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Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand
Correspondence
Wipa Chungjatupornchai
stwcj{at}mucc.mahidol.ac.th
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Synechococcus PCC 7942 is a prime example of photoautotrophic cyanobacteria. Synechococcus PCC 7942 (NZ_AADZ00000000) and Synechococcus PCC 6301 (NC_006576) possess nearly identical genomic sequences and therefore are considered as the same species. Results obtained with Synechococcus PCC 7942 or PCC 6301 would most likely be applicable to both strains. Synechococcus PCC 7942 and PCC 6301 (hereafter referred to as Synechococcus) have approximately 10 copies of the chromosome (Binder & Chisholm, 1990; Mori et al., 1996). Each chromosome contains two rRNA operons, rrnA and rrnB (Tomioka & Sugiura, 1984), which are separated by 600 kb and transcribed oppositely (Kaneko et al., 1996). The rrnA and rrnB operons have 100 % identical coding sequences organized in the following order: tandem promoters (P1, P2 and P3) (Asato, 2003), 16S gene, tRNAIle gene, tRNAAla gene, 23S gene, 5S gene and terminator region. Many bacteria also possess multiple rrn operons; for example there are seven rrn operons in Escherichia coli (Kiss et al., 1977), ten in Bacillus subtilis (LaFauci et al., 1986) and six in Lactococcus lactis (Tulloch et al., 1991).
rRNAs play an important role in protein synthesis. A growing number of regions in both 16S and 23S rRNA have been identified as having specific functions (for a review, see Green & Noller, 1997). It has been shown that 23S rRNA is a ribozyme that catalyses the peptidyltransferase step of protein synthesis (Nissen et al., 2000). In Synechococcus, rRNA synthesis is stimulated by a light-activated DNA-binding factor in the light but not in the dark (Asato, 1998). However, current knowledge concerning the structure–function relationship of rRNA in cyanobacteria is still limited, due to the lack of a genetic system for mutational analysis. The presence of a multicopy chromosome in cyanobacteria has restricted the mutational analysis of rRNA and the in vivo production of pure mutant ribosome populations. Genetic systems for expressing homogeneous mutated rRNA have been developed in the yeast Saccharomyces cerevisiae (Chernoff et al., 1994) and in E. coli (Asai et al., 1999).
In this work, we describe a strategy for developing a genetic system for expressing homogeneous mutated rRNA in cyanobacteria. We sequentially constructed and characterized Synechococcus mutant strains with chromosomal rrnA or rrnB inactivated and a final strain with all chromosomal rrn operons inactivated but carrying a replaceable multicopy plasmid containing a single rrn operon. Here we report an application of our system. We constructed and characterized a Synechococcus strain in which essentially all cellular 23S rRNA contained the mutation C2588A encoded by a plasmid. Position C2588 is equivalent to C2611 of the peptidyltransferase centre in domain V of E. coli 23S rRNA.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(Hanahan, 1983) and RL443 (Elhai & Wolk, 1988b) were grown in LB broth or on agar (Sambrook & Russell, 2001). Synechococcus PCC 7942 strain R2-SPc (Kuhlemeier et al., 1983) was grown in liquid or on solid (1·5 % Difco Bacto Agar) BG-11 medium (Williams, 1988) under constant illumination of 3000 lx (i.e. 38 µE m–2 s–1). Synechococcus cell growth was monitored as OD730. The OD730 of cell suspensions was linearly related to cell density (determined by flow cytometry) over the range of values used in the experiments (OD730<0·55). The doubling time of exponentially growing cultures at 30 °C was calculated from OD730 data. Synechococcus was transformed as described by Kuhlemeier et al. (1983). Triparental conjugation, used to transfer plasmid into Synechococcus, was performed according to Elhai & Wolk (1988b). Antibiotics were added at the following concentrations when required: 10 µg chloramphenicol (Cm) ml–1, 20 µg erythromycin (Em) ml–1, 10 µg kanamycin (Km) ml–1 and 50 µg spectinomycin (Spc) ml–1.
Construction of chromosomal rrn-inactivated strains.
In order to construct strains harbouring 
rrnA : : SpcR (designated A1) and rrnB : : CmR (designated B1), pDA-Spc and pDB-Cm were linearlized with NdeI and separately transformed into Synechococcus wild-type strain (WT). Since Synechococcus contains multiple copies of the chromosome, the transformants containing heterogeneous chromosomal rrn operons (i.e. wild-type and inactivated rrn operons) were segregated on BG-11 agar containing Spc at 50–150 µg ml–1 or Cm at 10–30 µg ml–1. The resulting A1 and B1 strains harbouring homogeneous chromosomal rrn operons were transformed with pRN-A or pRN-B to obtain strains A2 (rrnA : : SpcR/pRN-A), A3 (rrnA : : SpcR/pRN-B) and B2 (rrnB : : CmR/pRN-B). Strain A3 was transformed with NdeI-linearlized pDB-Cm to obtain strain AB1 (rrnA : : SpcR rrnB : : CmR/pRN-B). The resulting strains harbouring homogeneous inactivated rrn operon(s) with or without plasmid-borne rrn operon are shown in Table 1.
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. Three micrograms of total DNA was digested with PstI, electrophoresed on agarose gel and transferred to a nylon membrane (Hybond-N+, Amersham Biosciences). Probe I (485 bp) and probe II (490 bp) were amplified using primer sets 16F1 and 16R2; 23F1 and 23R1, respectively (Table 2, Fig. 1), and labelled with digoxigenin using the PCR DIG Probe Synthesis Kit (Roche Applied Science). The hybridized membrane was treated with antidigoxigenin–alkaline phosphatase Fab fragments and luminescent substrate CSPD as recommended by the manufacturer (Roche Applied Science). The treated membrane was exposed to X-ray film.
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; Plansangkate et al., 2004). The cDNA of the 23S rRNA gene, synthesized from total RNA with primer 23R3, was used as template for amplification of PCR product using primers 23F3 and 23R3. The resulting PCR and RT-PCR products of 1·6 kb were digested with AccI to generate fragments of 630 and 1002 bp. The 1002 bp fragment was further analysed by Sau96I digestion (see Results). The digested DNA was resolved in a 4·5 % agarose gel containing ethidium bromide. An image of the gel was captured with UVP (Life Science). The mutated C2611A of PCR and RT-PCR products was confirmed by automated sequence analysis (Perkin-Elmer, ABI Prism 3100). |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Synechococcus strains with the chromosomal rrn operons inactivated were sequentially constructed (see Methods). The chromosomal rrnA and rrnB operons were inactivated by deletion–insertion mutagenesis using cassettes encoding Spc and Cm resistance, respectively (Fig. 1). The resulting Synechococcus strains harbouring homogeneous inactivated rrn operon(s) with or without plasmid-borne rrn operon are shown in Table 1. Southern blot analysis using probes specific to regions of the 16S and 23S genes confirmed that strains with expected genotypes were obtained (Fig. 2). The intensity of hybridized bands was determined by densitometry. The results indicated that the ratio of a chromosomal rrn operon (either rrnA or rrnB) to a plasmid-borne rrn operon (either pRN-A or pRN-B) was 1 : 3 in strains A2, A3 and B2 (Fig. 2). The band intensity ratio of 1 and 3 ng of unlabelled probes used as control was also 1 : 3. We obtained the final strain AB1 with all chromosomal rrn operons inactivated but carrying the replaceable multicopy plasmid pRN-B containing a single rrnB operon (Fig. 2).
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). The lag time that was required to initiate cell growth upon returning to light was determined. The lag time of strains A1 and B1 was 3 h as compared to 2 h for the WT, representing a 50 % increase; however, the presence of plasmid-borne rrn operon in strains A2 and B2 restored the lag time to 2 h (Fig. 3). The results indicated that the two chromosomal rrn operons were necessary for Synechococcus to adapt rapidly for initiating cell growth on dark/light shift.
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) survived with no apparent deleterious effects. In order to investigate the effect of rrn inactivation on cell growth, the doubling time of the mutant strains was measured. This revealed that inactivation of chromosomal rrnA (strain A1) prolonged the doubling time (Table 3). The presence of plasmid-borne rrnA (strain A2), but not plasmid-borne rrnB (strain A3), restored the doubling time. In contrast, inactivation of chromosomal rrnB (strain B1) in the presence of plasmid-borne rrnB (strain B2) did not affect the doubling time. Similarly, the presence of only plasmid-borne rrnB in strain AB1 prolonged the doubling time, whereas only plasmid-borne rrnA in strain AB2 did not. The results suggested that the rrnA operon might produce a higher amount of rRNA and tRNA molecules than the rrnB operon. In addition, the doubling time of strain AB3 producing C2611A-mutated 23S rRNA was significantly longer than those of the AB2 and WT strains (Table 3).
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). To apply our system for experimental analysis of mutated rRNA, we constructed plasmids containing the mutation at position 2588 with respect to the first nucleotide of the 23S rRNA transcript (Douglas & Doolittle, 1984). Position C2588, equivalent to C2611 of the peptidyltransferase centre in domain V of E. coli 23S rRNA, is shown in Fig. 4(a). Plasmids containing wild-type C2611 (pKT-A) or mutated C2611 of the 23S rRNA gene (pKT-AC2611A, pKT-AC2611G and pKT-AC2611T) were transferred separately into strain AB1 by conjugation to replace pRN-B. The resulting EmR-KmS strains are AB2 and AB3, harbouring pKT-A and pKT-AC2611A, respectively. Strains AB2 and AB3 accounted for 35 % of the conjugants. However, attempts to select for an EmR-KmS strain harbouring pKT-AC2611G or pKT-AC2611T, but not pRN-B, were unsuccessful. The presence of wild-type C2611 and C2611A-mutated 23S rRNA was confirmed by PCR, RT-PCR (Fig. 4b, c) and DNA sequencing (data not shown). The results indicated that strains AB2 and AB3 expressed homogeneous wild-type and C2611A-mutated 23S rRNA, respectively.
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). The results indicated that C2611A-mutated 23S rRNA confers temperature-sensitive phenotypes in Synechococcus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Synechococcus has approximately 10 copies of the chromosome (Binder & Chisholm, 1990; Mori et al., 1996). Thus, the data suggested that there were approximately 30 copies of pRN-A or pRN-B in the cells. It has been shown that there were approximately 30 copies of RSF1010-derived plasmid in cyanobacterial cells (Ng et al., 2000). Therefore, there were approximately 30 copies of the RSF1010-derived plasmids pKT and pKT-AC2611A in strains AB2 and AB3, respectively. The data suggested that there were approximately 20 copies of the rrn operons in WT; 10 copies in strains A1 and B1; 40 copies in strains A2, A3 and B2; and 30 copies in strains AB1, AB2 and AB3.
Strains A1 and B1, harbouring an inactivated rrnA and rrnB operon respectively, survive with no apparent deleterious effects, similar to the findings of Golden et al. (1989). Analysis of the growth response on dark/light shift (Fig. 3) revealed that decreased rrn copy number (10 copies in strains A1 and B1) increased the lag time required for initiating cell growth upon returning to light, whereas increased rrn copy number (40 copies in A2 and B2) restored the lag time but did not achieve faster adaptation than the 20 copies in the WT. The results agree well with the feedback inhibition model in E. coli, in which increased rrn copy number does not lead to increased rRNA transcription; rRNA synthesis from individual operons is reduced to keep the total rRNA production unchanged (Jinks-Robertson et al., 1983) and depletion of functional rrn operons causes increased expression of the remaining intact copies (Condon et al., 1993).
The presence of 10 copies of the rrnA operon (strain B1) did not affect the doubling time, whereas the same copy number of the rrnB operon (strain A1) increased the doubling time. However, 40 copies of the rrnB operon (strain A3) did not restore the doubling time (Table 3). These results suggested that the rrnA operon might produce a higher amount of rRNA and tRNA molecules than the rrnB operon. The sequences downstream of nt –60, including the core promoter regions of the rrnA and rrnB operons, are 100 % identical (NZ_AADZ00000000 and NC_006576). However, we observed that the upstream sequence of the rrnA operon, but not the rrnB operon, includes the sequence 5'-TGCA-TCTCC-AGCA-3' (nt –123 to –111), which is highly homologous to the binding site for the LysR-type transcriptional activator of Synechococcus TGCA-N5-TGCA (Maeda et al., 1998). Whether the presence of this putative binding site for a LysR-type transcriptional activator leads to higher expression of the rrnA operon remains to be investigated. rrn operons with different promoter strengths have been found in E. coli (Condon et al., 1992).
We have successfully constructed the AB1 strain with all chromosomal rrn operons inactivated but carrying the replaceable plasmid pRN-B. Using this system, we obtained strains AB2 and AB3 carrying only pKT-A and pKT-AC2611A, respectively. However, attempts to obtain a strain harbouring only pKT-AC2611G or pKT-AC2611T, but not pRN-B, were unsuccessful. Thus, the mutation C2611G/U in the absence of wild-type 23S rRNA may be lethal to cells. The C2611A-mutated 23S rRNA in strain AB3 prolongs doubling time (Table 3) and confers temperature-sensitive phenotypes at 16 °C and 45 °C (Fig. 5). It has been reported that in Sacch. cerevisiae, a single C3993A mutation in the peptidyltransferase region of mitochondrial 21S rRNA confers a cold-sensitive phenotype and blocks the assembly of the 54S ribosomal subunit at 20 °C but not at 32 °C, suggesting that the nucleotide at position 3993 or base pairing between positions 3993 and 1950 may influence the interaction between the rRNA and a ribosome protein (Cui & Mason 1989). Positions C3993 and G1950 are equivalent to C2611 and G2057 of E. coli 23S rRNA, respectively. Positions 2611 and 2057 of the peptidyltransferase centre in domain V of E. coli 23S rRNA, Synechococcus 23S rRNA and Sacch. cerevisiae 21S rRNA are shown in Fig. 4(a). Disruption of the 2611–2057 base pair, resulting in disruption of the rRNA structure at the end of the stem preceding the single-stranded portion of the peptidyltransferase region, was found to confer resistance to macrolide antibiotics including Em (Vester & Douthwaite, 2001). However, the mutation C2611A/G/U has little effect on ErmE methylation (Villsen et al., 1999). Mutation at nt 2611 is associated with disparate phenotypes. Our results indicated that C2611G/U-mutated 23S rRNA seems to be lethal to Synechococcus, whereas the mutations C2611A/G/U in Sacch. cerevisiae mitochondria, C2611G/U in Chlamydomonas reinhardtii chloroplast, C2611A/G/U in Streptococcus pneumoniae and C2611U in E. coli cause a different pattern of resistance to macrolide antibiotics (Vester & Douthwaite, 2001; Franceschi et al., 2004). We are not able to determine whether the C2611A-mutated 23S rRNA in AB3 strain confers resistance to Em, since pKT-AC2611A contains an EmR gene (ermC) as a selectable marker.
We have successfully constructed a Synechococcus strain for expression of homogeneous engineered rRNA in cyanobacteria; this strain has all chromosomal rrn operons inactivated but carries a replaceable multicopy plasmid containing a single rrn operon. This system provides potential for the study of the structure and function of rRNA in photoautotrophs.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 17 November 2005;
revised 25 January 2006;
accepted 31 January 2006.
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