP158A encoding variant CRP protein CRPpc (with a T-to-A mutation at position 158 [T158A]). This protein is a mutant derivative of CRP that is defective in the transcriptional activation of class I and class II CRP-dependent promoters but not defective in DNA binding or bending (32, 33). Strain JMG158 displayed a nearly normal growth rate (Fig. 4). Therefore, the plasmid carrying variant CRPpc (T158A) was able to reverse the growth deficiency of SBS688 but not the effect of the Dcrp39 mutation on the expression of the gyrAlacZ fusion (Fig. 5). In contrast, after plasmid pYZCRP encoding the wild-type CRP was transferred to SBS688, this strain recovered both the normal growth rate and the ability to express the gyrA-lacZ fusion (Fig. 5). These results show that the observed growth deficiency in
FIG. 5. Effect of wild-type CRP and variant CRPpc (T158A) on gyrA-lacZ expression in the Dcrp39 background. Strain SBS688(pMGL25) was transformed with plasmid pBR322 (as a negative control), pYZCRP encoding wild-type CRP, and pYZCRP carrying variant CRPpc (T158A). For each transformant, overnight cultures were grown with kanamycin (40 mg/ml) and ampicillin (50 mg/ml). Cells were subcultured (1/200) in LB medium and incubated at 378C with agitation. At different intervals (30 min), aliquots were removed and cell density and b-galactosidase activity were measured. b-Galactosidase activity was compared with that of strain MC4100 (pMGL25), which was grown under the same conditions. b-Galactosidase activity was plotted versus growth (OD600). F, crp1; ■, Dcrp39; å, Dcrp39 (wild-type CRP); ç, Dcrp39 (CRP [T158A]).
the Dcrp39 mutant was not related to the downregulation of gyrA transcription. The expression of gyrA obtained in the crp mutant background was strong enough, despite its low level compared with that of the wild type, to produce levels of GyrA protein sufficient to assure the production of functional DNA gyrase. The data obtained also suggest that the growth-phasedependent induction of the gyrA promoter is mediated by CRP. This control could be mediated by a promoter(s) belonging to class I or II. It can be expected that such a strong effect of the Dcrp39 mutation on gyrA expression might have consequences on DNA topology. To evaluate these consequences, strains MC4100 (crp1) and SBS688 (Dcrp39) were transformed with reporter plasmid pACYC184 (8) and incubated at 378C in LB medium. Plasmid DNA of pACYC184 was extracted from both strains throughout the bacterial growth cycle. The linking number was measured with a chloroquine agarose gel (15, 16). Discernible differences in pACYC184 supercoiling were found after the plasmid was isolated from MC4100 or SBS688 at the period of the reentry to the growth cycle from the stationary phase. A decrease of the negative supercoiling (relaxation) of plasmid pACYC184 after it was isolated from background Dcrp39 (SBS688), compared with that of the wild-type crp (MC4100) strain, was apparent in chloroquine agarose gels (Fig. 6; compare lanes 1 to 4 with lanes 8 to 10). At later stages of the early exponential phase (at an optical density at 600 nm [OD600] of $0.2), the topoisomer distributions in both genetic backgrounds were similar (Fig. 6; compare lanes 5 to 7 with lanes 11 to 13). The results indicate that CRP may be involved in the modulation of DNA topology in the transition from the lag period to the exponential growth phase. We reassayed the b-galactosidase activity of the gyrA-lacZ fusion along this transition in a more detailed way (samples were taken every 15 min) to test the possibility of a CRP control on gyrA expression at the point of entry from the stationary phase to the growth cycle. As can be seen from Fig. 7, a decrease (twofold) in b-galactosidase activity compared with that in the wild type was observed in the Dcrp39 (SBS688) strain. As was expected, the introduction of plasmid pYZCRP (containing the wild-type crp gene) in the crp mutant strain was capable of reversing this phenomenon. This reversal was not observed in the presence of the crpT158A variant. These results suggest that the effect of CRP on DNA topology might be associated with the CRP-dependent regulation of gyrA expres-
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FIG. 7. gyrA-lacZ response to nutritional upshift and the effect of CRP. Overnight cultures of strains MC4100(pMGL25, pBR322), SBS688(pMGL25, pBR322), SBS688(pMGL25, pYZCRP), and SBS688(pMGL25, pYZCRP158A) were grown with kanamycin (40 mg/ml) and ampicillin (50 mg/ml). The cells were subcultured (1/400) in LB medium and incubated at 378C with agitation. At different intervals (15 min), aliquots were removed and cell density and b-galactosidase activity were measured. F, crp1; ■, Dcrp39; å, Dcrp39 (wild-type CRP); ç, Dcrp39 (CRP [T158A]).
sion. Interestingly, under the conditions of our experiments, the CRP mutant had a consistently slightly longer lag period (15 min) before growth resumed compared with that for the crp1 strain. It is tempting to speculate that this physiological effect is possibly a consequence of the alteration of DNA topology observed with the strain lacking CRP. In conclusion, the results obtained in this work strongly suggest that (i) the expression of gyrA in E. coli is growth phase dependent and under the positive control of the cAMP-CRP complex, (ii) this control seems to be mediated by one or more CRP-dependent promoters of class I or II, and (iii) there is an alteration of superhelical density upon reentry from the stationary phase to the growth cycle in the Dcrp39 mutant. Although more experimental data are necessary to understand the precise mechanism(s) by which the cAMP-CRP complex controls gyrA expression as well as the physiological role of this control, this work offers new perspectives of interest for further studies of CRP-mediated gene expression. We thank J. C. Pe´rez-Dı´az for his continual support and always helpful discussions and L. de Rafael for his corrections of our English. Strain SBS688 (Dcrp39) was obtained from P. L. Boquet via F. Moreno. The pYZCRP plasmid derivatives carrying wild-type CRP and variant mutant CRPpc (T158A) were obtained from R. Ebright via J. Pe´rez-Martı´n. Plasmids pSLS447 and pKOK6 were obtained from K. Bott and V. de Lorenzo, respectively. REFERENCES 1. Balke, V. L., and J. D. Gralla. 1987. Changes in the linking number of supercoiled DNA accompany growth transitions in Escherichia coli. J. Bacteriol. 169:4499–4506. 2. Barber, A. M., and V. B. Zhurkin. 1990. CAP binding sites reveal pyrimidinepurine pattern characteristic of DNA bending. J. Biomol. Struct. Dyn. 8:213– 232. 3. Barber, A. M., V. B. Zhurkin, and S. Adhya. 1993. CRP-binding sites: evidence for two structural classes with 6-bp and 8-bp spacers. Gene 130:1–8. 4. Berg, O. G., and P. H. von Hippel. 1988. Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites. J. Mol. Biol. 200:709–723. 5. Bolivar, R., R. Rodrı´guez, P. J. Greene, M. Betlach, H. Heyneker, H. W. Boyer, J. Crosa, and S. Falkow. 1977. Construction and characterization of new cloning vehicles. Gene 2:95–113.
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