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J. Biol. Chem., Vol. 282, Issue 22, 16267-16277, June 1, 2007

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Exoribonuclease R in Pseudomonas syringae Is Essential for Growth at Low Temperature and Plays a Novel Role in the 3' End Processing of 16 and 5 S Ribosomal RNA^*

From the Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India

Received for publication, June 12, 2006 , and in revised form, April 2, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The (3' 5') exoribonuclease RNase R interacts with the endoribonuclease RNase E in the degradosome of the cold-adapted bacterium Pseudomonas syringae Lz4W. We now present evidence that the RNase R is essential for growth of the organism at low temperature (4 °C). Mutants of P. syringae with inactivated rnr gene (encoding RNase R) are cold-sensitive and die upon incubation at 4 °C, a phenotype that can be complemented by expressing RNase R in trans. Overexpressing polyribonucleotide phosphorylase in the rnr mutant does not rescue the cold sensitivity. This is different from the situation in Escherichia coli, where rnr mutants show normal growth, but pnp (encoding polyribonucleotide phosphorylase) and rnr double mutants are nonviable. Interestingly, RNase R is not cold-inducible in P. syringae. Remarkably, however, rnr mutants of P. syringae at low temperature (4 °C) accumulate 16 and 5 S ribosomal RNA (rRNA) that contain untrimmed extra ribonucleotide residues at the 3' ends. This suggests a novel role for RNase R in the rRNA 3' end processing. Unprocessed 16 S rRNA accumulates in the polysome population, which correlates with the inefficient protein synthesis ability of mutant. An additional role of RNase R in the turnover of transfer-messenger RNA was identified from our observation that the rnr mutant accumulates transfer-messenger RNA fragments in the bacterium at 4 °C. Taken together our results establish that the processive RNase R is crucial for RNA metabolism at low temperature in the cold-adapted Antarctic P. syringae.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Regulated degradation of RNA within cells is mostly an outcome of coordinated and combined activities of endoribonucleases, exoribonucleases, and RNA helicases. In bacteria, few of these enzymes interact with each other to form the RNA degradosome complex (1–3). The degradosome has been shown to be involved in both mRNA and rRNA degradation (4, 5). In the Escherichia coli degradosome, RNase E (a 5' end-dependent endoribonuclease) associates with polynucleotide phosphorylase (PNPase,² a3' 5' exoribonuclease), RhlB (a DEAD box RNA helicase), and enolase (an enzyme of glycolytic pathway) to constitute the "core" complex. Additionally, DnaK, poly(A) polymerase and polyphosphate kinase have also been reported to be a part of this complex (6). A similar kind of RNA degrading complex has also been reported from Rhodobacter capsulatus (7). On the other hand, we have recently shown that exoribonuclease RNase R interacts with RNase E in the degradosome of the cold-adapted Antarctic bacterium Pseudomonas syringae Lz4W (8). This bacterium does have a pnp gene that is expressed but does not form a part of this complex.³ PNPase and RNase R differ in their mode of action, the former exhibiting phosphorolytic activity and the latter exhibiting hydrolytic activity.

RNase R is one of the eight exoribonucleases reported in E. coli and distributed widely in different prokaryotes (9). Although all of these exoribonucleases display 3' 5' activity on RNA substrate, RNase R is the most highly processive enzyme among them. The enzyme was first identified in E. coli crude cell extract, where RNase II contributes the bulk (98%) of the poly(A) RNA degrading activity, whereas RNase R contributes only residual (2%) activity (10). Subsequently, it was named RNase R because of its action on rRNA and shown encoded by the vacB gene, which is essential for virulence in Shigella flexneri and E. coli (11). It has been proposed that RNase R along with PNPase, encoded by rnr and pnp genes, respectively, are responsible for quality control of rRNA and that rnr pnp double mutants are inviable (12). Interestingly, PNPase was found important for growth at low temperature in E. coli and in the psychrotrophic Yersinia enterocolitica (13, 14), although the pnp mutants of P. putida were not cold-sensitive (15). RNase R, on the other hand, was shown to be cold-inducible and involved in tmRNA maturation in E. coli, but the rnr mutant was not cold-sensitive (16). Extensive study from Deutscher's group (17) has now established that RNase R can degrade RNA with secondary structures without the help of helicase in vitro and is proficient in degrading mRNAs with repetitive extragenic palindromic sequences in vivo. The same group shows that the RNase R level is elevated in response to stress conditions including starvation and entry into the stationary phase (18). The authors speculate that extensive remodeling of structured RNA occurs under stress conditions, which would require the highly processive activity of RNase R to work on the RNA substrates.

Our observation that phosphorolytic PNPase is replaced by hydrolytic RNase R in the degradosome of the psychrotrophic bacterium P. syringae Lz4W is puzzling (8). As a further step toward detailed characterization of the P. syringae degradosome machinery, knock-out mutants of RNase R (rnr), RhlE (rhlE), and the C-terminal degradosome-organizing region of RNase E (rne^C) would be useful. We report here our results on the rnr knock-out mutant of P. syringae, which was found to be severely cold-sensitive. Remarkably, our data suggest that RNase R is involved in the processing of 3' ends of 16 and 5 S rRNAs. The rnr mutants are not only defective for 16 and 5 S rRNA maturation at 4 °C but also accumulate unprocessed 16 S rRNA in the polysomes, probably making the translation machinery inefficient at low temperature. The mutant cells also accumulate degradation intermediates of tmRNA, suggesting that the important regulatory RNA is also a target of RNase R in the bacterium. We suggest that a combination of defects in RNA metabolism at low temperatures in the absence of RNase R lead to the cold-sensitive phenotype of the bacterium.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial Strains and Growth Conditions—P. syringae Lz4W and E. coli cells were routinely grown in Antarctic bacterial medium (ABM) (5 g liter^–1 peptone and 2.5 g liter^–1 yeast extract), and Luria-Bertani medium, respectively, as reported earlier (8). When required, the growth media were supplemented with antibiotics in following concentrations: 100 µg ml^–1 ampicillin, 50 µgml^–1 kanamycin, 20 µgml^–1 tetracycline, 100 µgml^–1 chloramphenicol, and 400 µgml^–1 rifampicin. Composition of minimal growth medium for P. syringae was: 0.12% Na₂HPO₄, 0.06% KH₂PO₄, 0.05% (NH₄)₂SO₄,1% succinic acid, 0.01% valine, 0.01% isoleucine, and 1 mM MgSO₄.⁴ For growth analysis, bacterial cells from overnight culture were inoculated into fresh medium at a dilution of 1:100, and the turbidity of the cultures at 600 nm (A₆₀₀) was measured at various time intervals. For complementation studies, the plasmids were introduced into P. syringae strain by conjugation with E. coli S17-1 (19).

Recombinant DNA Methods—General DNA recombinant techniques including isolation of genomic DNA, restriction analysis, PCR, ligation, and transformation etc. were performed as described (20). All of the restriction enzymes, T4 DNA ligase, and Klenow enzyme used in this study were from New England Biolabs. An Ominiscript RT kit (Qiagen) was used for reverse transcription, and oligonucleotides were purchased from Bio-Serve BioTech (Hyderabad, India).

For Southern hybridization, genomic DNA was isolated from both wild type and rnr mutant of P. syringae and digested with SalI enzyme. DNA fragments were separated on 1% agarose gel and then transferred onto Hybond N⁺ nylon membrane (Amersham Biosciences). PCR-amplified DNA of full-length rnr gene (2.7 kilobase pairs) was labeled with [-³²P]dATP using random primer labeling kit (Jonaki, BARC, India), and used as probe. DNA hybridization was carried out in 0.5 M sodium phosphate containing 7% SDS at 65 °C for 8–10 h. The membrane blots were washed twice with 2x and 0.1x salinated sodium citrate, respectively. Radioactive signals were developed using a phosphorimaging device (Fuji FLA3000).

Construction of rnr Knock-out Mutant of P. syringae—Plasmid pBSrnr-tet used for knocking out the rnr gene was constructed as follows. The rnr gene was first subcloned as KpnI-XbaI fragment into pBlueScript-KS vector, taking the fragment from earlier reported plasmid pMOSBlue containing P. syringae rnr gene (8). The resultant plasmid pBS-rnr was linearized with HincII, and a tetracycline resistance gene was cloned within the rnr to generate pBSrnr-tet plasmid. The source of tetracycline resistance gene (tet) cassette (2.4 kilobase pair) was pTc28 plasmid (19), from which it was cleaved out as XbaI-HindIII fragment and made blunt-ended using Klenow enzyme before ligating to the HincII site of pBS-rnr. Plasmid construct was confirmed by sequencing and PCR analysis. For rnr knockout, the pBSrnr-tet was electroporated into P. syringae following denaturation of the plasmid as described earlier (21), which apparently increases homologous recombination and hence gene disruption frequency. Recombinants were selected by tetracycline resistance, and the rnr knock-out mutation was confirmed by Southern and PCR analysis.

Plasmids for Complementation Analysis—For genetic complementation of rnr mutant, plasmids pGLrnr-his and pGLpnp-his were used. Construction of pGLrnr-his expressing His-tagged RNase R in P. syringae has been described earlier (8). For construction of pGLpnp-his, a similar strategy was followed. Briefly, the P. syringae PNPase gene (pnp) was amplified from the genomic DNA by PCR using the forward PnpFP1 (5'-TTCCAGTTCGGTCAGTCGACCGT-3') and reverse PnpRP2 (5'-ACGTCCTTGAT(C/G)GACAGCTTGAT-3') primers. The 2.1-kilobase pair PCR product was first cloned into the EcoRV site of the pMOSBlue plasmid (Amersham Biosciences) and subsequently into the expression vector pET28a (Novagen). The His-tagged version of the PNPase gene was then cleaved out of pETpnp-his and cloned into the broad host range plasmid vector pGL10 (22) to generate pGLpnp-his. Reading frame and the in-frame cloning of PNPase gene were confirmed by DNA sequencing. Expression of protein was confirmed by Western blot analysis, using either anti-His tag antibody or anti-PNPase antibodies.

RNA Isolation and Northern Hybridization Analysis—Wild type and rnr mutant strains of P. syringae were grown at 22 °C till the cultures attained A₆₀₀ =0.5. The cultures were then shifted to 4 °C, and every 24 h, 6 ml of culture were removed, of which 3 ml was centrifuged immediately. To the remaining 3 ml, rifampicin was added (final concentration, 400 µgml^–1), and incubation was continued at 4 °C for another 60 min. Total RNA was isolated from all the samples by hot phenol method (23), and the concentration of RNA was estimated by measuring the A₂₆₀ of all the samples. RNA (10 µg) from each sample was resolved on 1.2% agarose gel (12) in Tris-acetate-EDTA buffer (20) and transferred to Hybond N⁺ membrane by vacuum transfer using 5x salinated sodium citrate, 10 mM NaOH. The oligonucleotide probes (supplemental Table S1, p1–p4) used for Northern hybridization were 5' end-labeled with [-³²P]ATP using T4 polynucleotide kinase (New England Biolabs) following the manufacturer's protocol. The ³²P-labeled oligonucleotides were separated from unincorporated [-³²P]ATP by gel filtration on Sephadex G50 columns. The temperatures for oligonucleotide probe hybridization and washing of the Northern blot membrane are indicated in Table S1. Probe for tmRNA was prepared by amplifying the tmRNA gene by PCR of genomic DNA using a set of forward (5'-TTAGGATTCGACGCCGGT-3') and reverse (5'-TGGTGGAGCCGGGGGGATTTGAAC-3') primers and labeled by random primer labeling method as described for Southern hybridization probe.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Generation of rnr knock-out mutant of P. syringae and growth analysis. A, schematic representation of the construct pBSrnr-tet used for rnr disruption. A few important restriction sites have been marked. The numbers below the rnr gene refer to the nucleotide position of the RNase R open reading frame. B, Southern analysis using rnr specific probe as described under "Experimental Procedures." The probe hybridizes to a 7-kilobase pair DNA fragment in wild type, which is missing in the rnr mutant. Because of a single SalI site present in the tet cassette, two DNA bands of smaller size are visible instead of one in the mutant lane. C, Western analysis of the wild type and mutant cells using polyclonal anti-RNase R antibody. Note that RNase R is absent in the rnr mutant. D, growth of the bacterial strains at 22 °C. A600 of the cultures was measured every 2h. E, growth of the bacterial strains at 4 °C. A600 was taken every 24 h. kbp, kilobase pair.

Western Analysis—Protein blotting and immunodetection techniques for Western analysis were as described in Ref. 8. For checking low temperature induction of the RNase R, P. syringae cells were grown at 22 °C till A₆₀₀ reached 0.5 and then shifted to 4 °C. Culture (3 ml) was withdrawn at the indicated time, and cell extracts were prepared by sonication. Protein concentration was determined using Bio-Rad protein assay kit. Equal amount of proteins were loaded in each lane for SDS-PAGE separation and blotting onto Hybond-C membrane (Amersham Biosciences). Polyclonal anti-His antibody was from Santa Cruz Biotechnology. Antibody against the P. syringae PNPase was raised in rabbit against the purified recombinant protein as described for the polyclonal anti-RNase R antibody (8). Alkaline phosphatase-conjugated anti-rabbit goat IgG was used as secondary antibody.

Cell Viability Studies—Viability of cells was examined both by counting colony-forming units (CFU) in the cultures and also by differential fluorescent staining of live and dead cells under a fluorescent microscope (Zeiss). Briefly, the cells were grown at 22 °C till A₆₀₀ reached 0.5 followed by shifting to 4 °C. At every 24-h interval, aliquots of culture were withdrawn for measuring turbidity at A₆₀₀ and spreading of cells onto ABM-agar plates for growth at 22 °C. For each time point, CFUs on plates (in triplicates) were counted. The cells in cultures at each time point were also examined microscopically (19) by staining them with Syto9 and propidium iodide (PI) using LIVE/DEAD Baclight bacterial viability kit (Molecular Probes, Eugene, OR).

View larger version (37K): [in this window] [in a new window]   FIGURE 2. Analysis of cell death at low temperature. A, growth of the indicated strains was followed as a function of A600 of the cultures. The cells grown at 22 °C till 0.5 A600 were shifted to 4 °C, and A600 was measured every 24 h. B, loss of cell viability in rnr mutant as measured by counting CFU. The counting was done every 24 h following the low temperature (4 °C) shift, and the data were plotted. C, LIVE-DEAD staining of cells after shifting of cultures from 22 to 4 °C. The cells were stained with LIVE/DEAD Baclight bacterial viability kit that comprises Syto9 and propidium iodide. Syto9 stains live cells green, and PI stains dead cells red.

Analysis of Protein Synthesis—Protein synthesis was monitored by pulse chasing the growing cells of P. syringae in the presence of a mixture of ³H-labeled amino acids (Amersham Biosciences). Briefly, cells (A₆₀₀ 0.5) growing in ABM at 22 °C were shifted to 4 °C for incubation. At the various time points, the cells were harvested and suspended in minimal growth medium supplemented with a mixture of ³H-amino acids (50 µCi ml^–1; specific activity, 13–78 mCi mmol^–1) for 30 min (pulse) of labeling. The cells were then incubated (chase) for another 30 min in the richer medium by adding an equal volume of 2x ABM into the minimal medium. The cells were then lysed with Trislysozyme-EDTA buffer, and proteins were precipitated by adding equal volume of ice-cold 10% trichloroacetic acid. Protein pellet was spotted onto filter paper (Whatman), and radioactive counts were measured in a toluene-based scintillant (0.5% 2,5-diphenyl oxazole and 0.03% 1,4-bis(4-methyl-5-phenyl-2-oxazolyl) benzene dissolved in toluene) using a liquid scintillation counter (Packard Tricarb).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
rnr Knock-out Mutants of P. syringae Are Cold-sensitive—To assess the importance of RNase R in RNA metabolism of the cold-adapted P. syringae Lz4W, an rnr knock-out mutant strain (rnr:tet) was created. The strategy used for knocking out rnr is shown schematically in Fig. 1A. Integration of the tet cassette by homologous recombination and disruption of the rnr gene in mutant was confirmed by Southern hybridization (Fig. 1B) and by PCR analysis of genomic DNA (data not shown). Expectedly, the rnr gene-specific probe hybridized to only one SalI restriction fragment in the wild type but to two fragments in the mutant because of the location of a single SalI recognition site within the tet cassette (Fig. 1B). The lack of RNase R production because of inactivation of the rnr was confirmed by Western analysis of cell extract using anti-RNase R antibodies (Fig. 1C).

We then examined growth of the rnr mutant at 22 °C and at 4 °C. Growth analysis (Fig. 1, D and E) reveals that the mutant, although marginally affected at room temperature (22 °C), is severely defective for growth at 4 °C. The mutant strain failed to grow even after 15 days of incubation at low temperature. To confirm that the observed phenotype was only due to absence of functional RNase R, the rnr mutant was transformed with plasmid pGLrnr-his for expression of RNase R in trans. The mutant cells produced recombinant His-tagged RNase R and grew well at 4 °C, similar to wild type, but a mutant strain carrying pGL10 (empty vector) remained cold-sensitive. This confirmed that only the lack of RNase R is responsible for cold-sensitive phenotype of the rnr knock-out mutant.

Viability of rnr Mutants Decreases at 4 °C—The cold-sensitive phenotype of the rnr mutant can be either due to lack of growth or due to cell death. To test this, we checked the viability of cells by two different methods following a temperature downshift (22 to 4 °C) of the cultures. Fig. 2A represents the growth curve of the rnr mutant and wild type Lz4W after the shift of cells to 4 °C. The A₆₀₀ of the culture of rnr mutant increased during the first 24 h of incubation but subsequently declined slowly but continuously, suggesting a possible cell lysis as a result of cell death at 4 °C. CFU measurements of the cultures were also consistent with this, as shown in Fig. 2B. The CFU in the cultures increased in the first 24 h but then gradually declined, and at 144 h 95% of the mutant cells were dead. On the other hand, cultures of wild type or the complemented mutant, i.e. rnr^–(pGLrnr-his) strain did not show any decrease of CFU.

Cell death of rnr mutants at 4 °C was also confirmed by staining cells with vital dye Syto9 and PI that stain nucleic acids. With increase in the time of incubation at 4 °C, not only did the number of live cells (Syto9 stained, green) decrease but also the total number of cells in case of the rnr mutant, probably because of cell death and lysis (Fig. 2C). In wild type the number of living cells (green) expectedly increased because of cell division, but the frequency of PI-stained dead cells (red) within the cell population appears to remain constant even upon reaching the stationary phase. We also noticed that the size of wild type cells of P. syringae as reported earlier (19) decreases (from 2.8 µm length to 1.3 µm) when incubated at 4 °C, but cell size of the rnr mutant does not alter much. This makes mutant cells appear 2–3-fold bigger (3 µm) than the wild type cells at 4 °C under similar conditions. However, few cells (1%) in the mutant were 3–5-fold longer than the rest. Interestingly, cell elongation occurred in the pnp^ts rnr^– double mutants of E. coli defective for both PNPase and RNase R at the nonpermissive temperature (42 °C) but not in the single mutants of pnp and rnr (12). It was speculated in the report that cell elongation of the mutants could be due to defective cell division, which might be true of rnr mutant of P. syringae at low temperature too.

Increased Amount of PNPase Does Not Rescue Cold Sensitivity of rnr Mutant—In E. coli RNase R and PNPase have overlapping function, because each of the single mutants are viable, but the double mutation is lethal (12). Neither of the single mutants shows any defect in rRNA turnover. The fact that rnr mutants of P. syringae are cold-sensitive suggests that PNPase does not complement the RNase R function in the bacterium at 4 °C. However, we wanted to check whether increased amount of PNPase is capable of complementing the cold-sensitive phenotype of rnr mutant to any extent. For this, the rnr mutant strain was transformed with pGLpnp-his for expression of His-PN-Pase in trans. Expression of the recombinant PNPase was confirmed by Western analysis using anti-His antibodies that cross-reacted to the protein with expected 75-kDa molecular mass (Fig. 3A). The level of PNPase in this strain, as estimated from the Western blot analysis using anti-PNPase antibodies, was 2.5–3-fold higher compared with the wild type (Fig. 3A, right panel). Growth of the rnr^–(pGLpnp-his) strain was then compared with the wild type and rnr mutant carrying empty pGL10 plasmid at 4 °C. The rnr mutant expressing recombinant PNPase was equally growth defective, as was the rnr mutant (Fig. 3B). The recombinant His-PNPase was confirmed to be enzymatically active (supplemental Fig. S1). This suggests that increased amount PNPase is unable to complement the RNase R function in the bacterium at 4 °C.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Inability of PNPase overexpression to complement the cold sensitivity of rnr mutant. A, Western analysis showing expression of His-PNPase in the rnr mutant. In the left panel, the blot was probed with anti-His antibody. Lane M shows low molecular weight protein markers. Lane 1 was loaded with 15 µg of total cell lysate protein. In the right panel, the blot was probed with anti-PNPase antibodies. Each lane contained 15 µg of proteins of cell lysate. B, growth of rnr mutant expressing His-PNPase in trans at 4 °C. A600 was noted every 24 h. Both mutant and the mutant expressing His-PNPase failed to grow at 4 °C.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. Western analysis of RNase R expression. Immunoblots were probed with anti-RNase R polyclonal antibodies. A, testing for cold induction. Bacterial culture (A600 =0.5) grown at 22 °C was shifted to 4°, and cell aliquots were analyzed for RNase R expression at the indicated time. Each lane contains cell extracts with 10 µg of protein. Lane C contains the sample from cells at the 0 time point, immediately before the shift. B, testing growth phase specific expression. Cells at different growth phases corresponding to culture A600 values of 0.3, 0.7, 1.2, and 2.1 were harvested and analyzed in lanes 1-4, respectively. Two temperature (4 and 22 °C) grown cells were tested. Each lane contains cell extracts with 15 µg of protein.

View larger version (58K): [in this window] [in a new window]   FIGURE 5. Northern analysis of rRNA. A, schematic representation of rRNA operon of P. syringae to indicate the position of oligonucleotide probes used for Northern hybridization. Mature region of rRNAs and tRNAs are shown as boxes on the rRNA operon. Relative locations of the oligonucleotide probes (p1–p4) used in the Northern analysis have been shown as short thick lines below the operon. B, result of p1 oligonucleotide probe hybridization to RNAs from wild type (wt) and RNase R inactivated mutant (rnr). Probe p1 corresponds to sequence complementary to unprocessed residues of 3' end of 16 S rRNA. 22 °C grown cells were shifted to 4 °C, and RNAs were prepared from 0-h (in respect to 4 °C), 24-h, 48-h, and 72-h time points directly, or following 60 min of rifampicin treatment (marked as 60 on the lanes). 10 µg of RNA was loaded in each lane and separated by electrophoresis on 1.2% agarose gel. C, ethidium bromide stained gel used for Northern blot shown in B. D, Northern blot hybridization with the p2 oligonucleotide probe comprising residues complementary to mature 16 S rRNA. Experimental conditions were as in B. E, p1 probe hybridization result with RNAs prepared from the RNase R complemented mutant strain (rnr(pGLrnr-his)). The experimental conditions were same as in B. F, p4 oligonucleotide probe hybridization results showing accumulation of unprocessed 5 S rRNA in the rnr mutant of P. syringae. Northern hybridization conditions were as in B.

The p1 probe corresponding to the nucleotide sequence lying 11 bases downstream of the mature 3' end of 16 S rRNA, hybridized to the 16 S rRNA of the rnr mutant at low temperature. Fig. 5B shows accumulation of the incompletely processed 16 S rRNA population at various time points only in the rnr mutant after shifting to 4 °C. Fig. 5C shows the ethidium bromide-stained gel for RNA loading control. Although a certain amount of incompletely processed 16 S rRNA was noticeable at 22 °C, at the zero time point of both wild type and the mutant (lanes C with 0 min samples, Fig. 5B), it disappeared within 60 min after rifampicin treatment in both (lanes C, 60 min, Fig. 5B). This suggests that some other enzyme is capable of 16 S rRNA maturation at the higher temperature (22 °C), but it fails to do so at 4 °C. Interestingly, Fig. 5B also reveals that there is a increased degradation of 16 S rRNA precursors (smearing of the RNA on Northern blot) with time in the absence of RNase R, which occurs much earlier than the 72 h noted in Fig. 5D for the degradation of matured 16 S rRNA at 4 °C (see below). The processing of 16 S rRNA in the complemented rnr mutant, as shown in Fig. 5E, was normal.

In a control experiment when p2 oligonucleotide sequence corresponding to a matured region of 16 S rRNA was used as probe for hybridization, the 16 S rRNA profiles of the mutant and wild type were similar, except for one additional defect noticed in the mutant. This defect in rnr mutant was manifested by accumulation of the degradation intermediates of 16 S rRNA only at a later stage (after 72 h) of incubation at 4 °C (Fig. 5D). Thus, RNase R might also be involved in general turnover and/or quality control of 16 S rRNA. Accumulation of similar rRNA degradation intermediates was reported for the temperature-sensitive rnr pnp double mutants of E. coli when they were grown at nonpermissive temperatures (12). Interestingly, we did not observe any accumulation of unprocessed and/or degradation intermediates of 23 S rRNA in the rnr mutant of P. syringae when the probe p3 was used for Northern hybridization (data not shown). This suggests that 3' end processing of 23 S rRNA does not involve RNase R.

We also examined the status of the 3' end of 5 S rRNA in the rnr mutant at 4 °C. The probe p4 containing nucleotides beyond the matured 3' end of 5 S rRNA was used for hybridization analysis. It appears that the 5 S RNA population in the mutant cells hybridize to p4 probe (Fig. 5F). Thus, in addition to 16 S rRNA, processing of the 5 S rRNA is also defective in the rnr mutant of P. syringae at low temperature.

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Distribution of unprocessed 16 and 5 S rRNAs in ribosome fractions. Ribosomes were isolated from both wild type and rnr mutant cells grown at 22 °C, then shifted to 4 °C for 24 h, and separated on sucrose gradient. A, fractions (500 µl each) were collected from the gradient, and A260 values were measured for the plotting of ribosomal profile (upper panel). 40-µl aliquots from alternative fractions were then examined for distribution of rRNAs (lower panel). Because the ribosomal profiles were similar in the wild type and mutants, only the wild type profile is shown here. B and C, Northern hybridization of RNA in the ribosomal fractions (23 in case of wt and 22 in case of rnr mutant). Lanes from left to right represent fractions from top to bottom of the gradient. The last lanes in both wt and mutant panel contain ribosome pellet. Alternate fractions were used for Northern analysis. Probes were p1 (B) and p4 (C) specific for unprocessed 3' end residues of 16 and 5 S rRNAs, respectively. Negative hybridization result for only p1 but not for p4 probe in the fractions from wild type has been shown (top panel in B).

Nature of Unprocessed 16 S rRNA in the rnr^– Mutant—To further analyze the nature of unprocessed regions in the 16 S rRNAs of rnr null mutant, nucleotide sequences of the circular RT-PCR products of rRNAs was determined as described under "Experimental Procedures." Although the 16 S rRNAs from wild type produced one prominent product of 100 bp, the mutant strain produced an additional major product of 200 bp (supplemental Fig. S2). DNA sequence analysis of the cloned PCR products established that two types of unprocessed 16 S rRNAs (type I and II) accumulate in the rnr^– mutant strain, which have 36 additional residues beyond the 3' matured end (Fig. 7A and supplemental Fig. S3). The unprocessed 3' tail of 16 S rRNA in case of E. coli is 33 residues long (25, 26). The type II unprocessed 16 S rRNA surprisingly contained nucleotide extensions at the 5' end too (Fig. 7 and supplemental Fig. S3). The 65 nucleotide 5'-extended leader was similar to a 66-nucleotide leader sequence produced by the RNase E processing cleavage in the case of E. coli RNase G^– mutant (26). It is interesting to note that the 5' end unprocessed 16 S precursor that accumulates in E. coli RNase E^– and G^– double mutants also gets incorporated into ribosomes.

rnr Mutants Are Defective in Protein Synthesis at 4 °C—Although the rRNA processing defect did not lead to any apparent assembly defect of ribosomal particle, accumulation of 3'-unprocessed 16 S rRNA in polysomes is known to affect translation processes (25). To test this, we examined the protein synthesis activity of wild type and rnr mutant cells at 4 °C in a pulse-chase experiment using a mixture of ³H-labeled amino acids in growth medium. We found that, after 48 h of incubation at 4 °C, protein synthesis in rnr mutant cells is reduced to 36% of the wild type (Fig. 8). This suggests that rnr mutants with polysomes containing unprocessed 16 S rRNA are perhaps inefficient in protein synthesis. The reason why it takes 24 h before an effect on protein synthesis is evident in the mutant cells might be related to the time taken by the inactive ribosomes containing unprocessed rRNAs to critically outnumber the normal ribosomes already existing in the cytoplasm.

tmRNA Defect in Absence of RNase R—tmRNA, also known as SsrA, plays an important role in rescuing ribosomes that are stalled on mRNAs lacking stop codons (28). In the process, it tags the nascent peptides, which are then identified for degradation. This mechanism ensures that cells get rid of defective proteins, and the ribosomes become available for fresh rounds of translation. It has been shown in E. coli that processing of tmRNA becomes defective in absence of RNase R at the low temperature (16). RNase R was also implicated in degradation of tmRNA in Caulobacter crescentus (29). Therefore, we examined the status of tmRNA in the rnr mutant of P. syringae. We found that the processing of tmRNA was only mildly affected, but there was a strong defect in the degradation of tmRNA in the rnr mutant at 4 °C. Although only a trace amount of precursor tmRNA was detected in cells after 24 h, numerous degradation intermediates accumulated in all the time points of 4 °C incubated cells following the downshift of temperature (Fig. 9A). The complemented mutant, however, displayed normal turnover of tmRNA (Fig. 9B).

View larger version (18K): [in this window] [in a new window]   FIGURE 7. Nucleotide sequence and the putative secondary structures around the 3' and 5' termini of precursor 16 S rRNA. The junction region of ligated 5' and 3' ends of 16 S rRNAs from wild type and rnr– mutant of P. syringae were amplified by circular RT-PCR using appropriate primers and analyzed by nucleotide sequencing (supplemental Figs. S2 and S3). A depicts the two types of unprocessed precursor molecules (types I and II) with untrimmed 3' tails of 36 nucleotides that accumulate in rnr– mutant. Type II molecule has an additional 65 nucleotides leader sequence at the 5' end. Mature 3' end and the putative RNase E cleavage site have been marked on the sequence by vertical arrows. B shows the putative folded structures around the 5' and 3' terminal regions of precursor 16 S rRNA. Both mature and the unprocessed 5' and 3' ends observed in the study have been marked by arrows. A smaller arrow near the mature 5' end indicates the heterogeneity observed in some sequence. Residues shown beyond the unprocessed 5' end and the conserved box C sequence are taken from the DNA sequence of the rRNA operon to depict the duplex secondary structure of the region. Conserved box C sequence where the RNase III cleavage occurs has been marked as a rectangular box. The number of residues at the 3' extended tail (36 bases) and the 5' leader region (65 bases) of the defective 16 S rRNA determined in this study has also been indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A lot is known about rRNA maturation steps and ribosome biogenesis in bacteria, with data mostly from E. coli (25). The primary transcript of rRNA including 16, 23, and 5 S rRNAs, which sometimes contain tRNAs in the spacer region between the 16 and 23 S, and after 5S, is processed by a series of cleavage and trimming reactions to generate individual mature rRNAs. In E. coli, RNase III cleaves the 30 S primary transcript to produce the individual rRNA precursors, which undergo secondary processing reactions subsequently to generate the mature 5' and 3' termini of each rRNA. Apart from RNase III, three more endoribonuclease RNase E, RNase G (Caf A), and RNase P are involved in the maturation process. Although RNase P generates the mature 5' end of tRNAs (31), RNase E and RNase G are responsible for generation of the 5' ends of mature 16 S rRNA (26). Maturation of 5 S rRNA on the other hand involves the activities of both RNase E and the exoribonuclease RNase T (32, 33). RNase T is responsible for maturation of the 3' ends of both 23 and 5 S rRNA (34). Enzyme involved in 3' end maturation of 16 S rRNA has still not been elucidated in E. coli (26). Therefore, our observation that the rnr^– mutant of P. syringae accumulates immature 16 S rRNA molecules with unprocessed 3' end residues, most probably because of the lack of trimming by RNase-dependent 3' 5' exoribonuclease activity, is significant. By expressing RNase Rin trans from a plasmid in the mutant, unprocessed 3' end nucleotides of 16 and 5 S rRNAs can be removed (Fig. 5E).

View larger version (31K): [in this window] [in a new window]   FIGURE 8. Protein synthesis ability of the rnr mutant at 4 °C. Protein synthesis at 4 °C was measured at different time points by pulse chasing the cells with a mixture of 3H-labeled amino acids as described under "Experimental Procedures." Incorporation of radioactive amino acids into cells at zero time point was taken as control for normal protein synthesis ability. The values obtained for controls in wild type (80,000 ± 5000 cpm/106 cells) and mutant (75,000 ± 4000 cpm/106 cells) were taken as 100% for calculation and plotted as a bar diagram.

View larger version (42K): [in this window] [in a new window]   FIGURE 9. Defective tmRNA degradation in rnr mutant. RNAs (10 µg in each lane) were separated on 6% polyacrylamide, 8 M urea gel in 1x Tris-borate-EDTA buffer (20) and electroblotted onto Hybond N+ membrane. The blots were probed with 32P-labeled full-length tmRNA gene of P. syringae. Shown are phosphorimaging results of samples from wild type and rnr mutant that were incubated at 24, 48, and 72 h at 4 °C (A) and those from the RNase R complemented mutant (B). Lanes in the extreme left of panels A and B contain 32P-labeled RNA century marker (Ambion), and lanes C contain RNA samples from cells at 22 °C just before the shift to 4 °C. Lower panels in A and B show portions of the ethidium bromide stained gels used for Northern blotting to indicate the equal loading of RNA samples. RNA was quantified by measuring A260 values of the samples.

Unprocessed 16 S rRNA accumulates in polysomes of the rnr mutant of P. syringae, and the cells are defective in protein synthesis. This is consistent with the long standing view that 30 S ribosomes containing precursor 16 S rRNA are not biologically active and that maturation of 16 S rRNA is essential for protein synthesis in E. coli (36, 37). We propose that defective protein synthesis is one of the important factors causing death of P. syringae rnr mutants at low temperature. However, the intriguing aspect that needs to be investigated in future is to find out the significance of the accumulation of unprocessed 16 S rRNA in polysomes (Fig. 6). It is known in the case of E. coli that the assembly of rRNAs into ribosomal particles takes place so fast that most maturation processes take place in the ribosomes, and the process is more efficient under translating conditions (25). It is therefore likely that ribosomal particles containing 3'-untrimmed 16 S rRNAs are trapped in the translation-defective polysomes of the rnr mutant.

The highly defective tmRNA degradation observed in the rnr mutant of P. syringae is also significant. Although the mutant cells show accumulation of a precursor form of tmRNA at the early time point during low temperature shift, our data implicate the role of RNase R mostly in tmRNA degradation rather than in tmRNA processing. In this respect, tmRNA-processing defects seen in rnr mutants of E. coli during cold shock or tmRNA degradation defects in the stalked cells of C. crescentus rnr mutant are interesting (16, 29). Because tmRNA plays a pivotal role in not only rescuing stalled ribosomes on damaged mRNA but also in tagging the truncated defective peptides for proteolytic degradation (28), the defect of tmRNA metabolism in the rnr mutant of P. syringae might cause a physiological imbalance in the cell, perhaps contributing toward cell death at low temperature. Additionally, metabolic defects might arise in the mutant cells of P. syringae because of the known role of RNase R in mRNA degradation, which has not been examined in this study.

Low temperature-specific defects in growth and RNA metabolism observed in the rnr mutant of P. syringae underscore the fact that RNase R enzyme activity is essential at low temperature. But why does not the lack of RNase R cause much defect at higher temperature (22 °C) in the bacterium? It is possible that another enzyme carries out the RNase R-dependent functions at higher temperature. At 4 °C, RNAs might have more extensive and highly stabilized structures, and the organism needs a highly efficient and processive exoribonuclease like RNase R. At higher temperature (22 °C), a less processive enzyme might be sufficient to process/degrade RNAs with less extensive and less stabilized structures, and hence cells without RNase R would grow almost normally. There is of course a second possibility that the enzyme, which complements the RNase R activity at 22 °C but fails to express at a lower temperature (4 °C), has not been tested in the present study. However, our study clearly establishes that PNPase does not substitute the RNase R function in P. syringae. The third possibility, that RNase R works only at low temperatures, is probably not correct because RNase R expression levels remain similar at both low and high temperatures and the ability of RNase R to associate with ribosomal particles or the interaction with RNase E in degradosome does not alter with temperature changes.³ It is, however, to be noted that similar levels of expression of any protein do not ensure similar levels of activity. For example, RNA degradation activity of P. syringae RNase R at 22 °C is higher than at 4 °C under in vitro condition,³ and therefore, a similar level of RNase R at 4 °C might be critical for RNA metabolism at lower growth temperatures but not at higher temperatures. This explanation needs further investigation.

In conclusion, a significant finding of this study is that RNase R activity in the cold-adapted P. syringae is singularly important for growth at low temperature. Equally significant is our observation that the RNase R activity is required for 3' end processing of 16 and 5 S rRNAs, suggesting a previously unidentified role of RNase R, in addition to its role in tmRNA degradation in the cold-adapted bacterium. Our future aim is to understand why the deprivation of RNase R in cells shows only a low temperature specific defect, how many of the RNA metabolic defects are due to the sole function of RNase R, and how many of them are due to a defect in the activity of degradosomal complex in the bacterium.

	FOOTNOTES

 
^* This work was supported by the Volkswagen Foundation, by the Council of Scientific and Industrial Research (to M. K. R.), by a Council of Scientific and Industrial Research senior research fellowship (to R. I. P.), and by a Department of Biotechnology, Government of India postdoctoral fellowship (to M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and supplemental Figs. S1–S3.

¹ To whom correspondence should be addressed: Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India. Tel.: 91-4027192512; Fax: 91-4017160591; E-mail: malay{at}ccmb.res.in.

² The abbreviations used are: PNPase, polynucleotide phosphorylase; CFU, colony forming unit; PI, propidium iodide; rRNA, ribosomal RNA; ABM, Antarctic bacterial medium; RT, reverse transcription; tmRNA, transfer-messenger RNA.

³ R. I. Purusharth and M. K. Ray, unpublished results.

⁴ B. N. Sahu and M. K. Ray, unpublished observations.

	ACKNOWLEDGMENTS

 
We thank Prof. G. Klug and E. Evguenieva-Hackenberg for interest in the work and reading the manuscript and Prof. D. Schlessinger for sending the important reprints of works on rRNA processing, including an excellent review on the topic.

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