The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum
© Venkataramanan et al.; licensee BioMed Central Ltd. 2013
Received: 19 July 2013
Accepted: 14 November 2013
Published: 4 December 2013
Small non-coding RNAs (sRNA) are emerging as major components of the cell’s regulatory network, several possessing their own regulons. A few sRNAs have been reported as being involved in general or toxic-metabolite stress, mostly in Gram- prokaryotes, but hardly any in Gram+ prokaryotes. Significantly, the role of sRNAs in the stress response remains poorly understood at the genome-scale level. It was previously shown that toxic-metabolite stress is one of the most comprehensive and encompassing stress responses in the cell, engaging both the general stress (or heat-shock protein, HSP) response as well as specialized metabolic programs.
Using RNA deep sequencing (RNA-seq) we examined the sRNome of C. acetobutylicum in response to the native but toxic metabolites, butanol and butyrate. 7.5% of the RNA-seq reads mapped to genome outside annotated ORFs, thus demonstrating the richness and importance of the small RNome. We used comparative expression analysis of 113 sRNAs we had previously computationally predicted, and of annotated mRNAs to set metrics for reliably identifying sRNAs from RNA-seq data, thus discovering 46 additional sRNAs. Under metabolite stress, these 159 sRNAs displayed distinct expression patterns, a select number of which was verified by Northern analysis. We identified stress-related expression of sRNAs affecting transcriptional (6S, S-box & solB) and translational (tmRNA & SRP-RNA) processes, and 65 likely targets of the RNA chaperone Hfq.
Our results support an important role for sRNAs for understanding the complexity of the regulatory network that underlies the stress response in Clostridium organisms, whether related to normophysiology, pathogenesis or biotechnological applications.
Small non-coding regulatory-RNAs (sRNAs), discovered on the genome of all bacteria so far examined, have been established as an integral component of the regulatory system of the cell [1–3]. Unlike their counterparts in eukaryotes, which are about 20 nucleotides long, sRNAs in bacteria span a wider size range between 50 to 500 nts . Regulation of gene expression at post-transcriptional level by sRNAs has been established in both Gram-, such as Vibrio fischeri, Pseudomonas aeruginosa, and Escherichia coli[1, 7], and Gram+ bacteria, such as Bacillus subtilis, Listeria monocytogenes and Streptococcus pyogenes. Identification of sRNAs in bacteria has been carried out experimentally using whole genome microarrays, intergenomic tiling arrays, shotgun cloning and, recently, RNA deep sequencing (RNA-seq) [7, 11–14]. In silico prediction of sRNAs has been carried out using comparative genomic analyses by employing algorithms such as SIPHT , QRNA , ISI , and sRNAscanner . Experimental detection of sRNAs that are expressed only under specific culture conditions may not be successful at other conditions, while computational methods relying on sequence conservation may not identify species-specific sRNAs. Hence, a combination of the two approaches should be logically preferable.
A number of sRNAs have been identified to play an important role in the response to stress in Escherichia coli, such as in oxidative stress (OxyS) , cold shock (SraF, SraG and SraJ) , iron depletion (RyhB) [20–22] and sugar stress (SgrS) . The best and most celebrated case so far uncovered is the regulation of the major stress sigma factor, RpoS, in E. coli. RpoS orchestrates the cellular response to a variety of stresses and the transition to the stationary phase, and is regulated at the post-transcriptional level by several sRNAs. DsrA, RprA and ArcZ are positive regulators the RpoS expression, while OxyS is a negative regulator [24–26]. Yet, little is known regarding a role of sRNAs in the stress response of Gram+ prokaryotes, and nothing about the role of sRNAs in the response to chemical stress. Here we are focusing on the stress-responsive small RNome of Clostridium acetobutylicum, a model organism for the Clostridium genus and more broadly the anaerobic endospore formers . Clostridium organisms are Gram+, endospore-forming firmicutes capable of fermenting a very broad set of substrates and are of great importance in human and animal pathogenesis and health, cellulose degradation, non-photosynthetic CO2 fixation, bioremediation and biotechnology, such as for the production of solvents and other chemicals in the context of biofuel and biorefinery applications [27, 28].
The response to chemical stress, whether from autologous metabolites or allogeneic toxic chemicals (such as from carboxylic acids, high H+ concentrations (low pH), antibiotics, and solvents like ethanol and butanol), plays a major role in cell physiology. Chemical stress affects cell survival, metabolism, sporulation and pathogenesis in physiological milieus, such as the gut microbiome , and pathogenesis [19, 30–32], and the natural environment. Chemical stress is a major and well recognized problem in modern bioprocessing due to toxic substrates and desirable or undesirable toxic metabolites . Chemical stress in Clostridium organisms engages the general stress response, better known as the heat-shock protein (HSP) response, as well as more specialized responses. The HSP response involves strong upregulation of all major HSP proteins, including those of the GroESL and DnaKJ systems. Specialized responses include the acid resistance systems under acid stress [34–36], and changes in metabolic and biosynthetic programs in response to both acid and solvent stress [35, 37–39]. Thus, chemical stress is one of the broadest stress responses known in this and other prokaryotes, and as such, understanding the stress-related small RNome under chemical stress is of broad and general interest.
C. acetobutylicum carries out the biphasic ABE (acetone-butanol-ethanol) fermentation, which consists of an acidogenic exponential phase resulting in the production of butyrate and acetate, followed by the solventogenic stationary phase characterized by the production of acetone, butanol and ethanol, and driven by the reassimilation of the acids. Using a SIPHT-based comparative genomics method, we recently predicted the existence of 113 sRNAs in C. acetobutylicum, among which 31 were validated by either Q-RT-PCR or Northern analysis . The goal of this study is to identify sRNAs, at the genome scale, that respond to butanol and/or butyrate stress and possibly start assigning mechanistic roles for these sRNAs. sRNAs that modulate the stress response can be engaged to engineer strains tolerant to these toxic metabolites, as we and others have recently reported for both C. acetobutylicum and Escherichia coli[36, 41].
Results and discussion
A large set of temporal RNA-seq data is essential for discovery
Using RNA-seq, we aimed to identify sRNAs (previously predicted  and novel) that are differentially expressed under butanol and butyrate stress. To do so, we aimed to collect a large set of temporal data, which, based on our experience are more likely to lead to robust discovery outcomes [35, 38, 39, 42]. Cultures of C. acetobutylicum were grown in batch mode in 4-L bioreactors up to the mid-exponential phase of growth (O.D ~ 1.0), at which point the cultures were stressed with three different concentrations of butanol and butyric acid, respectively, in 3 biological-replicate experiments each. For butanol stress experiments, the cultures were stressed with 30 mM (low), 60 mM (medium) and 90 mM (high), while for butyric-acid stress, 30 mM (low), 40 mM (medium) and 50 mM (high) butyrate concentrations were used. These levels of metabolite stress were chosen based on prior studies [35, 38, 39] and preliminary experiments to achieve the desirable low, medium or strong metabolic response to the applied stress. Cultures were sampled at 15, 30, 60 and 75 min post stress for RNA isolation and sequencing. These sampling times, which are of the order of the doubling time of these cells, were meant to capture largely the direct and immediate impact of these stresses on gene expression and the small RNome. Following RNA isolation, mRNA and sRNA enrichment, cDNA generation, adapter ligations and indexing, libraries were deep sequenced using Illumina’s second generation HiSeq 2000 with a read length of 50 bp.
High sequencing depth was observed for all 84 sequenced libraries from samples representing 7 distinct culture conditions with 4 time points and 3 biological replicates each. On average, for each sequenced library, 18,162,979 total reads were obtained, which are indicative of a high sequencing depth (Additional file 1). From these, for each sequenced library, 9,537,317 reads were mapped into the genome with 884,618 distinct reads after discarding unreliable reads. 46.5% of the reads mapped to annotated genes, while 2% of the reads were mapped to the 113 sRNA we have previously predicted . The balance reads were mapped to structural RNA components (47%) and interoperonic (IOR; genomic DNA between operons ) and intergenic regions (IGR; DNA between annotated ORFs) (5.5%). These data suggest that the stress transcriptome is very rich in transcripts beyond those coded by ORFs and rRNA components.
Read count distribution and metrics for robust identification of sRNAs
What sRNAs are expressed and differentially expressed under metabolite stress
We examined the expression profiles of the 159 (113 previously identified and the newly identified 46) sRNAs aiming to identify which are expressed and differentially expressed under the various metabolite-stress conditions. 114 of the 159 sRNAs had a minimum expression of 50 read counts in 20% of the sequenced libraries, while 70 of the 159 sRNAs had read counts over 50 read counts for 90% of the sequenced libraries representing a very broad set of culture conditions. Expression of genes and sRNAs are specific to culture conditions and not all of them are expressed at all culture conditions. Thus, expression of over 60% of the predicted sRNAs under all culture conditions in this study provides strong support for an important role of sRNAs in orchestrating the cellular response to metabolite stress.
Butyrate stress gave rise to more (45) differentially upregulated sRNAs than butanol stress (33), while butanol stress had more differentially downregulated sRNAs (51) compared to butyrate (44). 42 sRNAs were differentially expressed under both metabolite stresses: 21 were upregulated and 21 were downregulated under both stresses (Figure 3B & 3C). Although the two metabolite stresses result in differential expression of specific sets of sRNAs that are stress and dose dependent, we found a considerable conservation of expression patterns for the two stressors among these sRNAs, thus suggesting a possible role of these sRNAs in the general stress response.
Northern analysis of select stress-related sRNAs
Patterns of expression: hierarchical clustering of sRNA expression under metabolite stress
Hfq binding motifs on clostridial sRNAs
In E. coli and a few other prokaryotes, it has been shown that activity of several sRNAs (and notably of many trans-acting sRNAs) requires the assistance of, or is enhanced by, the hexameric RNA chaperone Hfq [51–55]. Thus, we wanted to examine which of the 159 sRNAs in C. acetobutylicum might be Hfq targets, and if these putative targets might be responsive to metabolite stress.
sRNAs co-immunoprecipitated with Hfq contain the signature Hfq-binding motif and are designated as Hfq-associated sRNAs [11, 12, 55, 56]. This binding motif was discovered largely based on the E. coli sRNAs, but appears to be valid in other organisms [5, 21, 51, 52, 57] since the Hfq protein is well conserved among many prokaryotes. A structural CBLAST of the annotated C. acetobutylicum Hfq (CAC1834) with the two Hfq crystal structures, one from E. coli (3GIB_B) and the other from S. aureus (1KQ1_H), showed conservation in the sequence and the secondary structure of the Hfq monomeric unit (Additional file 3: Figure S1). Thus, we hypothesized that the binding motif of Hfq on sRNAs in E. coli might be preserved on sRNAs from C. acetobutylicum. This Hfq binding motif is characterized by U-rich regions, specifically a poly-U tail at the 3’ end of the sRNA (downstream of the Rho independent terminator), and the U-rich or the AU-rich region upstream of the rho-independent terminator or other secondary hairpin structure; the 5’ region of the sRNA is involved in (non-perfect) base-pairing with the target mRNA [52, 53, 55]. Using this model, we identified 65 potential Hfq-associated sRNAs in C. acetobutylicum. Among these 65 sRNAs, 20 sRNAs belonged to the 46 newly identified sRNA from the deep sequencing data (Additional file 4). We clustered these putative 65 Hfq-associated sRNAs and found most of them to be differentially expressed under both butanol and butyrate stress (Additional file 5: Figure S2). The Hfq gene (CAC1834) was found to be mildly differentially expressed (upregulated) only under butanol stress.
Identification of the putative Hfq binding module on 65 sRNAs may prove useful for deconvoluting the stress-responsive regulatory network in Clostridia, since the unstructured 5’ region of sRNAs that are targets of Hfq contains information that can be possibly used to identify potential target mRNAs of these sRNAs .
Differentially expressed sRNAs that can be related to physiological events of the metabolite-stress response: SRP RNA, 6S RNA, tmRNA, SAM RNA and solB(sCAP_176)
The data presented above showing a large number of sRNAs exhibiting differential expression under metabolite stress provides strong evidence that sRNAs are an integral part of the clostridial stress response system. While the detailed action of these sRNAs remains to be elucidated, there are several sRNAs whose action can be readily related to the phenotypic response of these cells to metabolite stress affecting various metabolic pathways as previously shown [35, 37, 39, 42, 60] and further confirmed by the present set of RNA-seq data as well as the accompanying large set of new microarray and proteomic data .
Both butanol and butyrate stress affect membrane physiology and homeostasis by reducing the transmembrane electrochemical potential and proton gradient (ΔpH) [33, 34, 62]. Bacteria respond to the toxicity of these metabolites by altering the membrane composition by increasing the percentage of saturation in the lipid tails and also by incorporating various integral membrane and transport proteins . We have previously shown that the signal recognition particle (SRP) system and upregulation of several membrane proteins are apparently important in imparting butyric-acid tolerance . The SRP, which consists of the SRP RNA and the Ffh protein, recognizes a motif on mRNAs coding for membrane proteins and, thus, transports the corresponding ribosomes to the membrane to synthesize the targeted proteins . In this light, upregulation of the 4.5S SRP-RNA (Figures 6 & 7) is consistent with its role in the biosynthesis and localization of membrane proteins and the role of membrane proteins in metabolite stress response and tolerance . The Ffh gene is not differentially expressed under metabolite stress (Figure 8), thus further supporting its role as a housekeeping protein.
C. acetobutylicum, like other Clostridium organisms and most prokaryotes, reorganizes its transcriptional and translational machineries during the transition from exponential to stationary phase of growth and under stress conditions [35, 37, 38, 42, 45, 47, 60, 65]. Downregulation of non-essential transcripts and overexpression of different transcript sets requires a quick turnover in the engagement of sigma factors. The 6S RNA (also known as SsrS RNA) has been shown to negatively regulate the transcripts under the control of the major sigma factor σ70 in E. coli and B. subtilis (where it is better known as σA) during the stationary phase of growth by interacting with the RNA polymerase holoenzyme . 6S RNA has been found to be important in cell survival under stress in both E. coli and B. subtilis[67, 68]. We have previously shown that the 6S RNA in C. acetobutylicum, which displays the conserved secondary structure (Figure 4A) of an asymmetric bubble , is expressed at high levels . Its Northern blot (Figure 4A) confirms its strong expression and displays multiple bands, which correspond to distinct processed RNA forms as in other prokaryotes [66, 69]. In contrast to previously reported 6S RNAs displaying two sRNA forms of distinct size, here the 6S sRNA displays three bands (Figure 4A). 6S RNA acts as a template for binding of σ70, and is thus capable, when upregulated, of titrating σ70 thus leading to downregulation of genes under σ70 control. This stress responsive role of 6S RNA has been established in E. coli[70, 71], and our data support that it has a similar role in C. acetobutylicum. It is notable that the 6S sRNA here contains the two characteristic central bubbles with a short stem loop attached . The two components of the σA (the σ70 in C. acetobutylicum) binding motif (UUGACA [-35] & UAUAAU [-10], which corresponds to the DNA motif TTGACA and TATAAT) are found to be perfectly preserved, one on each of the central asymmetric bubbles (Figure 4A), thus apparently regulating the transcriptional responses to metabolite and other stresses. It is interesting to note that in Legionella pneumophila, 6S RNA was found to regulate the expression of secretion system effectors, and stress response proteins such as GroES and RecA . As discussed, the GroESL system is one of the most upregulated HSP systems under a broad spectrum of stresses in Clostridium organisms.
The transfer-messenger RNA (tmRNA or SsrA RNA, which has both tRNA- and mRNA like properties) together with 3 proteins (small protein B [SmpB], elongation factor Tu [EF-Tu], and ribosomal protein S1) forms the tmRNP complex. The tmRNP complex is involved in the quality- control, so-called trans-translation process, recycling stalled ribosomes and facilitating the degradation of aberrant proteins and mRNAs [74, 75]. Trans-translation is especially important in the transition between growth phases and under stress conditions [75–77], whereby many ribosomes may stall on damaged or partially degraded mRNAs. In this context, the stress-induced upregulation (Figures 4B, 6 & 7) of the C. acetobutylicum tmRNA (sCAC834) confirms its role in the quality-control process of trans-translation. It is worth noting that tmRNA is one of the most highly expressed sRNAs in these experiments (blue plots of Figures 6 & 7), further confirming its critical roles for the trans-translation process under stress. Of note, none of the three proteins (CAC0716 – smpB, SsrA-binding protein; CAC1964 – rpsA, 30S ribosomal protein S1; & CAC3136 – tuf, elongation factor Tu) of the tmRNP complex appear to be differentially expressed under stress (Figure 8), thus suggesting that the tmRNA upregulation is controlling the trans-translation process under stress. This is the first experimental evidence for the expression and role of a tmRNA in a Clostridium organism. Deletion of tmRNA in Streptomyces coelicolor was shown [78, 79] to affect the translation of proteins that play a vital role in survival such as cell-cycle and stress proteins including the major HSP protein DnaK, a protein universally engaged in the stress response of Clostridium organisms as already discussed.
S-box (SAM) and T-box riboswitches regulate the expression of genes involved in the metabolism of cysteine and methionine in C. acetobutylicum and are typically found adjacent to the genes involved in sulfur amino acid metabolism . S-box, which is dependent on the concentration of s-adenosyl methionine (SAM), has been shown to regulate the expression of genes in methionine metabolism through transcriptional anti-terminator systems . In C. acetobutylicum, genes involved in sulfur metabolism were found to be upregulated (Figure 8) during high levels of acid stress. We note that an earlier study from our lab had reported that under acid stress, the genes involved in cysteine, methionine and serine metabolism were downregulated ; this difference between the two studies can be attributed to the role of proton concentration since in this present study, in contrast to the earlier one, we used pH control in the fermentation experiments.
Solventogenesis in C. acetobutylicum is controlled by the pSOL1-megaplasmid borne genes (adhE1(aad)-ctfA-ctfB) of the sol operon and the convergent monocistronic adc operon [81, 82]. Expression of the sol operon is dependent on Spo0A  but other genes are also involved in regulating its expression through a long 5’ UTR, which appears like a good target for sRNA regulation. solB (sCAP_176), located just upstream of the sol operon, has been identified as the putative repressor of the sol operon and thus of solventogenesis . Although expressed at very low levels (Figures 6 & 7), solB appears to be a very potent repressor: upon solB inactivation (originally achieved by inactivating the adjacent gene CAP0161 ), solvent formation starts earlier and leads to considerably higher levels of solvents . Thus, solB downregulation promotes sol mRNA expression and solvent production, and vice versa. Here, we found that solB is downregulated (Figure 6D) under butyrate stress, except for the first time point (15 min post stress). Accordingly, the sol-operon genes display a strong upregulation pattern (Figure 8). Butanol stress leads to a more complex pattern of solB expression (Figure 7E), thus leading to a largely opposite expression of the sol-operon genes, except for the first two time points of the high butanol stress (Figure 8). Schiel et al. (2010) reported a putative antisense binding of the solB repressor to the upstream region of the sol operon  (Additional file 6: Figure S3).
The goal of this study was to identify sRNAs that respond to butanol and/or butyrate stress, and also general stress, since, as we discussed, it was previously shown that toxic-chemical stress in Clostridium organisms engages both the general HSP systems as well as specialized systems. One can logically argue that the sRNAs that are differentially expressed under both butanol and butyrate stress would belong to the general stress response. In this sense, the putative roles of SRP RNA, 6S RNA, tmRNA and SAM RNA are part of the general stress response, but perhaps solB belongs to the specialized stress response (Figures 6, 7 & Additional file 7: Figure S4). The metabolite-stress sRNome in C. acetobutylicum was investigated using deep RNA sequencing, in combination with computational analyses. 46 novel sRNAs were identified. The sRNA expression patterns under different levels of butanol and butyrate stress strongly support a role of many sRNAs in orchestrating stress-related cellular changes to deal with the complex, pleiotropic effects of the toxic metabolite stress. This is further supported by the fact that 7.5% of the RNA-seq reads map to non-annotated IOR and IGR of the genome. This is the first comprehensive study of genome-scale expression of sRNAs in a Clostridium or any organism under metabolite stress. Use of extensive temporal RNA-seq data in combination with computational predictions and Northern-based assays are essential in reaching robust outcomes in identifying previously unexplored sRNAs. These data can be used for understanding the role of sRNAs in regulating growth and metabolism thus aiming to provide a more comprehensive understanding of the regulatory network of the cell, and how that network can be engineered for practical applications to produce chemicals and fuels or for remediation processes.
Strain and growth conditions
Three biological replicate cultures of C. acetobutylicum ATCC 824 were carried out in pH- controlled (pH > 5) batch fermentations in 4 L bioreactors (Bioflow II and 110, New Brunswick Scientific, Edison, NJ, USA) in a defined clostridial growth media . The cultures were stressed with butanol (30 mM, 60 mM and 90 mM) and butyric acid (30 mM, 40 mM and 50 mM) at mid-exponential phase of growth at an OD of 1.0 and were sampled at 4 different time points: 15 min, 30 min, 60 min and 75 min post stress. Parallel cultures (n = 3) that were exposed to neither stress were used as the non-stress controls.
RNA isolation and construction of cDNA libraries for RNA-seq
Samples for RNA isolation were collected by centrifugation at 5000 rpm at 4°C for 10 min and the pellets were stored at -80°C. RNA isolation was carried out using the Qiagen’s miRNeasy Mini kit . After RNA extraction, mRNA and sRNA were enriched by using Microbe Express kit from Ambion® kit as per the manufacturer’s protocol. The Ovation Prokaryotic RNA-Seq System (NuGEN® Technologies, Inc, San Carlos, CA) was used to synthesize cDNA from 500 ng of enriched RNA. In brief, 2 μL of first primer mix was added to the 500 ng of the RNA and incubated at 65°C for 5 min. Later, 10 μL of the master mix (first strand buffer and enzyme) were added to the above reaction for first strand synthesis followed by the purification of the first strand cDNA using the QiaQuick PCR purification kit (QIAGEN, Inc. Valencia, CA). The last step of cDNA synthesis was synthesizing the 2nd strand, which was then purified using the Minelute Reaction clean up kit (QIAGEN®) and eluted in 10 μL of elution buffer. The elution buffer was used to make up the volume of the cDNA to 50 μL. The resulting 50 μL of cDNA was used to construct libraries using the TruSeq DNA Sample preparation kit (Illumina®, San Diego, CA). In brief, the cDNA underwent end repair, 3’ end adenylation, adapter ligation and enrichment. Clean up of DNA fragments after each process were carried out using AMPure XP Beads. The fragment length of the libraries was checked using a Bioanalyzer before loading onto HiSeq 2000.
RNA sequencing and data analyses
Deep sequencing was performed using Illumina's HiSeq 2000 with a read length of 50 bp, generating individual library sequence files. Sequence files were processed to remove barcodes, trim adapters, and count read abundances using a set of custom perl, python, and MySQL scripts. Reads were mapped to the C. acetobutylicum genome using Tophat . Gene annotations were downloaded from NCBI, and predicted sRNA regions were included from Chen et al.. Differential expression analysis of sRNAs was performed using DESeq, part of the R Bioconductor package . Differentially expressed sRNAs were determined at a p-value ≤ 0.05, for a pairwise comparison between the control library set and any of the six stress groups (low, medium, and high butanol and low, medium, and high butyrate). Interoperonic regions were defined using previously predicted operons . The data was submitted to Gene Expression Omnibus (GEO) and can be accessed with the accession number GSE48349.
Prediction of novel sRNAs using RNA-seq data
The IORs used in this study were the same as those identified and used by Chen et al. for predicting the 113 sRNAs in C. acetobutylicum. Identification of new sRNAs was based on selecting interoperonic regions (IORs) with a minimal read count of 50. This metric was assigned based on the analysis of the RNA-seq data for mRNAs and sRNAs (Figure 1). Only IORs that met the criteria of a minimal read count were considered for further analysis. Computational prediction of sRNAs was performed using SIPHT based on the comparative analysis of the 21 Clostridium genomes in NCBI. The previously identified 113 sRNAs  were removed for predicting novel sRNAs. Thus, IORs expressed at a minimal read count of 50 and were also computationally predicted to contain sRNAs in the expressed IORs, were manually curated to eliminate false positives. False positives were defined as IORs, which had predominant expression only from the untranslated region (UTRs) of the neighboring genes, even though sRNAs were computationally predicted in those regions. Identification of false positives was carried out using a custom web viewer (generated using custom PHP scripts) by visually analyzing RNA-seq data.
Northern analysis of select sRNAs was performed as described previously using single stranded oligo DNA probes . The probes used in Northern analysis are listed in Additional file 2: Table S2. For each lane, 10 μg of total RNA was loaded in a 5% precast polyacrylamide Ready Gel TBE-urea (Bio-Rad, Hercules, CA), and was electrophoretically resolved along with molecular markers of single stranded RNA ranging from 50 nt to 1000 nt (New England Biolabs, Ipswich, MA). Following electrophoresis, the RNA was transferred to a BrightStar®-Plus positively charged nylon membrane (Ambion). Probes were labeled with ATP [γ32P] using Optikinase (USB, Cleveland, OH) and the unincorporated radioactive material was removed using Micro Bio-Spin Column (Bio-Rad, Hercules, CA). The prehybridization and hybridization of the membrane with labeled oligo probes was carried using the ULTRAhyb hybridization solution (Ambion) at 42°C.
Promoters and Rho-independent terminators of sRNAs
Promoter prediction in the upstream region of the sRNAs were carried out using PSSMs of B. subtilis promoter consensus sequences . For the Lactococcus promoters, we used the promoter HMM model from the PPP tool . Rho independent terminators were predicted using RNAmotif , Erpin  and Findterm (http://www.softberry.com). Secondary structures of sRNAs were predicted using Vienna RNAfold [92, 93].
The sRNA nomenclature for the newly identified sRNAs was done in the same manner as described previously .
sRNA identification protocol using high-throughput technologies
Quantitative reverse transcriptase polymerase chain reaction
Open reading frame
Position specific scoring matrix
Hidden Markov model
Host factor-I protein from Q8 bacteriophage
Transcriptional factor binding site
Signal recognition particle
Transfer and messenger RNA
Alcohol/aldehyde dehydrogenase 1
Co-enzyme A transferase
Small protein B
Elongation factor Tu
Ribosomal protein A.
We acknowledge the assistance of Dinesh Indurthi in training KPV for Northern analysis and members of Papoutsakis laboratory for their assistance during sampling of the cultures for RNA isolation. The work was supported by the genomic sciences grant from Department of Energy, USA (grant# DESC0007092).
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