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Journal of Bacteriology, June 2002, p. 3044-3052, Vol. 184, No. 11
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.11.3044-3052.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Glycerol-3-Phosphate-Induced Catabolite Repression in Escherichia coli

Tanja Eppler,¹ Pieter Postma,² Alexandra Schütz,³ Uwe Völker,³ and Winfried Boos^1*

Department of Biology, University of Konstanz, 78457 Konstanz,¹ Department of Microbiology, University of Marburg, 35032 Marburg, Germany,³ Swammerdam Institute for Life Sciences, University of Amsterdam, 1018 WS Amsterdam, The Netherlands²

Received 12 December 2001/ Accepted 11 March 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIA^Glc, as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIA^Glc phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIA^Glc. Isopropyl-ß-D-thiogalactopyranoside-induced overexpression of EIIA^Glc did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIA^Glc. A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIA^Glc is controlled by G3P and other phosphorylated sugars such as D-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and D-tagatose-1,6-bisphosphate aldolase.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Catabolite repression refers to the reduction in transcription of sensitive operons that is caused by certain carbon sources in the medium, most prominently by glucose (glucose effect). In Escherichia coli, phosphoenolpyruvate-dependent phosphotransferase system (PTS)-mediated uptake of glucose is crucial for this effect. The model largely accepted for E. coli focuses on the level of cyclic AMP (cAMP) synthesized by the membrane-bound adenylate cyclase (29, 30, 38). EIIA^Glc, an intermediate in the phosphorylation cascade of the PTS for glucose, in its phosphorylated form is thought to stimulate adenylate cyclase. The basal level of adenylate cyclase activity would be present in the absence of EIIA^Glc or in the presence of unphosphorylated EIIA^Glc. As a consequence, CAP, the catabolite activator protein (or cAMP receptor protein [CRP]) that is needed for the transcription of sensitive operons (22) is linked in its activity to the PTS (31). The glucose PTS is also responsible for inducer exclusion, i.e., inhibition of the different transport systems by unphosphorylated EIIA^Glc (31).

Even though participation of the PTS in catabolite repression and inducer exclusion in E. coli has been documented very well, the effects of non-PTS sugars are less clear. Thus, glucose-6-phosphate or gluconate exerts strong catabolite repression although it is not transported by the PTS. Recent studies on catabolite repression caused by non-PTS sugars concluded that it is the amount of CAP, as well as cAMP, that is altered in response to non-PTS sugars (14, 18, 34). In these studies, it was not determined whether or not metabolism is required for catabolite repression to occur.

Glucose-6-phosphate and other non-PTS carbon sources were also shown to cause inducer exclusion by influencing the phosphorylation state of EIIA^Glc, indicating that substrates can cause dephosphorylation of EIIA^Glc phosphate (EIIA^Glc-P) without being transported by the PTS. The dephosphorylation of EIIA^Glc-P brought about by glucose-6-phosphate, melibiose, lactose, and arabinose metabolism was nearly as strong as that of the PTS sugars tested. Other carbon sources (like glycerol, galactose, maltose, etc.) caused weaker dephosphorylation (12, 13). For glucose-6-phosphate, the authors showed that metabolism of the sugar phosphate is necessary for the dephosphorylation of EIIA^Glc-P and it was concluded that the phosphoenolpyruvate/pyruvate ratio determines the phosphorylation state of EIIA^Glc. Thus, inducer exclusion by non-PTS sugars could be explained by the temporary dephosphorylation of EIIA^Glc. Consequently, in the absence of metabolism, for instance, in a pgi mutant, glucose-6-posphate does not cause inducer exclusion. Thus, one might conclude that when metabolism of the non-PTS sugars is prevented, neither catabolite repression nor inducer exclusion should occur. However, this conclusion does not hold for the catabolite repression exerted by glycerol or glycerol-3-phosphate (G3P), a phenomenon that we recently studied by using the E. coli maltose system as a model (11). In that study, it was shown that glycerol has to be phosphorylated to G3P in order to exert catabolite repression but that further metabolism is not necessary.

The maltose system of E. coli (2) is a typical catabolite-sensitive regulon. It consists of 10 genes encoding proteins for the uptake and metabolism of maltose and maltodextrins. These genes are specifically controlled by MalT, the positive activator of all mal genes (32). Expression of malT, as well as of some, but not all, mal genes, is under control of the cAMP-CAP system (7, 8). Since malT expression is independent of maltose or maltodextrins as an inducer, its expression is independent of the effects of inducer exclusion, allowing the testing of catabolite expression exclusively. But malT expression is controlled not only by cAMP-CAP-dependent catabolite repression but also by Mlc, a global negative regulator of carbohydrate metabolism (9). In this paper, we present evidence that catabolite repression of malT exerted by glycerol or G3P involves the cAMP-CAP system, as well as EIIA^Glc of the PTS. Metabolism of G3P is not required, and dephosphorylation of EIIA^Glc is not the determining factor. Instead, we conclude that it is EIIA^Glc-P-dependent stimulation of adenylate cyclase that is inhibited by G3P or other phosphorylated sugars, such as glucose-6-phosphate. Two-dimensional (2D) gel electrophoresis was used to identify some of the proteins that are significantly affected by glycerol-mediated repression.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and growth conditions. The bacterial strains used in this study are listed in Table 1. P1 vir general transductions were done as described by Miller (26). Strain ET160 was constructed by mini-Tn 10::Cam mutagenesis (21) of strain ET18. Its isolation was based on its insensitivity to repression by glycerol. The E. coli K-12 strains were grown at 37°C in Tryptone broth (TB) or TB buffered with phosphate-based minimal medium (26). For inoculation, overnight cultures in TB medium were routinely used. If necessary, kanamycin (100 µg/ml), tetracycline (5 µg/ml), or chloramphenicol (15 µg/ml) was added.

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TABLE 1. Strains and plasmids used in this study

Enzymatic activity assays. For enzymatic activity assays, overnight cultures were permeabilized and ß-galactosidase activity was measured as previously described (26). Specific activity is given in micromoles of substrate per minute (units) per milligram of protein. The values given in the tables represent averages of two independent measurements, each done in duplicate. The individual measurements did not vary by more than 10%.

Preparation of cell extract for 2D gel electrophoresis. E. coli K-12 strain MC4100 was routinely grown at 37°C in TB with or without 0.4% (vol/vol) glycerol. Cultures were inoculated with an overnight culture grown in TB, and after 18 h of shaking, the bacteria were harvested by centrifugation. After washing with TE (10 mM Tris, 1 mM EDTA, pH 7.5), the pellet was resuspended in TE and cells were disrupted by being passed through a French press. After ultracentrifugation at 4°C and 100,000 x g for 60 min, the protein content of the supernatant fraction was determined as described by Bradford (3).

2D gel electrophoresis. For isoelectric focusing (IEF), proteins were solubilized in a rehydration solution containing 8 M urea, 2 M thiourea, 2% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS), 28 mM dithiothreitol (DTT), 1.3% (vol/vol) Pharmalytes (pH 3 to 10), and bromophenol blue. After rehydration for 24 h under low-viscosity paraffin oil, IPG strips (Amersham Pharmacia Biotech) covering a pH range of 4 to 7 or 3 to 10 were subjected to IEF with the following voltage-time profile: a linear increase from 0 to 500 V for 1,000 Vh, 500 V for 2,000 Vh, a linear increase from 500 to 3,500 V for 10,000 Vh, and a final phase of 3,500 V for 35,000 Vh for the pH range of 4 to 7 and 20,000 Vh for the pH range of 3 to 10. After IEF, the individual strips were consecutively incubated, for 15 min each time, in equilibration solutions A and B (50 mM Tris-HCl [pH 6.8], 6 M urea, 30% [vol/vol] glycerol, and 4% [wt/vol] sodium dodecyl sulfate [SDS] completed with DTT at 3.5 mg/ml [solution A] or iodoacetamide at 45 mg/ml instead of DTT [solution B]). In the second dimension, proteins were separated on SDS-10 to 12.5% polyacrylamide gels with the Investigator System (Genomic Solutions) at 2 W/gel. For routine use, proteins were stained with PhastGel BlueR in accordance with the manufacturer's (Amersham Pharmacia Biotech) instructions. After scanning, analysis of the 2D polyacrylamide gel electrophoresis (PAGE) images was done with the Melanie3 software package (Bio-Rad). Separate gels were analyzed under each condition, and only spots displaying the same pattern under all of the conditions were labeled.

Protein identification by peptide mass fingerprinting. Protein spots were excised from stained 2D gels, destained, and digested with trypsin (Promega). Peptides were extracted as described by Otto et al. (28). They were purified with C₁₈ tips in accordance with the manufacturer's (Millipore Inc.) instructions and eluted with 50% acetonitrile-0.1% (vol/vol) trifluoroacetic acid. Peptide solutions were mixed with an equal volume of saturated -cyano-3-hydroxycinnamic acid solution in 50% acetonitrile-0.1% (vol/vol) trifluoroacetic acid and applied to a sample template for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Peptide masses were determined in the positive ion reflector mode in a Voyager DE RP mass spectrometer (PerSeptive Biosystems) with internal calibration. Mass accuracy was usually in the range of 10 to 50 ppm. Peptide mass fingerprints were compared to databases by using the program MS-Fit (http://prospector.ucsf.edu). The searches considered oxidation of methionine, pyroglutamic acid formation at the N-terminal glutamine, and modification of cysteine by carbamidomethylation, as well as partial cleavage leaving one internal cleavage site.

Determination of the phosphorylation state of EIIA^Glc. The phosphorylation state of EIIA^Glc was determined as described by Hogema et al. (13). Cells were grown in phosphate-based minimal medium-buffered TB with or without 0.4% (vol/vol) glycerol or G3P, respectively. A 0.2-ml volume of logarithmic-phase cells (optical density at 600 nm, 0.5) was mixed with 20 µl of 10 M NaOH and vortexed for 10 s. After addition of 1 ml of cold ethanol and 180 µl of 3 M sodium acetate (pH 5.2), the samples were frozen at -70°C. The extracts were thawed and centrifuged in an Eppendorf microcentrifuge at 4°C. The pellets were washed in ethanol and resuspended in 200 µl of sample buffer. Samples (10 µl) were loaded onto SDS-15% polyacrylamide gels and electrophoresed. The proteins were transferred to nitrocellulose membranes and probed with polyclonal anti-EIIA^Glc antibody, followed by a horseradish peroxidase-conjugated secondary antibody. For detection, an ECL kit was used in accordance with the method of the manufacturer (Amersham Pharmacia).

Top Abstract Introduction Materials and Methods Results Discussion References

 
2D gel analysis of cells grown in TB with and without glycerol. To obtain an overview of the impact of glycerol-G3P-mediated catabolite repression, strain MC4100 was grown in TB with and without 0.4% glycerol. Under these conditions, catabolite repression by glycerol on a malT-lacZ fusion can be monitored (see Tables 4 to 7). Crude protein extracts of these outgrown cultures were separated by 2D gel electrophoresis (Fig. 1A, pHs 4 to 7, and B, pHs 3 to 10). Thirty protein spots, indicated by filled arrowheads, were produced in smaller amounts when the strain was grown in the presence of glycerol and are therefore subject to glycerol catabolite repression. Of these proteins, 12 prominent spots were identified by peptide mass fingerprinting and are listed in Table 2. Among these, proteins known to be subject to PTS-mediated catabolite repression can be found, including the maltose and galactose binding proteins, as well as enzymes taking part in the tricarboxylic acid (TCA) cycle. More surprising was the finding that the glutamine and oligopeptide binding proteins are also prominently subject to repression. The DNA control region of their operons does not give any hint of a binding site for the cAMP-CAP complex. The above-described analysis demonstrates that glycerol-mediated repression is pleiotropic and not restricted to the peculiarities of the maltose system. Twenty-seven spots were present more prominently in the glycerol-induced culture. Eighteen of those were analyzed and represent 12 different proteins. Ten of these were identified by peptide mass fingerprinting and are listed in Table 3. Two of them were identified as GroEL and DnaK by their correlation to Swiss2D-Page (http://www.expasy.ch/ch2d). Of the 10 proteins identified by peptide mass fingerprinting, TktA, FbaA, YeaD, and PflB appear as at least two isoforms on 2D SDS-PAGE. Of the 12 proteins identified as present in increased amounts after growth in the presence of glycerol, only one, GlpK, is part of the glp regulon. Attempts to find operator binding sites typical for GlpR, the negative repressor of the glp regulon, in front of the other nine genes only revealed the presence of a "half" palindromic consensus binding site for GlpR (39) in front of the tktA gene.

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TABLE 4. ß-Galactosidase specific activity of a malT-lacZ fusion in some glycerol mutants

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TABLE 7. Glucose-6-phosphate mediates repression by galactose

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FIG. 1. Influence of glycerol on the protein profile of E. coli MC4100. E. coli K-12 strain MC4100 was grown in TB or TB with glycerol for 18 h. Crude protein extracts were prepared and separated by 2D gel electrophoresis. Each gel was loaded with 300 µg of protein extract. After staining, the images were recorded and analyzed. Protein spots induced or repressed by glycerol are marked by open and filled triangles, respectively. Proteins identified by peptide mass fingerprinting are indicated. (A) Images of 2D gels covering a pH range of 4 to 7. (B) Sections of the alkaline part of 2D gels covering a pH range of 3 to 10. Mr is given in thousands.

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TABLE 2. Overview of glycerol-repressed proteins identified by peptide mass fingerprint matching

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TABLE 3. Overview of glycerol-induced proteins identified by peptide mass fingerprint matching

Titration of EIIA^Glc by glycerol kinase is not the cause of glycerol-mediated catabolite repression. In this report, we propose that it is the interference of G3P with the stimulation of adenylate cyclase by EIIA^Glc-P that causes catabolite repression. A plausible alternative explanation was that glycerol kinase, together with its product, G3P (or with glycerol), could bind to and titrate the unphosphorylated form of EIIA^Glc, thus reducing the amount of EIIA^Glc-P available for the stimulation of adenylate cyclase. This phenomenon, called inducer exclusion, occurs in cells induced for the glp system (33) when glucose is added. One GlpK tetramer is capable of binding four EIIA^Glc molecules, as deduced from the crystal structure (17). To determine whether this titration of EIIA^Glc by glycerol kinase could be the reason for glycerol-mediated repression in a glpD mutant, we measured the expression of a malT-lacZ fusion in a glpD glpK double mutant. Table 4 shows that this double mutation does not prevent repression by G3P. Moreover, EIIA^Glc overexpression in a glpFKX⁺ strain still allows twofold repression of malT-lacZ by G3P in Bre1161 (glp⁺), ET18 (glpD), or ET16 (glpK). Table 5 shows the ß-galactosidase activity of the malT-lacZ fusion strains harboring isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible and plasmid-encoded EIIA^Glc that were grown in TB and TB-G3P with increasing IPTG concentrations (for the induction of EIIA^Glc). As judged by Western blot analysis, at 30 µM IPTG, the amount of EIIA^Glc produced was about 20 times the chromosomal level. The data in Table 5 show that G3P in the growth medium maintained repression despite high levels of EIIA^Glc. We also tested two glpK alleles that had been characterized as resistant to EIIA^Glc and those that are resistant to inhibition by fructose-1,6-diphosphate (15). In each case, repression of malT-lacZ expression by glycerol and G3P was unchanged. This demonstrated that glycerol kinase itself is not involved and excluded the possibility that the titration of EIIA^Glc by GlpK is the basis of G3P-mediated repression. However, the observation that overexpression of EIIA^Glc, to some extent, lessened the repression by G3P points to an interaction of EIIA^Glc-P with adenylate cyclase as a target for G3P inhibition.

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TABLE 5. Influence of EIIAGlc overexpression on repression of malT-lacZ by G3P

G3P causes only weak dephosphorylation of enzyme EIIA^Glc-P. It has been suggested that non-PTS compounds like glucose-6-phosphate may elicit repression by altering the ratio of phosphorylated to unphosphorylated EIIA^Glc and, consequently, would cause catabolite repression by reducing adenylate cyclase activity that is no longer stimulated maximally by EIIA^Glc-P (13). The authors of that report used a method that makes it possible to separate the phosphorylated and unphosphorylated forms of EIIA^Glc by SDS-gel electrophoresis (27). In order to test the phosphorylation state of EIIA^Glc in response to the presence of glycerol or G3P in the growth medium, we investigated several isogenic mutants defective in glycerol metabolism (Fig. 2A). In wild-type strain Bre1161 grown in TB, EIIA^Glc was completely phosphorylated. There was weak dephosphorylation in the presence of glycerol and less in the presence of G3P. The glpD mutant, defective in aerobic G3P dehydrogenase, already showed small amounts of unphosphorylated EIIA^Glc in the absence of exogenous glycerol or G3P that were not increased by the addition of glycerol or G3P. There was weak dephosphorylation in the TB-G3P culture of the glycerol kinase (glpK) mutant by G3P but not by glycerol. Figure 2B shows the corresponding repression of malT-lacZ in these strains. The comparison shows that G3P does cause a little dephosphorylation of EIIA^Glc-P; however, the reduction in the levels of EIIA^Glc-P (and thus, the reduction in the activity of adenylate cyclase) is too small to account for the repression observed. In particular, in the glpD mutant, the proportion of the unphosphorylated to the phosphorylated form of EIIA^Glc does not coincide with the degree of repression. The conversion of a small proportion of EIIA^Glc-P to the unphosphorylated form upon addition of glycerol or G3P was fast, already appearing 0.5 min after the addition of G3P to TB-grown Bre1161 cells (Fig. 3A, first five lanes), with no further significant dephosphorylation thereafter. In TB-G3P-grown cells, further addition of G3P had no effect (Fig. 3A, last five lanes). In a glpD mutant, weak dephosphorylation of the enzyme EIIA^Glc was observed from the beginning on, whether the cells were grown in the presence of G3P or not, and there was no change after G3P was added (Fig. 3B).

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FIG. 2. Determination of the phosphorylation state of the enzyme IIAGlc in the presence of glycerol or G3P. (A) Isogenic strains Bre1161 (lanes 1 to 3), ET18 (glpD glpR; lanes 4 to 6), und ET16 (glpF glpK; lanes 7 to 9) were grown in TB alone (lanes 1, 4, and 7), in TB with 0.4% glycerol (lanes 2, 5, and 8), or in TB with 0.4% G3P (lanes 3, 6, and 9). Samples were taken during logarithmic growth at an optical density at 600 nm of 0.5. EIIAGlc and EIIAGlc-P were detected by Western blotting with EIIAGlc-specific antibodies. (B) ß-Galactosidase specific activity of MalT-LacZ in micromoles of substrate per minute and milligram of protein. wt, wild type.

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FIG. 3. Time-dependent dephosphorylation of EIIAGlc-P by the addition of G3P. Isogenic malT-lacZ fusion strains Bre1161 (A) and ET18 (glpD glpR) (B) were grown at 37°C in TB (first five lanes) or in TB with 0.4% G3P (last five lanes). At time 0, 0.2% G3P was added. Samples were taken at the indicated time points and analyzed by SDS-PAGE, followed by Western blotting with anti-EIIAGlc-P antibodies. The positions of EIIAGlc-P and EIIAGlc are indicated.

In contrast, the presence of glucose in the growth medium leads to complete dephosphorylation of EIIA^Glc-P (13). Depletion of EIIA^Glc-P in cells exposed to glucose has been the basis for the model of glucose-mediated repression in E. coli (31). This explanation cannot not be correct for G3P-mediated catabolite repression.

Influence of G3P on the EIIA^Glc-P-adenylate cyclase interaction. Adenylate cyclase is thought to contain a regulatory C-terminal domain that functions as the interaction site for stimulation by EIIA^Glc-P. A strain harboring an adenylate cyclase form lacking the last 48 C-terminal amino acids (35) appeared, to some extent, resistant to glucose. malT-lacZ expression in this mutant is less sensitive to glucose than in the strain harboring wild-type adenylate cyclase (Table 6). Also, malT-lacZ expression is resistant to repression by exogenous G3P. As expected, deletion of EIIA^Glc-P in this mutant abolishes the sensitivity of malT-lacZ expression to G3P, as well as its sensitivity to glucose. This is consistent with the notion that the EIIA^Glc-P-mediated stimulation of adenylate cyclase is the major target of repression by G3P.

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TABLE 6. malT-lacZ expression in cya* (800-848) strains

Repression of malT-lacZ expression by glucose-6-phosphate and galactose. Catabolite repression by non-PTS compounds has been observed for some time, glucose-6-phosphate being one of the most effective examples. The availability of a malT-lacZ fusion offered an opportunity to measure catabolite repression by glucose-6-phosphate free of inducer exclusion. The data are shown in Table 7. Glucose-6-phosphate strongly inhibited malT-lacZ. The introduction of a pgi mutation preventing the conversion of glucose-6-phosphate to fructose-6-phosphate and further glycolysis did not abolish the strong repression by glucose-6-phosphate. These findings are not in conflict with a recent report by Kimata et al. (20), who reported that a mutation in pgi prevented catabolite repression by glucose. In this case, loss of carbon flow through the glycolytic pathway led to the degradation of ptgG mRNA, thus preventing glucose uptake.

We also observed that the presence of galactose repressed the expression of malT-lacZ. In order to identify the inhibitory compound responsible, we introduced several mutations and tested their effects on the repression of malT-lacZ by galactose (Table 7). A mutation in galT encoding the enzyme that forms UDP-galactose and glucose-1-phosphate from galactose-1-phosphate and UDP-glucose is insensitive to galactose. Thus, galactose-1-phosphate is unable to exert catabolite repression. Likewise, when the transformation of glucose-1-phosphate to glucose-6-phosphate was prevented by a pgm mutation, allowing the accumulation of glucose-1-phosphate derived from external galactose, the strain was still insensitive to galactose. Thus, glucose-1-phosphate also cannot exert catabolite repression. However, when metabolism of glucose-6-phosphate to fructose-6-phosphate was prevented by a pgi mutation, repression by galactose was still observed. Thus, galactose-induced catabolite repression must be mediated by glucose-6-phosphate.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, non-PTS sugar-mediated catabolite repression was analyzed. Our model system used G3P as the repression-eliciting compound and malT as a representative of a catabolite repression-sensitive gene (8). The latter was chosen for its independence of inducer exclusion. We chose TB as a low catabolite repression medium and measured repression upon the addition of glycerol, G3P, and other compounds. Even though pH alterations do occur in this weakly buffered medium, addition of phosphate buffer to keep the pH constant over the entire growth period did not alter the transcriptional repression by G3P (11). It was important to be sure that assaying the effect of carbon sources on malT expression does indeed reflect catabolite repression and is not a peculiarity of the maltose system. Thus, we studied the glycerol-mediated repression of the majority of the E. coli proteins. The 2D gel electrophoretic analysis shown in Fig. 1 allowed us to conclude that at least 30 of the proteins visualized by this technique were reduced when the cells were grown to stationary phase in the presence of glycerol. Twelve of these proteins were identified by mass fingerprinting. Thus, the glycerol effect is not a peculiarity seen in the regulation of the maltose system but is pleiotropic in nature. As expected, among the proteins subject to this pleiotropic effect, we found those that are typically encoded by catabolite-repressible genes, such as enzymes of the TCA cycle and sugar binding proteins. Surprisingly, genes encoding the glutamine binding protein or the oligopeptide binding protein were also among those repressed, even though the promoters of their genes are not known to contain CAP binding sites. Possibly, the reduction in the amount of these proteins is indirect, due to a cAMP-CAP-dependent regulator. Also surprising was the number of proteins induced by glycerol. The only known glycerol-inducible genes of E. coli are the 12 glp genes that are under negative control by the GlpR repressor (23). Not all of these glp gene products would be seen. Membrane proteins were removed prior to gel electrophoresis by high-speed centrifugation, and proteins produced in small amounts would not be visible. Of the 27 proteins that were induced by glycerol, we could only identify one, GlpK, that genuinely belongs to the glp regulon. Nine additional proteins were identified by protein mass fingerprinting. The proteins identified mostly belong to the pathways for glycolysis and gluconeogenesis. It is unlikely that they are regulated by GlpR, the central regulator of the glp regulon, since no corresponding operator site was found in the control regions of their genes. They were probably induced by the metabolic products of glycerol metabolism. A surprise was the large amount of PflB, pyruvate formate lyase, present in the glycerol-induced culture. This enzyme is known to be induced only under anaerobic conditions. Possibly, the presence of the glycerol-metabolizing enzymes exerts an effective drain of available oxygen since in E. coli, glycerol can only be utilized oxidatively. We also noticed the clear induction of heat shock proteins GroEL and DnaK. We have no explanation for their appearance.

Previously, we had established that EIIA^Glc is necessary for glycerol-mediated repression and that glycerol has to be phosphorylated to G3P but that no further metabolism is needed. Since PTS sugar-mediated catabolite repression is based on the dephosphorylation of EIIA^Glc-P, we analyzed whether or not EIIA^Glc would undergo a dramatic change in its degree of phosphorylation. In contrast to the effect of glucose, this was not the case with glycerol or G3P. Clearly, addition of glycerol or G3P did result in the formation of small amounts of dephosphorylated EIIA^Glc that was completed within 1 min. However, the small amount of dephosphorylated EIIA^Glc was not related to the degree of repression. This was seen very clearly in the glpD mutant, which is unable to metabolize G3P. Here, dephosphorylated EIIA^Glc is already present prior to the addition of G3P and its amount is not further increased by the addition of exogenous G3P. However, the addition of G3P reduces malT-lacZ expression. glpD mutants do contain elevated levels of endogenous G3P, even when grown in the absence of glycerol or G3P. This is due to the function of dihydroxyacetone phosphate dehydrogenase providing G3P from dihydroxyacetone phosphate for phospholipid biosynthesis. Endogenous G3P, not removed by GlpD, must be the cause for the small amounts of dephosphorylated EIIA^Glc observed. It is unclear how dephosphorylation of EIIA^Glc by G3P comes about.

Since the minute reduction in the amount of EIIA^Glc-P upon addition of glycerol or G3P cannot account for repression (by reduction of stimulation of adenylate cyclase activity), what are other options to explain repression? Chagneau et al. (6) have recently concluded that the induction of glycerol kinase is responsible for repression. In their scheme, glycerol kinase would interact with dephosphorylated EIIA^Glc, removing EIIA^Glc-P from the equilibrium and thus reducing EIIA^Glc-P-mediated stimulation of adenylate cyclase. They reject the possibility that it is the activity of glycerol kinase (in forming G3P from glycerol) that causes repression, since repression by IPTG-induced glycerol kinase was also observed in the absence of exogenous glycerol. We feel that this mode of action is rather unlikely. As we have shown, the amount of unphosphorylated EIIA^Glc that would be able to react with glycerol kinase is very small and the effective concentration (for the stimulation of adenylate cyclase) of EIIA^Glc-P is hardly reduced by glp induction. Also, in the glycerol kinase-EIIA^Glc interaction scheme, overproduction of EIIA^Glc should eliminate glycerol-induced repression. We have shown here that this is not the case. Also, the use of a glpK allele whose product is insensitive to EIIA^Glc (15) still showed the same level of repression in response to glycerol or G3P. Thus, our explanation for the observation of Chagneau et al. (6) is that glycerol kinase-mediated formation of G3P causes repression. Their observation that repression by glycerol kinase also occurs in the absence of exogenous glycerol finds its explanation in the endogenous formation of glycerol that has been established for some time (37). Also, the interaction of glycerol kinase with EIIA^Glc that is the basis for their explanation requires binding of glycerol to glycerol kinase (17). Thus, even if interaction between these two proteins does occur to a minor extent under conditions of repression (15), it always necessitates the presence of glycerol that will invariably lead to the formation of G3P. The last argument is that glycerol-mediated repression requires glycerol kinase (for the formation of G3P) but repression by G3P does not and is the same in the wild type, a glpK mutant, and a glpK glpD mutant.

Our model to explain G3P-mediated catabolite repression is based on the knowledge that adenylate cyclase and EIIA^Glc-P, as well as G3P, are necessary players. In addition, we have to conclude that the amount of EIIA^Glc-P cannot be the regulating principle, as it is in glucose-mediated repression. Therefore, we propose that the stimulation of adenylate cyclase by EIIA^Glc-P is the target of repression by G3P. We postulate that G3P inhibits EIIA^Glc-P-mediated stimulation of adenylate cyclase, thus lowering the cAMP concentration. A recent publication described the catabolite repression of citrate fermentation genes in Klebsiella pneumoniae by glycerol and gluconate, among other compounds (25). The authors of that report also noted the discrepancy between the state of dephosphorylation of EIIA^Glc and the repressing effect of non-PTS sugars while maintaining the importance of the cAMP-CAP system in this type of catabolite repression.

What about other non-PTS sugars that elicit catabolite repression? Using glucose-6-phosphate, we found a strong repression of malT-lacZ that appears to be independent of glucose-6-phosphate glycolytic metabolism. Analysis of galactose-mediated repression revealed that the causative agent again was glucose-6-phosphate, whose further metabolism was not required for repression (Fig. 4 shows the galactose pathway).

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FIG. 4. Galactose mediates repression by the formation of glucose-6-phosphate (Glucose-6-P). The effects of the galT, pgm, and pgi mutations on the formation of glucose-6-phosphate are indicated.

Where do these sugar phosphates act as inhibitors? They could fit into a binding site in EIIA^Glc-P, altering its ability to stimulate adenylate cyclase. Alternatively, these sugar phosphates may mimic the phosphorylated histidine in EIIA^Glc-P, preventing the proper interaction with adenylate cyclase. A challenging biochemical approach involving adenylate cyclase and EIIA^Glc-P is necessary to elucidate the molecular mechanism of this type of repression.

 
We gratefully acknowledge the receipt of bacterial strains from Hiroji Aiba, Donald W. Pettigrew, and Erhard Bremer.

This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

 
* Corresponding author. Mailing address: Department of Biology, University of Konstanz, 78457 Konstanz, Germany. Phone: 49 7531 88 2658. Fax: 49 7531 88 3356. E-mail: Winfried.Boos{at}uni-konstanz.de.

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Journal of Bacteriology, June 2002, p. 3044-3052, Vol. 184, No. 11
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.11.3044-3052.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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