Heat Shock RNA Polymerase (Esigma 32) Is Involved in the Transcription of mlc and Crucial for Induction of the Mlc Regulon by Glucose in Escherichia coli*

Dongwoo ShinDagger, Sangyong Lim§, Yeong-Jae Seok, and Sangryeol Ryu||

From the Research Center for New Bio-Materials in Agriculture, Department of Food Science and Technology and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea and the  School of Biological Sciences, Seoul National University, Seoul 151-742, Korea

Received for publication, February 26, 2001, and in revised form, April 17, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mlc is a global regulator of carbohydrate metabolism. Recent studies have revealed that Mlc is depressed by protein-protein interaction with enzyme IICBGlc, a glucose-specific permease, which is encoded by ptsG. The mlc gene has been previously known to be transcribed by two promoters, P1(+1) and P2(+13), and have a binding site of its own gene product at +16. However, the mechanism of transcriptional regulation of the gene has not yet been established. In vitro transcription assays of the mlc gene showed that P2 promoter could be recognized by RNA polymerase containing the heat shock sigma factor sigma 32 (Esigma 32) as well as Esigma 70, while P1 promoter is only recognized by Esigma 70. The cyclic AMP receptor protein and cyclic AMP complex (CRP·cAMP) increased expression from P2 but showed negative effect on transcription from P1 by Esigma 70, although it had little effect on transcription from P2 by Esigma 32 in vitro. Purified Mlc repressed transcription from both promoters, but with different degrees of inhibition. In vivo transcription assays using wild type and mlc strains indicated that the level of mlc expression was modulated less than 2-fold by glucose in the medium with concerted action of CRP·cAMP and Mlc. A dramatic increase in mlc expression was observed upon heat shock or in cells overexpressing sigma 32, confirming that Esigma 32 is involved in the expression of mlc. Induction of ptsG P1 and pts P0 transcription by glucose was also dependent on Esigma 32. These results indicate that Esigma 32 plays an important role in balancing the relative concentration of Mlc and EIICBGlc in response to availability of glucose in order to maintain inducibility of the Mlc regulon at high growth temperature.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

When Mlc is overproduced on a multicopy plasmid in Escherichia coli grown in the presence of glucose, it causes reduction of acetate accumulation and E. coli makes large colonies (1). Mlc has been proposed to be a new global regulator of carbohydrate metabolism (2-5). It has been reported that Mlc regulates manXYZ encoding enzyme II of the mannose PTS (4), malT encoding the activator of maltose operon, and mlc itself negatively (2). Moreover, ptsG encoding enzyme IICB of the glucose PTS (EIICBGlc) and the pts operon encoding general PTS proteins also proved to be repressed by Mlc (3, 5-8). The Mlc regulon is also under the positive control of the CRP1·cAMP complex. It has been discovered that repression of the Mlc regulon is relieved in cells grown in the media containing glucose or other PTS sugars (7). It has been shown that the unphosphorylated EIICBGlc can sequester Mlc from its binding sites by direct protein-protein interaction to induce expression of the Mlc regulon in response to glucose (9-11).

The mlc gene encoding a 44-kDa Mlc protein is located around 35 min of chromosomal locus (1). This gene was also identified as the same allele of the dgsA gene (4, 12, 13). Decker et al. (2) have shown that mlc transcription starts from two promoters called upstream "+1" and downstream "+13" and there exists one Mlc-binding site centered at +16. In addition, a highly conserved CRP-binding site is present within the mlc promoter. However, the detailed mechanisms of transcriptional regulation of mlc have not yet been reported.

The majority of the E. coli promoters are recognized by the RNA polymerase containing the housekeeping sigma factor, sigma 70 (14, 15). Several genes that are necessary to respond to various environmental or nutritional changes require specific recognition by RNA polymerase associated with the alternative sigma factors, sigma 32 (16), sigma E (17), sigma 54 (18), or sigma S (19). The heat shock response in E. coli is mediated by Esigma 32 (20) and it is known that expression of at least 26 genes is induced by heat shock in E. coli (21). Many essential genes in E. coli have multiple promoters including one recognized by Esigma 32 in order to respond to various environmental conditions (22-24). It has been shown that the pts P0 promoter is recognized by Esigma 32 as well as Esigma 70 (25) as is expected for a system as central to carbohydrate metabolism as the PTS. In this work, we studied the transcriptional regulation of the mlc gene in vitro as well as in vivo and the role of Esigma 32 in maintaining glucose-dependent induction of the Mlc regulon at high growth temperature.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Cyclic AMP was obtained from Sigma, RNA polymerase saturated with sigma 70, nucleotide triphosphates, [gamma -32P]ATP, and [alpha -32P]UTP were purchased from Amersham Pharmacia Biotech. The cycle sequencing kit was from Epicentre Technologies (Madison, WI).

Bacterial Strains-- MC4100 (araD139 Delta argF-lacU169rpsL150 thiA relA1 flb5301 deoC1 ptsF25 rbsR) was used as a wild type strain in this study. The mlc strain SR505 is the same as KD413 (MC4100, mlc::Tn10Tet). To construct the rpoH-deleted mutant, SR701 (SR702, Delta rpoH), Delta rpoH allele of KY1603 (26) was transferred to SR702 (MC4100, suhX1 (6)) using P1 transduction. To study the effect of sigma 32 on mlc expression, a plasmid pKV10 in which the rpoH gene is under control of tac promoter was introduced into MC4100.

Plasmid Construction and Preparation-- Basic cloning protocols used were described in Sambrook and Russell (27). Polymerase chain reaction cloning of the mlc promoter was carried out using primers that have unique restriction sites in their sequences. The clone was verified by DNA sequencing. The supercoiled plasmid pMX, which contains the mlc promoter region, was made by inserting the DNA segment from base pair -272 to +105 (all of these numberings are based on the transcription start site of the P1 promoter of mlc in Fig. 1) between the EcoRI and the PstI sites in front of the rpoC terminator in plasmid pSA600 (28). Supercoiled DNA was prepared by Concert kit (Life Technologies, Inc.) in RNase-free condition for in vitro transcription assay.

Primer Extension-- Cells were grown aerobically at 30 °C in tryptone broth (1% Bacto-tryptone, 0.8% NaCl) either in the presence or absence of 0.2% glucose. At A600 = 0.5, growth temperature was shifted to 42 °C and incubation was continued for the designated time and total E. coli RNA was purified using Trizol reagent (Life Technologies, Inc.) to study the heat shock effects on mlc and ptsG transcription. Purified RNA was resuspended in sterile distilled water. To study mlc transcription, 32P-labeled primer MLC7 (5'-ATTTTAGTGATACTGGCAGGAGCCAGTTGC-3'), which is complementary to +162 to +192, was co-precipitated with 40 µg of total cell RNA. The primer PG1 (5'-AATTGAGAGTGCTCCTGAGTATGGGTGC-3', complementary to +74 to +102) and the primer P11 (6) were co-precipitated with 30 µg of total RNA to study ptsG and pts transcription, respectively. The pellet was resuspended in 20 µl of 250 mM KCl, 2 mM Tris-HCl, pH 7.9, and 0.2 mM EDTA. Primer extension reactions were done as described by Ryu and Garges (29).

Purification of RNA Polymerase Holoenzyme Containing sigma 32-- E. coli MC4100 cells harboring the pKV10 plasmid were grown on 2 × LB (1% Bacto-tryptone, 0.5% yeast extract, 2% NaCl) in a 5-liter fermenter at 30 °C in the presence of 1 mM isopropyl-1-thio-beta -D-galactopyranoside. The cells were harvested 10 min after a temperature shift from 30 to 42 °C at A600 = 2.5 in late exponential growth phase. RNA polymerase was purified by fast protein liquid chromatography according to Harger et al. (30) with some modifications as described by Sukhodolets et al. (31). Crude RNA polymerase was obtained from 10 g of wet cell paste by Polymin P precipitation and the drained pellet was solubilized with TGED (10 mM Tris, pH 7.9, 5% (v/v) glycerol, 0.1 mM EDTA, 0.1 mM dithiothreitol) containing 0.2 M NaCl (TGEDN). This enzyme solution was loaded on a single-stranded DNA-agarose column (Amersham Pharmacia Biotech) pre-equilibrated with TGEDN. After the column was washed with 3 column volumes of TGEDN, RNA polymerase was eluted with a linear salt gradient (from TGEDN to TGED containing 1.0 M NaCl) at a flow rate of 2 ml/min. The elution of RNA polymerase was monitored by the presence of beta beta ' on a sodium dodecyl sulfate-polyacrylamide gel. The fractions containing RNA polymerase (from 0.3 to 0.5 M NaCl gradient) were loaded onto a 5/5 Mono-Q column (Amersham Pharmacia Biotech), and RNA polymerase containing sigma 32 was eluted with a linear gradient of NaCl (0.3 M to 0.5 M in TGED) at 1 ml/min. The yield for Esigma 32 was 1.0~1.5 mg.

In Vitro Transcription Assay-- Reactions were done as described by Ryu and Garges (29) in a 25-µl volume containing the following: 20 mM Tris acetate, pH 8.0, 3 mM magnesium acetate, 200 mM potassium glutamate, 1 mM dithiothreitol, 1 mM ATP, 0.2 mM GTP, 0.2 mM CTP, 0.02 mM UTP, 10 µCi of [alpha -32P]UTP (800 Ci/mmol), 2 nM supercoiled DNA template, 20 nM RNA polymerase saturated with sigma 70 or sigma 32, 100 µg/ml bovine serum albumin, and 5% glycerol. Additional regulators such as 40 nM CRP, 100 µM cAMP, or 0-1.5 µM Mlc were added to the reaction as needed. All components except nucleotides were incubated at 37 °C for 10 min. Transcriptions were started by the addition of nucleotides and terminated after 10 min by the addition of 25 µl of formamide loading buffer (80% formamide, 89 mM Tris base, 89 mM boric acid, 2 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol). RNA was resolved by electrophoresis on an 8 M urea, 6% polyacrylamide gel. The amounts of transcripts were measured using a phosphoimage analyzer, BAS2500 (Fuji Photo Film Co.).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mlc Promoter Is Recognized by Both Esigma 70 and Esigma 32 in Vitro and Its Activity Is Modulated by CRP·cAMP and Mlc-- As shown in Fig. 1, there is a CRP-binding site centered at -58.5 and one Mlc-binding site centered at +16 within the mlc promoter region (2). The sequence of the P2 promoter region has a partial homology with a known consensus sequence (CTTGAAA, 11~16 base pairs, CCCATNT) of the promoters recognized by Esigma 32 (20). To investigate how the two promoters of the mlc gene are regulated, we performed in vitro transcription assays with supercoiled DNA template, purified proteins, and either Esigma 70 or Esigma 32. When Esigma 70 was used, transcripts were made from both the P1 and P2 promoters (Fig. 2, lanes 1 and 2). However, Esigma 32 could initiate transcription only from P2, as expected from the sequence of the promoter region of mlc shown in Fig. 1. No transcription from P1 by Esigma 32 was detected (Fig. 2, lanes 3 and 4). The specificity of Esigma 32 used for the in vitro transcription assay was confirmed by the lack of "rep" transcript that is transcribed only by Esigma 70. Interestingly, CRP·cAMP showed a negative effect on P1 transcription by Esigma 70, while it exerted a positive effect on P2 transcription directed by Esigma 70. However, CRP·cAMP had little effect on Esigma 32-directed transcription from P2.


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Fig. 1.   Nucleotide sequence of the promoter region of mlc. The transcription start points of P1(+1) and P2(+13) are shown with arrows. The -10 and -35 region of the P1 promoter is underlined and that of P2 is indicated with a line over the sequence. The known consensus -10 and -35 sequence recognized by Esigma 32 is shown in lowercase (20). The binding site of CRP and Mlc is indicated as box. The start codon of mlc, GTG, is also indicated. All of the numbering of this sequence is based on the transcription start point of the P1 promoter.


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Fig. 2.   The effect of CRP·cAMP on mlc transcription in vitro. The supercoiled DNA template, pMX, was used for the in vitro transcription by Esigma 70 (lanes 1 and 2) and Esigma 32 (lanes 3 and 4) in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of the CRP·cAMP complex. The transcripts from the plasmid origin of replication (106/107 nucleotides) are marked as rep. The 132-nucleotide transcript from P1 and the 120-nucleotide transcript from P2 are indicated.

The purified Mlc could inhibit transcription from both promoters but the degree of repression was dependent on the kind of sigma  factor and the promoter (Fig. 3). Transcription from the P2 promoter by Esigma 70 was most sensitive to the repression by Mlc, and half-repression of Esigma 70-directed P2 expression by Mlc was obtained at the level of 11.5 and 6.5 nM of the protein in the presence and absence of CRP·cAMP, respectively (Fig. 3A). However, about 100-fold more Mlc (0.7 and 0.6 µM of Mlc in the presence and absence of CRP·cAMP, respectively) was required in order to repress the P1 transcription (Fig. 3B). For half-repression of the Esigma 32-directed P2 promoter activity, 20 and 12.5 nM Mlc were required in the presence and absence of CRP·cAMP, respectively (Fig. 3C). Repression of P1 transcription and induction of Esigma 70-directed P2 transcription by CRP·cAMP shown in Fig. 2 were also observed in the presence of Mlc (compare lanes 1-4 with lanes 5-8 in Fig. 3, A and B).


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Fig. 3.   The autoregulatory effect of Mlc on its own gene in vitro. In vitro transcription was carried out using pMX as a DNA template. Forty nM of CRP·cAMP was added to the reactions as marked in the top of the figures. A, Esigma 70-directed transcription at low level of Mlc. The amount of Mlc used in each lane is following: lanes 1-4 and lanes 5-8 contains 0, 6.3, 12.5, and 25 nM Mlc in the reaction, respectively. B, Esigma 70-directed transcription at high level of Mlc. 0.25, 0.5, 1.0, and 1.5 µM Mlc was added to the lanes 1-4 and 5-8, respectively. C, Esigma 32-directed transcription in the presence of Mlc. Lanes 1-5 and 6-10 contain 0, 12.5, 25, 50, and 100 nM Mlc, respectively.

Transcription of mlc Was Modulated Less Than 2-fold by Glucose-- We studied further the regulation of mlc transcription in vivo by primer extension assay. The wild type strain MC4100 and its isogenic mutant strains were grown at 30 °C in the presence or absence of glucose then total RNA was extracted as described under "Experimental Procedures." Transcription from both P1 and P2 promoter was detected and the level of expression from two promoters was similar when the wild type cells were grown in the absence of glucose (Fig. 4A, lane 2). However, when cells were grown in the presence of glucose, transcription from P1 was increased slightly while that from P2 was not detectable (Fig. 4A, lane 1). These results establish that P1 and P2 promoter activity was dependent on CRP·cAMP in vivo in accordance with the in vitro transcription assay results as described above and that the overall expression level of mlc was changed less than 2-fold by glucose.


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Fig. 4.   In vivo transcription analysis and heat shock response of mlc. Primer extension analysis using 40 µg of total RNA per reaction was carried out as described under "Experimental Procedures." The start site of each promoter was marked with the name of each promoter. Sequence ladder was generated using the same end-labeled primer used for primer extension assay. (A) Wild type strain, MC4100 (lanes 1 and 2), MC4100 mlc::Tn10 (lanes 3 and 4), Delta rpoH strain (SR701, lanes 5 and 6), and MC4100 harboring pKV10 in which rpoH gene is under control of tac promoter (lanes 7 and 8) were grown at 30 °C in the presence or absence of glucose. In the case of MC4100 carrying pKV10, isopropyl-1-thio-beta -D-galactopyranoside was added to overexpress sigma 32. B, changes in mlc transcription in response to heat shock were monitored using primer extension analysis. Duration of heat shock was indicated on top.

We analyzed the mlc expression in SR505 (MC4100, mlc::Tn10) to test the autoregulatory effect of Mlc in vivo. The level of transcription could be analyzed in SR505 because Tn10 was inserted in the C-terminal region of the mlc gene. Transcription from both promoters was increased clearly when the mlc:: strain was grown in the absence of glucose (Fig. 4A, compare lanes 2 and 4), suggesting that Mlc acts as a repressor of the mlc promoter. A strong stimulatory effect of CRP·cAMP on mlc P2 promoter activity could also be seen when we compared the P2 promoter activities in SR505 grown in the presence and absence of glucose (Fig. 4A, lanes 3 and 4).

The P2 Promoter of mlc Is a Bona Fide Heat Shock Promoter-- In vitro transcription assay showed that the P2 promoter of mlc was recognized by Esigma 32 as well as Esigma 70. We analyzed expression of mlc in the Delta rpoH strain using primer extension assay. SR701 strain (MC4100, suhX1, Delta rpoH) can grow at 30 °C because it expresses a high level of GroELS due to an IS1 element inserted upstream of the groE gene (6, 26). Overexpression of GroELS did not affect mlc expression (data not shown). As shown on Fig. 4A (lanes 5 and 6), the expression level of mlc was decreased but the overall expression pattern was not changed in the Delta rpoH cell compared with wild type. However, P2 transcription was increased and its activity was not much affected by glucose in cells overexpressing sigma 32 (Fig. 4A, lanes 7 and 8). It is also interesting to note that even the activity of P1 that is not recognized by Esigma 32 in vitro was increased significantly when cells overexpressing Esigma 32 was grown in the presence of glucose (Fig. 4A, lane 7).

We also examined changes in the mlc expression upon heat shock by primer extension analysis. Growth temperature of a wild type strain, MC4100, was shifted from 30 to 42 °C, and cells were further incubated for the designated time as described under "Experimental Procedures." As shown on Fig. 4B, temperature upshift caused dramatic changes in the mlc expression. The mlc expression reached the maximum level 5 min after heat shock (Fig. 4B, lanes 3 and 4), when the intracellular concentration of Esigma 32 is known to be at the highest level (20, 32). When cells were grown in the presence of glucose (Fig. 4B, odd lanes), expression of both P1 and P2 was increased transiently after heat shock as in the cells overexpressing sigma 32 (Fig. 4A). The increased level of mlc expression from both P1 and P2 upon heat shock was decreased sharply after 15 min at 42 °C in the presence of glucose. Activation of mlc by heat shock was not dependent on the presence of glucose. The cells grown in the absence of glucose showed relatively little change in the P1 transcription but a high level of activation in the P2 transcription in response to heat shock (Fig. 4B, even lanes). However, heat shock effect was not observed in the Delta rpoH strain (data not shown), confirming that activation of mlc transcription by heat shock was dependent on Esigma 32. These results show that Esigma 32 can recognize the P2 promoter in vivo and Esigma 32-directed transcription from P2 becomes more prominent when cells were grown at a high temperature or overexpressing sigma 32.

Transcription of ptsG Is Increased by Heat Shock and Glucose Induction of ptsG Requires Esigma 32-- How does the highly increased level of mlc expression in heat-shocked cells affect glucose induction of Mlc regulon? To answer this question, we analyzed changes in the ptsG transcription upon heat shock. It has been known that transcription of the ptsG gene in E. coli encoding the major membrane-bound glucose transporter, EIICBGlc, is initiated from a major promoter, P1, and a minor promoter, P2, and that both P1 and P2 transcription is regulated negatively by Mlc (3, 5). There were no changes in P2 transcription of ptsG by heat shock (data not shown). But P1 transcription of ptsG was increased 5 min after heat shock when wild type cells were grown in the presence of glucose (Fig. 5A, compare lanes 1 and 3) despite that mlc expression was also increased at this condition as shown in Fig. 4B. However, ptsG P1 transcription was not changed by heat shock when cells were grown in the absence of glucose. Activation of ptsG P1 transcription by heat shock was also seen in the Delta mlc strain (Fig. 5A, lane 7), suggesting the possibility that Mlc was not involved in the activation of ptsG P1 by heat shock. In that expression of both EIICBGlc and its negative regulator Mlc was increased by heat shock, it seems that there is another mechanism(s) to maintain glucose induction of the Mlc regulon under the heat shock condition.


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Fig. 5.   Effects of heat shock RNA polymerase and Mlc on ptsG P1 and pts P0 transcription analyzed by primer extension analysis. A, heat shock effects on ptsG P1 transcription. Total RNA was extracted from cells grown at 30 °C to A600 = 0.5 in the presence or absence of glucose (lanes 1, 2, 5, 6, 9, and 10) or cells held at 42 °C for 5 min before RNA extraction (lanes 3, 4, 7, 8, 11, and 12). B, effects of heat shock RNA polymerase on glucose induction of pts P0 transcription. The pts P0 transcription was not induced by glucose in rpoH strain.

As shown on Fig. 5A, induction of ptsG P1 transcription by glucose was reduced significantly in the Delta rpoH strain. From these results, together with our previous data (9), it could be assumed that the Mlc regulon could not be induced by glucose because not enough EIICBGlc was available to sequester Mlc in the Delta rpoH strain. As expected, pts P0, one of several genes known to be under Mlc control (6-8), was not induced in the Delta rpoH strain (Fig. 5B). These results support the view that the expression of EIICBGlc and Mlc is balanced in response to glucose uptake even when the cells were heat-shocked and that Esigma 32 plays an important role in this regulation. However, it should be noted that heat shock still increased P1 transcription of ptsG in the Delta rpoH strain albeit to a lower degree when cells were grown in the presence of glucose (Fig. 5A).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been suggested that the mlc gene has two promoters, P1 and P2, which are separated by 12 bases and autoregulated by its product (2). Here, we report that the transcription of the mlc gene is regulated in a highly sophisticated manner and that heat shock sigma  factor, sigma 32, is involved in its transcription.

It is known that the expression level of several genes encoding transcriptional repressors such as galS (33), nagC (34), purR (35), and trpR (36) of E. coli is low and that their expression level is not modulated much in various growth conditions. It seems likely that both CRP·cAMP and Mlc work together in E. coli to maintain the level of Mlc optimum in response to availability of glucose. All genes known to be regulated negatively by Mlc, such as manXYZ, malT, ptsG, and pts, are also regulated positively by CRP·cAMP (2-8). In vitro transcription assay with Esigma 70 and CRP·cAMP showed two opposite effects on each promoter of mlc, that is the positive effect on P2 and the negative effect on P1 (Fig. 2). This can be explained based on the fact that the CRP-binding site of P2 centered at -71.5 to the transcription start site is more compatible for a functional CRP site (37) compared with that of P1 centered at -58.5 to the transcription start site. However, Esigma 32-directed P2 transcription was insensitive to CRP·cAMP. The P2 promoter of mlc should be a good model system to assess the effect of sigma  factor on transcription activation by CRP·cAMP because it has been known that CRP·cAMP activates transcription by direct protein-protein interaction with the alpha -subunit of RNA polymerase (38).

Repression of the P2 transcription when cells were grown in the presence of glucose implies that the action of CRP·cAMP is dominant over the self-repression by Mlc in the regulation of the mlc P2 promoter. The low binding affinity of Mlc to its own promoter that is 10 times weaker than that to ptsG or pts P0 promoters (9) seems to be a major reason for the low influence of Mlc on regulation of its own gene. In vitro transcription assay revealed that each promoter of mlc has a different sensitivity to Mlc (Fig. 3). When cells were grown in the absence of glucose, a similar level of expression from both P1 and P2 was observed even though the P2 transcription is more sensitive to Mlc repression probably because the intracellular concentration of Mlc is limiting in E. coli (3). The condition seems to be similar to the in vitro transcription condition where both CRP·cAMP and a small amount of Mlc were present as shown in lane 6 of Fig. 3A. However, P1 was as active as P2 when the mlc strain was grown in the absence of glucose. These results imply that the concentration of intracellular CRP·cAMP is lower than that of CRP·cAMP used for in vitro transcription reactions (40 nM) (39). When Mlc was induced and the concentration of CRP·cAMP was lowered by the addition of glucose in the growth medium, the P1 promoter was activated slightly while the P2 promoter was repressed because the P2 promoter is active only in the presence of CRP·cAMP. This situation is similar to the in vitro transcription condition where neither CRP·cAMP nor Mlc were present (lane 1 of Fig. 3A). Therefore, the addition of glucose in the growth medium resulted in the reduction of mlc expression by about half. These results also agree with the previous report by Decker et al. (2) that expression of mlc is reduced by half when cells were grown in the presence of glucose by measuring the beta -galactosidase activity of the mlc-lacZ fusion. Level of Mlc expression can vary precisely in response to the available sugars but the variation range is less than 2-fold in that the availability of unphosphorylated EIICBGlc may be more critical than the intracellular level of Mlc for induction of the Mlc regulon by glucose as shown in our previous report (9).

Esigma 32 is involved in the transcription of the mlc gene. In vitro transcription assay with Esigma 32 showed that Esigma 32 could recognize the P2 promoter of the mlc gene. Transcription of P2 was increased when sigma 32 was overexpressed (Fig. 4A). Moreover, mlc expression was increased upon heat shock. It is known that the intracellular concentration of sigma 32 in E. coli increases from 15-20-fold within 5 min then declines to a new steady-state level severalfold higher than the preshift level in response to temperature shift from 30 to 42 °C (20, 32). Transcription from P2 recognized by Esigma 32 was induced transiently to an extraordinary level upon heat shock when cells were grown in the absence of glucose (Fig. 4B, lane 4). The level of mlc transcription was changed parallel to the changes in intracellular concentration of sigma 32. In addition, P2 transcription was activated upon heat shock even when cells were grown in the presence of glucose. These results imply that the major RNA polymerase which activated the P2 transcription upon heat shock was Esigma 32 because Esigma 32 was less sensitive to Mlc than Esigma 70 and the P2 transcription by Esigma 32 was not dependent on CRP·cAMP as revealed by the in vitro transcription assay (Fig. 3). It is not clear why the P1 transcription was reduced in the Delta rpoH strain and activated by heat shock or when cells overexpressing sigma 32 were grown in the presence of glucose even though P1 promoter was not recognized by Esigma 32 in vitro. Because heat shock should exert pleiotropic effects by regulating transcription of various genes (21), further study on the mechanism of heat shock is needed for a better understanding of these phenomena.

To investigate whether the increased level of mlc expression resulting from heat shock can influence Mlc-dependent gene expression, we analyzed changes in ptsG expression by heat shock. The P1 expression of ptsG was increased significantly by heat shock only when cells were grown in the presence of glucose regardless of the presence of Mlc (Fig. 5A). These results suggest that activation of ptsG P1 by heat shock was not mediated by Mlc or CRP·cAMP even though Mlc repression might be dominant over activation of ptsG P1 by heat shock. We have reported that the unphosphorylated form of EIICBGlc sequesters Mlc from its target promoters upon glucose uptake by direct protein-protein interaction (9). Therefore, glucose is required to maximize the level of dephosphorylated EIICBGlc necessary to sequester Mlc that is increased by heat shock. However, contrary to the case of mlc P2 transcription in which Esigma 32 plays a major role in its regulation, it is likely that additional factors independent of Esigma 32 are involved in regulation of the ptsG expression because ptsG P1 expression was increased partially upon heat shock even in the Delta rpoH strain grown in the presence of glucose. It means that two separate mechanisms involving Esigma 32-dependent and Esigma 32-indendent activation of ptsG P1 may work additively for full activation of ptsG P1 by glucose when cells were heat-shocked. The importance of Esigma 32 in glucose induction of the Mlc regulon was manifested by the fact that glucose induction of ptsG could not be observed in the Delta rpoH strain. The inability of glucose to activate ptsG expression resulted in an insensitivity of the pts P0 promoter to glucose in Delta rpoH strain. We are trying to elucidate the mechanism of activation of ptsG transcription by heat shock in the absence of Esigma 32 in order to understand the general role of Esigma 32 in regulation of genes involving carbohydrate metabolism.

    ACKNOWLEDGEMENTS

We are grateful to Dr. C. Park for providing KD413 and Dr. T. Yura for providing bacterial strains and a pKV10.

    FOOTNOTES

* This work was supported in part by Grant 2000-2-20200-006-3 from the Basic Research Program of the Korea Science & Engineering Foundation.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 Supported by the Brain Korea21 Project

§ Supported by the Brain Korea21 Project.

|| To whom correspondence should be addressed: Dept. of Food Science and Technology and School of Agricultural Biotechnology, Seoul National University, Suwon, 441-744 Korea. Tel.: 82-31-290-2584; Fax: 82-31-293-4789; E-mail: sangryu@snu.ac.kr.

Published, JBC Papers in Press, May 4, 2001, DOI 10.1074/jbc.M101757200

    ABBREVIATIONS

The abbreviations used are: CRP, cyclic AMP receptor protein; PTS, phosphoenolpyruvate: carbohydrate phosphotransferase system.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Hosono, K., Kakuda, H., and Ichihara, S. (1995) Biosci. Biotechnol. Biochem. 59, 256-261[Medline] [Order article via Infotrieve]
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