Deep sequencing analysis of the mazei Go¨1 transcriptome in response to nitrogen availability

Dominik Ja¨ gera,1, Cynthia M. Sharmab,1, Jens Thomsena, Claudia Ehlersa,Jo¨ rg Vogelb, and Ruth A. Schmitza,2

aInstitut fu¨r Allgemeine Mikrobiologie, Christian-Albrechts-Universita¨t Kiel, 24118 Kiel, Germany; and bRNA Biology Group, Max-Planck-Institut fu¨r Infektionsbiologie, D-10117 Berlin, Germany

Edited by Norman R. Pace, University of Colorado, Boulder, CO, and approved October 8, 2009 (received for review August 10, 2009) Methanosarcina mazei and related mesophilic are the only Besides transcriptional regulation, small regulatory RNAs organisms fermenting , , and to (sRNAs) have increasingly been implicated in a variety of and , contributing significantly to green- adaptive cellular responses to biotic and abiotic stresses and house gas production. The biochemistry of these metabolic pro- development in bacteria (9, 10). In comparison, there are few cesses is well studied, and sequences are available, yet studies on the role of sRNAs in archaea. Computational and little is known about the overall transcriptional organization and experimental analysis discovered C/D-box and H/ACA-box the noncoding regions representing 25% of the 4.01-Mb genome small nucleolar RNAs (snoRNAs) with predicted functions in of M. mazei. We present a genome-wide analysis of transcription ribosomal RNA maturation in Sulfolobus sulfataricus, S. acido- start sites (TSS) in M. mazei grown under different nitrogen caldarius, Archaeaglobus fulgidus, and Pyrococcus (11–14), as well availabilities. Pyrosequencing-based differential analysis of pri- as stable antisense RNAs and sRNA from intergenic -mary vs. processed 5؅ ends of transcripts discovered 876 TSS across regions (IGRs), which are likely to be involved in posttranscrip the M. mazei genome. Unlike in other archaea, in which leaderless tional control of gene expression (15–18). More recently, several mRNAs are prevalent, the majority of the detected mRNAs in M. dozen sRNAs were predicted in the high salt-adapted species .(mazei carry long untranslated 5؅ regions. Our experimental data Haloferax volcanii (19 predict a total of 208 small RNA (sRNA) candidates, mostly from Although all previous experimental approaches in archaea MICROBIOLOGY -intergenic regions but also antisense to 5؅ and 3؅ regions of mRNAs. relied on cDNA cloning followed by Sanger sequencing, high In addition, 40 new small mRNAs with ORFs of <30 aa were throughput sequencing of cDNA [RNA-seq (20)] has now identified, some of which might have dual functions as mRNA and revolutionized transcriptome analysis and sRNA discovery in regulatory sRNA. We confirmed differential expression of several many organisms (21–23). In the present work we used a recently sRNA genes in response to nitrogen availability. Inspection of their developed differential RNA-seq method selective for newly promoter regions revealed a unique conserved sequence motif initiated transcripts. We present a genome-wide map of tran- associated with nitrogen-responsive regulation, which might serve scriptional start sites (TSS) and sRNA output in M. mazei and as a regulator binding site upstream of the common IIB recognition provide insight into transcriptional regulation in response to element. Strikingly, several sRNAs antisense to mRNAs encoding nitrogen in this organism. transposases indicate nitrogen-dependent transposition events. This global TSS map in archaea will facilitate a better understand- Results ing of transcriptional and posttranscriptional control in the third We analyzed cDNA libraries derived from M. mazei cultures domain of life. growing under different nitrogen availabilities [i.e., from cells ϩ growing exponentially under nitrogen sufficiency (NH4 ;NS methanoarchaea ͉ noncoding RNAs ͉ transcription ͉ transposition ͉ libraries) or under nitrogen-fixing conditions with molecular long 5’UTRs nitrogen as the sole nitrogen source (N2; NF libraries)]. For each of the 2 conditions, 2 differential cDNA libraries were con- Methanosarcina mazei strain Go¨1 is a representative methane- structed. The NS(Ϫ) and NF(Ϫ) libraries were generated from producing archaeon of ecologic significance because of its role the original RNA pool and covered both primary (carrying 5Ј in biogenic methane production in various anaerobic habitats on triphosphate group) and processed (5Ј monophosphate) tran- Earth (1). The genome sequences of M. mazei and its close scripts; whereas in the cognate NS(ϩ) and NF(ϩ) libraries, relatives Methanosarcina acetivorans and newly initiated transcripts with primary 5Ј end were enriched by have recently become available and have revealed an unexpected enzymatic treatment. After library construction, Ϸ185,000 low proportion of coding region (74.2% in M. acetivorans, cDNAs from ammonium conditions (NS libraries) and Ϸ43,000 75.15% in M. mazei, and 79.2% in M. barkeri) (2–4). The cDNAs from nitrogen-fixing conditions (NF libraries) were biochemical basis of has been analyzed in sequenced. The obtained cDNA reads varied in length from 1 to considerable detail (5, 6). In contrast, little is known about global 280 bp, and disregarding cDNAs Ͻ18 bp, between 85% and 94% regulatory networks that ensure survival in periods of nutrient starvation or stress in this important group of archaea. More Author contributions: J.V. and R.A.S. designed research; D.J., C.M.S., and C.E. performed than 50 predicted transcriptional regulators were annotated in research; J.V. contributed new reagents/analytic tools; D.J., C.M.S., J.T., and R.A.S. analyzed the genome of M. mazei. Strikingly, most of them seem to be data; and R.A.S. wrote the paper. closely related to bacterial proteins (2), whereas the basic The authors declare no conflict of interest. components of the archaeal transcription and translation ma- This article is a PNAS Direct Submission. chineries generally are more similar to those of eukaryotes (7). 1D.J. and C.M.S. contributed equally to this work. A recent genetic study (8) discovered the first global transcrip- 2To whom correspondence should be addressed at: Institut fu¨r Allgemeine Mikrobiologie, tional regulator of M. mazei, the nitrogen regulator NrpR, which Universita¨t Kiel, Am Botanischen Garten 1–9, 24118 Kiel, Germany. E-mail: was experimentally demonstrated to globally repress transcrip- [email protected]. tion of nitrogen fixation and assimilation genes in response to the This article contains supporting information online at www.pnas.org/cgi/content/full/ nitrogen source. 0909051106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909051106 PNAS Early Edition ͉ 1of5 Downloaded by guest on September 27, 2021 Fig. 1. Visualization of the clone distribution of cDNAs mapped to the chromosomal glnA1 region. An extraction of the screenshot of the Integrated Genome Browser (Affymetrix) of the mapped cDNAs is shown for nitrogen fixation (NFϩ) and ammonium conditions (NSϩ)oftheM. mazei glnA1 region. The y axis indicates the relative score of clone numbers per nucleotide. The nitrogen-regulated TSS and a potential second TSS of glnA1 are indicated. The respective Northern blot analysis is shown (Right). Fig. 2. Transcription of spRNA09 and sRNA60.(A) Chromosomal localization; (B) cDNA clone distribution for nitrogen fixation (NFϩ) and ammonium ϩ of them were unequivocally mapped to the M. mazei genome. conditions (NS ); (C) confirmatory Northern blot analysis; the calculated fold induction (NF vs. NS) is given below the blot. The number of cDNA hits for each nucleotide position for both strands were calculated and the data visualized using the Inte- grated Genome Browser (Affymetrix). Of the cDNAs, 26.4% detected under nitrogen sufficiency, whereas 99 were solely (NF) and 31.6% (NS) mapped to intergenic regions (IGRs) of present under nitrogen-fixing conditions, indicating differential ϩ the genome in the enriched ( ) libraries, as compared with 21% transcription in response to nitrogen (Table S5). Ϫ and 28%, respectively, in the cognate nonenriched ( ) libraries, In addition, 40 sRNAs candidates located in IGRs contain indicating depletion of processed rRNAs and tRNAs in favor of very short ORFs potentially encoding peptides Ͻ30 aa, 15 of IGR transcripts. Furthermore, a strong enrichment of cDNAs which are preceded by a consensus ribosome binding site. A high clustering toward the TSS of several mRNA genes analyzed in number of these small ORFs are conserved in other Methano- previous studies was observed [e.g., of grpE and glnA1 (24, 25)]; sarcina strains (Table S6), and several exist in multiple homo- Ј the 5 ends of the grpE and glnA1 cDNA clusters exactly match logues in the M. mazei genome. Most of the associated peptides the previously identified TSS (Table S1). Fig. 1 illustrates TSS Ͼ20 aa contained a transmembrane helix, indicating membrane identification and nitrogen source-dependent transcription reg- association. For 16 candidates the flanking RNA region up- ulation of glnA1 (encoding glutamine synthetase). Nitrogen- stream of the ORF also showed high conservation within the responsive regulation of glnA1 was previously shown by quanti- Methanosarcina strains, suggesting a potential dual function as tative RT-PCR analysis (25) and is verified here by Northern blot both mRNA and RNA regulator. Interestingly, 7 of the short probing (Fig. 1). A second, constitutive TSS associated with ORF loci were associated with a cis-encoded antisense RNA glnA1 was found upstream of the nitrogen-regulated TSS. Over- overlapping the 5Ј (n ϭ 5) or 3Ј (n ϭ 2) ORF region (Fig. 2). all, 876 potential TSS were identified, confirming transcription For a randomly chosen Ϸ400-kb fragment of the chromosome (coverage of Ͼ5 cDNAs) for 586 of the originally annotated (nt 11,700–407,000) all IGR transcripts identified by the pyro- 3,371 ORFs. Inspection of cDNAs from noncoding regions sequencing approach were subjected to confirmatory Northern identified 42 new ORFs encoding small proteins (30–99 aa) blot analysis. Of 36 predicted transcripts (sRNAs and new (Table S2), which had failed identification in the original genome ORFs), 16 could be verified (Table S7), indicating that a sequence analysis owing to the criteria of automated annotation transcript has to be covered by at least 5 cDNAs to be detectable (2). Overall, the majority (60%) of all primary transcripts on Northern blots. Outside the above selected region, an addi- detected here are differentially transcribed in response to ni- tional 41 sRNA candidates with high cDNA coverage were trogen; 287 transcripts were exclusively present under ammo- analyzed by Northern blot and verified with few exceptions nium conditions and 253 exclusively under nitrogen fixation (Table S8). For Ͼ85% the transcript length determined was conditions (see Tables S2–S6). consistent with the length predicted by cDNA coverage; in Binding boxes for general transcription factors can be pre- several cases, however, the primary transcript seemed to be dicted in an appropriate distance to the TSS of 53% [IIB processed into 2 or 3 smaller products. In addition, the Northern recognition element (BRE)] and 75% (TATA-box) of all pri- blot analysis confirmed differential expression (Ն1.5-fold regu- mary transcripts, with the consensus sequence depicted in Fig. lation) of 18 sRNAs, as summarized in Tables S7 and S8 and S1. For Ϸ60% of all detected mRNAs a ribosome binding site exemplarily depicted in Fig. 3. with the consensus sequence AGGAGG was identified, consis- tent with previous computational predictions (26). Most inter- Identification of N-Associated Regulator Binding Sites in sRNA Pro- estingly, 521 mRNAs contained long 5Ј UTRs up to 500 nt, with moters. We inspected the 5Ј regions including 500 bp upstream an average size of 150–200 nt (Table S3). Such long 5Ј UTRs may of TSS of sRNA candidate genes with significantly higher be targeted by posttranscriptional regulators. Sixty-six mRNAs transcript levels under nitrogen limitation (cDNA libraries). possess 5Ј UTRs of Ͻ10 nt and are thus designated leaderless Two contained the operator sequence of the global nitrogen mRNAs (Table S4). repressor (NrpR) in their promoter regions (Fig. 4), overlapping TSS (sRNA154) or the BRE (sRNA159). In addition, a unique Small Noncoding RNAs. Our analysis predicted 208 candidate conserved putative binding site for a transcriptional regulator sRNAs from IGRs, including such that overlapped in antisense was identified in the promoter region of sRNA030, sRNA089, orientation with 5Ј regions (n ϭ 43) or 3Ј regions (n ϭ 5) of sRNA090, and sRNA106, consisting of 2 motifs located up- and mRNAs, and were thus designated cis-encoded antisense RNAs downstream of the BRE and TATA-box (AGGAGGCA-N27- (asRNAs). Strikingly, 36 sRNA candidates were exclusively AAAGCTA). Genome-wide searches for this sequence motif

2of5 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909051106 Ja¨ger et al. Downloaded by guest on September 27, 2021 Fig. 5. Sequence-logo of promotor regions of sRNAs candidates differen- tially transcribed in response to nitrogen. The regions upstream of the TSS (up to 500 nt) were aligned using the ClustalW multiple alignment tool (50) and the consensus visualized with WebLogo (51).

Fig. 3. Expression of selected sRNA candidates in response to nitrogen. Total RNA was isolated from M. mazei grown under nitrogen fixation (NF) and scriptional regulators, and mechanisms of translation initiation ammonium conditions (NS) and subjected to Northern blot analysis. Blots showing stable sRNAs transcripts, the lower line the 5S rRNA transcripts of the in methanoarchaea) and of regulation in response to (nitrogen) respective RNA preparations. The calculated fold induction (NF vs. NS) of at stress. Given the newly identified ORFs, the protein-coding gene least 2 independent experiments is given below the blots. number of M. mazei increased by Ϸ1.3% (from 3,371 previously annotated). Several unique features of the transcriptional and posttranscriptional regulation, as well as of the genome organi- identified 8 additional sRNA candidate genes covered by the zation, of M. mazei have been discovered in this study. cDNA libraries with a similar motif in their promoter region (see First, the genome-wide identification of 876 TSS demonstrates Fig. 5 for consensus sequence), 6 of which showed significantly that the vast majority of the covered mRNAs contained a long higher transcript levels in the NF cDNA libraries, indicating 5Ј UTR (up to 500 nt) and a ribosome binding site. Long 5Ј association with nitrogen-responsive regulation. UTRs in methanoarchaea were completely unexpected, because previous genomic and experimental analyses had predicted Antisense RNAs to 5؅ UTRs of Transposase Genes. The exceptionally archaeal mRNAs to either be leaderless or possess rather short MICROBIOLOGY large number of insertion sequence (IS) elements and trans- 5Ј UTRs (27). In addition, a recent systematic screen showed that posase genes in the M. mazei genome (2) has been considered the majority of haloarchaeal transcripts might be leaderless (28), evidence that the high content (30%) of M. mazei ORFs with and a very recent RNA-seq analysis (52) demonstrated that closest homologues in the bacterial domain were acquired by leaderless mRNAs are the rule in S. solfataricus P2. In contrast, lateral gene transfer. Our cDNA libraries covered 13 mRNAs of the high number of long 5Ј UTRs argue for extensive posttran- transposase genes, validating expression of these mobility loci. scriptional regulation at 5Ј UTRs in methanoarchaea (e.g., at the Ј Intriguingly, the 5 UTRs of 6 of these transposase genes overlap level of transcript stability) by regulatory proteins or RNAs, or with an asRNA candidate. Northern blot analysis discovered through riboswitches. To date, evidence for regulatory functions differential transcription of several transposase asRNAs in re- of archaeal UTRs has been limited to translational regulation by sponse to nitrogen (e.g., asRNA036) (Figs. 3 and 6), indicating 3Ј UTR and short 5Ј UTR in (29), and in silico posttranscriptional (antisense-mediated) control of transposases prediction of 1 riboswitch candidate in Thermoplasma spp. and therefore nitrogen-dependent regulation of potential trans- resembling bacterial THI-element responsible for thiamine- position events in M. mazei. Thus, it is tempting to speculate that mediated regulation (30). lateral gene transfer via transposition events may occur in Only Ͻ10% of the 5Ј UTRs identified in M. mazei here are response to changes of environmental conditions. present upstream of bacterial-like genes assumed to have been Discussion acquired by lateral gene transfer (2), and almost all of them contain a BRE and a TATA-box in their promoters. Collectively, Our TSS map covering Ϸ20% of all predicted ORFs of this these findings strongly indicate that 5Ј UTRs are specific for ecologically important archaeal model organism M. mazei pro- vides remarkable insight into general archaeal transcription and global regulation in response to nitrogen or general stress. The comprehensive information will facilitate future studies eluci- dating general aspects of the archaeal transcription and trans- lational machinery (promoter elements, binding sites for tran-

Fig. 6. Transcription of MM2686 and the corresponding cis-encoded as- RNA36. cDNA clone distribution for nitrogen fixation (NFϩ) and ammonium Fig. 4. Promoter region of sRNA154 (A) and sRNA159 (B). The NrpR binding (NSϩ) conditions of the M. mazei transposase gene (MM2686) region is shown boxes are indicated by gray boxes. for the forward and reverse strand.

Ja¨ger et al. PNAS Early Edition ͉ 3of5 Downloaded by guest on September 27, 2021 stream of the BRE and TATA-box of 12 sRNA candidates (Fig. 5). On the basis of our observation that the BRE of those sRNA candidates may have low affinity to transcription factor IIB (TFB), we hypothesize that a yet-unknown activator protein is binding to box I. Binding of this yet-to-be identified factor would then recruit the general transcription factors, TFB and TATA- box binding protein, and consequently RNA polymerase. Be- cause the motif has not been identified in promoters of mRNA genes, it might be recognized by an sRNA-specific regulator. Overall, the strong conservation of the identified sRNA candi- dates in , and the large number of differen- tially transcribed sRNAs in response to nitrogen, argue for a prominent regulatory function of sRNAs in the nitrogen and in general stress responses of methanoarchaea. Thus, together with the predictions by Straub et al. (19) in Haloarchaea, sRNA- mediated regulation might play a much more prominent role in archaea than appreciated. We identified a high number of asRNA candidates in opposite orientation to transposase transcripts in M. mazei, indicating that transposon mobility might be regulated by an RNA-antisense mechanism as previously demonstrated for the Tn10 and Tn30 transposons in E. coli (36, 37). Recently, 8 transposase asRNAs were identified in S. sulfataricus, suggesting that this archaeon is using a similar strategy to control mobility of its multiple Fig. 7. Genomic map showing the localization of the verified IGR transcripts. insertion elements at the posttranscriptional level (17). Most The 400-kb fragment (nt 11,700–407,000) thoroughly analyzed is enlarged. strikingly, several of the transposon asRNAs identified here in Red, new ORFs; green, spRNAs; dark blue, asRNAs; and light blue, sRNAs. M. mazei are differentially expressed in response to nitrogen, predicting a potential link between nitrogen availability and transposition events in M. mazei. Regulation of transposition— methanoarchaeal ORFs, and the few bacterial-like ORFs with well studied in bacteria but much less so in archaea—was known long 5Ј UTRs have been placed under the control of an archaeal to occur mostly in response to stresses (e.g., UV light or promoter after acquisition. temperature) and to take place at the transcriptional and Second, we identified 248 sRNA candidates including cis- posttranscriptional level, as well as by modulating transposase encoded antisense RNAs from IGRs scattered across the genome. activity (38). However, there was only 1 prominent example for Of 77 randomly selected sRNA candidates, 57 have been verified on direct control of transposase gene transcription in response to Northern blots (summarized in Fig. 7), allowing us to estimate a nutrient starvation, by ␴S in Pseudomonas putida (39). Given our total number of Ϸ180 sRNAs on the basis of the empirical threshold observations, M. mazei seems to lend itself as a model to (5 cDNAs per verifiable transcript). Although all genes coding for elucidate the predicted relationship of transposition events and the core small nucleolar ribonucleoprotein (snoRNP) are present in nitrogen availability. Moreover, taking into account the fluctu- M. mazei, and 34 snoRNAs were in silico predicted [University of ations of nitrogen availability in multiple ecosystems, nitrogen- California-Santa Cruz Archaeal Genome Browser (31)], only 1 of stimulated genetic exchange mediated by transposons might our sRNA candidates (spRNA26) was identified as a putative have pronounced ecologic consequence. snoRNA using the Snoscan server 1.0 (11) and SnoReport (32); Finally, in recent years, small ORFs of a size too short for this seemingly low coverage of snoRNAs might be due to the automated genome annotation (usually Ͻ150 nt) have been detection limit or our protocol for cDNA library construction. discovered at a staggering rate in various bacterial , Comparative genome analysis demonstrated that 30% of the including E. coli (40), Pseudomonas aeruginosa (41), and marine identified asRNA and 21% of sRNA candidates in M. mazei were cyanobacteria (42). In addition, many of the corresponding small conserved in all 3 Methanosarcina species (Table S5). Less than peptides were experimentally validated in E. coli (43, 44). Our 3% of the sRNAs candidates showed homology to bacterial analysis in M. mazei identified small ORFs in 40 sRNA candi- IGRs, and only 2 asRNAs are antisense to mRNAs of bacterial- dates (Table S6), the majority of which is conserved in the 3 like ORFs in M. mazei, which is a striking result considering that Methanosarcina strains. Some of those sRNAs might have a dual Ϸ30% of the identified ORFs in M. mazei are proposed to be function, given that the flanking 5Ј RNA regions of the small acquired by lateral gene transfer. Thus, it is tempting to speculate ORFs also showed conservation. At present, no such small that the majority of sRNAs in M. mazei function as regulators of peptide has been demonstrated in M. mazei; consequently, it archaeal gene expression, unlike in other archaea in which Ϸ30% remains to be shown whether the oligopeptide and/or the of these sRNA candidates were snoRNAs (16, 17). noncoding part of the respective sRNA are functional. The expression of 135 of the sRNA candidates is affected by Dual function of an sRNA was originally identified in Staph- nitrogen availability, as validated for 18 sRNAs on Northern ylococcus aureus; RNAIII encodes a 26-aa delta-hemolysin blots. To date, there are very few examples for sRNAs indirectly peptide and also acts as a translational regulator of trans- involved in nitrogen regulation in bacteria (33). A single sRNA, encoded virulence and transcription factor mRNAs (45, 46). GlmY, has been demonstrated to be transcribed by the ␴54-RNA Moreover, the 5Ј region of the Ϸ220-nt E. coli SgrS RNA polymerase in Escherichia coli, but independently of the global encodes a functional peptide, whereas the 3Ј region contains a nitrogen transcription regulator NtrC (34). Consistent with regulatory RNA domain that targets ptsG mRNA by base pairing nitrogen-regulated transcription of ORFs by the global nitrogen (47). Besides such potential dual functions, some of the short regulator NrpR in M. mazei, 2 of the verified nitrogen-regulated ORFs identified in M. mazei were associated with a cis-encoded sRNAs contained the NrpR operator in their promoters (8, 35). asRNA overlapping the 5Ј or 3Ј region of the short ORF (Fig. Furthermore, a new motif (box I), which seems to be associated 2). Because a subset of bacterial cis-encoded antisense RNAs with nitrogen-responsive regulation, was detected up- and down- promotes degradation and/or represses translation of mRNAs

4of5 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909051106 Ja¨ger et al. Downloaded by guest on September 27, 2021 that are toxic at high levels [so-called type I toxin–antitoxins (5ЈP) ends. The details of this protocol, which includes treatment with Termi- (48)], it is tempting to speculate that the identified mRNA– nator 5ЈP-dependent exonuclease (Epicenter) to deplete processed RNAs, will asRNA pairs in M. mazei are archaeal examples for type I be published elsewhere. For each library, graphs representing the number of toxin–antitoxins. mapped reads per nucleotides were calculated and visualized using the Inte- grated Genome Browser software from Affymetrix. TSS were detected by Methods higher cDNA coverage of the 5Ј end of a given RNA in the library constructed with nuclease-treated RNA. The cDNA sequencing raw data are available at RNA Preparation. M. mazei strain Go¨1 was grown under anaerobic conditions our institution’s web site (www.uni-kiel.de/mikrobio/data.html). at 37 °C with an atmosphere of 80% N2 plus 20% CO2 in 70-mL sealed tubes in minimal medium that contained 150 mM methanol plus 40 mM acetate as carbon source and 10 mM ammonium chloride (N sufficiency); for nitrogen- Northern Blot Analysis. RNA was separated in 6% Tris-borate-EDTA polyacryl- fixing conditions the gas atmosphere served as sole nitrogen source (25). amide (PAA) gels containing 7 M urea, or in agarose gels. Ten micrograms (PAA Cultures were grown until cells reached a turbidity of 0.18–0.21 (N fixation) or gel) or 20 ␮g (agarose gel) RNA were loaded per lane; pUC Marker Mix 8 0.5–0.6 (N sufficiency) at 600 nm, which corresponds to the respective midex- (Fermentas) served as a size marker. After separation, RNA was transferred onto ponential growth phase. Cells were harvested at 4 °C and RNA isolated by HybondXL membranes (GE Healthcare) by electroblotting and cross-linked to the phenol extraction followed by DNase I treatment (49). Before cDNA construc- membrane. Membranes were prehybridized in Rapid-hyb buffer (GE Healthcare) 32 tion, differential transcription of the glnK1 gene was verified by quantitative at 42 °C, followed by hybridization with 10 pmol [␥- P]-ATP end-labeled oligode- RT-PCR as described previously (25). oxynucleotides (Table S9) for 2 h. After washing 3 times for 15 min in 5ϫ,1ϫ, and 0.5ϫ SSC–0.1% SDS solutions (42 °C), signals were visualized on a phosphorim- cDNA Library Construction and TSS Analysis. cDNA libraries were prepared and ager (FLA-5000 Series, Fuji) and quantified with AIDA software (Raytest). analyzed on a Roche FLX sequencer as previously described (22). In addition, we used a unique treatment protocol to take into account the 5Ј triphosphate ACKNOWLEDGMENTS. This research was supported by the Deutsche For- end characteristic for primary transcripts (most mRNAs and sRNAs), whereas schungsgemeinschaft as part of the priority program SPP 1258, ‘‘Sensory and processed RNA including abundant rRNA and tRNA have 5Ј monophosphate Regulatory RNAs in Prokaryotes.’’

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