The ptsG mRNA encoding the major glucose transporter is rapidly degraded in an RNase E-dependent manner in response to the accumulation of glucose 6-P or fructose 6-P when the glycolytic pathway is blocked at its early steps in Escherichia coli. RNase E, a major endonuclease, is associated with polynucleotide phosphorylase (PNPase), RhlB helicase and a glycolytic enzyme, enolase, which bind to its C-terminal scaffold region to form a multienzyme complex called the RNA degradosome. The role of enolase within the RNase E-based degradosome in RNA decay has been totally mysterious. In this article, we demonstrate that the removal of the scaffold region of RNase E suppresses the rapid degradation of ptsG mRNA in response to the metabolic stress without affecting the expression of ptsG mRNA under normal conditions. We also demonstrate that the depletion of enolase but not the disruption of pnp or rhlB eliminates the rapid degradation of ptsG mRNA. Taken together, we conclude that enolase within the degradosome plays a crucial role in the regulation of ptsG mRNA stability in response to a metabolic stress. This is the first instance in which a physiological role for enolase in the RNA degradosome has been demonstrated. In addition, we show that PNPase and RhlB within the degradosome cooperate to eliminate short degradation intermediates of ptsG mRNA.
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The degradation of mRNA is mediated by the combined action of endo- and exoribonucleases in Esherichia coli (Coburn and Mackie, 1999; Grunberg-Manago, 1999; Regnier and Arraiano, 2000; Carpousis, 2002; Kushner, 2002). A major endoribonuclease, RNase E, plays a central role in the decay of many mRNAs by initiating endonucleolytic cleavage. The mRNA fragments resulting from RNase E cleavage are thought to be further degraded endonucleolytically and trimmed by the 3′ to 5′ exonucleases, polynucleotide phosphorylase (PNPase) and RNase II, assisted by poly(A) polymerase. RNase E is part of a multiprotein complex called the RNA degradosome, which also contains PNPase, RhlB and enolase as major components (Carpousis et al., 1994; Miczak et al., 1996; Py et al., 1996). RhlB is a member of the DEAD-box family of ATP-dependent RNA helicases (Py et al., 1996) whereas enolase is a glycolytic enzyme that catalyses the interconversion of 2-phospho- d-glycerate and phosphoenolpyruvate (Fraenkel, 1996). RNase E itself is a large protein (1061 amino acid residues), composed of three distinct domains, an N-terminal catalytic region, a central RNA-binding domain (RBD) and a C-terminal region carrying the binding sites for PNPase, RhlB and enolase (McDowall and Cohen, 1996; Vanzo et al., 1998; Carpousis, 2002).
The discovery of the RNA degradosome raised an attractive idea that it may act as a general RNA decay machine and that the components of the degradosome may cooperate during the decay and/or processing of many RNAs. It is easy to imagine that both RNase E and PNPase within the degradosome act in a concerted fashion during the degradation of an RNA and that RhlB helicase unwinds secondary structure to permit access by RNase E and/or PNPase during the degradation of an RNA. In fact, there is evidence that the major components of the degradosome can functionally interact each other. For example, PNPase and RNase E were shown to act together in the degradation of an anti-sense RNA I of ColE1-type plasmids (Xu and Cohen, 1995). RhlB helicase was shown to stimulate degradation of the malEF transcript by PNPase in vitro (Py et al., 1996; Coburn et al., 1999). On the other hand, the role of enolase in the degradosme has remained to be completely mysterious.
Various truncated RNase E proteins lacking the scaffold region have been used to study functional roles of degradosome formation in mRNA degradation. The best-characterized such mutant is rne 131 (smbB131), which was first isolated as an extragenic suppressor that alleviates temperature-sensitive colony formation and anucleate cell production caused by a mukB mutation (mukB106) (Kido et al., 1996). The rne 131 mutant encodes a polypeptide lacking the last 477 C-terminal amino acid residues of RNase E. It was hypothesized that the elevated level of the mutant MukB protein results from the stabilization of the mukB mRNA by the rne 131 mutation (Kido et al., 1996). Subsequently, the rne 131 mutation was shown to cause stabilization of both bulk and several specific mRNAs (Lopez et al., 1999; Nogueira et al., 2001). The stabilization of bulk and a number of individual mRNAs in the rne 131 strain has been observed in a recent DNA microarray-based global analysis of mRNAs (Bernstein et al., 2004). The microarray analysis has also revealed that the rhlB, pnp and eno mutations cause prolongation of the median mRNA half-life more or less (Bernstein et al., 2004). These results support a view that degradosome assembly is involved in mRNA decay in vivo. However, the situation is complicated by the fact that the truncated RNase E encoded by the rne 131 lacks both the C-terminal scaffold and the central RBD (Kido et al., 1996; Vanzo et al., 1998). In other words, the properties of the rne 131 mutation do not prove that the degradosome assembly itself really affects mRNA decay in vivo because any effects manifested by the rne 131 mutation could result from the lack of either the scaffold region or the RBD or both. In fact, Ow et al. (2000) revealed by using a series of truncated RNase E proteins that the removal of the scaffold region alone only slightly affected normal mRNA decay whereas mRNA decay was significantly impaired when both the scaffold region and RBD were deleted. They concluded based on these results that the degradosome assembly is not required for normal mRNA decay. However, their results do not exclude the possibility that the degradosome assembly may play roles in the regulation of mRNA stability under certain physiological conditions.
Previously, we demonstarted that the ptsG mRNA encoding the major glucose transporter (IICBGlc) is dramatically destabilized when the glycolytic pathway is blocked in its early stages (Kimata et al., 2001). The rapid degradation of ptsG mRNA in response to the block of glycolysis is mediated by RNase E (Kimata et al., 2001). We also found that the accumulation of either glucose 6-P or fructose 6-P is responsible for the RNase E-mediated destabilization of ptsG mRNA (Morita et al., 2003). In this article, we addressed the question whether the RNA degradosome assembly and/or its components are involved in the regulation of ptsG mRNA degradation in response to the accumulation of glucose 6-P. We demonstrate that the rapid degradation of ptsG mRNA is abolished when the C-terminal scaffold region of RNase E is deleted. The depletion of enolase but not the disruption of pnp and rhlB encoding PNPase and RhlB, respectively, eliminates the rapid degradation of ptsG mRNA. We conclude that enolase within the assembled degradosome plays a critical role in the regulation of ptsG mRNA degradation in response to phosphosugar stress. This is the first instance in which a physiological role of enolase in the RNA degradosome has been demonstrated.
Alterations of the chromosomal rne gene and expression of truncated RNase E proteins
Ow et al. (2000) analysed the effects of rne truncations by using strains harbouring a plasmid carrying various rne alleles but lacking a chromosomal rne. To investigate whether the RNase E-based degradosome assembly is involved in the regulation of ptsG mRNA degradation, we constructed strains in which the chromosomal rne gene was manipulated to encode either RNase E844-FLAG, RNase E701-FLAG, or RNase E598-FLAG (Fig. 1A). These C-terminally FLAG-tagged truncated RNase E proteins retain the N-terminal catalytic region, which is essential for cell viability, but lack part or all of the C-terminal scaffold region and the central RBD. RNase E844-FLAG and RNase E701-FLAG are missing the last 217 and 360 amino acid residues corresponding to the PNPase binding site and the entire scaffold region respectively. RNase E598-FLAG is missing the last 477 amino acid residues corresponding to both the scaffold region and the RBD. We also constructed a control strain in which the rne gene was altered to encode FLAG-tagged full-length RNase E (RNase E-FLAG). The advantage of our system compared with the previous analysis is the use of isogenic strains with single-copy chromosomal RNase E alleles with FLAG epitope rather than mutations on plasmid. This allows us to analyse simultaneously both the degradosome composition and a more accurate effect of RNase E truncation on mRNA stability.
Western blot analysis using antibody to the FLAG tag revealed that all of the FLAG-tagged RNase E proteins were clearly detected (Fig. 1B, lanes 2–5) whereas no signal was detected in the control strain expressing the non-tagged wild-type RNase E (Fig. 1B, lane 1). The expression level of the RNase E proteins increased when the C-terminal regions were deleted. The increase was dramatic when both the C-terminal scaffold region and the central RBD were removed (Fig. 1B, lane 5). These results are in agreement with previous reports (Ow et al., 2000; Lee et al., 2002; Ow and Kushner, 2002), indicating that the removal of C-terminal region leads to the loss of RNase E autoregulation. In addition, our experiments clearly indicate that the C-terminal FLAG tag does not perturb the RNase E autoregulation.
Properties of strains carrying rne truncations
Kushner and coworkers reported that truncation of the C-terminal scaffold region alone caused a moderate growth reduction whereas removal of both the C-terminal scaffold region and RBD impaired further the growth rate resulting in the loss of viability at 44°C (Ow et al., 2000; Ow and Kushner, 2002). We examined the growth properties of our strains carrying the truncated rne alleles in LB medium. All strains except TM529 carrying the rne598-FLAG exhibited growth rates that were almost identical to the wild-type control at 37°C (Fig. 2A). Strain TM529 lacking both the C-terminal scaffold region and RBD showed a slightly reduced growth rate (Fig. 2A) but it was still viable even at 44°C (data not shown). The discrepancy concerning the effect of RNase E truncations on growth properties between two systems may arise from the different strain backgrounds and/or different natures of truncated rne alleles. We observed that the 9S rRNA processing activity was normal in cells expressing the FLAG-tagged RNase E variants as expected (data not shown). Previous work showed that both bulk RNA and several specific mRNAs were significantly stabilized in strains expressing RNase E lacking both the C-terminal scaffold region and RBD (Lopez et al., 1999; Nogueira et al., 2001; Bernstein et al., 2004) whereas the removal of the scaffold region alone only slightly affected mRNA stability (Ow et al., 2000). To examine whether the C-terminal truncation of RNase E affects the stability of ptsG mRNA, the expression of ptsG mRNA in a series of strains was analysed by Northern blotting. The levels of ptsG mRNA in the four strains carrying the different rne alleles were essentially identical (Fig. 2B), suggesting that the removal of the scaffold region and RBD does not affect the stability of ptsG mRNA, at least under normal conditions.
Analysis of degradosome assembly in strains expressing FLAG-tagged RNase E variants
In order to examine the ability of FLAG-tagged RNase E variants to interact with the three other major components of the RNA degradosome, strains carrying pnp-HA or rhlB-HA alleles encoding HA-tagged PNPase or RhlB, respectively, were constructed and these alleles were transferred to the rne-FLAG and rne701-FLAG strains. Neither growth nor expression of FLAG-tagged RNase E variants was affected by the introduction of the pnp-HA or rhlB-HA alleles (data not shown). Extracts prepared from cells grown to mid-log phase were incubated with anti-FLAG M2-agarose beads and proteins bound to the agarose beads were analysed by Western blotting. Anti-FLAG and anti-enolase antibodies were used to detect RNase E and enolase, respectively, whereas anti-HA antibody was used to detect PNPase and RhlB. As expected, both RNase E-FLAG and RNase E701-FLAG proteins were efficiently recovered in the bound fraction (Fig. 3, top, lanes 4–9). The pull-down assay demonstrated that PNPase, RhlB and enolase were co-eluted with RNase E-FLAG, indicating that the FLAG-tagged full-length RNase E retains the ability to form the RNA degradosome (Fig. 3, middle and bottom, lane 6). On the other hand, none of three other major degradosome components were co-eluted with RNase E701-FLAG, indicating that the truncated RNase E no longer retains the ability to form the degradosome (Fig. 3, middle and bottom, lane 9). These results confirm that the C-terminal scaffold region of RNase E is responsible for the interaction with PNPase, enolase and RhlB to form the RNA degradosome. Neither PNPase, enolase nor RhlB was retained by the agarose beads as expected (Fig. 3, lane 3). The data in Fig. 3 also show that the failure of degradosome formation does not affect the levels of enolase and PNPase whereas the level of RhlB helicase appears to be reduced by the RNase E truncation (Fig. 3, lanes 1, 4 and 7).
The destabilization of ptsG mRNA in response to glucose 6-P accumulation no longer occurs when the scaffold region of RNase E is removed
To examine whether the failure of degradosome assembly affects the degradation of ptsG mRNA in response to the accumulation of glucose 6-P, the rne701-FLAG or rne-FLAG allele was transferred to the Δpgi strain lacking phosphoglucose isomerase that catalyses the interconversion of glucose 6-P and fructose 6-P. We showed previously that the rapid degradation of ptsG mRNA occurs in the Δpgi background in the presence of glucose because of the accumulation of glucose 6-P (Kimata et al., 2001; Morita et al., 2003). The expression pattern of ptsG mRNA in both pgi+ and Δpgi cells grown in LB medium containing 1% glucose was analysed by Northern blotting. The ptsG mRNA was again stably expressed in all pgi+ strains (Fig. 4A, lanes 1, 3 and 5); it became unstable in Δpgi rne-FLAG (Fig. 4A, lane 4), as in the cells carrying the wild-type rne allele (Fig. 4A, lane 2). Interestingly, the full-length ptsG mRNA was stably expressed when the rnE701-FLAG allele was transferred into the Δpgi background (Fig. 4A, lane 6). The stabilization of ptsG mRNA in the Δpgi background was also observed when the rne598-FLAG allele but not the rne844-FLAG allele was transferred to the Δpgi strain (data not shown). These results imply that the C-terminal scaffold region of RNase E is necessary for the rapid degradation of ptsG mRNA in response to the accumulation of glucose 6-P. Another interesting observation is that a significant amount of short degradation intermediates of ptsG mRNA accumulated in the Δpgi rnE701-FLAG cells (Fig. 4A, lane 6). This suggests that degradosome assembly also plays a role in the elimination of short degradation intermediates.
Neither PNPase nor RhlB helicase is required for the rapid degradation of ptsG mRNA
The results described above strongly suggest that any one or more components assembled within the RNA degradosome play a critical role in the destabilization of ptsG mRNA in response to the accumulation of glucose 6-P. Therefore, we examined the effect of each of three major proteins associated with RNase E on ptsG mRNA degradation by manipulating the corresponding genes. We first constructed strains in which pnp or rhlB was disrupted in the pgi+ and the Δpgi backgrounds and tested the effect of individual mutations on the ptsG mRNA by Northern blotting. As shown in Fig. 4B, the destabilization of ptsG mRNA occurred normally in the Δpgi background when either pnp or rhlB was disrupted, indicating that neither PNPase nor RhlB is required for the destabilization of the full-length ptsG mRNA in response to accumulation of glucose 6-P. However, a significant amount of the degradation intermediates of ptsG mRNA accumulated. This was particularly true in the ΔpgiΔpnp cells (Fig. 4B, lane 4) where the amount was similar to that in the Δpgi rne701-FLAG cells (Fig. 4A, lane 6). The accumulation of the intermediates was also observed in the ΔpgiΔrhlB cells although less significantly (Fig. 4B, lane 6). These data strongly suggest that PNPase and RhlB assembled in the RNA degradosome cooperate to degrade efficiently short RNAs generated by the endonucleolytic cleavage by RNase E although they are not involved in the destabilization of the full-length ptsG mRNA itself. Our observation is consistent with a recent finding that RhlB assembled within the degradosome along with poly(A) polymerase facilitates the degradation of small mRNA decay intermediates containing REP-stabilizers by PNPase (Khemici and Carpousis, 2004).
The ptsG mRNA under the control of the bla promoter is destabilized in response to the accumulation of glucose 6-P
Before testing the possible role of enolase in the degradation of ptsG mRNA, we constructed strains in which the ptsG on the chromosome was under the control of the constitutive bla promoter by the modified method of Datsenko and Wanner (2000) using pTM27 as a template for polymerase chain reaction (PCR) amplification (Fig. 5A). This is because the transcription of ptsG is strictly dependent on CRP-cAMP and mutations in several genes for glycolytic enzymes reduce the ptsG expression presumably by affecting the CRP-cAMP pathway (Morita et al., 2003). The replacement of the ptsG promoter by the constitutive bla promoter would eliminate this potential problem. The chimeric Pbla-ptsG gene was designed to reproduce the ptsG mRNA including 5′ leader region (Fig. 5A). Northern blot analysis revealed that the full-length ptsG mRNA was stably detected when the Pbla-ptsG allele was expressed in the pgi+ background (Fig. 5B, lane 1) while it became unstable in the Δpgi background (Fig. 5B, lane 2). The introduction of the ams1 mutation encoding a temperature-sensitive RNase E suppressed the destabilization of ptsG mRNA in the Δpgi Pbla-ptsG cells at non-permissive temperature but not at 30°C (Fig. 5B, lanes 3 and 4). Thus, the response of ptsG mRNA to the accumulation of glucose 6-P was not affected by the replacement of the ptsG promoter by the bla promoter.
Construction and growth property of strains in which the eno gene is under the control of the arabinose-inducible promoter
To examine the possible role of enolase in the degradation of ptsG mRNA, we tried to disrupt the eno gene by the one-step gene disruption method. Our initial attempt to disrupt the eno gene was unsuccessful because cells apparently require enolase to grow in LB medium. In fact, it is known that cells carrying a mutation in eno do not grow in rich medium (Irani and Maitra, 1977). Therefore, we constructed a strain in which the eno gene is under the control of an arabinose-inducible promoter PBAD by the modified method of Datsenko and Wanner using pIT801 as a template for PCR amplification (Fig. 6A). The PBAD-eno allele was transferred into the Pbla-ptsG and the Δpgi Pbla-ptsG strains. The PBAD-eno allele could support cell growth on LB agar plates containing arabinose but not without arabinose (Fig. 6B, left). On the other hand, the PBAD-eno strains grow normally on M9 agar plates containing both succinate and glycerol even without arabinose (Fig. 6B, right). These results are consistent with the early report that a mixture of glycerol and succinate could support the growth of cells lacking enolase (Irani and Maitra, 1977).
The rapid degradation of ptsG mRNA no longer occurs in the absence of enolase
To examine the effect of the depletion of enolase on the degradation of ptsG mRNA, PBAD-eno Pbla-ptsG cells were grown in the M9 minimum medium containing succinate plus glycerol with or without arabinose. Glucose was added to the culture and the incubation was continued for 30 min. Then, total proteins and RNAs were prepared. Western blot analysis revealed that a significant amount of enolase was expressed when cells were grown in the presence of arabinose but no enolase was detected in cells grown without arabinose in both the pgi+ and the Δpgi backgrounds (Fig. 7A). Northern blotting indicated that the full-length ptsG mRNA was stably expressed when the pgi+PBAD-eno Pbla-ptsG cells were grown in the presence and the absence of arabinose (Fig. 7B, lanes 1 and 2). As expected, the full-length ptsG mRNA was no longer observed when the Δpgi PBAD-eno Pbla-ptsG cells were grown in the presence of arabinose because of the rapid degradation of ptsG mRNA (Fig. 7B, lane 3). An important observation was that the full-length ptsG mRNA became stable in the Δpgi PBAD-eno Pbla-ptsG cells grown in the absence of arabinose (Fig. 7B, lane 4). We confirmed that glucose 6-P accumulates upon glucose addition in the Δpgi PBAD-eno Pbla-ptsG cells grown with or without arabinose (Fig. 7C). These results establish that enolase is certainly required for the rapid degradation of ptsG mRNA in response to the accumulation of glucose 6-P. In addition, we observed that enolase depletion eliminates the rapid degradation of ptsG mRNA when pgk, located upstream of the eno in the glycolytic pathway, was disrupted (Fig. 7B, lane 6), indicating that the effect of enolase depletion does not result from the accumulation of 2-P-glycerate and/or 3-P-glycerate.
In the present work, we examined whether or not RNA degradosome assembly affects the rapid degradation of ptsG mRNA in response to the accumulation of glucose 6-P. This question is particularly intriguing because the glycolytic enzyme enolase is one of the major components of RNA degradosome (Py et al., 1996). We focused on a truncated RNase E (RNase E701-FLAG) lacking the entire C-terminal scaffold region but retaining the central RBD and the N-terminal catalytic region (Fig. 1). We verified that RNase E701-FLAG no longer retains the ability to associate with PNPase, RhlB and enolase (Fig. 3). We demonstrated that the prevention of degradosome assembly has no significant effect on the decay of ptsG mRNA under normal conditions (Fig. 2B). However, we found that the destabilization of ptsG mRNA in response to the accumulation of glucose 6-P no longer occurs in cells expressing RNase E701-FLAG (Fig. 4A). These results clearly establish that the C-terminal scaffold region of RNase E and therefore the assembly of the degradosome are required for the destabilization of ptsG mRNA in response to the specific metabolic stress. The present study strongly suggests that the degradosome assembly plays important roles in the regulation of mRNA stability in general under stress conditions such as the accumulation of phosphosugars rather than in normal conditions.
The highlight in the present study is the finding that the destabilization of ptsG mRNA in response to the accumulation of glucose 6-P no longer occurs in cells carrying the wild-type rne allele when enolase but not PNPase or RhlB is depleted (Figs 4B and 7). These results definitely establish that enolase is required for the destabilization of ptsG mRNA in response to the metabolic stress. Enolase could play its role in the RNase E-mediated destabilization of ptsG mRNA independently of the degradosome assembly. This could be possible, for example, if the depletion of enolase somehow prevents the accumulation of glucose 6-P in the Δpgi cells even in the presence of glucose. However, this is apparently not the case because glucose 6-P normally accumulates in the Δpgi cells (Fig. 7C). The simplest possibility would be that enolase integrated in the degradosome modulates RNase E action on ptsG mRNA under metabolic stress. In any case, this is the first instance in which a functional role of enolase in mRNA decay has been shown.
How does enolase integrated in the degradosome stimulate the rapid degradation of ptsG mRNA in response to phosphosugar stress? Although we do not have any definite answers to this question at this moment, it would be worthwhile to speculate several possibilities in connection with related observations. First, it should be noted that RNase E and therefore the RNA degradosome seem to be predominantly located near the cytoplasmic membrane (Liou et al., 2001). Our preliminary results suggest that the localization of ptsG mRNA to the inner membrane coupled with co-translational membrane targeting of IICBGlc is essential for the destabilization of ptsG mRNA in response to the accumulation of glucose 6-P. Thus, one possible mechanism by which enolase stimulates ptsG mRNA degradation would be to modulate the membrane localization of RNase E. It is certainly interesting to test whether the depletion of enolase or the lack of degradosome assembly affects RNase E localization. Alternatively, the enolase integrated in the degradosome may directly affect the activity of RNase E by a totally unknown mechanism without changing its localization. Increasing numbers of proteins have been found to have two or more different functions (Jeffery, 1999). In particular, it is known that glycolytic enzymes such as phosphoglucose isomerase and glyceraldehydes-3-phosphate dehydrogenase often exhibit additional functions other than their major enzymatic activities in various organisms. The role of enolase in the regulation of the ptsG mRNA degradation is an additional example for the multiple functions of ‘moonlighting’ proteins. In any case, if the enolase protein integrated in the degradosome is involved in the regulation of ptsG mRNA degradation, one may expect to isolate enolase mutants that are specifically defective in the ability to interact with RNase E. Such mutations may lead to the stabilization of ptsG mRNA without losing its activity of enolase even under metabolic stress. Isolation and characterization of enolase mutants would certainly contribute to our understanding of the mechanism by which enolase facilitates the degradation of ptsG mRNA in response to the metabolic stress.
Another important finding in this study is that the removal of the C-terminal scaffold region of RNase E leads to the accumulation of short mRNA fragments corresponding to decay intermediates (Fig. 4A). The accumulation of short mRNA fragments was also observed strongly in a strain lacking PNPase and moderately in a strain lacking RhlB helicase (Fig. 4B). These results led us to conclude that PNPase and RhlB helicase assembled within the degradosome cooperate to degrade efficiently mRNA decay intermediates. Recently, it has been reported that RhlB helicase assembled in the degradosome along with poly(A) polymerase is required for the degradation of small mRNA decay intermediates containing REP-stabilizers (Khemici and Carpousis, 2004). Our finding suggests that PNPase and RhlB helicase assembled in the RNA degradosome are acting more widely to efficiently degrade mRNA decay intermediates.
Although we have demonstrated that enolase integrated in the degradosome plays a crucial role in the rapid degradation of ptsG mRNA in response to the accumulation of glucose 6-P degradation, it has not been worked out yet how the ptsG mRNA is destabilized specifically when glucose 6-P and/or fructose 6-P accumulate. We have observed that the destabilization of ptsG mRNA no longer occurs in cells lacking Hfq (our unpublished result). This suggests that Hfq is an additional player involved in the regulation of the decay of ptsG mRNA. Hfq was originally identified as a host factor required for phage Qb replication (Franze de Fernandez et al., 1968) and has been shown to be implicated in post-transcriptional regulation by affecting stability and/or translation of several mRNAs (Tsui et al., 1997; Vytvytska et al., 1998). Recent studies have revealed that a number of small regulatory RNAs associate with Hfq to regulate target mRNAs in E. coli (Gottesman, 2002; Masse and Gottesman, 2002; Moller et al., 2002; Zhang et al., 2002; Masse et al., 2003; Vecerek et al., 2003; Valentin-Hansen et al., 2004). Among them, RyhB is particularly intriguing because it is known to stimulate the degradation of mRNAs encoding Fe-binding or Fe-storage proteins presumably by pairing with its target mRNAs in response to Fe depletion (Masse and Gottesman, 2002; Masse et al., 2003). It has been demonstrated that degradation of target mRNAs is coupled with RyhB turnover and their degradation is dependent on RNase E (Masse et al., 2003). Furthermore, it should be noted that degradation of target mRNAs is slowed down in degradosome mutants (Masse et al., 2003). These properties of RyhB-targeted mRNAs are strikingly analogous to those of ptsG mRNA. Thus, it is quite possible that a specific small regulatory RNA may act together with Hfq in the regulation of ptsG mRNA. In fact, Vanderpool and Gottesman (2004) have just discovered that RyaA (SgrS), an Hfq-binding small RNA, is indeed involved in the destabilization of ptsG mRNA. They have shown that RyaA is expressed in response to phosphosugar accumulation and that RyaA expression is correlated with greatly reduced levels of ptsG mRNA. The downregulation of ptsG mRNA by RyaA apparently results from the degradation of message presumably mediated by RyaA–ptsG pairing. They also found that the expression of ryaA is regulated by a novel transcriptional activator, YabN (SgrR), that may activate ryaA transcription in response to high intracellular levels of phosphosugars although how phosphosugar accumulation modulates YabN remains to be studied.
Media and growth conditions
Cells were grown aerobically at 37°C unless specified in LB medium or M9 minimum medium supplemented with indicated sugars. Antibiotics were used at the following concentrations when needed: ampicillin (50 µg ml−1) and chloramphenicol (15 µg ml−1). Bacterial growth was monitored by determining the optical density at 600 nm.
Bacterial strains and plasmids
The E. coli K-12 strains and plasmids used in this study are listed in Table 1. IT1568 (W3110 mlc) was used as a parent wild-type strain. TM224 (Δpgi) was constructed from TM162 (Δpgi::cat) by eliminating the cat marker by using an FLP expression plasmid pCP20. TM388 (Δpnp::cat) and TM390 (ΔrhlB::cat) were constructed from IT1568 by one-step gene inactivation protocol (Datsenko and Wanner, 2000). The Δpnp::cat and ΔrhlB::cat alleles were moved to TM224 by P1 transduction to construct TM389 (ΔpgiΔpnp::cat) and TM391 (ΔpgiΔrhlB::cat) respectively. TM338 (rne-FLAG-cat), TM527 (rne844-FLAG-cat), TM528 (rne701-FLAG-cat) and TM529 (rne598-FLAG-cat) were constructed from IT1568 according to the modified Datsenko-Wanner protocol using pSU313 harbouring the FLAG-tag sequence (Uzzau et al., 2001). Similarly, TM409 (pnp-HA-cat) and TM411 (rhlB-HA-cat) were constructed using pSU314 harbouring the HA-tag sequence. The FLAG-tagged rne allelles were moved to TM224 to construct TM339 (Δpgi rne-FLAG-cat), TM524 (Δpgi rne844-FLAG-cat), TM525 (Δpgi rne701-FLAG-cat) and TM526 (Δpgi rne598-FLAG-cat). The cat marker of TM411 was eliminated to construct TM415 (rhlB-HA). The pnp-HA-cat allele of TM409 was moved to TM415 to construct TM420 (rhlB-HA pnp-HA-cat). The cat marker of TM420 was eliminated to construct TM432 (pnp-HA rhlB-HA). The rne-FLAG-cat and rne701-FLAG-cat alleles were moved to TM432 to construct TM433 (pnp-HA rhlB-HA rne-FLAG-cat) and TM434 (pnp-HA rhlB-HA rne701-FLAG-cat) respectively. TM449 (cat-Pbla-ptsG) and TM447 (cat-PBAD-eno) were constructed according to the modified Datsenko-Wanner protocol (Uzzau et al., 2001) using pTM27 and pIT801 respectively. The cat-Pbla-ptsG allele was moved to TM224 to construct TM453 (Δpgi cat-Pbla-ptsG). The ams1 allele of GW20 was moved to TM453 to construct TM467 (Δpgi cat-Pbla-ptsG ams1). The cat marker of TM449 and TM453 was eliminated to construct TM454 (Pbla-ptsG) and TM461 (Δpgi Pbla-ptsG). The cat-PBAD-eno allele of TM447 was moved to TM454 and TM455 to construct TM465 (Pbla-ptsG cat-PBAD-eno) and TM466 (Δpgi Pbla-ptsG cat-PBAD-eno) respectively. The cat marker of TM466 was eliminated to construct TM509 (Δpgi Pbla-ptsG PBAD-eno). The Δpgk::cat allele of TM61 was moved to TM509 to construct TM510 (Δpgi Pbla-ptsG PBAD-enoΔpgk::cat).
Table 1. Bacterial strains and plasmids used in this study.
Derivative of pIT801 carrying FRT-cat-FRT-rrnB T-PBAD
Derivative of pIT800 carrying FRT-cat-FRT-Pbla
To construct pIT800, a 1.1 kb DNA fragment carrying the cat gene flanked by FRT sites was amplified from pKD3 by PCR and cloned between EcoRI and PstI sites of pTWV228 (Takara). To construct pIT801, a 300 bp DNA fragment carrying the araBAD promoter (PBAD) was amplified from pBAD33 (Guzman et al., 1995) by PCR and cloned between PstI and HindIII sites of pIT800. To construct pTM26, a 100 bp DNA fragment carrying the rrnB terminator was amplified from pKK3535 (Brosius et al., 1981) by PCR and cloned into PstI site of pIT801. To construct pTM27, a 300 bp DNA fragment carrying the bla promoter (Pbla) was amplified from pBR322 by PCR and cloned between PstI and SphI sites of pIT800.
Cells were grown in 200 ml of LB medium to A600 of 0.6, harvested and washed with 10 ml of 50 mM Tris-HCl pH 8.0, 5 mM EDTA. The cell pellets were suspended in ice-cold 5 ml of IP buffer (20 mM Tris-HCl pH 8.0, 0.1 M KCl, 5 mM MgCl2, 10% glycerol, 0.1% Tween20, 10 mM β-mercaptoethanol, 0.2 mM PMSF). The cell suspension was sonicated and centrifuged at 10 000 g for 1 h at 4°C. The supernatant (crude extract) was incubated with 50 µl of anti-FLAG M2-agarose suspension (Sigma) for 15 min at 4°C. The mixture was filtered by using a mini-chromatography column (Bio-Rad). The filtrate was used as unbound fraction. The proteins bound to the beads trapped on the column were eluted with 50 µl of IP buffer containing 1 mg ml−1 FLAG peptide (Sigma) and used as bound fraction. The following amounts of protein samples were analysed by Western blotting using anti-FLAG, anti-HA and anti-enolase antibodies: crude extract (10 µl), unbound fraction (10 µl) and bound fraction (1 µl).
Bacterial cells grown in LB medium containing appropriate antibiotics(s) or carbon(s) were harvested and suspected in 100 ml of SDS-PAGE loading buffer (62.5 mM Tris-HCL pH 6.8, 2% SDS, 10% glycerol, 5%β-mercaptoethanol, 0.1% bromophenol blue). The sample was heated at 100°C for 5 min. Indicated amounts of protein samples were subjected to a 12% or 15% polyacrylamide-0.1% SDS gel electrophoresis and transferred to Immobilon membrane (Millipore). The membranes were treated with anti-FLAG monoclonal antibody (Sigma), anti-HA (Santa Cruz biotechnology) and anti-enolase (gift from Dr M. Wachi) polyclonal antibodies. Signals were visualized by the ECL system (Amersham).
Total RNAs were isolated from cells grown to mid-log phase as described (Aiba et al., 1981). For Northern blot analysis, indicated amounts of total RNAs were resolved by 1.2% agarose gel electrophoresis in the presence of formaldehyde and blotted on to Hybond-N+ membrane (Amersham Biosciences). The mRNAs were visualized using digoxigenin (DIG) reagents and kits for non-radioactive nucleic acid labelling and detection system (Roche Molecular Biochemicals) according to the procedure specified by the manufacturer. The 305 bp DIG-labelled DNA probe corresponding to the 5′-ptsG region was used for ptsG mRNA.
Determination of intracellular glucose 6-P
Bacterial cells were grown in LB medium to A600 of 0.6 unless otherwise specified. The protocols of sampling and determinant are performed as previously described (Morita et al., 2003).
We are grateful to Susan Gottesman and Carin Vanderpool for communicating results before publication and for comments on the manuscript. We thank Jacqueline Plumbridge, Philippe Regnier and Marc Dreyfus for comments on the manuscript. We also thank Masaaki Wachi for providing anti-enolase antibody. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Ajinomoto, Co., Inc. T.M. is grateful to Masami Oguchi for continuous encouragement during this work.