Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA

REPORT Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA JESÚS DE LA CRUZ 1,2 and AGUSTÍN VIOQUE 1 1Instituto de Bioquímica Vegetal y Fotosíntesis,Universidad de Sevilla-CSIC, Américo Vespucio s/n,E-41092 Sevilla,Spain ABSTRACT tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans -translation reaction that contributes to the recycling of stalled ribosomes at the 39end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperaturesensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells ( D ssrA ) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of D ssrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the D ssrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics. Keywords: antibiotics; ssrA ; Synechocystis ; tmRNA; translation INTRODUCTION Bacterial cells contain a panoply of stable nontranslated RNAs such as ribosomal and transfer RNAs (rRNAs and tRNAs;reviewed in Wassarman et al+, 1999)+Amongst these RNAs,the so-called tmRNA(also known as SsrAor 10Sa RNA) was one of the first to be identified in Escherichia coli (Jain et al+, 1982);however,its function has remained elusive for a decade The tmRNA is encoded by the ssrA gene,which is highlyconservedinalleubacteria(Knudsen etal+, 2001)+ In vivo,tmRNA is tightly associated to the SmpB protein and less tightly to other proteins (Karzai et al+, 1999;Karzai & Sauer,2001)+Different evidence indicates that tmRNA plays a role in translation (for a review,see Muto et al+, 1998;Karzai et al+, 2000)+tmRNA can form in vivo a tRNA half molecule consisting of a amino acid acceptor arm and a TCC stem-loop (Komine et al+, 1994)+The presence of this tRNA-like structure is further supported by the fact that the precursor of tmRNA(pre-tmRNA),as all pre-tRNAs,is 59-end processed by RNase P,and tmRNA is aminoacylated by alanyl-tRNA synthetase (Komine et al+, 1994)+In addition,thealanylated-tmRNAinteractswith elongation factor Tu (EF-Tu) and GTP forming a ternary complex (Rudinger-Thirion et al+, 1999) and is found associated with 70S ribosomes (Tadaki et al+, 1996)+Finally, tmRNAs have a short reading frame coding for about 11 amino acids (Knudsen et al+, 2001),which are added tothe C-terminusofan incompletenascent proteintranslated from truncated mRNAs (Keiler et al+, 1996)+ Based on some of these observations,Sauer and coworkers proposed the “ trans -translation” model for the biological function of tmRNA (Keiler et al+, 1996)+ Reprint requests to:Agustín Vioque,Instituto de Bioquímica Vegetal y Fotosíntesis,Universidad de Sevilla-CSIC,Centro de Investigaciones Científicas Isla de la Cartuja,Avda+Américo Vespucio s/n, E-41092 Sevilla,Spain;e-mail:vioque@us+es+ 2Present address:Departamento de Genética,Facultad de Biología,Universidad de Sevilla,Reina Mercedes 6,E-41012 Sevilla, Spain+ RNA (2001), 7 :1708–1716+Cambridge University Press+Printed in the USA+ Copyright © 2001 RNA Society+ DOI:10+1017+S1355838201011281 1708 Cold Spring Harbor Laboratory Press on March 24, 2023 - Published by rnajournal.cshlp.orgDownloaded from According to this model,alanine-charged tmRNAassociated with EF-Tu enters theAsite of a ribosome stalled at the 39end of a truncated mRNA lacking a stop codon+Then,the noncoded alanine is incorporated to the nascent polypeptide chain through a cycle of translationelongation,before the mRNA-like domain of tmRNA replaces the truncated mRNA on the ribosome+Then, this domain is used as a template to add a short peptide to the nascent polypeptide before translation terminates and a tagged protein is released (for details, see Keiler et al+, 1996;Muto et al+, 1998)+The short peptide acts as a recognition sequence that directs the protein to quick degradation by specific proteases (Gottesman et al+, 1998;Levchenko et al+, 2000)+Therefore,tmRNA has a double function:First,ribosomes that are stalled at truncated mRNAs lacking a termination codon are released+Second,the prematurely truncated proteins are rapidly degraded,avoiding accumulation+Both function have been separated genetically by engineering cells that carry a ssrA gene coding for an altered tag that is resistant to proteases,or alternatively,that cannot be aminoacylated (Keiler et al+, 1996;Withey & Friedman,1999;Huang et al+, 2000)+ From these studies,it seems clear that ribosome release and not protein tagging is the relevant function of tmRNA (Keiler et al+, 1996;Withey & Friedman,1999; Huang et al+, 2000)+In addition,it has been demonstrated that tmRNA-mediated trans -translation occurs quite frequently in normally growing cells and not only at truncated mRNAs lacking a termination codon (Abo et al+, 2000)+Tagging by tmRNA occurs at rare codons in an mRNA or even at the natural termination codon (Roche & Sauer,1999,2001)+This suggests that ribosomepausingor stalling byanumberof differentcauses can activate tmRNA function+However,several questions remain unsolved:How does tmRNA recognize stalled ribosomes,what is the mechanism of incorporation of the noncoded alanine,and how does the ribosome resume in-frame translation at the mRNA-like domain of tmRNA? Moreover,despite its importantbiological function and its ubiquitous presence in the eubacterial kingdom,the precise physiological role of tmRNA still remains elusive,as it is,in general,dispensable for cell viability and it has been proved to be essential only in Neisseria gonorrhoeae and Mycoplasma (Hutchison et al+, 1999; Huang et al+, 2000)+However,cells lacking a functional tmRNA exhibit a variety of subtle phenotypes such as slow growth at high temperature or under different stresses,reduced motility,inhibition of phage growth, an increase in the activity of an unidentified protease, enhanced activity of several repressors or reduced pathogenesis (Karzai et al+, 2000;Muto et al+, 2000; and references therein)+Interestingly,Sugita and coworkers could not inactivate the ssrA gene in the cyanobacterium Synechococcus sp+strain PCC6301, suggesting an essential function for this gene (Watanabe et al+, 1998)+For this reason,we undertook the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp+strain PCC6803,in the context of an ongoing project on the analysis of stable RNAs from this cyanobacterium+Herein,we report that Synechocystis cellsdisruptedfor the ssrA gene (D ssrA cells) show no growth defect phenotype under normal laboratory conditions+However,aD ssrA strain is not viable when cultivated at low concentrations of several protein synthesis inhibitors+In addition,global protein synthesis is impaired at low concentration of chloramphenicol in a D ssrA strain but not in a wild-type strain+We conclude that tmRNA has a role in recycling stalled ribosomes when they are blocked by those protein synthesis inhibitors+We assume that the first elongation cycle that incorporates the noncoded alanine to nascent polypeptide chains is resistant to the bound antibiotic+ RESULTS AND DISCUSSION tmRNA is dispensable for growth To study the cellular function of Synechocystis tmRNA, we first disrupted the ssrA gene by replacing most of the gene sequence with a kanamycin resistance (Km r ) cassette (Fig+1A)+Although the ssrA gene is not part of a polycistronic unit (Kaneko et al+, 1996),the cassette was inserted in both orientations to control for hypothetical polar effects+The disrupted copy of ssrA could be completely segregated as tested by southern blot (data not shown)+Therefore,cells lacking a functional ssrA gene seem to be viable+ To further confirm the nonessential role of tmRNA, we determined its steady-state levels by northern blot and primer extension analyses in wild-type and D ssrA strains+As seen in Figure 1B,C,disruption of the ssrA gene resulted in the loss of mature tmRNA+In addition, we detected an additional band in the wild-type strain by primer extension analysis (Fig+1C,lane 1),which we attribute to the precursor of tmRNA (Tous et al+, 2001)+Altogether,these experiments indicate that ssrA is a dispensable gene in Synechocystis ,in disagreement with the data from Sugita and coworkers for Synechococcus (Watanabe et al+, 1998)+ Disruption of ssrA causes no apparent growth defect To understand the nonessential role of tmRNA,the growth rate of the D ssrA strain was compared to that of the wild-type strain+As seen in Figure 2A,when cells were cultivated in nitrate containing BG-11 medium at 308C (see Materials and Methods),the growth rates were identical for wild-type and D ssrA strains+In addition,D ssrA cells were as motile as wild-type cells and we could not detect any morphological difference when tmRNA and translation inhibitors 1709 Cold Spring Harbor Laboratory Press on March 24, 2023 - Published by rnajournal.cshlp.orgDownloaded from cells were observed under the light microscope (data not shown)+We conclude that Synechocystis tmRNAis totally dispensable under normal laboratory growth conditions+ tmRNA requirement under several stresses: Sensitivity to protein synthesis inhibitors To estimate differences in growth rates under different stressfulconditions,serialdilutions of cultures fromwildtype and D ssrA cells were spotted on BG11 medium plates,and plates were incubated at 308C (see Materials and Methods)+Stressful conditions were:exposure to UV irradiation (2,000 KJ{s 21 )for2to10min, DTT (250 mM),2-mercaptoethanol (5 mM),treatment at 258C,378C,and 458C for 30 min+In all conditions, no differences in the survival or the growth rates of the wild-typeand D ssrA strainswere found(data notshown)+ Because tmRNA plays a role on translation (see Introduction),we also measured growth rates of both strains in the presence of low concentrations of antibiotics affecting protein synthesis+As seen in Table 1 and Figure 2B,the D ssrA strains do not grow in the presence of sublethal concentrations for the wild-type strain of chloramphenicol,erythromycin,lincomycin, spectinomycin,spiramycin,and tylosin+These data indicate that the survival rate of the wild-type strain is 10to 1000-fold higher than that of the D ssrA strains (Fig+2B)+Interestingly,all these drugs except spectinomycin have been described to bind to the peptidyl transferase center,which has been localized within domain V of the 23S rRNA (Gale et al+, 1981; Rodríguez-Fonseca et al+, 1995;Nissen et al+, 2000; Poulsen et al+, 2000)+Furthermore,all these drugs except erythromycin and spectinomycin inhibit the peptidyl transferase reaction (Gale et al+, 1981)+ReFIGURE 1. Disruption of the Synechocystis ssrA gene+A: Schematic representation of the Synechocystis genomic region where the ssrA gene is located+A Hin cII Kmrcassette (1+3 kb) containing the neomycin phosphotransferase gene ( npt ) was inserted between the blunt-ended Hin dIII and Cla I sites in both orientations (D ssrA-1 and D ssrA-2 )+B: Northern blot analysis;total RNA was extracted from wild-type and null ssrA (D ssrA-1 and D ssrA-2 ) cells and hybridyzed with a Sma INco I fragment containing the ssrA gene (upper panel) or a rnpB gene probe (bottom panel;Vioque,1992)+C: Primer extension using the oligonucleotide 10SAPE (see Materials and Methods) on total RNA extracted from wild-type and null ssrA (D ssrA-1 and D ssrA-2 ) cells+Arrows points to the extension products identifying the putative transcription start site (pre-tmRNA) and the 59end of mature tmRNA (tmRNA) only in wild-type cells+For the primer extension shown in lanes 3 and 4,a 10-fold molar excess of cold primer was mixed with the radiolabeled primer before annealing+Asequencing reaction on the wild-type ssrA gene using the same primer is also shown+ 1710 J. de la Cruz and A. Vioque Cold Spring Harbor Laboratory Press on March 24, 2023 - Published by rnajournal.cshlp.orgDownloaded from cently,it has been shown that erythromycin binds to the polypeptide tunnel,suggesting a simple sterichindrance mechanism of inhibition of elongation (Gabashvili et al+, 2001)+Spectinomycin also inhibits translocation,but it binds to specific sites on 16S rRNA (Bilgin et al+, 1990;Carter et al+, 2000)+ FIGURE 2. tmRNAis required for survival in the presence of protein synthesis inhibitors+A: Growth comparison of wild-type and D ssrA strains+Cultures were inoculated at an initial concentration of 0+5mg{mL21chlorophyll+Cells were grown on nitrate containing BG11 medium for up to 12 days and growth rate was measured as the concentration of chlorophyll per milliliter of culture+Wild-type (d),D ssrA-1 (n) and D ssrA-2 (,) strains+B: Cells from wild-type (wt),null ssrA (D ssrA-1 and D ssrA-2 ) and null crtO (D crtO ) strains were grown to 3 OD750 units+Serial dilutions (dilution factor 1 to 103) were spotted onto nitrate containing BG11 medium plates at the indicated concentrations of antibiotics+Plates were incubated as described in Materials and Methods for 7 days+Control:no antibiotic added;Cm:chloramphenicol;Er:erythromycin;Lc: lincomycin;Sp:spectinomycin;Sy:spiramycin;Ty:tylosin+ tmRNA and translation inhibitors 1711 Cold Spring Harbor Laboratory Press on March 24, 2023 - Published by rnajournal.cshlp.orgDownloaded from As also shown in Table 1,antibiotics affecting translation initiation or EF-Tu and EF-G-mediated steps during translocation (except erythromycin and spectinomycin) do not differentially affect the D ssrA strain as compared to the wild-type strain+In addition,the D ssrA strains grow better than the wild-type strain in the presence of any concentration of kanamycin,neomycin,or streptomycin+This growth advantage is due to the function of the neomycin phosphotransferase gene present in the Km r cassette introduced during the transformation to the D ssrA cells+This is demonstrated by the control D crtO strain (Fernández-González et al+, 1997), which is a Km r mutant that preserves a functional ssrA gene+As expected,the D crtO strain showed also a growthadvantageonplatescontainingkanamycin,neomycin,or streptomycin (data not shown)+Finally,D ssrA and wild-type cells are equally affected by other antibiotics that do not interfere with protein synthesis such as ampicillin or rifampicin (Table 1)+We conclude that tmRNA is essential for growth in the presence of low concentrations of chloramphenicol,lincomycin,spiramycin,tylosin,erythromycin,and spectinomycin+ Protein synthesis is impaired in D ssrA cells pretreated with a low dose of chloramphenicol To better define the altered resistance of D ssrA cells to the drugs mentioned in the previous section,we analyzed the wild-type and D ssrA strains for protein synthesis rate by in vivo incorporation of [ 35 S]methionine (see Materials and Methods)+As shown in Figure 3A, when no antibiotic was added,the relative amount of [ 35 S]methionine incorporated into proteins was reproducibly similar in both wild-type and D ssrA strains+ However,when cells weretreatedwith 1 mg{mL 21 chloramphenicol for 30 min,more than a 50% reduction in [ 35 S]methionine incorporation into proteins was observed in D ssrA cells when compared to the wild-type strain (Fig+3A)+Furthermore,we could not detect any significative effect on the [ 35 S]methionine uptake (data not shown)+To investigate if the translation rate of a set of proteins is more affected than other ones by chloramphenicol,we analyzed SDS/PAGE profiles of 35 Slabeled proteins from both wild-type and D ssrA cells treated or not with the drug+As shown in Figure 3B,no differenceswerefoundalong the profiles (compare lines 1 and 3 with 2 and 4)+Altogether,these results indicate that tmRNA is required for optimal protein synthesis in the presence of sublethal concentrations of chloramphenicol+This requirement is not specific for a set of proteins,therefore strongly suggesting that global impairment of protein synthesis is the primary cause of the lethality of the D ssrA strains when grown in the presence of low concentrations of protein synthesis inhibitors+ CONCLUSIONS In this work,we have demonstrated that tmRNA is essential for survival in the presence of sublethal concentrations of several protein synthesis inhibitors+In these conditions,only a fraction of ribosomes would bind the antibiotic and becomes blocked on a mRNA+ Assuming that these blocked ribosomes can recruit charged tmRNA and moreover,assuming that the first elongation cycle that incorporates the noncoded alanine is resistant to the bound antibiotic,which must be displaced,then ribosomes have the opportunity to be released+This ability is not present in D ssrA cells;therefore,blocked ribosomes accumulate,translation is impaired,and cells do not grow+ tmRNA-mediatedtagginghasbeen described in three circumstances:(1) when ribosomes reach the end of a truncated mRNA (Keiler et al+, 1996),(2) when ribosomes stall at rare codons (Roche & Sauer,1999),and (3) at natural stop codons (Roche & Sauer,2001)+ In these cases,the ribosomal A-site is empty+Our results provide evidence that ribosomes stalled by some protein synthesis inhibitors can also be a substrate of tmRNA+However,in the presence of peptidyl transferase inhibitors,the ribosomalA-site can be occupied by the correct aminoacyl-tRNAor the tmRNA+In the presence of erythromycin and spectinomycin,the action of tmRNAon stalled ribosomes is more difficult to explain because peptidyl-tRNA is in the ribosomal A-site+In TABLE 1+Effect of different antibiotics on the growth rate of wildtype and D ssrA cells from Synechocystis sp+PCC6803+ Inhibitor Function affected Growth difference a Ampicillin Cell wall synthesis None Rifampicin Transcription None Kasugamycin Initiation (fMet-tRNA binding) None Kanamycin Translation accuracy 1 Neomycin Translation accuracy 1 Streptomycin Translation accuracy 1 Tetracycline aa-tRNA{EF-Tu{GTP binding None Chloramphenicol Peptide bond formation 2 Lincomycin Peptide bond formation 2 Spiramycin Peptide bond formation 2 Tylosin Peptide bond formation 2 Erythromycin Elongation 2 Spectinomycin Translocation (EF-G{GTP interaction) 2 Fusidic acid Translocation (EF-G{GDP release) None Thiostrepton Translocation (EF-G{GDP turnover) None Puromycin Elongation None Relative growth rate was compared by dotting serial dilutions of exponentially growing cells on plates containing the indicated antibiotics at sublethal concentrations for the wild-type strain (see Materials and Methods)+ a None:similar growth of wild-type and D ssrA strains;1:better growth of the D ssrA strain;2:better growth of the wild-type strain+Note that the better growth of the D ssrA strains in kanamycin,neomycin,and streptomycin is not related to the loss of tmRNA function but to the introduced Km r cassette following transformation+ 1712 J. de la Cruz and A. Vioque Cold Spring Harbor Laboratory Press on March 24, 2023 - Published by rnajournal.cshlp.orgDownloaded from any case,our data strongly suggest that tmRNA participates in a productive elongation cycle in the presence of any of the above mentioned inhibitors,most likely by displacing the antibiotic+Protein synthesis antibiotics had been and still are extremely useful tools in the dissection of the complex mechanisms of translation+Further in vivo and in vitro experiments using these antibiotics may be very useful to elucidate the mechanism of tmRNA-mediated trans -translation+ Finally,there is a great interest in the development of newtoolsagainstpathogenicmicroorganisms+Although tmRNA is not essential for growth,our results indicate thatinhibition of tmRNAactivitycould increasethesensitivity to translation antibiotics of clinical relevance+If our results can be generalized for pathogenic bacteria, tmRNA could be an attractive target for the development of new specific drugs,as tmRNAis not present in animal cells+Thus,a combined delivery of a tmRNAinhibitorandadocumentedantibioticatalowerdosewould be more effective against pathogenic microorganisms+ MATERIALS AND METHODS Strains, media, and growth conditions Synechocystis sp+strain PCC6803 was grown in nitrate containing BG-11 medium (Rippka et al+, 1979)+For plates,the medium was solidified with 1% (w/v),separately autoclaved agar (Difco)+Cultures were grown at 308C in the light (50– 100 meinsteins{m 22 {s 21 ;white light fluorescent lamps),with shaking (80–100 rpm) for liquid cultures+Growth was monitored by OD 750 or chlorophyll content that was determined as FIGURE 3. Protein synthesis is impaired in D ssrA cells+A: Protein synthesis rates in wild-type (wt) and D ssrA cells+ Cultures (3 OD750 units) were incubated in the absence or presence of chloramphenicol (1 mg{mL21) for 30 min+[35S]methionine was added either immediately (0 min,upper panel) or 30 min after the addition of chloramphenicol (30 min,upper panel)+Incubation with [35S]methionine was for 15 min+The radioactivity present in the TCA-insoluble material was counted and divided by that taken up into cells+The translation rate for each strain is the average of at least three independent experiments and the standard deviations are given as bars+B: Detection of 35S-labeled proteins in wild-type and D ssrA cells+ Cultures (3 OD750 units) were incubated in the absence or presence of chloramphenicol (1 mg{mL21) for 30 min and then with [35S]methionine for 15 min+One OD750 unit was lysed by heating at 608C in Laemmli buffer+Equal amounts of radioactivity (20,000 cpm) were subjected to SDS/PAGE and 35S-labeled proteins were detected by autoradiography+Size markers are indicated on the left+ tmRNA and translation inhibitors 1713 Cold Spring Harbor Laboratory Press on March 24, 2023 - Published by rnajournal.cshlp.orgDownloaded from previouslydescribed(Mackinney,1941)+Antibiotic plates were prepared by adding the drugs from stock solutions into the medium before pouring the plates+Antibiotics were used at a range of concentrations between 1 ng{mL 21 and the concentration at which wild-type cells die+The following antibiotics were used:ampicillin (sodium salt),chloramphenicol, erythromycin,fusidic acid (sodium salt),kanamycin sulfate, kasugamycin,lincomycin hydrochloride,neomycin,puromycindihydrochloride,rifampicin,spectinomycindihydrochloride, spiramycin,streptomycin sulfate,tetracycline hydrochloride, thiostrepton,and tylosin tartrate+All antibiotics except kasugamycin were of analytical grade+Kasugamycin was a generous gift from Dr+A+Jiménez+ Synechocystis D crtO strain is a Km r disruptant of the b-carotene ketolase gene (FernándezGonzález et al+, 1997) . Escherichia coli DH5awas used for recombinant DNAtechniques and grown in Luria–Bertani medium with or without an appropriate antibiotic+ DNA manipulation All recombinant DNAexperiments were performed according to standard procedures (Sambrook et al+, 1989)+Southern blotting was done as previously described (Sambrook et al+, 1989)+To clone the ssrA gene,two primers,based on the published coordinates from the Synechocystis sp+PCC6803 chromosome sequence (Kaneko et al+, 1996),were synthesized:10SAF,59-CGCTTGTGGATCCTGTCCCAG-39(a Bam HI site is underlined) and 10SAR,59-TAACCGCTCGAG TAAAGTACTGTTACTGG-39(chromosome homology region is in bold type and a Xho I site is underlined)+PCRs were performed by a standard procedure (Sambrook et al+, 1989)+ The PCR product was cloned as a 1+5-kb Bam HIXho I fragmentintopBluescriptKS(1)(Stratagene)digested with Bam HI and Xho I to generate pBS-ssrA+ Inactivation of ssrA in Synechocystis cells To inactivate the ssrA gene,pBS-ssrA was digested with Hin dIII and Cla I,then treated with Klenow DNA polymerase and dephosphorylated+The resulting blunt-ended open plasmid lacking most of the ssrA gene sequence was ligated to a 1+3-kb Hin cII fragment containing a Km r cassette (C+K1) from Tn5 (Elhai & Wolk,1988)+Two plasmids containing the cassette in different orientation (pBS-D ssrA-1 and pBS-D ssrA-2 , respectively) were chosen for Synechocystis transformation+ Cells were transformed using the method from Chauvat et al+ (1986),except that cells were spread out onto nitrocellulose filters (Nucleopore REC-85,Whatman)+Km r transformants were selected on 50 mg{mL 21 Km-containing BG-11 plates+ To facilitate segregation of the mutant chromosomes,the Km r transformants were grown for three rounds on 100,200,and 300 mg{mL 21 Km-containing BG-11 plates+Correct integration and total segregation of the mutant strains were checked by southern blot analysis+Two segregants harboring the Km r cassette in different orientations (D ssrA-1 and D ssrA-2 ) were chosen for further experiments+ RNA analyses Total Synechocystis RNAwas isolated from liquid cultures as previously described (Navarro & Florencio,1996)+Denatured RNA(5 mg) wassizefractionatedon7%(w/v)polyacrylamide8 M urea gels+RNA was transferred to and immobilized on Hybond N 1 nylon membranes (Amersham-Pharmacia) as previously described (Kressler et al+, 1997)+Probes were labeled with a DNA labeling kit (Ready to Go,AmershamPharmacia)+Prehybridization and hybridization were done as previously described (Kressler et al+, 1997)+ Primer extension was done on the same RNA samples that were used for northern analysis according to GarcíaDomínguez et al+(2000)+Oligonucleotide 10SAPE (59-CTA GGCTGCTATGGCTACC-39) was used as a primer+To identify the positions of the primer extension stop,plasmid-borne ssrA was sequenced with the same primer+AMV reverse transcriptaseandRNAguardwere purchased fromAmershamPharmacia+ Chloramphenicol inhibition of protein synthesis A modified procedure from Xu et al+(2000) was used to determine protein synthesis rates;2mL of chloramphenicol dissolved in ethanol (1 mg{mL 21 final concentration) or 2 mLof ethanol were added to 1-mL cultures of Synechocystis wildtype and D ssrA-1 strain (3 OD 750 units) and incubated for different times under the growth conditions described above+ Labeling was done using 10 mCi L-[ 35 S]methionine (1,000 Ci/mmol) for 15 min+Then,half of each sample was added to 0+5 mL ice-cold BG11 medium plus 1 mg{mL 21 methionine, filtered onto a Whatman GF/C paper,and washed three times with 5 mL BG11 medium;the filter was finally immersed in scintillation cocktail (5uptake sample)+The remaining half of each sample was incubated with TCA and SDS [final concentrations:10% (w/v) TCA,1% (w/v) SDS] for1hat48C, then filtered onto Whatman GF/C paper and washed three times with 5 mL 10% (w/v) TCA,followed by an additional wash with 5 mL acetone+The filter was finally immersed in scintillation cocktail (5incorporation sample)+The radioactivity of the samples was measured by counting and quench corrected+The protein synthesis rate was expressed as the incorporationdividedbythe uptake tocompensateforchanges in label uptake among the samples+ Protein analyses ProteinconcentrationwasdeterminedbytheBradfordmethod using ovoalbumin as a standard (Bradford,1976)+Protein cell extracts were prepared by adding Laemmli buffer to 3 OD 750 units of cells and heating for 15 min at 608C (Ausubel et al+, 1994)+In vivo protein labeling experiments were performed as described above using cultures of Synechocystis wildtype and D ssrA-1 strains+At3OD 750 units,the cultures were divided in two halves;one was incubated for at least 10 min with 2 mLof chloramphenicol dissolved in ethanol (1 mg{mL 21 final concentration) and the other was treated with 2 mL ethanol for the same period of time+Then,1 mLof each half was incubated with 10 mCi L-[ 35 S]methionine (1,000 Ci/mmol) for 15 min+Cells were washed twice by centrifugation with 1 mL of BG11 medium supplemented with 1 mg{mL 21 methionine and resuspended in 50 mL of Laemmli buffer+Labeled proteins were analyzed by SDS/PAGE (equal amounts of radioactivity was loaded in each well) followed by autoradiography+ 1714 J. de la Cruz and A. Vioque Cold Spring Harbor Laboratory Press on March 24, 2023 - Published by rnajournal.cshlp.orgDownloaded from ACKNOWLEDGMENTS We thanks Dr+A+Jiménez (CBM,Madrid) for providing kasugamycin,L+López-Maury for help during the preparation of Figure 2,Dr+M+A+Vega-Palas for fruitful discussions,and Dr+ D+Kressler(Biozentrum,Basel)forcriticalreading of themanuscript+J+d+l+C+acknowledges financial support from the Spanish Government (Contrato de Reincorporación,Ministerio de Educación y Cultura)+This work was supported by grants from the Human Frontier Science Organization (RG291/ 1997),Dirección General de Enseñanza Superior (PB970732),and Junta de Andalucía (CVI215)+ Received July 19, 2001; returned for revision August 5, 2001; revised manuscript received September 21, 2001 REFERENCES Abo T,Inada T,Ogawa K,Aiba H+2000+SsrA-mediated tagging and proteolysis of LacI and its role in the regulation of lac operon+ EMBO J 19 :3762–3769+ Ausubel FM,Brent R,Kingston RE,Moore DD,Seidman JG,Smith 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