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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Background: We have shown recently that read-through of a normal stop codon by a suppressor tRNA in specific genes possessing a Rho-independent terminator leads to SsrA-mediated tagging of extended proteins in Escherichia coli cells. Miscoding antibiotics such as kanamycin and streptomycin reduce translational fidelity by binding to the 30S ribosomal subunit. The aim of the present study was to address how miscoding antibiotics affect the read-through of stop codons and SsrA-mediated protein tagging.

Results: Miscoding antibiotics caused translational read-through of stop codons when added to the culture medium at sublethal concentrations. Under the same conditions, the drugs enhanced SsrA-mediated tagging of bulk cellular proteins, as observed in cells carrying an ochre suppressor tRNA. Translational read-through products generated from the crp gene in the presence of the antibiotics was efficiently tagged by the SsrA system, presumably because the ribosome reached the 3′ end of the mRNA defined by the terminator hairpin. The SsrA-defective cells were more sensitive to the miscoding antibiotics compared to the wild-type cells.

Conclusion: We conclude that the SsrA system contributes to the survival of cells by dealing with translational errors in the presence of low concentrations of miscoding antibiotics.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial SsrA RNA, also known as tmRNA or 10Sa RNA, is involved in a unique process called trans-translation, together with an associated protein SmpB by acting as a tRNA and an mRNA, to rescue the stalled ribosome on an mRNA and to tag the nascent polypeptide with a C-terminal peptide that leads to its degradation (Karzai et al. 2000; Gillet & Felden 2001). A large number of tagged cellular polypeptides can be detected when Escherichia coli cells express a mutant SsrA RNA encoding a protease-resistant tag sequence, indicating that endogenous targets for the SsrA system are produced quite frequently, even in normally growing cells (Abo et al. 2000; Roche & Sauer 1999, 2001). When and how are mRNA targets for SsrA RNA produced in cells? SsrA tagging was initially demonstrated for proteins translated from truncated mRNAs lacking an in-frame stop codon because the ribosome would stall at the 3′ ends of such mRNAs (Keiler et al. 1996). Recent studies have suggested that SsrA tagging could occur in cases other than truncated mRNAs. For instance, it has been shown that a run of rare codons on an mRNA induces the SsrA tagging, presumably because the deficiency of cognate aminoacyl-tRNAs may lead to ribosome stalling (Roche & Sauer 1999). In this case, it remains obscure whether the ribosome stalling itself leads to tagging at internal sites of the mRNA or if stalling leads to tagging by generating somehow truncated mRNAs (Karzai et al. 2000; Roche & Sauer 1999). More recently, it has been suggested that SsrA-mediated protein tagging occurs even at positions corresponding to regular stop codons in several genes (Roche & Sauer 2001; Hayes et al. 2002).

So far we found two situations where ribosomes may reach the 3′ end of an mRNA where the SsrA acts to cause tagging of endogenous proteins. Firstly, we showed that a strong DNA binding protein could produce truncated mRNAs by prematurely blocking transcription when it binds to specific sites within or near protein coding sequences (Abo et al. 2000). For example, the binding of LacI to the lac operators results in truncated lacI mRNAs which are recognized by the SsrA system. The SsrA-mediated tagging and proteolysis of LacI may play a role in the regulation of lac operon expression. Secondly, we have shown that read-through of a normal stop codon in specific genes possessing a Rho-independent terminator allows ribosomes to reach the 3′ end of mRNA, resulting in SsrA-mediated tagging and degradation of extended proteins (Ueda et al. 2002). The read-through of normal stop codons caused by suppressor tRNAs is a typical representation of this situation. Thus, the SsrA system may play an important role in dealing with unwanted translational read-through in cells carrying suppressor tRNAs.

Read-through of stop codons could occur not only in the presence of suppressor tRNAs but also when translational fidelity is reduced for some reason. For example, the presence of aminoglycoside antibiotics such as kanamycin and streptomycin, which are known to reduce the fidelity of translation by binding to the 30S ribosomal subunit (Jerinic & Joseph 2000; Puglisi et al. 2000a,b) is expected to cause the read-through of stop codons. In this study, we examined how miscoding antibiotics affect the read-through of stop codons and trans-translation. We found that the drugs enhance SsrA-mediated protein tagging by causing the read-through of normal stop codons when added at sublethal concentrations. We demonstrated that the inhibitory effects of miscoding antibiotics on cell growth are magnified in the absence of SsrA RNA, suggesting that the SsrA system confers the cells a partial resistance to miscoding antibiotics by dealing with translational errors caused by low concentrations of drugs.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Miscoding antibiotics cause translational read-through

The aminoglycosides are known to bind to the 30S subunit of bacterial ribosome, resulting in translational misreading, including stop codons (Jerinic & Joseph 2000; Puglisi et al. 2000a,b). To detect the read-through of stop codons caused by the drugs, plasmids pST513 and pST523 carrying crp-TAA-crr and crp-TAG-crr fusion genes, respectively, were constructed. In these fusion genes, the ORF of cAMP receptor protein (CRP) is followed by the IIAGlc ORF in the same frame (Fig. 1A). The read-through of the crp stop codon in the fusion genes should generate CRP-IIAGlc fusion proteins. The plasmids were introduced into either suppressor-free or supC or supE cells. The transformants were grown in LB medium and the whole cell extracts were subjected to Western blot analysis using anti-CRP and anti-IIAGlc antibodies. The CRP-IIAGlc fusion proteins were not detected in suppressor-free cells (Fig. 1B,C, lanes 2 and 7), indicating that translation termination occurred efficiently at both UAA and UAG stop codons. On the other hand, the CRP-IIAGlc fusion protein was produced along with the normal size CRP in the supC cells carrying pST513 as a result of translational read-through (Fig. 1B,C, lane 1). The CRP-IIAGlc fusion protein was produced more efficiently in the supE cells carrying pST523 (Fig. 1B,C, lane 6), reflecting the higher suppression activity of the supE compared to the supC. Thus, the crp-stop codon-crr model system is useful for monitoring the read-through of stop codons. We then examined how antibiotics affect the translational read-through at the crp-stop codon-crr fusion genes in suppressor-free cells when they are added at sublethal concentrations where the cell growth is partially inhibited. The CRP-IIAGlc fusion proteins were clearly produced in the presence of kanamycin or streptomycin (Fig. 1B,C, lanes 3, 4, 8 and 9), indicating that the miscoding antibiotics caused translational read-through at the crp-stop codon-crr genes to some extent. The translational read-through at the crp-TAG-crr was more significant than that at the crp-TAA-crr. In contrast, only a faint band corresponding to CRP-IIAGlc fusion protein was detected in the presence of chloramphenicol (Fig. 1B,C, lanes 5 and 10). Chloramphenicol does not belong to miscoding drugs, since it primarily acts to inhibit peptide bond formation on the 50S ribosomal subunit (Jerinic & Joseph 2000; Puglisi et al. 2000a,b).

image

Figure 1. Translational read-through in the crp-stop codon-crr model system caused by miscoding antibiotics. (A) Schematic drawing of the crp-TAA-crr or crp-TAG-crr fusion gene carried by pST513 or pST523. The nucleotide and amino acid sequences of the junction region of crp and crr are shown, along with the restriction enzyme sites. (B) and (C) Western blot analysis of read-through products at crp-stop codon-crr model system. W3110 cells harbouring pST513 or pST523 were grown in the LB medium containing 50 µg/mL of ampicillin without any additional drug (lanes 2 and 7), and with 3 µg/mL of streptomycin (lanes 3 and 8), 2 µg/mL of kanamycin (lanes 4 and 9), or 2 µg/mL of chloramphenicol (lanes 5 and 10). CA167 cells harbouring pST513 (lane 1) and TG1 cells harbouring pST523 (lane 6) were also grown as supC and supE controls. Cells were harvested at an OD600 of roughly 0.6, and the total proteins equivalent to OD600 = 0.01 were subjected to 12% SDS-PAGE followed by Western blotting using anti-CRP (B) and anti-IIAGlc (C) antibodies. The positions of the CRP and CRP-IIAGlc fusion proteins are indicated by arrowheads. The asterisk indicates the position of the cellular IIAGlc. Abbreviations used for antibiotics are: K, kanamycin; S, streptomycin; and C, chloramphenicol.

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Miscoding antibiotics enhance the SsrA-mediated tagging of cellular proteins

The observation that miscoding antibiotics cause read-through of stop codons raised the possibility that these drugs may enhance SsrA-mediated tagging of cellular proteins, as is observed in cells carrying a suppressor tRNA (Ueda et al. 2002). We previously used a system to monitor the protein tagging using a plasmid-borne mutant ssrA gene encoding SsrADD RNA, which causes the addition of a protease resistant ‘DD-tag’ instead of the regular protease sensitive ‘AA-tag’ (Roche & Sauer 1999; Abo et al. 2000). The DD-tagged polypeptides can be detected by anti-DD-tag antibodies. Here, we modified the system in which strain TA371 carrying the ssrADD gene on the chromosome instead of the wild-type ssrA gene was used. We then analysed the effect of miscoding antibiotics on the SsrA-mediated tagging of cellular proteins in TA371 cells. Cells were grown in the LB medium in the presence or absence of antibiotics at low concentrations. TA431, carrying the ssrADD in the supC background was also constructed and grown in the LB medium in the absence of antibiotics. The cells were harvested and the whole cell extracts were monitored for SsrA-mediated protein tagging by Western blotting using anti-DD-tag antibodies. The presence of kanamycin or streptomycin significantly stimulated the SsrA-mediated tagging of bulk proteins in TA371, resulting in a number of enhanced bands (Fig. 2A, lanes 2–7). The extent of SsrA-mediated protein tagging caused by the drugs was comparable to that caused by an ochre suppressor tRNA (Fig. 2A, lane 10). The pattern of enhanced tagged bands in the presence of the drugs was similar but not completely the same as that observed in the supC cells. On the other hand, the tagging of cellular proteins was only slightly affected by the addition of chloramphenicol (Fig. 2A, lanes 8 and 9). We also analysed the cell extracts by SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining (Fig. 2B). The presence of the drugs did not significantly affect the profile of total proteins, except for a few bands.

image

Figure 2. SsrA-mediated tagging of cellular proteins in the presence of antibiotics. The crude lysates equivalent to OD600 = 0.005 and 0.1 were analysed by Western blotting using anti-DD tag antibody (A), and by SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining (B), respectively. TA371 carrying ssrADD (lanes 1–9) or TA431 carrying ssrADD and supC (lane 10) cells were grown in LB medium without the drug (lanes 1 and 10), and with increased concentrations of streptomycin (lanes 2–4), kanamycin (lanes 5–7) and chloramphenicol (lanes 8 and 9). Cells were harvested during the late-log phase and total proteins equivalent to OD600 = 0.01 or 0.1 were subjected to a 12% SDS-polyacrylamide gel electrophoresis and analysed by Western blotting using anti-DD tag antibody (A) or by Coomassie Blue staining (B), respectively. The positions of the molecular weight markers are shown on the right.

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Miscoding antibiotics cause SsrA-mediated tagging of read-through products at the crp gene

We showed previously that the read-through at the crp gene generated several incomplete extended CRPs, in addition to the full-length extended CRP with additional 22 amino acid residues (CRP+22), and that the incomplete extended CRPs but not the full-length extended CRP were recognized by the SsrA system (Ueda et al. 2002). This is because the major 3′ end of crp mRNA is defined by the Rho-independent terminator located before the next stop codon (Ueda et al. 2002; see also Fig. 3A). A mass spectrometric analysis of the read-through products revealed that a major incomplete extended CRP was CRP+13 and a major DD-tagged CRP, which was detected in cells expressing SsrADD RNA, was CRP +13-DD (Ueda et al. 2002). If miscoding antibiotics cause read-through of the stop codon in the normal crp gene, the incomplete extended CRP should be tagged by the SsrA system, as was observed in cells carrying an ochre suppressor tRNA (Ueda et al. 2002). To test this, pHA7 containing the crp gene was introduced into TA371 (ssrADD) and TA431 (ssrADDsupC). Cells were grown in LB medium in the presence or absence of antibiotics and the whole cell extracts were subjected to Western blot analysis using anti-CRP and anti-DD-tag antibodies. As expected, the untagged extended CRP+22 (band II) and DD-tagged CRP+13-DD (band III) were clearly produced in addition to the normal CRP (band I) of 209 amino acid residues in TA431 (ssrADDsupC) cells harbouring pHA7 in the absence of drugs (Fig. 3B, lanes 5 and 10). It should be noted that the mobility of the DD-tagged band III in the SDS-PAGE gel is somehow slower than that expected from its molecular weight. Interestingly, the same extended bands were also detected in TA371 cells harbouring pHA7 when kanamycin (Fig. 3B, lanes 2 and 8) or streptomycin (Fig. 3B, lanes 3 and 8) was present. Little read-through proteins were produced in the presence of chloramphenicol (Fig. 3B, lanes 4 and 9), or in the absence of antibiotics (Fig. 3B, lanes 1 and 6). We conclude that miscoding antibiotics cause a translational read-through at the crp gene, thereby leading to SsrA tagging of extended proteins, as was observed in the supC cells. Western blot analysis of TA371 cells harbouring pHA7am containing the crp gene with the TAG stop codon was also carried out. Essentially the same results were obtained, although the translational read-through and tagging of extended proteins in the presence of miscoding antibiotics were more prominent in this case (Fig. 3B, lanes 11–20). It is known that TAA gives the most efficient translation termination (Tate & Mannering 1996). Thus, the translational read-through by miscoding drugs occurs more efficiently at a less efficient TAG stop codon.

image

Figure 3. SsrA-mediated tagging of extended CRP in the presence of miscoding antibiotics. (A) Schematic drawing of the crp gene, crp mRNAs and extended CRP proteins. The primary crp transcript is made beyond the Rho-independent terminator and then processed back to the hairpin. Read-through of the normal crp stop codon leads to the production of ‘full-length’ extended CRP (CRP+22) when the ribosome reaches the next in-frame stop codon that is located 22 codons downstream from the normal crp stop codon. The incomplete extended CRP made from the processed crp mRNA lacking the next stop codon is tagged by the SsrA system resulting in CRP+13-DD as a principal product. (B) TA371 cells harbouring pHA7 (lanes 1–4 and 6–9) or pHA7am (lanes 11–14 and 16–19) were grown in the LB medium containing 50 µg/mL of ampicillin without any additional drug (lanes 1, 6, 11 and 16) and with the indicated concentrations of streptomycin (lanes 2, 7, 12 and 17), kanamycin (lanes 3, 8, 13 and 18), or chloramphenicol (lanes 4, 9, 14 and 19). TA431 cells (lanes 5 and 10) and TG1 cells containing pSsr-DD (lanes 15 and 20) were also grown as supC and supE controls, respectively. Cells were harvested at an OD600 of roughly 0.6 and total proteins equivalent to OD600 = 0.01 were subjected to the 12% SDS-PAGE followed by Western blotting using anti-CRP or anti-DD-tag antibodies.

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SsrA-deficient cells exhibit an increased sensitivity to miscoding antibiotics

The enhancement of SsrA-mediated tagging by miscoding antibiotics suggests that the SsrA system plays a role in cell survival in the presence of low concentrations of drugs. To test this possibility, the sensitivity of the cells to the drugs was evaluated by monitoring the growth of W3110 (ssrA+), TA331 (ssrA), and TA371 (ssrADD) cells in the LB medium containing various concentrations of representative antibiotics. A significant difference in sensitivity to streptomycin was observed between the three strains (Fig. 4, upper). For example, the growth of TA331 was significantly inhibited in the presence of 2.5 µg/mL of streptomycin, whereas W3110 cells grew normally under the same conditions. When 5.0 µg/mL of streptomycin was used, the growth of TA331 was almost completely inhibited, while W3110 could still grow to some extent. Cells carrying SsrADD RNA were slightly more sensitive to streptomycin compared to ssrA+ cells. Similar results were obtained with kanamycin (Fig. 4, middle). In contrast, only a moderate difference was observed among the three strains with respect to the sensitivity when chloramphenicol was used (Fig. 4, lower). We also briefly examined the sensitivities of ssrA+ (W3110) and ssrA (TA331) cells to the series of antibiotics listed in Table 1. All drugs completely inhibited the growth of both cells when added at higher concentrations. However, when lower concentrations of the drugs were used, it became clear that the ssrA cells were always more sensitive to miscoding antibiotics than the ssrA+ cells. For example, the ssrA+ cells could still grow significantly in the presence of the minimum concentration of each miscoding drug that could almost completely inhibit the growth of the ssrA cells (Table 1). On the other hand, no or only a little difference in sensitivity to the drugs was observed between two cells with non-miscoding antibiotics, except puromycin. Taken together, we conclude that the SsrA system plays a role in cell survival in the presence of low concentrations of miscoding antibiotics, presumably by dealing with the translational problems caused by these drugs. It remains to be studied whether puromycin affects the SsrA tagging of cellular proteins.

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Figure 4. Growth curves of E. coli cells in the presence or absence of antibiotics. An aliquot (5 µL) of overnight culture of W3110 (ssrA+), TA331 (ssrA) or TA371 (ssrADD) was inoculated into 5 mL of LB medium containing indicated concentrations of streptomycin (upper), kanamycin (middle) and chloramphnenicol (lower), and the cell growth was monitored by measuring the OD600 of the culture at every 30 min. The concentrations of antibiotics (µg/mL) added to the culture medium are indicated in the left side of each plot.

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Table 1.  Effects of various antibiotics on cell growth
AntibioticConcentration (µg/mL)Relative cell growth (−/+ drug)§
ssrA+ΔssrA
  • Asterisks are antibiotics that are known to cause misreading (Jerinic & Joseph 2000).

  • The minimum concentrations of antibiotics required to inhibit almost completely the growth of ΔssrA cells are shown. These values were evaluated as follows. An overnight culture of TA331 was 1000-fold diluted into 200 µL of LB medium containing antibiotics prepared by twofold serial dilution in a 96-well microtitre plate. The plate was incubated at 37 °C for 12 h, and the optical density at 550 nm (OD550) of the culture was measured.

  • §

    The 1000-fold diluted overnight cultures (200 µL) of W3110 (ssrA+) and TA331 (ΔssrA) were prepared in the presence of antibiotics at the concentrations indicated in a 96-well microtitre plate. The plate was incubated at 37 °C for 12 h, and the optical density at 550 nm of the culture was determined. The %OD550 in the presence of each drug relative to that in the absence of the drug were calculated. The mean values of three independent experiments are shown.

kanamycin*  2.525.80.8
gentamycin*  1.2518.24.3
hygromycin B* 20.042.4< 0.1
streptomycin*  2.526.9< 0.1
G418*  1.2539.4< 0.1
paromomycin*  5.048.91.7
puromycin 80.024.91.2
spectinomycin 20.0< 0.1< 0.1
fusidic acid320.06.26.6
erythromycin160.010.64.1
nalidixic acid 10.01.11.1
tetracycline  0.64.18.7
ampicillin  2.5< 0.10.8
chloramphenicol  2.54.00.8
novobiocin320.03.82.6
rifampicin 10.01.41.4

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The aminoglycoside antibiotics bind to the 30S ribosomal subunit, resulting in conformational changes within the ribosome and therefore reducing the selectivity of the codon–anti-codon interaction which in turn leads to misreading of the genetic code (Carter et al. 2000; Jerinic & Joseph 2000; Puglisi et al. 2000a,b). The major finding described in this report is that streptomycin and kanamycin, typical translational miscoding antibiotics, enhance the SsrA-mediated tagging of cellular proteins by causing read-through of the normal stop codons when they are added at low concentrations. We have recently found that the read-through of the normal stop codon in specific genes possessing a Rho-independent terminator generates mRNA targets for the SsrA system, presumably because the ribosome may reach the 3′ ends of these mRNAs (Ueda et al. 2002). Thus, the ability of kanamycin and streptomycin to induce an efficient read-through of stop codons can easily explain why these drugs enhance the SsrA-mediated protein tagging. Translational fidelity would be reduced in other physiological and/or genetic conditions that affect the decoding process. For example, mutations affecting either ribosome component or release factors that reduce the efficiency of translational termination may facilitate the read-through of a stop codon. The SsrA system would be expected to enhance protein tagging in these conditions.

Another important finding shown here is that the lack of the SsrA system enhances the sensitivity of the cells to miscoding antibiotics. The increasing concentrations of miscoding antibiotics in the culture medium may cause an inhibition of the translation elongation process itself, in addition to the errors in the decoding process (Puglisi et al. 2000a,b). Thus, the SsrA system has nothing to do for the cell survival when miscoding antibiotics are present at higher concentrations. Our results, however, suggest that the SsrA system contributes, at least partly, to the resistance of cells to the miscoding antibiotics at sublethal concentrations. When translational read-through occurs in a gene and when there is no additional stop codon after the normal stop codon before its transcriptional terminator, the ribosome may be jammed at the 3′ end of the mRNA. We believe that the SsrA system would rescue this situation by releasing the stalled ribosome from the 3′ end of mRNA. The accumulation of abnormal extended proteins due to the translational read-through could be also responsible for the elevated sensitivity of the ssrA cells to miscoding drugs. However, it should be noted that the sensitivity of cells having SsrADD RNA is rather close to that of the wild-type cells. This suggests that the SsrA system confers enhanced drug resistance to the wild-type cells primarily through the ribosome-rescue function, rather than the through the degradation of abnormal proteins. Our results are consistent with previous observations that the SsrA variants possessing the protease-resistant tag sequences could rescue the growth deficiency of Mu and lambdoid phages in the ssrA strains (Retallack et al. 1994; Withey & Friedman 1999). The importance of the ribosome recycling effect of SsrA RNA was also shown in the growth of N. gonorrhoeae (Huang et al. 2000) and in the stress tolerance of B. subtilis (Muto et al. 2000).

So far we have focused on the translational read-through at stop codons regarding the effect of miscoding antibiotics because it certainly generates mRNA targets for the SsrA system. It should be noted that an earlier study suggested that the A-site is left unoccupied when the ribosome is stalled by aminoglycosides (Hausner et al. 1988). This raises the possibility that the drugs may cause internal tagging within a protein coding region by allowing the SsrA–SmpB complex to access the A-site. This event is analogous with that observed at a run of rare codons within the ORF, coupled with tRNA scarcity (Roche & Sauer 1999). In addition, the lowered selectivity caused by the drugs may also allow the entrance of the SsrA-SmpB complex into the A-site at any codons resulting in internal tagging. In addition, we cannot exclude the possibility that SsrA RNA may also contribute to enhanced resistance to the drugs by some mechanisms other than by trans-translation.

The SsrA RNA has been shown to be widely involved in cellular activities because cells lacking this molecule exhibit a variety of phenotypes such as slower growth (Komine et al. 1994; Oh & Apirion 1991; Roche & Sauer 2001), reduced motility (Komine et al. 1994), inhibition of phage growth (Julio et al. 2000; Karzai et al. 1999; Ranquet et al. 2001; Retallack et al. 1994), induction of Alp protease activity (Kirby et al. 1994), the enhanced activity of several repressors (Retallack & Friedman 1995), reduced pathogenesis (Julio et al. 2000). In addition, the SsrA system has been shown to be essential for the growth of N. gonorrhoeae (Huang et al. 2000), the proper response of lac operon expression to inducer (Abo et al. 2000), and in the stress tolerance of B. subtilis (Muto et al. 2000). The finding that cells lacking SsrA RNA are more sensitive to miscoding antibiotics has revealed an additional aspect regarding the physiological role of the SsrA system. In the natural situation, bacteria would occasionally be exposed to low concentrations of various antibiotics that are produced by other micro-organisms. The SsrA system certainly provides the cells with a survival advantage under such conditions.

A recent paper has shown that Synechocystis lacking SsrA RNA is more sensitive to protein synthesis inhibitors such as chloramphenicol (De La Cruz & Vioque 2001). The increased sensitivity to chloramphenicol in Synechocystis cells lacking SsrA RNA appears to be rather moderate. A similar moderate increase in the sensitivity to chloramphenicol was also observed in our experiments on E. coli cells lacking SsrA RNA (see Fig. 4 and Table 1). The weak increase in sensitivity to chloramphenicol of SsrA-defective Synechocystis and E. coli cells is consistent with our view that the SsrA system is dealing with the problems caused by translational read-through, because chloramphenicol is also able to cause a weak read-through, although far less significantly compared to miscoding drugs. Unfortunately, it was not possible to test the sensitivity of Synechocystis cells to miscoding drugs in their work because the SsrA-defective cells exhibited a resistance to kanamycin and streptomycin due to a kanamycin resistance cassette that was used for disruption of the ssrA gene (De La Cruz & Vioque 2001). It would certainly be interesting to test the effect of miscoding drugs on the growth of SsrA-defective Synechocystis cells without the drug resistance marker.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Strains and plasmids

The E. coli K-12 strains and plasmids used in this study are listed in Table 2. The gene knock-out system described by Datsenko & Wanner (2000) was used to manipulate of the chromosomal ssrA gene. Primers 5′-CATTGGGGCTGATTCTGGATTCGACGGGATTTGCGAAACCCAAGGTGCATGTGTAGGCTGGAGCTGCTTC-3′ (ΔssrA1) and 5′-AGGACTTCATCGGATGACTCTGGTAATCACCGATGGAGAATTTTGGTGGACATATGAATATCCTCCTTA-3′ (ΔssrA2) were used to amplify the cat gene flanked by FRT sites of pKD3 by PCR. The PCR product was then gel-purified, introduced into BW25113 cells carrying a Red helper plasmid pKD46 and Cmr transformants were isolated. The ΔssrA::FRT-catFRT region of the resulting strain was transferred to W3110 to obtain TA330 by P1 transduction. TA330 was transformed with pCP20, and the cat gene flanked by FRT sites was excised to obtain TA331. The kan gene flanked by FRT sites carried by pKD4 was amplified by PCR using primers 5′-AAAAGCTTGTGTAGGCTGGAGCTGCTTC-3′ (H3-P1) and 5′-AAAAGCTTCATATGAATATCCTCCTTA-3′ (H3-P2), digested with HindIII, and cloned into the HindIII site of pSsr-DD. The region containing ssrADD and FRT-kan-FRT was PCR amplified with the primers 5′-CATTGGGGCTGATTCTGGATT-3′ (ssrA-5) and 5′-AGGACTTCATCGGATGACTCTGGTAATCACCGATGGAGAATTTTGGTGGAAGTCACGACGTTGTA-3′ (ΔssrA3), and used to transform BW25113 carrying pKD46. The Kmr transformant carrying the ssrADD gene was screened by anti-DD-tag Western blotting. The ssrADD-FRT-kan-FRT region was transferred to the W3110 and CA167 (supC) by P1 transduction to obtain strains TA370 and TA430, respectively. The FRT-flanked kan of TA370 and TA430 was excised from the chromosome as described above to obtain TA371 and TA431, respectively.

Table 2.  Bacterial strains and plasmids used in this study
Strain/plasmidRelevant genotype and propertySource
Strain
 W3110wild-typeLaboratory stock
 CA167supCBrenner & Beckwith (1965)
 TG1supESambrook et al. (1989)
 TA330W3110 ΔssrA::FRT-cat-FRTThis work
 TA331W3110 ΔssrA::FRTThis work
 TA370W3110 ssrADD-FRT-kan-FRTThis work
 TA430CA167 ssrADD-FRT-kan-FRTThis work
 TA371W3110 ssrADD-FRTThis work
 TA431CA167 ssrADD-FRTThis work
 BW25113An efficient host for gene inactivation systemDatsenko & Wanner (2000)
Plasmid
 pHA7Derivative of pBR322 carrying the crp expressed from bla promoterAiba et al. (1982)
 pHA7amDerivative of pHA7 carrying the crp in which TAA stop codon is changed to TAGUeda et al. (2002)
 pHA7MDerivative of pHA7 in which a MluI site is introduced near the last codon of crpAbo et al. (2000)
 pBR322DDerivative of pBR322 in which EcoRV site is removedAbe et al. (1999)
 pHA7DDerivative of pBR322D carrying the crp expressed from bla promoterThis work
 pIT520Derivative of pBR322 carrying the crr expressed from bla promoterInada et al. (unpublished)
 pIZ3Derivative of pHA7D in which the crr gene is placed downstream of crpThis work
 pIZ3ADerivative of pIZ3 in which an AflII site is introduced within crrThis work
 pST513Derivative of pBR322 carrying the crp-TAA-crr fusion geneThis work
 pST523Derivative of pBR322 carrying the crp-TAG-crr fusion geneThis work
 pKD46A Red recombinase expression plasmidDatsenko & Wanner (2000)
 pKMA template plasmid carrying FRT-cat-FRTDatsenko & Wanner (2000)
 pKD4A template plasmid carrying FRT-kan-FRTDatsenko & Wanner (2000)
 pCP20A FLP expression plasmidDatsenko & Wanner (2000)
 pSsr-DDDerivative of pSTV28 containing a variant ssrA encoding SsrADD RNAAbo et al. (2000)

Plasmid pST513 or pST523 carrying a crp-crr fusion gene interrupted by either TAA or TAG stop codon was constructed as follows. The 963 bp HindIII-EcoRI fragment containing the crp gene of pHA7 was cloned into the HindIII-EcoRI region of pBR322D to obtain pHA7D. The HpaI-HpaI 747 bp fragment including the crr gene was ligated with the HindIII linker (5′-CAAGCTTG-3′) and digested with HindIII. The resulting fragment was cloned into the HindIII site of pBR322 to construct pIT520. The AluI fragment containing the crr gene was excised from pIT520 and cloned into the EcoRV site of pHA7D to obtain pIZ3. The AflII recognition site was introduced within the crr gene (at the position corresponding to the 7th and 8th codons of crr gene) of pIZ3 by PCR mutagenesis to obtain pIZ3A. The crp region of pHA7M in which a MluI site was introduced near the last codon of crp was PCR amplified with primers 5′- GCAATTTAACTGTGATAAAC-3′ (pBR-H3) and 5′-AGACTTAAGGGTACCTTACCC GCTACGCGTGCCGTAAAC-3′ (crp-3-SGtaaCRR) or 5′-AGACTTAAGGGTACCTTACCCGCTACGCGTGCCGTAAAC-3′ (crp-3-SGtagCRR), digested with HindIII and AflII, then cloned into the HindIII-AflII region of pIZ3A to obtain pST513 or pST523, respectively. Plasmid pHA7am, in which the TAA stop codon of crp is replaced by TAG was constructed by PCR mutagenesis using pHA7.

Growth conditions and the effects of antibiotics

Cells were grown aerobically at 37 °C in Luria-Bertani (LB) medium. Bacterial growth was monitored by determining the optical density at 600 nm (OD600). The effects of representative antibiotics on the growth curve of three isogenic strains (ssrA+, ΔssrA and ssrADD) were evaluated by monitoring the OD600 of cultures grown in the presence and absence of antibiotics every 30 min. To systematically accesses the inhibitory effects of various antibiotics, twofold serially diluted antibiotic solutions were added to a 1000-fold diluted overnight culture of test strains in a 96-well microtitre plate. The plate was incubated aerobically at 37 °C for 12 h, and the optical density of the culture at 550 nm was determined. The antibiotics used in this study were purchased from Wako or Sigma.

Western blotting

Anti-DD-tag, anti-CRP and anti-IIAGlc antibodies were as previously described (Abo et al. 2000; Ishizuka et al. 1993). Bacterial cells grown in LB medium containing the appropriate antibiotic(s) were harvested at mid-log phase and suspended in 100 µL SDS–polyacrylamide gel electrophoresis 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. The total cellular proteins at the indicated amounts were subjected to a 0.1% SDS−12% polyacrylamide gel electrophoresis and transferred to an Immobilon membrane (Millipore). The polypeptides detected by the antibodies were visualized by ECL system (Pharmacia). The total cellular proteins were also analysed by a 0.1% SDS−12% polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Drs Kirill Datsenko and Barry Wanner (Purdue University) for providing us with the gene inactivation system. We also thank Drs Toshifumi Inada (Nagoya University), Yoshikazu Nakamura (University of Tokyo), Hachiro Inokuchi (Kyoto University) for discussions and suggestions. This study was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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