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• W2083715812 abstract "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. 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. 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.Py B. Higgins C.F. Krisch H.M. Carpousis A.J. Nature. 1996; 381: 169-172Crossref PubMed Scopus (473) Google Scholar, 2.Vanzo N.F. Li Y.S. Py B. Blum E. Higgins C.F. Raynal L.C. Krisch H.M. Carpousis A.J. Genes Dev. 1998; 12: 2770-2781Crossref PubMed Scopus (273) Google Scholar, 3.Liou G.G. Jane W.N. Cohen S.N. Lin N.S. Lin-Chao S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 63-68Crossref PubMed Scopus (144) Google Scholar). The degradosome has been shown to be involved in both mRNA and rRNA degradation (4.Bessarab D.A. Kaberdin V.R. Wei C.L. Liou G.G. Lin-Chao S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3157-3161Crossref PubMed Scopus (60) Google Scholar, 5.Khemici V. Carpousis A.J. Mol. Microbiol. 2004; 51: 777-790Crossref PubMed Scopus (134) Google Scholar). In the Escherichia coli degradosome, RNase E (a 5′ end-dependent endoribonuclease) associates with polynucleotide phosphorylase (PNPase, 2The 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.2The 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. 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.Rauhut R. Klug G. FEMS Microbiol. Rev. 1999; 23: 353-370Crossref PubMed Google Scholar). A similar kind of RNA degrading complex has also been reported from Rhodobacter capsulatus (7.Jager S. Fuhrmann O. Heck C. Hebermehl M. Schiltz E. Rauhut R. Klug G. Nucleic Acids Res. 2001; 29: 4581-4588Crossref PubMed Scopus (84) Google Scholar). 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.Purusharth R.I. Klein F. Sulthana S. Jager S. Jagannadham M.V. Evguenieva-Hackenberg E. Ray M.K. Klug G. J. Biol. Chem. 2005; 280: 14572-14578Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). This bacterium does have a pnp gene that is expressed but does not form a part of this complex. 3R. I. Purusharth and M. K. Ray, unpublished results.3R. I. Purusharth and M. K. Ray, unpublished results. 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.Deutscher M.P. Li Z. Prog. Nucleic Acids Res. Mol. Biol. 2001; 66: 67-105Crossref PubMed Scopus (118) Google Scholar). 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.Deutscher M.P. J. Bacteriol. 1993; 175: 4577-4583Crossref PubMed Google Scholar). 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.Cheng Z.F. Zuo Y. Li Z. Rudd K.E. Deutscher M.P. J. Biol. Chem. 1998; 273: 14077-14080Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). 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.Cheng Z.F. Deutscher M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6388-6393Crossref PubMed Scopus (159) Google Scholar). Interestingly, PNPase was found important for growth at low temperature in E. coli and in the psychrotrophic Yersinia enterocolitica (13.Zangrossi S. Briani F. Ghisotti D. Regonesi M.E. Tortora P. Deho G. Mol. Microbiol. 2000; 36: 1470-1480Crossref PubMed Scopus (70) Google Scholar, 14.Goverde R.L. Veld J.H. Kusters J.G. Mooi F.R. Mol. Microbiol. 1998; 28: 555-569Crossref PubMed Scopus (76) Google Scholar), although the pnp mutants of P. putida were not cold-sensitive (15.Favaro R. Deho G. J. Bacteriol. 2003; 185: 5279-5286Crossref PubMed Scopus (18) Google Scholar). 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.Cairrao F. Cruz A. Mori H. Arraiano C.M. Mol. Microbiol. 2003; 50: 1349-1360Crossref PubMed Scopus (130) Google Scholar). Extensive study from Deutscher’s group (17.Cheng Z.F. Deutscher M.P. Mol. Cell. 2005; 17: 313-318Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) 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.Chen C. Deutscher M.P. J. Biol. Chem. 2005; 280: 34393-34396Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). 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.Purusharth R.I. Klein F. Sulthana S. Jager S. Jagannadham M.V. Evguenieva-Hackenberg E. Ray M.K. Klug G. J. Biol. Chem. 2005; 280: 14572-14578Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). 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 (rneC) 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. 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.Purusharth R.I. Klein F. Sulthana S. Jager S. Jagannadham M.V. Evguenieva-Hackenberg E. Ray M.K. Klug G. J. Biol. Chem. 2005; 280: 14572-14578Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). 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. 4B. N. Sahu and M. K. Ray, unpublished observations. 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.Regha K. Satapathy A.K. Ray M.K. Genetics. 2005; 170: 1473-1484Crossref PubMed Scopus (24) Google Scholar). Recombinant DNA Methods−General DNA recombinant techniques including isolation of genomic DNA, restriction analysis, PCR, ligation, and transformation etc. were performed as described (20.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 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 [α-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 2× and 0.1× 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.Purusharth R.I. Klein F. Sulthana S. Jager S. Jagannadham M.V. Evguenieva-Hackenberg E. Ray M.K. Klug G. J. Biol. Chem. 2005; 280: 14572-14578Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). 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.Regha K. Satapathy A.K. Ray M.K. Genetics. 2005; 170: 1473-1484Crossref PubMed Scopus (24) Google Scholar), 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.Hinds J. Mahenthiralingam E. Kempsell K.E. Duncan K. Stokes R.W. Parish T. Stoker N.G. Microbiology. 1999; 145: 519-527Crossref PubMed Scopus (100) Google Scholar), 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.Purusharth R.I. Klein F. Sulthana S. Jager S. Jagannadham M.V. Evguenieva-Hackenberg E. Ray M.K. Klug G. J. Biol. Chem. 2005; 280: 14572-14578Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). 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.Bidle K.A. Bartlett D.H. J. Bacteriol. 1999; 181: 2330-2337Crossref PubMed Google Scholar) 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.Ray M.K. Sitaramamma T. Kumar G.S. Kannan K. Shivaji S. Curr. Microbiol. 1999; 38: 143-150Crossref PubMed Scopus (7) Google Scholar), 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.Cheng Z.F. Deutscher M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6388-6393Crossref PubMed Scopus (159) Google Scholar) in Tris-acetate-EDTA buffer (20.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and transferred to Hybond N+ membrane by vacuum transfer using 5× salinated sodium citrate, 10 mm NaOH. The oligonucleotide probes (supplemental Table S1, p1–p4) used for Northern hybridization were 5′ end-labeled with [γ-32P]ATP using T4 polynucleotide kinase (New England Biolabs) following the manufacturer’s protocol. The 32P-labeled oligonucleotides were separated from unincorporated [γ-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. Circular RT-PCR for Mapping of 5′ and 3′ Ends of rRNA−The precise 5′ and 3′ ends of the 16 S rRNA and 16 S rRNA precursors were determined using circular RT-PCR method. Briefly, total RNA was circularized using T4 RNA ligase (New England Biolabs) in the presence of RNase inhibitor RNasin (Promega). Then reverse transcription reaction was performed with reverse transcriptase (Ominiscript RT kit; Qiagen) for 1 h at 42 °C as per the manufacturer’s instructions, in the presence of antisense primer corresponding to a region in the 5′ end of mature 16 S rRNA (primer 16 SRU1, same as p2 in Table S1). The resultant cDNAs spanning the junction of 5′- and 3′-ligated ends were then amplified by PCR using the 16 SRU1 and outwardly directed 16 SFU1 (5′-GGGGTGAAGTCGTAACAAGGTAGCCG-3′) corresponding to a region at the 3′ end of mature 16 S rRNA. The amplified PCR products were separated and visualized by ethidium bromide staining on 1.5% agarose gel. DNA sequence was determined either directly following the extraction and purification of PCR products from agarose gel or following their cloning in pMOS-Blue plasmid. Big-Dye™ terminatior cycle sequencing kit (ABI) was used for sequencing reaction. Western Analysis−Protein blotting and immunodetection techniques for Western analysis were as described in Ref. 8.Purusharth R.I. Klein F. Sulthana S. Jager S. Jagannadham M.V. Evguenieva-Hackenberg E. Ray M.K. Klug G. J. Biol. Chem. 2005; 280: 14572-14578Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar. 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.Purusharth R.I. Klein F. Sulthana S. Jager S. Jagannadham M.V. Evguenieva-Hackenberg E. Ray M.K. Klug G. J. Biol. Chem. 2005; 280: 14572-14578Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). 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.Regha K. Satapathy A.K. Ray M.K. Genetics. 2005; 170: 1473-1484Crossref PubMed Scopus (24) Google Scholar) by staining them with Syto9 and propidium iodide (PI) using LIVE/DEAD Baclight bacterial viability kit (Molecular Probes, Eugene, OR). Isolation and Separation of Ribosome−Ribosomes were isolated and separated on sucrose gradient in presence of 10 mM or 0.3 mm MgCl2 depending upon the experimental requirement as described earlier (23.Ray M.K. Sitaramamma T. Kumar G.S. Kannan K. Shivaji S. Curr. Microbiol. 1999; 38: 143-150Crossref PubMed Scopus (7) Google Scholar). Ribosomes were prepared either directly from 22 °C grown cells or following their shift to low temperatures for different periods. Just before harvesting, the cells were treated with chloramphenicol (100 μgml–1) for 10 min (24.Charollais J. Pflieger D. Vinh J. Dreyfus M. Iost I. Mol. Microbiol. 2003; 48: 1253-1265Crossref PubMed Scopus (170) Google Scholar). The cells were harvested and resuspended in ribosome buffer (10 mm Tris-Cl, pH 7.5, 30 mm NH4Cl, 10 mm MgCl2, and 6 mm β-mercaptoethanol) containing lysozyme (1 mg ml–1) and subjected to sonication. Cell debris was removed by centrifugation at 12,000 × g. Supernatant was then recentrifuged at 70,000 rpm in a Beckman table top ultracentrifuge (TLA 100.3 rotor). The ribosomal pellet was suspended in ribosome buffer, and 25 A260 unit equivalents of ribosomes were loaded onto 5–40% sucrose gradient for centrifugation at 35,000 rpm in a SW41 rotor (Beckman) for 2.5 h. Fractions of 500 μl each were collected from the gradient, and A260 was measured. A gradient maker (Haake-Buchler) was used for both preparation of the sucrose gradient and collection of the fractions from the tubes. When required, the pooled fractions of 30, 50, and 70 S, and polysomes were treated with Triton X-100 (1%) or with 200 μm poly(U) and pelleted by centrifugation before further analysis. 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 2× 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). 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.Regha K. Satapathy A.K. Ray M.K. Genetics. 2005; 170: 1473-1484Crossref PubMed Scopus (24) Google Scholar) 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 pnpts 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.Cheng Z.F. Deutscher M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6388-6393Crossref PubMed Scopus (159) Google Scholar). 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.Cheng Z.F. Deutscher M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6388-6393Crossref PubMed Scopus (159) Google Scholar). 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. RNase R Is Not Cold-inducible in P. syringae−Because the rnr mutant of P. syringae is cold-sensitive, we checked" @default.
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• W2083715812 date "2007-06-01" @default.
• W2083715812 modified "2023-09-30" @default.
• W2083715812 title "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" @default.
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