Accumulation of Glucose 6-Phosphate or Fructose 6-Phosphate Is Responsible for Destabilization of Glucose Transporter mRNA in Escherichia coli*

Teppei Morita, Waleed El-Kazzaz, Yuya Tanaka, Toshifumi Inada, and Hiroji AibaDagger

From the Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan

Received for publication, January 8, 2003, and in revised form, February 10, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previously we found that a mutation in either pgi or pfkA, encoding phosphoglucose isomerase or phosphofructokinase A, respectively, facilitates degradation of the ptsG mRNA in an RNase E-dependent manner in Escherichia coli (1). In this study, we examined the effects of a series of glycolytic genes on the degradation of ptsG mRNA and how the mutations destabilize the ptsG mRNA. The conditional lethal mutation ts8 in fda, encoding fructose-1,6-P2 aldolase just downstream of pfkA in the glycolytic pathway, caused the destabilization of ptsG mRNA at the nonpermissive temperature. Mutations in any other gene did not destabilize the ptsG mRNA; rather, they reduced the ptsG transcription mainly by affecting the cAMP level. The rapid degradation of ptsG mRNA in mutant strains was completely dependent upon the presence of glucose or any one of its compounds, which enter the Embden-Meyerhof glycolytic pathway before the block points. A significant increase in the intracellular glucose-6-P level was observed in the presence of glucose in the pgi strain. An overexpression of glucose-6-phosphate dehydrogenase eliminated both the accumulation and the degradation of ptsG mRNA in the pgi strain. In addition, accumulation of fructose-6-P led to the rapid degradation of ptsG mRNA in a pgi pfkA mutant strain lacking glucose-6-P. We conclude that the RNase E-dependent destabilization of ptsG mRNA occurs in response to accumulation of glucose-6-P or fructose-6-P.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In bacteria, a number of sugars represented by glucose are transported into the cells coupled with their phosphorylation by the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS)1 (2-4), whereas the translocation of some other sugars such as lactose is catalyzed by non-PTS transport systems. In either case, the incorporated sugars are metabolized primarily by the Embden-Meyerhof glycolytic pathway and by the pentose phosphate pathway to produce numerous intermediary metabolites as well as energy in cells (5). The PTS in Escherichia coli consists of two common cytoplasmic proteins, enzyme I and HPr (histidine-containing protein of the PTS), as well as an array of sugar-specific enzyme II complexes (EIIs). The glucose-specific EII (glucose transporter) consists of cytoplasmic protein IIAGlc and membrane receptor IICBGlc encoded by crr and ptsG, respectively. The phosphoryl group from phosphoenolpyruvate is transferred sequentially to enzyme I, HPr, the EIIs, and finally glucose as it is translocated across the membrane.

In addition to sugar transport and phosphorylation, the PTS plays important regulatory roles in a variety of cellular activities. This is particularly evident for the glucose-specific PTS. For example, IIAGlc regulates both the transport of non-PTS sugars and the activity of adenylate cyclase depending on its phosphorylation state (2-4). The former process, called inducer exclusion, is fully responsible for the glucose-lactose diauxie that is a prototype of catabolite repression (6, 7). A striking recent discovery regarding the regulatory function of PTS is that IICBGlc, depending on its phosphorylation state, interacts with Mlc to modulate the cellular localization and activity of this global repressor protein (8-10).

The expression of the ptsG gene encoding IICBGlc is regulated by two global control systems at the level of transcription initiation (7, 11, 12). First, it is under positive control by CRP-cAMP; hence, the ptsG expression is absolutely dependent on this complex. Second, the transcription of the ptsG gene is negatively regulated by a global repressor, Mlc. Recent studies have established that external glucose induces ptsG transcription by modulating the Mlc-mediated regulatory pathway (7, 11, 12). When glucose is transported into the cell, IICBGlc is dephosphorylated and binds Mlc, resulting in the sequestration of Mlc at the membrane (8-10). Another unexpected discovery regarding the regulation of ptsG expression is that a mutation in either pgi or pfkA, encoding glycolytic enzyme phosphoglucose isomerase or phosphofructokinase A, respectively, leads to a rapid degradation of ptsG mRNA and that RNase E is responsible for the degradation of ptsG mRNA (1). Thus, ptsG expression also is regulated at the level of mRNA degradation, presumably in response to the glycolytic flux. However, it remains unclear how the glycolytic flux is involved in the destabilization of ptsG mRNA, and little is known about the mechanism of the stimulation of ptsG mRNA degradation except that RNase E is a major player in this process.

In this study, we first investigated whether mutations in glycolytic genes other than pgi and pfkA affect the stability of ptsG mRNA. We found that the conditional lethal mutation ts8 in fda, encoding fructose-1,6-P2 aldolase (Fda) (13), stimulates degradation of ptsG mRNA but that mutations in any other glycolytic gene did not affect ptsG mRNA degradation. Then we addressed the question of how the mutations destabilize ptsG mRNA. The degradation of ptsG mRNA in mutant cells occurred only in the presence of glucose or any one of the glycolytic intermediates upstream of the block. More specifically, the degradation of ptsG mRNA was associated with elevated levels of glucose-6-P and/or fructose-6-P. We propose that accumulation of glucose-6-P or fructose-6-P is responsible for ptsG mRNA degradation. The physiological relevance of this novel feedback regulatory system at the post-transcriptional step is discussed below.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains, Plasmids, and Growth Conditions-- The bacterial strains used in this study are listed in Table I. A series of disruption mutants were constructed by the one-step gene inactivation protocol that is based on the high efficiency of the phage lambda  Red recombinase (14). The FLP recognition target-flanked resistance gene was eliminated by using an FLP expression plasmid, pCP20 (14). To transfer the ts8 fda allele to other backgrounds by P1 phage, the cat gene was inserted 2.7 kb downstream of the chromosomal ts8 fda gene in CAG417. IT1568 carrying a frameshift mutation in the mlc gene was isolated spontaneously. The zwf gene including the promoter region was amplified by PCR using primers 5'-CCCAAGCTTGTGCCGCACTTTGCGCGCT-3' and 5'-CCCAAGCTTGGCCTGTAACCGGAGCTCA-3'. The amplified DNA fragment was digested with HindIII and cloned into HindIII-digested pBR322 to construct plasmid pTM6. Cells were grown at 37 °C in LB medium or TB medium (15) supplemented with kanamycin (15 µg/ml), tetracycline (15 µg/ml), chloramphenicol (15 µg/ml), and/or ampicillin (50 µg/ml) when needed. Bacterial growth was monitored by determining the optical density at 600 nm.


                              
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Table I
Bacterial strains used in this study

Northern Blot Analysis-- Total cellular RNAs were isolated from exponentially growing cells as described (16). The RNAs were resolved by 1.2% agarose gel electrophoresis in the presence of formaldehyde and blotted onto a Hybond-N+ membrane (Amersham Biosciences) as described (17). The mRNAs were visualized using digoxigenin reagents and kits for nonradioactive nucleic acid labeling and detection systems (Roche Molecular Biochemicals) according to the procedure specified by the manufacturer. The 305-bp digoxigenin-labeled DNA probe corresponding to the 5'-ptsG region was used.

Determination of Intracellular cAMP-- Bacterial cells were grown in LB medium to A600 = 0.6. Two milliliters of the culture were taken and centrifuged at 12,000 × g for 20 s at 4 °C. The pellet was suspended in 40 µl of 5 mM NaCl and immediately heated at 100 °C for 5 min. After adding 160 µl of ethanol, the mixture was chilled at -70 °C for 30 min and centrifuged at 14,000 × g for 30 min at 4 °C. The resulting supernatant was dried up, dissolved in 20 µl of H2O, and used for cAMP assay. The determination of cAMP by gel mobility shift assay has been described previously (18). The cellular concentration of cAMP was calculated on the assumption that an A600 of 1.4 corresponds to 109 cells/ml (15) and that the volume of a cell is 2 × 10-12 ml (19).

Determination of Intracellular Glucose-6-P-- Bacterial cells were grown in LB medium to A600 = 0.6 unless otherwise specified. One milliliter of the culture was centrifuged at 10,000 × g for 2 min at room temperature. The pellet was suspended in 100 µl of H2O, immediately added to 50 µl of 5 M HClO4, and chilled on ice. After adding 100 µl of 2.5 M K2CO3, the mixture was centrifuged at 14,000 × g for 10 min at 4 °C. The supernatant was used for the glucose-6-P assay. The assay was performed according to the method of Hogema et al. (20). The intracellular concentration of glucose-6-P was calculated as in the case of the cAMP assay.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ts8 fda Mutation Stimulates the Degradation of ptsG mRNA at the Nonpermissive Temperature-- Among glycolytic genes other than pgi and pfkA, we first focused on fda, which encodes Fda just downstream of pfkA in the Embden-Meyerhof glycolytic pathway (Fig. 1). It is known that the Fda activity in a strain carrying a temperature-sensitive fda allele (ts8 or h8) was essentially undetectable at 42 °C (13). The ts8 mutation was shown to inhibit stable RNA synthesis and cell growth depending on glucose metabolism at the nonpermissive temperature (21). To examine whether the ts8 mutation affects the expression and/or degradation of ptsG mRNA, the ts8 allele was transferred to a W3110 background, and the expression of ptsG mRNA in the presence of glucose was analyzed by Northern blotting using a ptsG DNA probe. The full-length ptsG mRNA was expressed stably in the ts8 fda strain at 30 °C as in the wild-type strain, whereas the pgi mutation dramatically reduced the full-length ptsG mRNA, resulting in an extensive smear because of the stimulation of RNase E-mediated mRNA degradation (Fig. 2, lanes 1-3). When the temperature was shifted to 42 °C, the ts8 fda strain gave a smear of degradation intermediates of ptsG mRNA (Fig. 2, lane 4), whereas the temperature upshift to 42 °C did not affect the Northern pattern in the wild-type and pgi strains (data not shown). This implies that the loss of Fda activity leads to the rapid degradation of ptsG mRNA in the presence of glucose. The introduction of an rne allele, ams1 (22, 23), encoding a temperature-sensitive RNase E into the ts8 fda mutant prevented the rapid degradation of ptsG mRNA at the nonpermissive temperature (Fig. 2, lane 5). Thus, the ptsG mRNA is destabilized in an RNase E-dependent manner in the ts8 fda strain at 42 °C as in the case of the pgi and pfkA strains (1).


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Fig. 1.   Relevant metabolic pathways of several sugars in E. coli. The pathways shown illustrate glycolysis.


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Fig. 2.   Effects of mutations in glycolytic genes on the expression of ptsG mRNA. W3110 (lane 1), TM54 (lane 2), TM84 (lane 6), TM61 (lane 7), TM81 (lane 8), and TM154 (lane 9) were grown in LB medium with 1% glucose at 37 °C. Cellular RNAs were prepared at A600 = 0.6. In the case of TM191 (lanes 3 and 4) and TM234 (lane 5), cells were grown in LB medium with 1% glucose at 30 °C to A600 = 0.5, and the temperature was shifted to 42 °C (lanes 4 and 5) or kept at 30 °C (lane 3). Cellular RNAs were prepared after further incubation for 10 min. The RNA samples (15 µg) were subjected to Northern blot analysis. WT, wild type.

Mutations in Glycolytic Genes Downstream of fda Affect the Expression but Not the Degradation of ptsG mRNA-- To examine the effects of mutations in other glycolytic genes on the degradation of ptsG mRNA, a series of deletion mutants of glycolytic genes downstream of fda were constructed. We successfully disrupted tpiA, pgk, gpm, and pyk encoding triose-phosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase, respectively, although we failed to disrupt gap and eno encoding glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively. The ptsG mRNA expression in each mutant was analyzed by Northern blotting. As shown in Fig. 2, each of these mutations affected the expression of ptsG mRNA to various extents. However, the Northern patterns of the ptsG mRNA in these mutant strains were essentially the same as those of the wild-type, indicating that these mutations do not affect significantly the degradation of ptsG mRNA (Fig. 2, lanes 6-9). We conclude that mutational blocks only in early stages of the glycolytic pathway produce a signal that leads to the rapid degradation of ptsG mRNA. The effects of mutations in the glycolytic genes on ptsG expression also were analyzed by Western blotting. The level of IICBGlc was correlated with that of the full-length ptsG mRNA in each strain (data not shown). It is likely that the mutations in the glycolytic genes downstream of fda reduce the ptsG expression at the level of transcription initiation presumably by affecting the CRP-cAMP and/or Mlc pathways because ptsG transcription is regulated by these two global systems (7, 11, 12). However, the introduction of the mlc mutation did not affect the ptsG mRNA level in each mutant strain in the presence of glucose, suggesting that the Mlc pathway is not involved in the reduction of ptsG expression (data not shown). We then determined the intracellular cAMP levels in each mutant strain both in the presence and absence of glucose. We observed that the reduction in the intracellular cAMP level by glucose was more significant in the tpiA, gpm, and pyk strains than in the wild-type strain (Table II). Thus, the reduced expression of ptsG mRNA in the tpiA, gpm, and pyk strains particularly in the presence of glucose appears to be at least in part due to the reduction of the intracellular cAMP level. It remains to be studied why ptsG expression is reduced in the pgk strain, in which no significant reduction of cAMP was observed.


                              
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Table II
Intracellular cAMP levels in exponentially growing cells
Intracellular cAMP levels were determined as described under "Experimental Procedures." Each value is the average of three independent experiments.

The Destabilization of ptsG mRNA in Mutant Strains Is Dependent upon Glucose Uptake and Metabolism-- The ptsG mRNA is highly expressed in the mlc strain without the addition of glucose to the medium, and the ptsG mRNA expression is moderately reduced in the presence of external glucose because of the reduction in cAMP and CRP levels (11). When the ptsG mRNA was analyzed in the pgi mlc double mutant growing in LB medium, rapid degradation of ptsG mRNA occurred in the presence of external glucose as expected (Fig. 3, lane 4). However, a significant degradation of ptsG mRNA also was observed even without the addition of external glucose (Fig. 3, lane 3). This raises a question as to whether the rapid degradation of ptsG mRNA is dependent upon glucose metabolism. To test this, we introduced the ptsI mutation into the pgi mlc strain and examined the expression of ptsG mRNA by Northern blotting. The ptsG mRNA was stabilized in the pgi mlc ptsI triple mutant where the uptake of glucose is prevented (Fig. 3, lanes 5 and 6). It is apparent that the degradation of ptsG mRNA observed in the pgi mlc double mutant growing in LB medium without external glucose was due to trace amounts of endogenous glucose in the LB medium. In fact, the rapid degradation of ptsG mRNA no longer occurred in the mlc pgi and mlc pfkA double mutant strains without the addition of glucose when TB medium was used (Fig. 4, A and B, lane 1). The addition of glucose caused a dramatic degradation of ptsG mRNA in both strains growing on TB medium (Fig. 4, A and B, lane 2). These results clearly indicate that the destabilization of ptsG mRNA in mutant strains occurs depending on the glucose uptake and metabolism.


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Fig. 3.   Effect of ptsI mutation on the degradation of ptsG mRNA. IT1568 (lanes 1 and 2), TM162 (lanes 3 and 4), and TM145 (lanes 5 and 6) were grown in LB medium with and without 1% glucose to A600 = 0.6. Cellular RNAs were prepared, and 15 µg of each of the RNA samples was subjected to Northern blot analysis.


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Fig. 4.   Effects of various carbon sources on the degradation of ptsG mRNA. TM162 (A) and IT1165 (B) were grown in TB medium with and without 0.1% compounds indicated to A600 = 0.6. Cellular RNAs were prepared, and 15 µg of each of the RNA samples was subjected to Northern blot analysis. The compounds used are glucose (Glc), lactose (Lac), mannitol (Mtl), xylose (Xyl), fructose (Fru), glycerol (Gly), glucose-6-P (G6P), and fructose-6-P (F6P).

The Destabilization of ptsG mRNA Occurs Only in the Presence of Glycolytic Intermediates Upstream of the Block-- To test whether glucose generated internally also could lead to the destabilization of ptsG mRNA in the mutant strains, the effect of the addition of lactose on the degradation of ptsG mRNA was examined. The addition of lactose in the culture medium stimulated the degradation of ptsG mRNA in both the pgi mlc and pfkA mlc strains (Fig. 4, A and B, lane 3). Thus, internally produced glucose, which is believed to be phosphorylated by glucokinase encoded by glk (5), also leads to the destabilization of ptsG mRNA in the mutant strains. We also examined the effect of the addition of several glycolytic intermediates and/or carbon sources on the ptsG mRNA degradation in pgi mlc and pfkA mlc double mutant strains. The addition of glucose-6-P, which is taken up by UhpT (24), caused the rapid degradation of ptsG mRNA in both strains (Fig. 4, A and B, lane 8). On the other hand, the addition of fructose-6-P, which also is taken up by UhpT, failed to cause the rapid degradation of ptsG mRNA in the pgi mlc strain (Fig. 4A, lane 9), although it caused the degradation of ptsG mRNA in the pfkA mlc strain (Fig. 4B, lane 9). The addition of mannitol and xylose, which are taken up by mannitol-specific PTS and non-PTS xylose permease, respectively (25), and then converted to fructose-6-P, also caused the rapid degradation of ptsG mRNA in the pfkA mlc (Fig. 4B, lanes 4 and 5) but not in the pgi mlc (Fig. 4A, lanes 4 and 5) strains. However, the addition of fructose, which enters the Embden-Meyerhof pathway past fructose-6-P, did not affect the stability of ptsG mRNA in either strain (Fig. 4, A and B, lane 6). Likewise, the stability of ptsG mRNA in mutant strains was not affected by the addition of glycerol (Fig. 4, A and B, lane 7), which also enters the central carbon metabolism past fructose-1,6-P2. We conclude that the mutational block in the glycolytic pathway destabilizes ptsG mRNA depending on the accumulation of glycolytic intermediates up to the block points.

Elevated Glucose-6-P Level Is Associated with the Rapid Degradation of ptsG-- Among glycolytic intermediates, glucose-6-P would be the most likely participant in the destabilization of ptsG mRNA because this intermediate is expected to accumulate at least in the pgi strain growing in the glucose medium. In fact, it is known that the glucose-6-P level in the pgi strain markedly increases when grown in the presence of glucose (26, 27). To examine a link between the glucose-6-P level and the destabilization of ptsG mRNA, we determined the intracellular levels of glucose-6-P in various strains growing in LB medium containing exogenous glucose. The glucose-6-P level in the pgi strain was about 8 times higher than that in the wild-type strain (Table III, lines 1 and 2). A moderate but still significant accumulation of glucose-6-P was observed in the pfkA strain (Table III, line 3). On the other hand, little or no increase in the glucose-6-P level was observed in other mutant strains (Table III, lines 4-7). A large increase in the glucose-6-P level was observed in the mlc pgi but not in the mlc and the mlc pgi ptsI strains when cells were grown in LB medium with exogenous glucose (Table IV, lines 2, 4, and 8). As expected, the accumulation of glucose-6-P in the mlc pgi strain occurred even when cells were grown in LB medium without exogenous glucose because of trace amounts of glucose in the medium (Table IV, line 3). However, no increase in the glucose-6-P level was observed in the pgi strain when cells were grown in TB medium without exogenous glucose (Table IV, line 5). All of these results are entirely consistent with a view that accumulation of glucose-6-P is a degradation signal for the ptsG mRNA at least in the pgi strain.


                              
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Table III
Intracellular levels of glucose 6-phosphate
Cells were grown in LB medium containing 1% glucose. Intracellular levels of glucose 6-phosphate were determined as described under "Experimental Procedures." Each value is the average of three independent experiments.


                              
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Table IV
Glucose 6-phosphate levels in cells growing in various conditions
Cells were grown in LB or TB medium in the presence of the indicated sugars. Intracellular levels of glucose 6-phosphate were determined as described under "Experimental Procedures". Each value is the average of three independent experiments.

The Addition of Glucose Rapidly Leads to the Accumulation of Glucose-6-P and the Degradation of ptsG mRNA in the pgi Strain-- To examine how rapidly glucose-6-P accumulates to stimulate the degradation of ptsG mRNA after the addition of glucose, both mlc and mlc pgi cells were grown to early log phase in TB medium, glucose was added at a final concentration of 1%, and the incubation was continued. The glucose-6-P level was monitored at various times after the addition of glucose. The glucose-6-P level started to increase immediately after the addition of glucose, reaching the plateau level (about a 2-fold increase) characteristic of cells grown in the presence of glucose after less than 3 min in the mlc cells (Fig. 5A). On the other hand, the glucose-6-P level continuously increased up to 9-fold for 10 min in the mlc pgi cells (Fig. 5A). The expression of ptsG mRNA also was examined at various times after the addition of glucose by Northern blotting. It was expected that the addition of glucose would affect the ptsG expression at the levels of both transcription and mRNA degradation. In fact, the ptsG mRNA level was moderately reduced by the addition of glucose (Fig. 5B, lanes 1-5). This effect of glucose, called catabolite repression, is apparently a result of the reduction of both cAMP and CRP levels (11, 28, 29). Remarkably, the full-length ptsG mRNA was eliminated almost completely within 5 min after the addition of glucose in the mlc pgi cells (Fig. 5B, lanes 6-10). It is apparent that the dramatic decrease of ptsG mRNA in the mlc pgi cells is because of the degradation of the full-length ptsG mRNA level. The data clearly indicate that the addition of glucose rapidly leads to the accumulation of glucose-6-P, resulting in the stimulation of the degradation of ptsG mRNA.


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Fig. 5.   Effects of the addition of glucose on the glucose-6-P level and on the degradation of ptsG mRNA. IT1568 (mlc) and TM162 (mlc pgi) were grown in TB medium to A600 = 0.4. Glucose was added at a final concentration of 1%, and the incubation was continued. Intracellular glucose-6-P levels were determined at the indicated times as described under "Experimental Procedures" (A). Each value is the average of three independent assays. Cellular RNAs also were prepared at the indicated times and analyzed by Northern blotting using 15 µg of each of the RNA samples (B).

Accumulation of Fructose-6-P Can Be an Additional Metabolic Signal for the Degradation of ptsG-- It is rather complicated to specify the degradation signal in the pfkA strain, in which not only glucose-6-P but also fructose-6-P is expected to accumulate. To access a possible role of accumulation of fructose-6-P in the degradation of ptsG mRNA in the pfkA strain, an mlc pgi pfkA triple mutant was constructed. The effects of glucose, glucose-6-P, and fructose-6-P on the degradation of ptsG mRNA were examined in cells growing in TB medium. As expected, the addition of glucose (Fig. 6, lane 2) or glucose-6-P (Fig. 6, lane 3) caused a rapid degradation of ptsG mRNA because of the accumulation of glucose-6-P in the triple mutant strain. Interestingly, the destabilization of ptsG mRNA also occurred when fructose-6-P was added (Fig. 6, lane 4). Under this condition, cells should be virtually devoid of glucose-6-P because the interconversion from fructose-6-P to glucose-6-P is prevented. In fact, we observed that the glucose-6-P level was not elevated in the presence of fructose-6-P (Table IV, line 11), whereas it dramatically increased in the presence of glucose (Table IV, line 10). This implies that accumulation of fructose-6-P could act as an additional signal to stimulate the degradation of ptsG mRNA. On the other hand, the addition of fructose or fructose-1,6-P2 did not cause the rapid degradation of ptsG mRNA in the ts8 fda strain at the nonpermissive temperature, suggesting that the increased fructose-6-P rather than the accumulation of fructose-1,6-P2 itself is responsible for the destabilization of ptsG mRNA in the fda strain (data not shown).


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Fig. 6.   Effects of glycolytic intermediates on the degradation of ptsG mRNA in the mlc pgi pfkA triple mutant. Cells were grown in TB medium with and without 0.1% compounds indicated to A600 = 0.6. Cellular RNAs were prepared, and 15 µg of each of the RNA samples was subjected to Northern blot analysis. G6P, glucose-6-P; F6P, fructose-6-P.

Overexpression of Glucose-6-P Dehydrogenase Prevents the Rapid Degradation of ptsG mRNA-- If the increased intracellular pool of glucose-6-P is responsible for the rapid degradation of ptsG mRNA, an overexpression of glucose-6-P dehydrogenase (Zwf) is expected to affect the degradation of ptsG mRNA because it may stimulate the flow from glucose-6-P to 6-P-gluconolactone resulting in a reduction in the glucose-6-P level. To test this idea, a multicopy plasmid (pTM6) carrying the zwf gene was constructed and introduced into the pgi and pfkA strains. As expected, the glucose-6-P level was reduced dramatically in both strains growing in glucose medium when Zwf was overexpressed by introducing pTM6 (Table IV, lines 13 and 14). Interestingly, the rapid degradation of ptsG mRNA in both strains was prevented completely under the same conditions (Fig. 7, lanes 2 and 4), whereas the introduction of a control plasmid did not affect the degradation (Fig. 7, lanes 1 and 3). This strongly supports the argument that the increased level of glucose-6-P is surely responsible for the rapid degradation of ptsG mRNA in the pgi strain. The fact that the rapid degradation of ptsG mRNA was prevented by the introduction of pTM6 suggests that overexpression of Zwf may eliminate the accumulation of not only glucose-6-P but also fructose-6-P. The introduction of pTM6 caused only a slight decrease in the glucose-6-P level in the wild-type strain (Table IV, line 12).


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Fig. 7.   Effect of overexpression of glucose-6-P dehydrogenase on the degradation of ptsG mRNA. TM54/pBR322 (lane 1), TM54/pTM6 (lane 2), TM238/pBR322 (lane 3), and TM238/pTM6 (lane 4) were grown in LB medium with 1% glucose to A600 = 0.6. Cellular RNAs were prepared, and 15 µg of each of the RNA samples was subjected to Northern blot analysis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

One of the questions addressed in the present study is whether mutations in other glycolytic genes affect the expression and/or degradation of ptsG mRNA. We demonstrate that the ts8 mutation in fda, just downstream of pfkA in the glycolytic pathway, destabilizes the ptsG mRNA at the nonpermissive temperature in glucose-grown cells as in the pgi and pfkA mutations. On the other hand, mutations downstream of fda did not affect significantly the stability of ptsG mRNA, although these mutations somehow reduced ptsG expression at the level of transcription initiation by affecting primarily the intracellular cAMP level. Thus, we conclude that a mutational block in only the early stages of the glycolytic pathway leads to the destabilization of ptsG mRNA in glucose-grown cells.

An important conclusion of the present study is that the rapid degradation of ptsG mRNA in the mutant strains is completely dependent on the presence of glucose or any one of the compounds that enters the Embden-Meyerhof glycolytic pathway before the block points. These results led us to propose that the accumulation of early glycolytic intermediates due to the metabolic block triggers the stimulation of RNase E-mediated degradation of ptsG mRNA. Several lines of evidence indicate that accumulation of glucose-6-P or fructose-6-P is responsible for the rapid degradation of ptsG mRNA in the pgi strain. First, a large increase in glucose-6-P levels was observed in the pgi strain in the presence of glucose where the rapid degradation of ptsG mRNA occurs. Second, overexpression of glucose-6-P dehydrogenase eliminated both the accumulation of glucose-6-P and the degradation of ptsG mRNA in the pgi strain. Third, the ptsG mRNA was destabilized in the pgi pfkA mlc triple mutant strain lacking glucose-6-P when fructose-6-P was supplied. In addition, we observed that the degradation of ptsG mRNA was stimulated by the addition of a nonmetabolizable glucose analog, alpha -methyl glycoside, in the wild-type strain (1). This means that accumulation of phosphorylated alpha -methyl glycoside also could act as a signal to break down the ptsG mRNA. On the other hand, the data suggested that accumulation of fructose-1,6-P2 itself may not be a signal for the rapid degradation of ptsG mRNA in the ts8 fda strain.

The rapid degradation of ptsG mRNA in response to the accumulation of glucose-6-P or fructose-6-P illuminates an elaborate feedback regulatory mechanism in carbon central metabolism. The physiological relevance of this feedback regulation at the mRNA degradation step is clear because accumulation of metabolic intermediates sometimes would be deleterious to cells (21). It is known that the accumulation of hexose phosphates such as glucose-6-P and fructose-6-P damages DNA resulting in a dramatic increase in mutation frequency (26, 27). The down-regulation of ptsG expression through mRNA degradation certainly prevents glucose uptake by avoiding too much accumulation of hexose phosphates in mutant cells. It would be interesting to know whether the expression and stability of other sugar transport-related mRNAs also are affected by the accumulation of early glycolytic intermediates. We tested the effect of the pgi or pfkA mutation on the expression of several other mRNAs. So far, no clear evidence for the destabilization of other mRNAs by the pgi or pfkA mutation has been obtained.

Are there any situations in which the metabolic perturbation in the early stages of the Embden-Meyerhof glycolytic pathway occurs in the wild-type cells? One clear case for this is when cells incorporate nonmetabolizable glucose analogs such as alpha -methyl glucoside. The metabolic block that leads to the accumulation of glucose-6-P and/or fructose-6-P also may occur if certain substances inhibit the activity of phosphoglucose isomerase, phosphofructokinase A, or fructose-1,6-P2 aldolase. For example, it is known that Zn2+ acts as an inhibitor of the rat liver phosphofructokinase (30). Furthermore, it is known that the levels of glycolytic intermediates including glucose-6-P, fructose-1,6-P2, and phosphoenolpyruvate vary markedly depending on the nature of carbon and nitrogen sources and also after the addition of a new nutrient to cultures (31). It is certainly interesting to examine whether and how these changes in the levels of metabolites affect the degradation of ptsG mRNA.

Another important aspect of our study is that the metabolic flow in the glycolytic pathway regulates the stability of a specific mRNA of a solute transporter gene. Although the degradation of mRNA is an important step for controlling gene expression (32-36), little is known about how a particular environmental and/or internal signal regulates the stability of a specific mRNA in E. coli cells. The acceleration of ptsG mRNA degradation in response to the metabolic blocks in the early stages of the Embden-Meyerhof pathway certainly offers a good opportunity to investigate both the mechanism and the role of regulation of gene expression at the mRNA degradation level. RNase E is believed to play a central role in the degradation of a variety of mRNAs in E. coli cells (37, 38). The enzyme exists as a macromolecular complex called degradosome, a multi-enzyme RNA degradation complex that also contains polynucleotide phosphorylase, RhlB helicase, enolase, and other minor components (39-42). The presence of enolase in the degradosome complex suggests that this glycolytic enzyme would be involved somehow in the regulation of degradation of ptsG mRNA in response to the metabolic blocks in the glycolytic pathway.

Although the present study has clarified an important aspect regarding the regulation of ptsG mRNA degradation, the molecular mechanism underlying this phenomenon remains largely unknown. There are several important questions that should be addressed with a priority. First, how does the accumulation of glucose-6-P or fructose-6-P lead to the rapid degradation of ptsG mRNA by RNase E? The increased level of glucose-6-P in mutant strains may stimulate the metabolic flows from glucose-6-P to glucose 1-phosphate and 6-P-gluconolactone catalyzed by phosphoglucomutase (pgm) and glucose-6-P dehydrogenase (zwf), respectively (Fig. 1). The increased flows in these two pathways would alter the levels of downstream metabolites that somehow could affect the stability of ptsG mRNA. If this were the case, mutations in the pgm and/or zwf genes should affect the degradation of ptsG mRNA in the pgi strain. However, the rapid degradation of ptsG mRNA in the presence of glucose still occurred in the pgm pgi or zwf pgi double mutant strains.2 This indicates that the accumulation of glucose-6-P itself but not the possible metabolic change in the pentose phosphate pathway and in the glycogen biosynthetic pathway is responsible for the rapid degradation of ptsG mRNA. It is highly possible that some molecules that transduce the signal to RNase E also monitor the accumulation of glucose-6-P or fructose-6-P. It is certainly important to identify additional genetic components that might be involved in this process. One important molecule that could modulate the ptsG mRNA degradation would be host factor I, an Hfq that mediates RNA-RNA interaction (43-45). In fact, host factor I is known to modulate different aspects of RNA metabolism including the stability of several mRNAs (46-48). Second, because the rapid degradation of mRNA in response to the metabolic block is observed so far specifically in ptsG mRNA, there should be some specific features within ptsG mRNA that are recognized by RNase E and/or some other factors. Further genetic and biochemical studies are necessary to answer these questions and to understand fully the molecular mechanism underlying this novel feedback regulation in the central metabolic pathway.

    ACKNOWLEDGEMENTS

We thank C. A. Gross for providing the ts8 fda strain (CAG417). T. M. is grateful to Masami Oguchi for continuous encouragement during this work.

    FOOTNOTES

* This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Ajinomoto Co., Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-52-789-3653; Fax: 81-52-789-3001; E-mail: i45346a@nucc.cc.nagoya-u.ac.jp.

Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M300177200

2 T. Morita, W. El-Kazzaz, Y. Tanaka, T. Inada, and H. Aiba, unpublished results.

    ABBREVIATIONS

The abbreviations used are: PTS, phosphoenolpyruvate-dependent sugar phosphotransferase system; EII, enzyme II complex of the PTS; CRP, cAMP receptor protein; Fda, fructose-1,6-diphosphate aldolase; TB, tryptone broth; LB, Luria-Bertani broth.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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