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EMBO J. Jul 17, 2000; 19(14): 3762–3769.
PMCID: PMC313975

SsrA-mediated tagging and proteolysis of LacI and its role in the regulation of lac operon

Abstract

SsrA RNA of Escherichia coli, also known as 10Sa RNA or tmRNA, acts both as tRNA and mRNA when ribosomes are paused at the 3′ end of an mRNA lacking a stop codon. This process, referred to as trans-translation, leads to the addition of a short peptide tag to the C-terminus of the incomplete nascent polypeptide. The tagged polypeptide is then degraded by C-terminal-specific proteases. Here, we focused on endogenous targets for the SsrA system and on a potential regulatory role of SsrA RNA. First, we show that trans-translation events occur frequently in normally growing E.coli cells. More specifically, we report that the lacI mRNA encoding Lac repressor (LacI) is a specific natural target for trans-translation. The binding of LacI to the lac operators results in truncated lacI mRNAs that are, in turn, recognized by the SsrA system. The SsrA-mediated tagging and proteolysis of LacI appears to play a role in cellular adaptation to lactose availability by supporting a rapid induction of lac operon expression.

Keywords: lac operon/Lac repressor/tmRNA/transcriptional roadblock/trans-translation

Introduction

SsrA RNA or 10Sa RNA encoded by the ssrA gene is a small stable RNA found first in Escherichia coli (Subbarao and Apirion, 1989). The RNA is also named tmRNA because it has characteristics of both tRNA and mRNA (Atkins and Gesteland, 1996; Jentsch, 1996; Keiler et al., 1996). The sequence analysis and in vitro studies revealed that the E.coli SsrA RNA could form an alanyl tRNA-like structure and can be charged with alanine (Komine et al., 1994). The property of SsrA RNA as mRNA was first suggested by the observation that a mammalian protein expressed in E.coli had a short tag sequence encoded by the ssrA gene at its C-terminus (Tu et al., 1995). Following these observations and based on their own studies, Keiler et al. (1996) proposed an intriguing mechanism whereby SsrA RNA acts both as tRNA and mRNA. This model, referred to as trans-translation (Atkins and Gesteland, 1996; Jentsch, 1996), suggests that when ribosomes are stalled at the 3′ end of a truncated mRNA without an in-frame stop codon, SsrA RNA charged with alanine binds in the A site of the ribosome, donates alanine to the growing polypeptide chain and then acts as an mRNA to provide the coding sequence for a short peptide. The final translation products are chimeric polypeptides in which a specific 11 residue tag (AANDENYALAA) is attached to the C-terminus of aberrant polypeptides. The tag sequence targets the products of trans-translation for degradation through several C-terminal specific proteases such as ClpAP, ClpXP, HflB and Tsp (Keiler et al., 1996; Gottesman et al., 1998; Herman et al., 1998).

Since the proposal of the trans-translation model, major studies on SsrA RNA have been performed primarily from the mechanistic point of view, resulting in several important findings. First, trans-translation has been shown to occur not only at the 3′ ends of stop codon-less mRNAs but also at a run of rare codons (Roche and Sauer, 1999). Secondly, a unique RNA-binding protein, SmpB, has been newly identified and shown to be required for SsrA activity (Karzai et al., 1999). Thirdly, structural and/or sequence determinants of SsrA RNA itself that are responsible for the trans-translation reaction were partly elucidated (Nameki et al., 1999a,b; Williams et al., 1999). In addition, RNase E, known to participate in processing of rRNA precursors and degradation of a variety of mRNAs, has been shown to be involved in the maturation of SsrA RNA (Lin-Chao et al., 1999).

The wide distribution of SsrA RNA in many bacterial species certainly suggests an important role of this unusual RNA in cellular activity (Williams, 2000). In fact, bacterial cells lacking a functional SsrA RNA exhibit a variety of phenotypes such as slower growth (Oh and Apirion, 1991; Komine et al., 1994), reduced motility (Komine et al., 1994), inhibition of phage growth (Retallack et al., 1994; Karzai et al., 1999; Julio et al., 2000), induction of Alp protease activity (Kirby et al., 1994), enhanced activity of several repressors (Retallack and Friedman, 1995) and reduced pathogenesis (Julio et al., 2000). In addition, it has been shown that SsrA RNA is essential for the growth of Neisseria gonorrhoeae (Huang et al., 2000). The major biological relevance of SsrA-mediated tagging and proteolysis is believed to be providing both a way to clear the ribosome stalled on mRNAs and a quality-control mechanism that allows the cell to eliminate potentially harmful truncated polypeptides (Atkins and Gesteland, 1996; Jentsch, 1996; Keiler et al., 1996). However, it is largely unknown how the SsrA-mediated tagging and proteolysis is related to various cellular phenotypes mentioned above. This is, in part, due to the lack of information on natural mRNA substrates for the SsrA system. In fact, studies on trans-translation have been carried out only on foreign or artificial mRNAs both in vivo (Keiler et al., 1996; Karzai et al., 1999; Roche and Sauer, 1999) and in vitro (Himeno et al., 1997). Thus, no endogenous mRNA substrates for the SsrA system have been identified yet. The identification of such endogenous targets would certainly be useful to gain insight into the molecular mechanisms underlying various phenotypes observed in the SsrA-defective cells and to understand the full physiological significance of trans-translation, including a potential regulatory role.

We report here that the trans-translation event occurs quite frequently in cells. Then, we demonstrate that the lacI mRNA encoding Lac repressor (LacI) is a specific endogenous target for the SsrA-mediated tagging and proteolysis. The SsrA-mediated tagging and proteolysis of LacI plays a role in the regulation of the expression of the lac operon. This is the first instance in which a specific phenotype of SsrA-defective cells may have been linked via a specific target mRNA and protein to the trans-translation model.

Results

Effect of ssrA mutation on the expression of an artificially tagged model protein

As the first step to evaluate the physiological role of the SsrA system, we examined the effect of ssrA mutation on the expression of an artificially tagged model protein in E.coli cells. The crp gene, encoding the cyclic-AMP receptor protein (CRP), on a plasmid, was genetically manipulated to produce a tagged CRP (CRP-AA) that has a degradation tag at its C-terminus (Figure 1A). Another mutant crp gene encoding CRP-DD, in which two aspartates are substituted for two alanines at the very C-terminus of the tag sequence, was also constructed (Figure 1A). The tag sequence with two aspartates at its C-terminus (DD-tag) is known to be resistant to degradation by cellular proteases (Keiler et al., 1996; Gottesman et al., 1998; Herman et al., 1998). Plasmids carrying the wild-type or variant crp genes were introduced both into PP47 (crp) and PP47 ssrA cells. The expression of CRP proteins in two cells was analyzed by western blotting using anti-CRP polyclonal antibody (Figure 1B). The intact CRP and CRP-DD proteins were highly expressed at equivalent levels in both the ssrA and its parent cells (Figure 1B, lanes 1, 2, 5 and 6). On the other hand, the level of CRP-AA in cells with the wild-type ssrA gene was markedly reduced in comparison with CRP or CRP-DD (Figure 1B, lanes 3 and 4). More interestingly, little CRP-AA was detected with anti-CRP in the ssrA cells (Figure 1B, lane 4). Northern blot analysis revealed that the levels of crp-AA mRNA were comparable to levels of crp-DD in both cells (Figure 1C). Thus, the differences in steady-state protein levels are not due to the differences in mRNA levels. Instead, the results can be easily explained by assuming that CRP-AA is unstable and degraded more efficiently in the ssrA cells than in cells with the wild-type ssrA gene. The simplest interpretation would be that cells containing SsrA RNA produce abundant endogenous tagged polypeptides through trans-translation, which could compete with CRP-AA for proteases, while in the absence of SsrA RNA there is less competition for the proteases due to the lack of endogenous tagged polypeptides and CRP-AA is more susceptible to the tag-specific proteases.

figure cdd359f1
Fig. 1. Expression of artificially tagged CRPs in wild-type and ssrA cells. (A) Structure of CRP-AA and CRP-DD. Amino acid sequences of the C-terminal portion of CRP derivatives are shown along with corresponding nucleotide sequences. Asterisks ...

Direct evidence for tagging of endogenous proteins

In order to examine directly whether the SsrA-mediated tagging of cellular proteins indeed occurs, we constructed plasmids pSsr-AA and pSsr-DD, carrying the wild-type and mutant ssrA genes, respectively (Figure 2A). The mutant ssrA gene (ssrADD) encodes a variant SsrADD RNA containing a protease-resistant tag sequence in which two alanines at the very C-terminus of the tag sequence have been changed to two aspartates (Roche and Sauer, 1999). These plasmids were introduced into ssrA cells and the total proteins were analyzed by western blotting using anti-DD-tag polyclonal antibody. A large number of polypeptides were detected by anti-DD-tag antibody when cells carried pSsr-DD (Figure 2B, lane 3) while most of these bands were absent in ssrA cells harboring a control plasmid pSTV28 (lane 1) and in cells carrying pSsr-AA (lane 2). These results clearly indicate that many endogenous cellular proteins were tagged through the SsrA system, resulting in chimeric proteins. The proteins with the wild-type tag sequence would be subsequently degraded by proteases whereas the proteins with the DD-tag may escape from the proteolysis. We conclude that the SsrA-mediated trans-translation occurs quite frequently in normally growing cells.

figure cdd359f2
Fig. 2. (A) Schematic drawing of alanyl-SsrAAA and alanyl-SsrADD RNAs. The nucleotides mutated in the tag-coding region of SsrADD are underlined. (B) Detection of tagging of cellular proteins. Total proteins equivalent to OD600 = ...

Truncated LacI proteins are generated in ssrA cells

Potential targets for trans-translation, incomplete mRNAs, can be produced by premature transcription termination before a stop codon or by nuclease cleavage of mRNAs. For example, one can imagine that premature transcription termination may occur when a strong DNA-binding protein binds within an open reading frame (ORF). The E.coli lac system appears to represent this situation. In the lac operon, there are two auxiliary operators, O2 and O3, located 410 bp downstream and 82 bp upstream of the transcription start site, respectively, in addition to the major operator, O1 (Müller-Hill, 1996). It is known that the LacI tetramer binds simultaneously to O1 and to either O2 or O3, forming a DNA loop, to give a stronger repression (Oehler et al., 1990). It should be noted that O3 overlaps the coding sequence for the C-terminus of LacI (Figure 3). Thus, LacI binding to O3 is expected to cause premature termination of lacI transcription by forming an obstacle to transcription elongation. In fact, an early study suggested that this might be the case (Sellitti et al., 1987). This raises a possibility that the lacI mRNA can be a specific target of the SsrA system.

figure cdd359f3
Fig. 3. (A) Schematic drawing of the E.coli lacPO region. O1 and O3 are shown as open boxes. O3 partially overlaps the lacI ORF. Closed boxes indicate the parts of lacI and lacZ ORFs. The arrow indicates the transcription start site of lacP. ( ...

To examine whether the binding of LacI to the lac operators produces truncated lacI mRNAs and then truncated LacI proteins, we constructed a pBR322-derived pIT613 that contains the entire lac region under the control of the lacIq promoter. pIT613 was introduced into ssrA cells harboring compatible plasmids pSTV28, pSsr-AA or pSsr-DD. The expression of LacI protein in these cells was analyzed by western blotting using anti-LacI polyclonal antibody. In addition to the intact LacI protein, several prominent bands were observed below the intact LacI in cells with pIT613 in the absence of the ssrA gene (Figure 4, lane 2, shown by dotted arrows). These bands were not detected in cells carrying both pIT613 and pSsr-AA (Figure 4, lane 3), suggesting that they are efficiently removed by the SsrA system. No protein was detected with anti-LacI antibody in cells carrying a control plasmid pBR322 instead of pIT613, as expected (Figure 4, lane 1).

figure cdd359f4
Fig. 4. Western blot analysis of the LacI protein produced in cells harboring pIT613. MC4100ΔssrA cells harboring pIT613 were transformed with pSTV28 (lanes 2 and 7), pSsr-AA (lanes 3 and 6) or pSsr-DD (lanes 4 and 5). ...

Detection of SsrA-mediated LacI tagging

In order to examine whether the SsrA-mediated tagging of LacI occurs, total proteins of the ssrA cells harboring both pIT613 and pSsr-DD were also analyzed by western blotting using anti-LacI antibody (Figure 4, lane 4). In addition to the intact LacI protein, a prominent band, which has a higher molecular weight than the intact LacI, and several other weak bands were observed. These bands presumably represent tagged LacI proteins in which the protease-resistant DD-tag peptide was attached to the C-terminal portion of incomplete LacI proteins. If this is the case, the bands should also be recognized by anti-DD-tag antibody in western blotting. In fact, the bands recognized by anti-LacI antibody cross-reacted with anti-DD-tag antibody, indicating that the truncated LacI proteins are efficiently tagged with SsrADD RNA (Figure 4, lane 5). Interestingly, three strong signals to anti-DD-tag antibody were observed near the position of intact LacI (Figure 4, indicated by a bracket). The largest band corresponds exactly to the extended LacI that is recognized by anti-LacI antibody and separable from the intact LacI on the gel. The remaining two prominent DD-tagged proteins exhibit almost the same electrophoretic mobility as the intact LacI (Figure 4). These two bands are likely to be tagging products of shorter truncated LacI proteins observed below the intact LacI in ssrA cells carrying both pIT613 and pSTV28. Furthermore, the tagging of another longer truncated LacI, which was not completely separable from the intact LacI on the gel (Figure 4, lane 2), presumably leads to the production of the extended LacI. The exact assignment of these bands remains to be studied. In any case, however, the anti-DD-tag specific bands were no longer observed when cells carried pSsr-AA (Figure 4, lane 6) or pSTV28 (Figure 4, lane 7). In addition, they were not observed in cells harboring both pSsr-DD and a control plasmid pBR322, except a few, less significant smaller bands (Figure 4, lane 8). These results strongly suggest that truncated LacI proteins are indeed produced and efficiently tagged with the SsrADD RNA by a trans-translation mechanism and that the major tagging sites would be close to the C-terminus of LacI. Few tagged bands were detected when the LacI was co-expressed with the wild-type SsrA RNA (Figure 4, lane 6), suggesting that the LacI proteins containing the wild-type tag sequence were efficiently removed by proteolysis. Although a number of DD-tagged endogenous cellular proteins are produced in cells harboring pSsr-DD as shown in Figure 2, only a few bands were detectable due to the smaller amount of total proteins analyzed (Figure 4, lane 8).

Effects of IPTG and operator mutations on LacI tagging

If the binding of LacI to its operators is responsible for the production of truncated lacI mRNAs that are the potential substrates for the SsrA system, one would expect that inactivation of LacI by isopropyl-β-d-thiogalactopyranoside (IPTG) eliminates the LacI tagging. In fact, the presence of IPTG in the culture medium significantly reduced the production of the extended LacI recognized by anti-LacI antibody (Figure 5, lanes 1 and 2) and a series of tagged LacI proteins recognized by anti-DD-tag antibody including three major bands (lanes 3 and 4), indicating that LacI tagging depends on the binding of LacI to the lac operator(s).

figure cdd359f5
Fig. 5. Effect of IPTG on LacI tagging. MC4100ΔssrA cells harboring both pIT613 and pSsr-DD were grown in the presence (lanes 2 and 4) or the absence (lanes 1 and 3) of 1 mM IPTG. Total proteins equivalent to OD600 = ...

The dependency of LacI tagging on the binding of LacI to its operators was further analyzed by mutating the lac operators. As expected, the mutation of O3 significantly reduced the extent of LacI tagging (Figure 6, lane 5), indicating that LacI–O3 interaction is required for efficient tagging. Interestingly, the tagging was more significantly reduced when O1 was disrupted (Figure 6, lane 3), suggesting that the LacI tagging is dependent upon the specific binding of LacI to both O1 and O3. When O3 was replaced with the ideal operator (Sadler et al., 1983), the LacI tagging was markedly enhanced and it was still sensitive to IPTG (Figure 6, lanes 7 and 8). Taken together, we conclude that the O3–LacI–O1 repression loop (Oehler et al., 1990) is primarily responsible for the production of truncated lacI mRNAs and therefore for the LacI tagging (Figure 7).

figure cdd359f6
Fig. 6. Effect of operator mutations on LacI tagging. The pIT613 derivatives containing the lacO variants indicated were introduced into MC4100ΔssrA cells harboring pSsr-DD. The nucleotide sequences of lacO variants are shown in Figure  ...
figure cdd359f7
Fig. 7. Model for trans-translation acting on the lacI mRNA. Formation of the O1–LacI–O3 repression DNA loop may cause premature termination of transcription resulting in truncated lacI mRNAs that would be recognized by the SsrA system. ...

Tagging in cells harboring a plasmid containing the wild-type lacI gene

So far we focused on cells harboring pIT613 because the LacI protein is efficiently expressed from the lacIq promoter, and this facilitated the analysis of the SsrA-mediated LacI tagging. To examine whether the SsrA-mediated LacI tagging also occurs when LacI is expressed from the wild-type lacI promoter, we analyzed proteins produced in cells harboring pIT455 carrying the native lac region by western blotting using anti-LacI antibody. Although LacI is poorly expressed from the wild-type lacI promoter, the use of a large amount of total proteins allowed us to detect LacI as a major band along with other non-specific cellular bands with anti-LacI antibody (Figure 8). We first examined whether the major truncated LacI proteins could be produced in the absence of SsrA RNA. The presumed truncated LacI proteins were detected along with several non-specific bands (Figure 8, lane 2). These bands were apparently missing in the presence of the wild-type SsrA RNA, suggesting that they were removed by the SsrA system (Figure 8, lane 3). When LacI was co-expressed with SsrADD, extended tagged LacI was clearly detected (Figure 8, lane 4). This band was significantly reduced in the presence of IPTG (Figure 8, lane 5). The extended tagged LacI was no longer detected when LacI was co-expressed with the wild-type SsrA RNA (Figure 8, lane 3) or when LacI alone was expressed (Figure 8, lane 2). These results indicate that the SsrA-mediated LacI tagging and proteolysis indeed occur even when LacI was expressed from the wild-type lacI promoter. We also performed western blotting using anti-DD-tag antibody. However, the identification of tagged LacI proteins with anti-DD-tag antibody was difficult due to the presence of a large number of cellular tagged proteins under these experimental conditions (data not shown).

figure cdd359f8
Fig. 8. LacI tagging in the wild-type lacI gene. MC4100ΔssrA harboring pIT455 was transformed with vector plasmid pSTV28 (lane 2), pSsr-AA (lane 3) or pSsr-DD (lanes 4 and 5). MC4100ΔssrA harboring both pBR322 and ...

Effect of the ssrA mutation on the induction of the lac operon

Does SsrA-mediated LacI tagging and proteolysis play any role in the regulation of lac operon expression? It was reported previously that the level of β-galactosidase was consistently 10-fold lower in ssrA cells in comparison with the wild-type cells at concentrations of IPTG ranging from 0 to 4 mM (Retallack and Friedman, 1995). We re-examined the effect of the ssrA mutation on lac operon expression. We observed that the level of β-galactosidase was 2- to 3-fold lower in the ssrA strain than in the wild-type strain when a low concentration (<0.01 mM) of IPTG was used, whereas essentially no difference in the β-galactosidase activity between two strains was observed at a high concentration (>0.1 mM) of IPTG (data not shown). Thus, the effect of the ssrA mutation on the expression of the lac operon in an equilibrium state apparently varied depending on the IPTG concentration. However, we found that the ssrA mutation affected the kinetics of induction of the lac operon upon IPTG addition. Namely, cells lacking SsrA showed a significant delay in the induction of the lac operon upon the addition of IPTG (Figure 9A). Furthermore, an extended lag phase (a delay in lac operon expression) upon the exhaustion of glucose in glucose–lactose diauxic growth was also observed in ssrA cells (Figure 9B). These results suggest that the SsrA system allows the cells to use lactose rapidly in response to the environmental conditions (lactose availability) by modulating LacI expression and/or activity (see Discussion).

figure cdd359f9
Fig. 9. Effect of the ssrA mutation on lac operon expression. (A) Expression of the lac operon in response to the addition of IPTG. W3110 (squares) and its ssrA derivative (circles) were grown in LB medium. IPTG was added to the culture at OD ...

Discussion

In the present work, we first found that a plasmid-encoded chimeric CRP protein containing the C-terminal degradation tag appears to be degraded less effectively in the presence of the wild-type SsrA RNA than in the absence of this RNA. This was presumably due to the production of a plenitude of tagged cellular proteins through trans-translation in cells with the wild-type ssrA gene. In fact, we demonstrated directly by western blotting that many cellular polypeptides can be tagged with a mutant SsrADD encoding a protease-resistant DD-tag. The DD-tagged cellular proteins have been reported previously (Karzai et al., 1999), although the tagging was shown only in higher molecular weight proteins in the previous study. An important conclusion of our experiments is that a trans-translation event could occur quite frequently on a variety of cellular mRNAs, resulting in a large number of tagged polypeptides in normally growing cells.

Then we tried to identify the potential natural targets for trans-translation and this led us to the major conclusion that lacI mRNA is one of the targets for trans-translation. We found that truncated LacI proteins were significantly produced in the ssrA cells but not in cells with the wild-type ssrA gene. The use of a variant ssrA gene encoding SsrADD allowed us to detect the tagged LacI proteins directly by western blotting. We showed that the tagging occurred preferentially at the C-terminus of LacI because the major tagged proteins were larger or similar in size compared with the intact LacI. Furthermore, we demonstrated that the LacI tagging was dependent on the binding of LacI to both O1 and O3, because inactivation of LacI by IPTG or the disruption of one of two operators markedly reduced the LacI tagging. Our data clearly demonstrate a typical situation where SsrA RNA participates in tagging and proteolytic degradation of incompletely synthesized polypeptides. A strong DNA-binding protein such as LacI bound to sites within or near the ORF may cause premature transcription termination resulting in the truncated mRNAs that can be recognized by the SsrA system.

In addition, our results suggest that the SsrA-mediated tagging and proteolysis of LacI plays a role in the regulation of expression of the lac operon in response to lactose availability. How does SsrA affect lac expression? It was originally proposed that SsrA RNA, by binding several DNA-binding proteins including LacI and other regulatory proteins, may reduce their free concentrations and thereby reduce their activities (Retallack and Friedman, 1995). However, this competition model fails to explain more recent results regarding SmpB, a newly identified RNA-binding protein essential for the SsrA-mediated tagging activity, because smpB cells exhibit the same phenotype as ssrA cells even though they have a normal level of SsrA RNA (Karzai et al., 1999). We assume that the lack of SsrA-mediated tagging and subsequent proteolysis of truncated LacI is responsible for the delayed induction of the lac operon in the ssrA cells. For example, one could argue that the total concentration of LacI would be higher in ssrA cells due to the accumulation of truncated LacI proteins, which may lead to the delayed induction of the lac operon. More interestingly, our preliminary experiments suggest that some truncated LacI proteins lacking several amino acid residues at their C-termini exhibit an inducer-insensitive property (Is phenotype) (T.Abo, T.Inada and H.Aiba, unpublished data). The production of the truncated LacI proteins including the inducer-insensitive variant in the repressed state could explain the observed effects of ssrA mutation on the kinetic nature of lac operon expression, namely the significant delay of induction in the ssrA strain. This is because the truncated LacI proteins would be produced only in the absence of inducer and they will be diluted out over several generations after IPTG addition. This explains well why lac operon expression is barely affected by the ssrA mutation under the induced state.

The most distinctive phenotype of SsrA-defective cells is a dramatic reduction in the ability to support the growth of λ phage (Retallack et al., 1994; Withey and Friedman, 1999) and mu phage (Karzai et al., 1999). In this respect, it should be noted that the ssrA mutation has been shown to cause a delay in the induction of phage P22 lysogen after mitomycin treatment in Salmonella (Julio et al., 2000). Thus, there is an apparent similarity between the lac and P22 systems with regard to the kinetics of induction. It is interesting to examine whether the SsrA system is involved in the regulation of phage repressor proteins in quantity and/or in quality as in the case of LacI.

Materials and methods

Media and growth conditions

Cells were grown aerobically at 37°C in Luria–Bertani (LB) medium (Miller, 1972). Antibiotics were used at the following concentrations: ampicillin (50 µg/ml), kanamycin (50 µg/ml) and chloramphenicol (30 µg/ml). Bacterial growth was monitored by determining the optical density at 600 nm.

Strains and plasmids

The E.coli K-12 strains and plasmids used in this study are listed in Table I. P1 phage produced from W3110 ΔssrA::kan (Komine et al., 1994) was used to disrupt the ssrA gene of PP47 (crp) (Aiba et al., 1981) and MC4100 (Δlac) (Casadaban, 1976). To construct mutant crp genes encoding CRP-AA or CRP-DD, a MluI site was first introduced near the last codon of CRP ORF in plasmid pHA7 (Aiba et al., 1982). The resulting plasmid was designated pHA7M. DNA fragments encoding ANDENYALAA and ANDENYALDD (see Figure 1 for their nucleotide sequences) were prepared by annealing complementary synthetic oligonucleotides and inserted into the MluI site of pHA7M. The resulting plasmids were designated pHA7M-AA and pHA7M-DD, respectively. The EcoRI–BamHI fragments of pHA7M, pHA7M-AA and pHA7M-DD were subcloned into pSTV28 (Takara) to construct plasmids pCRP, pCRP-AA and pCRP-DD, respectively (Figure 1A). pIT613 containing the lacIZYA in which the lacI is transcribed from the lacIq promoter was constructed by inserting the 1.9 kb HindIII–EcoRV fragment containing the lacIq promoter and the lacI′ ORF derived from pTTQ18 (Stark, 1987), a 1.5 kb EcoRV–EcoRV fragment containing ‘lacI–lacZ’ and a 10.5 kb EcoRV–PstI fragment with PstI–SalI linker attached containing lacZYA, both derived from pXX701 (Niki et al., 1988), into pBR322. The pIT613 derivatives with operator mutations were constructed by PCR mutagenesis. pIT455 was constructed by inserting a 12 kb PstI fragment containing the lacIZYA region derived from pXX701 into pBR322. The 707 bp DNA fragment containing the ssrA gene was amplified by PCR using primers 5′-CCGGAATTCTGTTGACCAGTTCCTCACCG-3′ and 5′-CCCAAGCTTAATGGGCCTAAAAGGTTCGG-3′ from the chromosomal DNA of W3110, digested with EcoRI and HindIII, and then cloned into pSTV28 to construct pSsr-AA. pSsr-DD was constructed by PCR mutagenesis from pSsr-AA.

Table I.
Bacterial strains and plasmids used in this study

Western blotting

Anti-CRP polyclonal antibody was described previously (Ishizuka et al., 1993). Anti-LacI and anti-DD-tag polyclonal antibodies were prepared by immunizing the rabbit with LacI protein purified based on the method described (Müller-Hill et al., 1971) and a synthetic peptide, AANDENYALDD, respectively. Bacterial cells grown in LB medium containing appropriate antibiotic(s) were harvested at OD600 = 0.8 and suspended in 100 µl 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. Total cellular proteins of the indicated amount were subjected to 12% acrylamide–0.1% SDS gel electrophoresis and transferred to Immobilon membrane (Millipore). The polypeptides detected by the antibodies were visualized by an ECL system (Pharmacia).

Northern blot analysis

Cells were grown aerobically at 37°C in LB medium and total RNAs were extracted as described (Aiba et al., 1981). The total RNAs were resolved by 1.0% agarose gel electrophoresis in the presence of formamide and blotted onto Hybond-N+ membrane (Amersham) as described (Sambrook et al., 1989). The crp mRNAs were visualized using the Gene Images labeling and detection system (Amersham). The HindIII–EcoRV fragment derived from pHA7 was used as a probe for crp mRNA.

β-galactosidase assay

β-galactosidase activity was determined with permeabilized cells by the method of Miller (1992).

Acknowledgements

We thank Dr H.Inokuchi (Kyoto University) for providing W3110 ΔssrA::kan. This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.

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