BACKGROUND
since the discovery of the first small non-coding rna  in  <dig>  this class of untranslated transcripts of  <dig> to  <dig> nucleotides  in length has become more and more evident in transcriptional and posttranscriptional regulation in prokaryotes  <cit> .
in addition to tmrna,  <dig> s rna, 6s rna, and rnasep, which are related to house-keeping gene expression, a number of additional srnas was identified.
these are broadly classified in two major populations,  cis-encoded antisense srnas, oriented antisense to their target genes and  trans-encoded srnas situated in distinct locations from their targets  <cit> .
srna-mediated posttranscriptional regulation was characterized in a variety of cell processes, e.g.
transposition  <cit> , bacterial virulence  <cit> , quorum sensing  <cit> , plasmid replication  <cit> , function of toxin-antitoxin systems  <cit> , and responses to different stress conditions  <cit> .
cis- and trans-encoded srnas each function in a different manner by interacting with short regions of mrna transcripts via perfect and imperfect sequence complementarity, respectively  <cit> .
the main mechanisms of srna-mediated control of gene expression are: repression  <cit>  or activation of translation  <cit> , mrna degradation  <cit>  or stabilization  <cit> , and target mimicry  <cit> .
srnas may act in different ways on different targets, e.g.
the e. coli ryhb srna is a translational activator of shia and a repressor of sodb mrna  <cit> .
in addition, 5'-untranslated regions of bacterial mrna were found that regulate transcription attenuation and translation initiation in response to levels of specific metabolites or intracellular temperature  <cit> .
these mrna regions are called riboswitches.
the metabolite effectors are generally able to mediate changes between alternative secondary structures by binding to a metabolite sensing domain of the rna which prevents translation.
in contrast, rna thermometers are structures sensitive to temperature shifts.
usually, these elements are located in the 5'-utr  including the ribosomal binding site .
increasing of the temperature permits destabilization of the secondary structure and releases the rbs for translation  <cit> .
for example rose-like rna thermometers  are widespread in alpha- and gamma-proteobacteria and two putative candidates were found in sinorhizobium meliloti  <cit> .
to date, genome-wide profiling of srnas by experimental approaches was undertaken in several gram-positive and gram-negative bacteria  <cit> .
however, comprehensive experimental srna screening data is not yet available for the group of alpha-proteobacteria, with the exception of a tiling microarray-based transcriptome study in caulobacter crescentus  <cit> .
our study aimed at a genome-wide discovery of srnas in sinorhizobium meliloti that belongs to the rhizobiales of the alpha-proteobacteria.
s. meliloti exists either in symbiosis with its leguminous host plants  or in a free-living lifestyle.
the bacteria associate with the plant root and induce the formation of nodules that become colonized by the bacteria via infection threads.
inside the nodule, the bacteria differentiate into bacteroids that are capable of nitrogen-fixation to the benefit of the host plant  <cit> .
the genome of s. meliloti is composed of one chromosome  and two megaplasmids, psyma  and psymb   <cit> .
in addition to  <dig> s rna, tmrna, and rnasep  <cit> , the cis-encoded antisense srnas inca and suhb were previously identified in s. meliloti and related alpha-proteobacteria  <cit> .
inca mediates the posttranscriptional repression of the replication initiation protein-encoding gene repc, located in the repabc operon.
this highly conserved operon is essential for replication, segregation and copy number of many extrachromosomal replicons in alpha-proteobacteria, e.g.
the symbiotic megaplasmids in s. meliloti, the tumor inducing plasmid in a. tumefaciens and the second chromosome in brucella  <cit> .
suhb was first discovered in a. tumefaciens in opposite orientation to the suhb gene encoding an inositol-monophosphatase  <cit> .
in s. meliloti, four suhb paralogs were identified  <cit> .
three recent studies primarily applied bioinformatics approaches to the identification of srna candidates in s. meliloti.
del val et al.
<cit>  employed a genome wide computational analysis of s. meliloti intergenic regions leading to  <dig> candidates, eight of which were experimentally confirmed.
ulvé et al.
<cit>  discovered  <dig> novel srnas combining several computational approaches with microarray as well as northern and dot blot hybridizations for validation.
computational predictions and microarray hybridization experiments were also combined by valverde et al.
<cit>  to screen the intergenic regions resulting in  <dig> candidates that were confirmed as novel small non-coding rnas by northern blot and/or microarray hybridizations.
in this study, we have performed a comprehensive experimental screening for srnas in s. meliloti applying deep sequencing technologies as well as oligonucleotide microarray and chip hybridizations.
this approach resulted in  <dig>  transcription units that are novel candidates for trans-encoded srnas, cis-encoded antisense srnas, sense srnas or mrna leader transcripts suggesting that in s. meliloti about 3% of the genes encode trans-encoded srnas and about 2% antisense transcripts.
expression patterns provided further information on conditions of expression of a number of srna candidates.
sequence conservation analyses suggest strong similarities of a subset of s. meliloti srnas to regions in related alpha-proteobacteria.
RESULTS
srnas identified by deep sequencing
data generation by  <dig> and illumina/solexa sequencing of cdnas
a considerable proportion of srnas is probably only transcribed at high levels under specific conditions.
to increase the probability of discovery of these srnas by our screening approach, small rna fractions from a number of conditions were pooled for deep sequencing.
these included exponential and stationary growth phases as well as shifts to low or high temperature, to low or high ph, to high salt concentration, and addition of h2o <dig> to cause oxidative stress .
two small rna samples which were enriched for primary  and processed transcripts   were fractionated to a size range of  <dig> to  <dig> nt and subjected to  <dig> gs flx titanium sequencing.
a total of  <dig>  and  <dig>  reads were generated from sample  <dig> and  <dig>  respectively.
following the mapping to the s. meliloti  <dig> reference genome, the reads were matched to their original strand taking advantage of the sequence of the 5'-rna adapter used in sample preparation.
approximately 70% of all reads were mapped to rrna- or trna-encoding genes, or to repeat regions .
excluding the reads that did not map to the genome, the remaining  <dig>  reads were assigned to either intergenic regions , open reading frames , or regions overlapping both .
in addition to  <dig> sequencing of the pool of small rna fractions obtained from the different conditions, the pool of total rna was subjected to illumina/solexa sequencing .
approximately  <dig>  million reads  were mapped to the reference genome .
after removal of the reads that mapped to repeat regions, including the rrnas and trnas, the remaining  <dig>  reads were uniquely mapped either to orfs or igrs.
transcript definition
low abundant reads that may have originated from transcriptional background and mrna degradation were filtered-out by the following selection criteria employed to determine the contigs for further analyses.
we defined a contig by a seed region of length l, covered by at least c reads.
the seed region was extended on either side as long as read coverage was at least c. for  <dig> reads, we used: l = 50- <dig>  c =  <dig>  c =  <dig>   <dig>  contigs were identified, with the majority  associated to coding regions and the remaining  <dig> contigs mapping to igrs .
for the shorter illumina/solexa reads we used: l =  <dig>  c =  <dig>  c=  <dig>   <dig>  contigs were identified, with the majority  located inside coding regions and the remaining  <dig> contigs mapping to igrs .
these contigs were taken as the observed units of transcription in further analyses.
transcript classification
with respect to their positions relative to the neighboring or overlapping orfs, contigs were grouped into five classes:  trans-encoded srnas,  cis-encoded antisense srnas,  mrna leader sequences,  sense srnas overlapping with orfs, and  transcripts completely covering an orf  .
we identified eight such orfs that were previously identified as genes encoding small proteins    <cit> .
putative srna genes were included in the gendb s. meliloti genome project  <cit> http://www.rhizogate.de.
rna type and subtype: class of transcripts according to the classification in figure  <dig>  replicon: c, chromosome; a, psyma; b, psymb.
number: number of identified srnas per class, subclass and replicon.
*candidates derived from illumina/solexa sequencing that did not allow for identifying the dna strand.
therefore, subclassification is missing for these candidates.
candidates for mrna leader transcripts are defined as short rnas that probably represent a stable derivative of the 5'-part of the mrna of a protein-encoding gene.
this may be a processed form or a prematurely terminated mrna due to attenuation or riboswitch activity.
since the transcription start sites of the majority of the protein-encoding genes are unknown, it cannot be excluded that in some cases a putative mrna leader rather represents an srna transcribed from a promoter different from that of the mrna.
sense srnas represent short stable transcripts whose sequences are located within mrna sequences.
most of these sense srnas were probably processed from mrnas.
cis-encoded antisense srnas were further sub-classified into three groups depending on their relative positions in antisense to the 5'-  and 3'-utr , or inside an orf  .
sense srnas were further divided into candidates situated inside another orf , transcripts overlapping the 3'-utr and the orf , and regions overlapping only the 3'-utr .
sense transcripts overlapping the corresponding orf, but with a 5'-utr that was considered to be too short for translation initiation, were classified as type  <dig> sense srnas and not as putative mrna leader transcripts .
trans-encoded srnas were sub-classified as type  <dig> if their orientation is antisense to both neighboring orfs, and else as type  <dig> .
since the illumina/solexa sequencing did not allow for identification of the transcribed strand, only trans-encoded srnas from this approach were considered for further analyses  .
taken together, both deep sequencing approaches revealed  <dig> putative trans-encoded srnas.
from these  <dig> were exclusively derived from the illumina/solexa sequencing data.
srna candidates associated to transposable element that often occur in multiple copies are not included in this number and are considered separately for the following analyses.
transcription start sites and 3'-end information retrieved from deep sequencing data
information on 5'- and 3'-ends of transcripts in sample  <dig> enriched for primary transcripts and sample  <dig> enriched for processed transcripts were retrieved from the  <dig> sequencing data  .
this analysis indicated the positions of transcription start sites and 3'-ends of a number of srnas.
two-thirds of the trans-encoded srnas displayed one or two distinct 5'-ends.
the remaining transcripts possessed more than two or highly variable 5'-end positions which did not allow for identifying the transcription start sites.
the proportion of transcripts with one or two distinct 5'-ends was even higher in the class of cis-encoded antisense srnas.
only 23% of srnas from this class displayed multiple or highly variable 5'-ends.
a similar distribution of 5'-ends was observed in the class of mrna leader transcripts.
in the class of sense srnas, the proportion of transcripts with one or two defined 5'-ends was 72%.
ends: 5' and 3'; number of defined 5'/3'-ends per transcript; s <dig>  s <dig>  s1&2: number of transcripts in sample  <dig>   <dig> and both samples, which match the defined 5'/3'-end criteria; total: number of transcripts in all samples which match the defined 5'/3'-end criteria.
primary 5'-ends were identified for 74%, 46%, and 60% of the trans-encoded srnas, antisense srnas, and mrna leader transcripts, respectively.
in contrast, only 18% of the transcripts classified as sense srnas displayed a primary 5'-end indicating that the majority of these transcripts was processed from mrnas of the associated protein-encoding gene.
comparison of the primary transcript-enriched sample  <dig> and the processed transcript-enriched sample  <dig> revealed several different situations with respect to the derived transcript ends .
candidates possessing identical 5'-ends were identified in both samples .
alternative 5'-ends that may have been caused by posttranscriptional processing of the transcripts or activities of alternative promoters were observed for  <dig> srna candidates .
we also found a number of srnas with 5'-ends varying by only one or two nucleotides .
upstream the transcription start sites of  <dig> srna candidates the σ <dig> consensus promoter motif cttgac-n17-ctatat  <cit>  was predicted , further confirming the identified primary 5'-ends of these srnas.
two promoters of this type were predicted upstream of smelb <dig>  only  <dig> sense srna candidates were preceded by this consensus promoter motif, further supporting the assumption that most of these transcripts were processed from mrnas.
in all classes, about 60% of srnas exhibited multiple or highly variable 3'-ends .
the remaining 40% comprised  <dig> and  <dig> srnas with one or two defined 3'-ends, respectively .
in some cases more complex situations were observed.
an example is the  <dig> nt region comprising smelc111a and smelc111b  located in the intergenic region between smc <dig> and smc <dig>  this region displayed three putative transcription start sites detected in sample  <dig> and two 5'-ends that were only found in the processed transcript-enriched sample  <dig>  furthermore, several 3'-ends were found in this region.
this results in separate srna transcripts differing in length.
thus, smelc111a and smelc111b probably occur as separate srnas and were classified as trans-encoded srna and leader transcript, respectively.
in  <dig> cases we found clusters of at least two srna candidates separated by less than  <dig> nt.
among these were nine clusters composed of two and two clusters comprising three trans-encoded srnas.
only one protein-encoding gene   <cit>  was found to be associated with two antisense srnas  , while approximately 18% of the sense srna-associated orfs include at least two candidates.
interestingly, multiple srna candidates were found associated with six of seven genes of the rhizobactin operon.
characteristics of srna classes
the deep sequencing approaches revealed  <dig> trans-encoded srnas,  <dig> cis-encoded antisense srnas,  <dig> mrna leader transcripts, and  <dig> sense srna candidates.
distribution of srna candidates on the three replicons shows a prevalence of trans-encoded srnas, mrna leader sequences and sense srnas on the chromosome .
between 8% and 22% more srnas than expected from an equal distribution of srnas  were found on this replicon.
in contrast, 9% more cis-encoded antisense rnas than expected from an equal distribution  were identified on megaplasmid psymb.
srna candidate regions were almost evenly distributed over the whole genome .
thus, clustering was only observed at the gene or intergenic region level.
about half of all antisense srna candidates overlap with the 3'-end of the coding region and the 3'-utr .
18% overlap with the 5'-utr  and 26% are located on the opposite strand inside the orf  .
the majority of sense srna candidates are located within the coding region of the associated gene   or overlap the 3'-utr and the orf  .
transcription units in repeats and transposable elements
a number of recent studies revealed non-coding transcripts within transposable elements or repeat regions  <cit> .
hence, the transcriptional activity of these regions in the s. meliloti genome  <cit>  was analyzed.
this suggested repeat regions from two groups showing transcriptional activity.
the first group comprises  <dig> repeat regions,  <dig> to  <dig> nt in length, with transcriptional activity .
these are associated with transposase genes, either in antisense , in sense orientation  or as leader transcript  .
an example for this group is transposon trm <dig>  that occurs in several copies on the chromosome as well as on the megaplasmids.
it possesses a type  <dig> antisense srna of  <dig> nt which overlaps the 5'-end of the transposase gene .
antisense srnas with similar features were found in trm <dig> and trm <dig>  the partial transposases trm20c and trm5n are associated with a type  <dig> antisense srna.
furthermore, trm <dig>  trm <dig>  sma <dig> and sma <dig> possess sense srnas within the transposase-encoding region or overlapping its 3'-end.
interestingly, a putative mrna leader was identified upstream of sma <dig> which presumably contains two distinct mrna leader-like transcription start sites .
the second group comprises  <dig> repetitive extragenic palindromic  elements,  <dig> repeats and three regions which include both rep elements and repeats in s. meliloti  <cit>  with transcriptional activity .
identification and expression profiling of srnas by oligonucleotide microarray and chip hybridizations
as a complementary approach to the deep sequencing strategy, oligonucleotide microarray and affymetrix symbiosis chip hybridizations were carried out using arrays that contain probes representing coding regions and intergenic regions  .
rna for these experiments was obtained from identical stress conditions and growth phases as previously applied for deep sequencing.
in the oligonucleotide microarray hybridizations, signals derived from fractions composed of small  and long  rnas were compared .
this strategy allowed for identifying sequence regions predominantly present in the small rna fraction excluding sequence regions that were also represented in the long rna fraction at a considerable level.
applying a cut-off of ≥  <dig> to the ratio of signals derived from the small rna to signals from the long rna fraction  <dig>  srna candidate regions were identified .
among these were the  <dig> s rna, the inca srnas, and a number of trnas confirming the applicability of this strategy.
according to the standard procedure of classification ,  <dig> candidates were classified as putative trans-encoded srnas .
<dig> of these were mapped to intergenic regions oriented in antisense to neighboring genes but not overlapping the 5'- or 3'-utrs and thus classified as type  <dig> trans-encoded srna candidates, whereas  <dig> were classified as type  <dig> sharing the same orientation as at least one of the two neighboring genes.
the remaining candidates fell into the groups of cis-encoded antisense srnas and mrna leader transcripts .
in support of the oligonucleotide microarray-based analysis, affymetrix symbiosis chip hybridizations were carried out using the small rna fractions from the experiments described above.
signals were classified as small non-coding rna candidates when exhibiting the following characteristics:  a signal intensity at least two fold higher than the background, and  a distance of less than  <dig> nt between two probes with a positive signal.
candidates listed in  were classified following the standard procedure .
comparison of the microarray with the affymetrix symbiosis chip data revealed  <dig> trans-encoded srnas,  <dig> cis-encoded antisense srnas, and  <dig> mrna leader candidates identified by both approaches.
the candidates from the oligonucleotide microarray hybridizations were clustered on the basis of their expression profiles under the eight growth conditions tested .
information on expression patterns of  <dig> trans-encoded srnas with unambiguous 5'- and 3'-ends identified by deep sequencing could be retrieved from the microarray data .
expression patterns could also be deduced for  <dig> cis-encoded antisense srnas and  <dig> mrna leader sequences that are also shared by both data sets .
only  <dig> of these trans-encoded srna candidates appeared to be generally expressed in all conditions.
the other candidates from this srna class were detected only in a subset of conditions with the largest cluster comprising  <dig> candidates only found in a single condition.
clustering of the expression patterns of the putative cis-encoded antisense srnas shared by both data sets revealed  <dig> candidates expressed in all conditions, whereas  <dig> candidates showed expression in two to seven conditions, and  <dig> candidates in only one condition.
from the putative mrna leader transcripts identified by both approaches,  <dig> were predominantly detected in the small rna fraction in two to five conditions, whereas  <dig> were identified in only one condition.
none of these leader transcripts was detected in all conditions.
validation of selected candidates
prediction of peptide-encoding srnas
since a short transcript may have dual functions as regulatory srna and mrna  <cit> , srna candidate sequences were screened for coding regions of at least  <dig> nt preceded by a putative ribosome binding site .
results of this analysis are summarized in .
among the trans-encoded srnas, six candidates carry such a coding region, of which three are preceded by a predicted rbs.
the class of antisense srnas includes  <dig> candidates with a coding region, of which one possesses a putative rbs.
in case of sense srnas,  <dig> candidates were found that may encode a peptide and  <dig> of these also carried an upstream rbs motif.
different types of coding regions were found, orfs that overlap the corresponding gene or are located within the coding region of this gene.
the reading frame of most of the predicted srna orfs differs from the reading frame of the corresponding gene, but  <dig> share the same reading frame and hence the same stop codon with this gene.
a similar situation was found in the class of mrna leader transcripts.
here,  <dig> candidates include an orf,  <dig> of these with a potential rbs.
these orfs do not include those starting with the start codon of the corresponding gene because these lack an inframe stop codon on the srna sequence.
only orfs were included that map to the 5'-utr or overlap the coding region of the corresponding gene in a different reading frame.
analysis of srna candidates by 5'-race and northern hybridizations
srna candidates selected from different classes were further analyzed by 5'-race and northern hybridizations .
race experiments confirmed the 5'-ends derived from the  <dig> sequencing data of the trans-encoded srnas smelb <dig>  smela <dig>  smelb <dig>  smelc <dig>  smelb <dig>  and smelb <dig>  the cis-encoded antisense srna smela <dig>  the mrna leader transcript smela <dig>  and the intragenic sense srna smelb <dig>
northern hybridizations were carried out for six candidates  using total rna obtained from the exponential growth phase in complex medium, after shifting the culture to higher or lower temperature, and after adding salt to the culture.
hybridizations of the trans-encoded srnas smelb <dig>  smelc <dig>  smelb <dig>  and smelb <dig> showed signals in all conditions with just small variations in signal strength.
in contrast, smela <dig> and smela060/smela <dig> showed differential expression.
smela <dig> was only detected at a very low level in complex medium and in the same medium at decreased temperature.
however, it was strongly detected after the shift from 30°c to 40°c and at a medium level in gms medium.
smela <dig> and smela <dig> encode two srnas of the same length that differ by just one nucleotide.
these srnas were detected only after the temperature shift to 40°c and after adding  <dig> mm nacl to a culture in gms medium.
microarray hybridization data is available for smela060/smela <dig> represented by oligonucleotide probes on the array.
these results are in good agreement with the results of the northern hybridization, with the highest ratios obtained under heat shock  and salt shock conditions , and values slightly above the threshold  for srna detection in the other conditions.
this indicates that expression patterns can be retrieved from the microarray data.
the srna lengths of smelb <dig> and smelb <dig>  estimated from the northern hybridizations are shorter than those derived from the sequencing data.
this might be explained by incomplete denaturation of these srnas or by degradation processes during rna isolation.
in case of smelb <dig> a weak band at the expected size was detected that may correspond to the full length contig derived from the sequencing data.
northern hybridization of smel <dig> resulted in two close bands which may have originated from alternative 3'-ends found by cdna sequencing.
two dominant bands were also detected for smela <dig> and smela060/smela <dig>  the former are also indicated by the sequence coverage  whereas no hint on the origin of the additional band could be found for smela060/smela <dig>  interestingly, a structure composed of three stem-loops with identical sequence motifs in the loops was predicted for smela <dig> .
shorter transcripts suggested by northern hybridizations and cdna sequencing were predicted to lack one or two of the stem-loop substructures .
stable structures corresponding to predicted structural domains of the full-length transcripts were also predicted for shorter transcripts associated to the srnas smelc <dig>  smelb <dig>  and smelb <dig>  .
this is not the case for the putative mrna leader transcript smela <dig>  the structure predicted for the short 5'-transcript differs from that of the full-length rna  .
z-scores computed for the srna candidates  significantly differ from zero indicating that these srnas form a well-defined secondary structure .
predicted structures and z-scores of the srnas analyzed by northern and 5'-race are shown in figures  <dig> and  <dig>
bioinformatics analysis of srna candidates
blast homology search
blast homology searches for the  <dig>  transcripts from  <dig> sequencing were carried out at different cut-offs.
we report here on results for an e-value cut-off at 7e- <dig>  which is the most stringent cut-off for which all our transcripts match against their origin genome.
the match covering at least 70% of the transcript length was an additional requirement.
data for this setting is shown in table  <dig>  under these criteria, we obtained about  <dig>  hits in alpha-proteobacteria, and about  <dig>  in other bacteria.
hits were classified as "known" if there was annotation information associated with the matched region.
as expected, the class of sense transcripts comprised the highest percentage of "known" matches , followed by mrna leaders .
the low percentage for antisense and trans-encoded transcripts  can be explained by the fact that only few transcripts of this type are annotated in the databases.
data for other blast cut-offs are given in .
results of the blast search with srna trancripts against sequence databases with an e-value cut-off 7e- <dig> and the requirement that the hit covers at least 70% of the query are shown.
hits are classified as known  if annotation information is associated with the database sequence.
hit counts are given in taxonomic order.
hits to the taxonomic group do not include the hits to the subgroup which is listed above it.
for the alpha-proteobacteria , the percentage of "known" versus "unknown" hits for each class of transcript is listed.
y, known; n, unknown.
structural analysis
we investigated whether our transcripts appear to have a well-defined secondary structure.
rather than looking at individual minimum free energy foldings, we computed accumulated boltzmann probabilities of all  foldings of the rna which exhibit the same abstract shape  <cit> .
we determined the dominant shape for each sequence and computed z-scores against a background distribution taken from the s. meliloti genome  .
the result is shown in figure  <dig>  which shows that classes of trans-encoded, sense and antisense trancripts tend to have positive z-scores, centered around + <dig>  this implies that for these classes a well-defined secondary structure may be associated with their function.
leader transcripts showed no bias.
standard rfam homology search
from the  <dig> non-coding srnas from s. meliloti already known in rfam, belonging to  <dig> families, our approach confirmed  <dig> by direct positional matching, although with corrections in their precise position.
these were also returned by standard rfam search with regular cut-offs .
beyond these known transcripts,  <dig> candidates were classified as - heretofore unknown - members of  <dig> different rna families, excluding three families of hiv sequences attracting  <dig> hits, which were considered false positives.
<dig> of the remaining families included known members from s. meliloti.
the only "new" families resulting from this search were rf <dig>  and rf <dig> , the latter of which appears biologically implausible.
hence, members of only one new family were identified applying a standard rfam search.
refined rfam homology search
we performed a simple but complete evaluation of the family models presently available in rfam, comparing scores obtained from the covariance models to scores from plain sequence models generated from the same sequence alignments .
we observed that most of the covariance models strongly rely on sequence similarity.
they practically behave as hmm sequence models, with the encoded structure only contributing to the score in a negligible way.
a diverged sequence with a conserved structure will rarely pass the model thresholds, explaining the lack of generalization.
as known srnas are scarce in the class of alpha-proteobacteria, we sought a way to extract more information from rfam models.
we designed a pipeline using rnasifter  <cit>  that combines independent evidence of structural conservation with an rfam search below the suggested cut-offs .
by this procedure, we obtained   <dig> families and  <dig> families above cut-offs of  <dig> *t and  <dig> *t, respectively, where t is the default cut-off of the family.
these candidates require individual analysis; an encouraging observation about the refined search strategy is the correct classification of smela <dig> as a group ii intron, which was neither observed with the standard rfam search nor applying a blast search.
the list of new families is given in .
discussion
identification of srna candidates in s. meliloti
here we report on the first comprehensive screen for srnas in an alpha-proteobacterium applying deep sequencing and microarray hybridizations.
the symbiotic nitrogen-fixing soil bacterium s. meliloti was analyzed as representative of the rhizobiales.
our study suggested a total of  <dig>  srna candidates that were classified as trans-encoded srnas , cis-encoded antisense srnas , mrna leader transcripts , and sense srnas overlapping coding regions .
these results are in good agreement with a number of recent studies that reported genome-wide screens for prokaryotic srnas by cdna sequencing and hybridization to tiling microarrays  <cit> .
the first studies of this type were reported for e. coli, but similar studies were also published for other gram-negative bacteria  and gram-positive bacteria .
in the group of alpha-proteobacteria, only one global srna screening study was published so far, which was a tiling microarray-based srna screen in c. crescentus  <cit>  that resulted in  <dig> novel srna candidates.
for alpha-proteobacteria, only one pilot study applying pyrosequencing of cdna derived from total rna depleted for rrna sequences of s. meliloti was reported  <cit> .
but this approach aimed at identifying transcripts of new protein-encoding genes and delivered just  <dig>  reads derived from non-rrna sequences that indicated  <dig> new orfs.
depending on the methods and strategies of data analysis applied, and the selection criteria for srna candidates, these studies suggested up to several hundred srna candidates in different bacteria.
this indicates that not only mirna genes in eukaryotes but also srna genes in bacteria represent a significant proportion of the genome, although only a small proportion of these candidates was further validated.
srna discovery by deep sequencing
for discovery of srnas in s. meliloti we have combined two deep sequencing technologies.
<dig> sequencing of cdnas turned out to be the most valuable data resource.
sequence coverage defined 5'- and 3'-ends of the srna candidates which are of great advantage for both structural and comparative analyses.
moreover, enrichment of primary 5'-ends of transcripts allowed for identifying transcriptional start sites of srna candidates with several of these validated by 5'-race.
data from enrichment of processed 5'-ends was ambiguous with a very high proportion of 5'-ends present in both the primary and the processed transcript-enriched samples.
this effect may have been caused by a pyrophosphatase activity which eliminates the pyrophosphate from the 5'-end of primary transcripts making these transcripts accessible for the sequencing procedure followed in the processed transcript-enriched sample.
pyrophosphatase activity was recently described in several bacteria, such as the nudix enzymes rpph in e. coli and bdrpph in bdellovibrio bacteriovorus  <cit> .
nevertheless, a number of srna candidate regions displayed alternative 5'-ends that may have been caused by posttranscriptional processing of the transcripts.
processing of transcripts at the 5'-end is a well characterized mechanism, an example of which is the srna gady in e. coli  <cit> .
we also found a number of srnas with transcription start sites varying by only one or two nucleotides.
these were probably generated by the activity of a single promoter with a diffuse transcription start site, as was previously described for the sral srna  <cit> .
alternatively, transcripts with more than one distinct 5'-end may be products of alternative promoters.
alternative 3'-ends observed in this study might be generated by endo- or exonucleolytic cleavage of the full length transcript.
examples of srnas processed by such mechanisms are micx in vibrio cholerae  <cit>  and arcz  in e. coli and salmonella  <cit> .
the former is processed by rnase e into two transcripts with different 3'-ends whereas the primary and 5'-processed transcripts of the latter undergo 3'-exonucleolytic degradation.
another possible mechanism that results in alternative 3'-ends is the presence of two or more termination sites.
for instance, gcvb of e. coli possesses two termination sites, resulting in two distinct transcript sizes  <cit> .
illumina/solexa sequencing of cdna derived from total rna revealed only few additional trans-encoded srna candidates.
the deep sequencing data from this study suggests an average length of the srna candidates of about  <dig> nt.
this is in good agreement with results from several other studies that deduced a typical size range of srnas of  <dig> to  <dig> nt  <cit> .
filtering and classification of srna candidates
the stringency of criteria for selection of srna candidate regions based on cdna sequencing data has a great effect on the number of candidates derived from this approach.
in this study, we have based the  <dig> sequence data analyses on strict criteria that defined seed regions of  <dig> to  <dig> nt in length with a coverage of at least  <dig> reads per nucleotide.
selection criteria were relaxed to a minimum of five reads per nucleotide to complete the contigs.
definition of a lower coverage would have resulted in a higher number of candidate regions with an increasing number of false positives from transcriptional background and mrna degradation.
classification of srna candidates in putative trans-encoded srnas, cis-encoded antisense srnas, mrna leader transcripts, and sense srnas overlapping coding regions is ambiguous.
the main difficulty is the definition of a gene region including 5'- and 3'-utr, since promoter and terminator predictions are not well established in s. meliloti.
therefore, the classification was based on the estimated minimal length of 5'- and 3'-utrs which might have resulted in misclassification of a number of candidates.
moreover, the majority of sense srnas completely or partially overlapping coding regions probably are stable mrna degradation products.
several previous studies provided also evidences for the presence of antisense srna and mrna leader transcripts in addition to the intergenic trans-encoded srnas  <cit> .
cis-encoded antisense srna transcripts appear to be very dominant in synechocystis sp.
pcc  <dig>  georg et al.
<cit>  suggested that about 10% of all genes in this organism are influenced by antisense rnas.
recently, evidence for  <dig> antisense rnas that may affect about 3% of the protein-encoding genes was obtained by parallel sequencing in vibrio cholerae  <cit> .
such a high proportion of antisense transcripts as in synechocystis sp.
pcc  <dig> was not observed in our study in s. meliloti applying deep sequencing.
only about 2% of the protein-encoding genes seem to be partly transcribed in antisense direction.
in contrast, our microarray experiments suggested about 11% of genes with overlapping antisense transcripts.
such discrepancy may be organism-specific, but probably is more influenced by employing different discovery strategies.
in this study, a multi-tiered approach to srna identification was taken applying different complementary identification strategies.
illumina/solexa sequencing was carried out for cdna derived from total rna which resulted in a proportion of 87% of reads mapping to rrna genes or repeat regions.
usage of total rna and the inability to retrieve strand information from this sequencing data did not allow for identifying srnas associated to coding regions, but identified intergenic srna genes and revealed information on transcription units of protein-encoding genes .
in contrast, the  <dig> sequencing approach including size fractionation and primary 5'-end enrichment provided rich information on short transcripts encoded in intergenic regions as well as antisense srnas and leader transcripts associated to coding regions.
a microarray approach cannot provide exact information on 5'- and 3'-ends of srnas and is limited by the genome coverage with oligonucleotide probes.
the sm14koli microarray used in this study contained probes distributed on both strands of intergenic regions at irregular distances.
coding regions were just represented by a single probe.
the strategy of comparing short rna to long rna fractions was therefore tailored to predominantly identify intergenic srna genes.
in comparison to the high number of microarray hybridizations covering different conditions, only a low number of hybridizations of short rna to the affymetrix symbiosis chip were performed resulting in only few reliable srna candidates.
because of the different strengths of the applied strategies, a reasonable comparison of identified candidates is only possible for the class of trans-encoded srnas .
about 40% of the trans-encoded srnas identified by the  <dig> sequencing approach were also identified by the illumina/solexa sequencing and/or the microarray approaches.
only  <dig> candidates were identified by all approaches.
it has to be noted that these numbers change with relaxing the strict criteria applied in analysis of the deep sequencing data.
classification and determination of srna ends is more reliable based on the  <dig> sequencing-derived dataset.
because of the short illumina/solexa reads as well as distribution and length of microarray probes, the definition of the regions is less accurate based on these approaches.
this explains discrepancies in classification of a few srna regions and mapping of some srna regions identified by one method to multiple regions identified by one or both of the other approaches.
deep sequencing data confirmed previously reported srnas
in addition to  <dig> s rna, tmrna, trnas and rrnas, our deep sequencing approach confirmed  <dig> previously published srna candidates.
the tmrna  <cit> , the two inca antisense transcripts  <cit> , and two rose-like elements   <cit>  were found in studies dedicated to one or few srnas, whereas the other candidates resulted from global screening approaches  <cit>  .
overlap, percent overlap of the candidates from the cited studies or database compared to the region covered by a srna candidate in this study.
n, strand was not or not undoubtedly determined.
id; rna id ; type, rna type; st., strand; ov., overlap ; id , rna id corresponding to the given reference; north., experimentally validated by northern blot, given are the references for the northern hybridizations.
rna id: *, solexa candidate.
rna type:  <dig>  trans-encoded srna;  <dig>  cis-encoded antisense srna;  <dig>  mrna leader;  <dig>  sense srna;  <dig>  open reading frame.
the presumed location of the srnas predicted by ulvé et al.
<cit> , derived from the location of the dot blot probes.
thus an overlap analysis to our candidates could not be performed.
among the mrna leader transcripts were two candidates  that corresponded to the predicted rose-like elements upstream of ibpa and smb <dig> encoding heat shock proteins in s. meliloti  <cit> .
it has to be noted that the ibpa rose element was represented by only eight reads  in the sequencing data and thus marginally failed our filter for candidate selection.
maclellan et al.
<cit>  characterized the cis-encoded antisense srna gene inca situated within the repabc operon on each megaplasmid.
inca is located within the small intergenic region between repb and repc in opposite orientation to these coding regions.
high sequence coverage of both inca srnas was also evident in the sequencing data .
the s. meliloti tmrna  was identified because of its homology to sra from bradyrhizobium japonicum  <cit> .
northern blot analysis and mapping of both 5'- and 3'-ends identified two pieces, a  <dig> nt mrna-like domain and a  <dig> nt trna-like domain, both highly stable, whereas the premature form was unstable.
both transcripts were also identified in our study with a high  <dig> sequence coverage .
the s. meliloti strain used in this study harbours three classes of group ii introns, rmint <dig> , smb21477/smb <dig> , and sma <dig>   <cit> .
transcript smela <dig> probably corresponding to the intron lariat rna was identified by the  <dig> sequencing approach.
our sequencing data supported  <dig> srna candidates that resulted from the previous genome-wide screens of which  <dig> were confirmed by northern hybridizations  <cit>  .
from the  <dig> trans-encoded srnas identified in our study,  <dig> have previously been predicted .
this high percentage of re-discovery of known srna candidates increases the confidence in the  <dig> new putative srnas.
in the classes of antisense srnas, mrna leader transcripts, and sense srnas, only few known candidates were re-identified.
these were  <dig> out of  <dig> antisense srnas,  <dig> out of  <dig> mrna leader transcripts, and  <dig> out of  <dig> sense srnas.
hence, our approach resulted in a high number of new candidates.
furthermore, additional information on 5'- and 3'-ends as well as expression patterns under several growth and stress conditions could be derived for a number of previously described candidates from the sequencing and microarray data.
in several cases our srna regions differed in orientation from, only partially overlapped, or were located inside the regions predicted in other studies .
our approach also confirmed  <dig> rfam annotated transcripts of s. meliloti  <dig> .
putative srna functions in s. meliloti
putative functions of srnas
trans-encoded and cis-encoded antisense srnas predominantly seem to act via base pairing mechanisms  <cit> .
the former usually are located in intergenic regions and share only poor and incomplete complementarity with target sequences, which complicates computational target predictions.
in contrast, the latter share extended regions of complementarity to an overlapping sense encoded gene.
thus, in most cases antisense srnas modulate expression of the overlapping sense gene on transcriptional or posttranscriptional level, although trans targets may also be affected  <cit> .
the majority of antisense srnas identified in this study are complementary to the 3'-utr of the corresponding gene.
it is well established that this type of antisense rnas is involved in posttranscriptional regulation of the steady-state amount.
thus, either mrna stabilization by masking of potential ribonuclease recognition sites or mrna degradation by formation of a ribonuclease sensitive rna duplex   <cit>  are probably mediated by the  <dig> antisense srnas mapping to the 3'-utr.
only  <dig> cis-encoded antisense srnas were located in small intergenic regions in antisense to both neighboring genes which presumably form an operon structure.
this type of srna may mediate transcription termination inside the operon structure resulting in down-regulation of the downstream gene expression  <cit> .
microarray hybridizations supported a number of srna candidates identified by the deep sequencing approach and provided further information on expression patterns in different growth phases and stress conditions.
a group of  <dig> trans-encoded srna candidates,  <dig> antisense srnas, and  <dig> mrna leader candidates were found only in one of the eight conditions tested.
this indicates a specific expression pattern and provides hints to the biological context of their function.
among these were the antisense srna smela <dig> found under oxidative stress as well as smelc <dig> and smelb <dig> found under salt stress conditions.
<dig> of the mrna leader candidates were only found in response to heat stress.
the majority of cis-encoded antisense srnas have been described in plasmids, phages and transposons  <cit> .
such srnas probably inhibiting translation of the transposase are encoded by tn <dig>  and tn <dig>   <cit> .
transposon-encoded antisense rnas are quite stable which may explain the high sequence coverage of the majority of the transposable element-related srna candidates found in our study.
another group of repeats that were represented in the small rna fraction analyzed in this study were rep elements  <cit> .
although these elements are widely distributed within the genome, their functions remain obscure.
it has been shown that their presence can affect the level of gene expression  <cit> .
it was speculated that these elements play a role in gene transcription termination.
another proposed role is in relation to chromosomal structure, because both dna gyrase and dna polymerase i can bind to rep elements  <cit> .
functional classification via database search
an important observation from this study is that searching rfam family models with over  <dig>  srna candidates only re-discovered those sequences from s. meliloti already known and stored in rfam.
there was not a single case of an unclassified transcript that was predicted to be a member of a certain family, except for the ire family and some apparent false positives.
this is in strong contrast to the state of the art with protein-coding genes.
it can be explained in part by the fact that identification of srnas in bacteria is still at its outset, and in part by the fact that current rfam models are very strongly based on sequence similarity rather than conserved structure.
alternative ways to construct structural family models have been recently suggested  <cit> , but an implementation is not yet available.
our refined rfam search strategy yielded family associations which are still awaiting validation.
the blast search suggested a number of homologous regions, mainly in the rhizobiales but also in other alpha-proteobacteria, indicating that srna candidates derived from the comprehensive experimental screen in s. meliloti may support the computational prediction of srnas in related bacteria.
CONCLUSIONS
genome-wide experimental screening for srnas
several genome-wide screens for srnas applying parallel sequencing of cdnas and hybridizations of tiling microarrays have recently been published.
apart from a single study that used tiling oligonucleotide chips to discover srnas in c. crescentus, our study is the first comprehensive approach to discover srnas in an alpha-proteobacterium.
therefore, this data set will be a valuable resource for comparative studies in related bacteria.
identification of homologous srnas and assignment to rna families
assigning srna candidates to functional rna families with present bioinformatics tools is a difficult task.
a recent study  <cit>  shows that even human expertise in describing structural patterns does not take us much beyond a simple blast search.
the study argues for an effort in developing curated databases of aligned secondary structures, as a prerequisite for increasing the power of comparative analysis.
another perspective is that the use of structural models from locomotif  <cit> , which include the thermodynamics of rna folding and can handle simple cases of pseudoknots, may generalize better than the combinatorial pattern descriptions via rnamotif  <cit>  used in the cited study.
perspectives for functional analyses
global experimental screening strategies have delivered a high number of srna candidates in all bacteria studied so far.
in contrast to these well-developed discovery strategies, approaches to identify targets of srnas in high throughput are still in the infancy.
experimental approaches mainly rely on transcript or protein profiling in srna mutant or over-expression strains, and in vitro analysis of srna-mrna interaction.
in contrast to computational predictions of eukaryotic microrna targets, predictions of bacterial srna targets are complicated by poor and incomplete complementarity.
a first small step towards unravelling the functions of the srnas in s. meliloti is provided by the expression patterns of a subset of srnas derived from this study.

 since the discovery of the first small non-coding rna in this class of untranslated transcripts of to nucleotides in length has become more and more evident in transcriptional and posttranscriptional regulation in prokaryotes. in addition to tmrna, s rna, 6s rna, and rnasep, which are related to house-keeping gene expression, a number of additional srnas was identified. these are broadly classified in two major populations, cis-encoded antisense srnas, oriented antisense to their target genes and trans-encoded srnas situated in distinct locations from their targets. srna-mediated posttranscriptional regulation was characterized in a variety of cell processes, e.g. transposition, bacterial virulence, quorum sensing, plasmid replication, function of toxin-antitoxin systems, and responses to different stress conditions. cis- and trans-encoded srnas each function in a different manner by interacting with short regions of mrna transcripts via perfect and imperfect sequence complementarity, respectively. the main mechanisms of srna-mediated control of gene expression are: repression or activation of translation, mrna degradation or stabilization, and target mimicry. srnas may act in different ways on different targets, e.g. the e. coli ryhb srna is a translational activator of shia and a repressor of sodb mrna. in addition, 5'-untranslated regions of bacterial mrna were found that regulate transcription attenuation and translation initiation in response to levels of specific metabolites or intracellular temperature. these mrna regions are called riboswitches. the metabolite effectors are generally able to mediate changes between alternative secondary structures by binding to a metabolite sensing domain of the rna which prevents translation. in contrast, rna thermometers are structures sensitive to temperature shifts. usually, these elements are located in the 5'-utr including the ribosomal binding site. increasing of the temperature permits destabilization of the secondary structure and releases the rbs for translation. for example rose-like rna thermometers are widespread in alpha- and gamma-proteobacteria and two putative candidates were found in sinorhizobium meliloti. to date, genome-wide profiling of srnas by experimental approaches was undertaken in several gram-positive and gram-negative bacteria. however, comprehensive experimental srna screening data is not yet available for the group of alpha-proteobacteria, with the exception of a tiling microarray-based transcriptome study in caulobacter crescentus. our study aimed at a genome-wide discovery of srnas in sinorhizobium meliloti that belongs to the rhizobiales of the alpha-proteobacteria. s. meliloti exists either in symbiosis with its leguminous host plants or in a free-living lifestyle. the bacteria associate with the plant root and induce the formation of nodules that become colonized by the bacteria via infection threads. inside the nodule, the bacteria differentiate into bacteroids that are capable of nitrogen-fixation to the benefit of the host plant. the genome of s. meliloti is composed of one chromosome and two megaplasmids, psyma and psymb. in addition to s rna, tmrna, and rnasep, the cis-encoded antisense srnas inca and suhb were previously identified in s. meliloti and related alpha-proteobacteria. inca mediates the posttranscriptional repression of the replication initiation protein-encoding gene repc, located in the repabc operon. this highly conserved operon is essential for replication, segregation and copy number of many extrachromosomal replicons in alpha-proteobacteria, e.g. the symbiotic megaplasmids in s. meliloti, the tumor inducing plasmid in a. tumefaciens and the second chromosome in brucella. suhb was first discovered in a. tumefaciens in opposite orientation to the suhb gene encoding an inositol-monophosphatase. in s. meliloti, four suhb paralogs were identified. three recent studies primarily applied bioinformatics approaches to the identification of srna candidates in s. meliloti. del val et al. employed a genome wide computational analysis of s. meliloti intergenic regions leading to candidates, eight of which were experimentally confirmed. ulvé et al. discovered novel srnas combining several computational approaches with microarray as well as northern and dot blot hybridizations for validation. computational predictions and microarray hybridization experiments were also combined by valverde et al. to screen the intergenic regions resulting in candidates that were confirmed as novel small non-coding rnas by northern blot and/or microarray hybridizations. in this study, we have performed a comprehensive experimental screening for srnas in s. meliloti applying deep sequencing technologies as well as oligonucleotide microarray and chip hybridizations. this approach resulted in transcription units that are novel candidates for trans-encoded srnas, cis-encoded antisense srnas, sense srnas or mrna leader transcripts suggesting that in s. meliloti about 3% of the genes encode trans-encoded srnas and about 2% antisense transcripts. expression patterns provided further information on conditions of expression of a number of srna candidates. sequence conservation analyses suggest strong similarities of a subset of s. meliloti srnas to regions in related alpha-proteobacteria.  srnas identified by deep sequencing data generation by and illumina/solexa sequencing of cdnas a considerable proportion of srnas is probably only transcribed at high levels under specific conditions. to increase the probability of discovery of these srnas by our screening approach, small rna fractions from a number of conditions were pooled for deep sequencing. these included exponential and stationary growth phases as well as shifts to low or high temperature, to low or high ph, to high salt concentration, and addition of h2o to cause oxidative stress. two small rna samples which were enriched for primary and processed transcripts were fractionated to a size range of to nt and subjected to gs flx titanium sequencing. a total of and reads were generated from sample and respectively. following the mapping to the s. meliloti reference genome, the reads were matched to their original strand taking advantage of the sequence of the 5'-rna adapter used in sample preparation. approximately 70% of all reads were mapped to rrna- or trna-encoding genes, or to repeat regions. excluding the reads that did not map to the genome, the remaining reads were assigned to either intergenic regions, open reading frames, or regions overlapping both. in addition to sequencing of the pool of small rna fractions obtained from the different conditions, the pool of total rna was subjected to illumina/solexa sequencing. approximately million reads were mapped to the reference genome. after removal of the reads that mapped to repeat regions, including the rrnas and trnas, the remaining reads were uniquely mapped either to orfs or igrs. transcript definition low abundant reads that may have originated from transcriptional background and mrna degradation were filtered-out by the following selection criteria employed to determine the contigs for further analyses. we defined a contig by a seed region of length l, covered by at least c reads. the seed region was extended on either side as long as read coverage was at least c. for reads, we used: l = 50- c = c = contigs were identified, with the majority associated to coding regions and the remaining contigs mapping to igrs. for the shorter illumina/solexa reads we used: l = c = c= contigs were identified, with the majority located inside coding regions and the remaining contigs mapping to igrs. these contigs were taken as the observed units of transcription in further analyses. transcript classification with respect to their positions relative to the neighboring or overlapping orfs, contigs were grouped into five classes: trans-encoded srnas, cis-encoded antisense srnas, mrna leader sequences, sense srnas overlapping with orfs, and transcripts completely covering an orf. we identified eight such orfs that were previously identified as genes encoding small proteins. putative srna genes were included in the gendb s. meliloti genome project http://www.rhizogate.de. rna type and subtype: class of transcripts according to the classification in figure replicon: c, chromosome; a, psyma; b, psymb. number: number of identified srnas per class, subclass and replicon. *candidates derived from illumina/solexa sequencing that did not allow for identifying the dna strand. therefore, subclassification is missing for these candidates. candidates for mrna leader transcripts are defined as short rnas that probably represent a stable derivative of the 5'-part of the mrna of a protein-encoding gene. this may be a processed form or a prematurely terminated mrna due to attenuation or riboswitch activity. since the transcription start sites of the majority of the protein-encoding genes are unknown, it cannot be excluded that in some cases a putative mrna leader rather represents an srna transcribed from a promoter different from that of the mrna. sense srnas represent short stable transcripts whose sequences are located within mrna sequences. most of these sense srnas were probably processed from mrnas. cis-encoded antisense srnas were further sub-classified into three groups depending on their relative positions in antisense to the 5'- and 3'-utr, or inside an orf. sense srnas were further divided into candidates situated inside another orf, transcripts overlapping the 3'-utr and the orf, and regions overlapping only the 3'-utr. sense transcripts overlapping the corresponding orf, but with a 5'-utr that was considered to be too short for translation initiation, were classified as type sense srnas and not as putative mrna leader transcripts. trans-encoded srnas were sub-classified as type if their orientation is antisense to both neighboring orfs, and else as type. since the illumina/solexa sequencing did not allow for identification of the transcribed strand, only trans-encoded srnas from this approach were considered for further analyses. taken together, both deep sequencing approaches revealed putative trans-encoded srnas. from these were exclusively derived from the illumina/solexa sequencing data. srna candidates associated to transposable element that often occur in multiple copies are not included in this number and are considered separately for the following analyses. transcription start sites and 3'-end information retrieved from deep sequencing data information on 5'- and 3'-ends of transcripts in sample enriched for primary transcripts and sample enriched for processed transcripts were retrieved from the sequencing data. this analysis indicated the positions of transcription start sites and 3'-ends of a number of srnas. two-thirds of the trans-encoded srnas displayed one or two distinct 5'-ends. the remaining transcripts possessed more than two or highly variable 5'-end positions which did not allow for identifying the transcription start sites. the proportion of transcripts with one or two distinct 5'-ends was even higher in the class of cis-encoded antisense srnas. only 23% of srnas from this class displayed multiple or highly variable 5'-ends. a similar distribution of 5'-ends was observed in the class of mrna leader transcripts. in the class of sense srnas, the proportion of transcripts with one or two defined 5'-ends was 72%. ends: 5' and 3'; number of defined 5'/3'-ends per transcript; s s s1&2: number of transcripts in sample and both samples, which match the defined 5'/3'-end criteria; total: number of transcripts in all samples which match the defined 5'/3'-end criteria. primary 5'-ends were identified for 74%, 46%, and 60% of the trans-encoded srnas, antisense srnas, and mrna leader transcripts, respectively. in contrast, only 18% of the transcripts classified as sense srnas displayed a primary 5'-end indicating that the majority of these transcripts was processed from mrnas of the associated protein-encoding gene. comparison of the primary transcript-enriched sample and the processed transcript-enriched sample revealed several different situations with respect to the derived transcript ends. candidates possessing identical 5'-ends were identified in both samples. alternative 5'-ends that may have been caused by posttranscriptional processing of the transcripts or activities of alternative promoters were observed for srna candidates. we also found a number of srnas with 5'-ends varying by only one or two nucleotides. upstream the transcription start sites of srna candidates the σ consensus promoter motif cttgac-n17-ctatat was predicted, further confirming the identified primary 5'-ends of these srnas. two promoters of this type were predicted upstream of smelb only sense srna candidates were preceded by this consensus promoter motif, further supporting the assumption that most of these transcripts were processed from mrnas. in all classes, about 60% of srnas exhibited multiple or highly variable 3'-ends. the remaining 40% comprised and srnas with one or two defined 3'-ends, respectively. in some cases more complex situations were observed. an example is the nt region comprising smelc111a and smelc111b located in the intergenic region between smc and smc this region displayed three putative transcription start sites detected in sample and two 5'-ends that were only found in the processed transcript-enriched sample furthermore, several 3'-ends were found in this region. this results in separate srna transcripts differing in length. thus, smelc111a and smelc111b probably occur as separate srnas and were classified as trans-encoded srna and leader transcript, respectively. in cases we found clusters of at least two srna candidates separated by less than nt. among these were nine clusters composed of two and two clusters comprising three trans-encoded srnas. only one protein-encoding gene was found to be associated with two antisense srnas, while approximately 18% of the sense srna-associated orfs include at least two candidates. interestingly, multiple srna candidates were found associated with six of seven genes of the rhizobactin operon. characteristics of srna classes the deep sequencing approaches revealed trans-encoded srnas, cis-encoded antisense srnas, mrna leader transcripts, and sense srna candidates. distribution of srna candidates on the three replicons shows a prevalence of trans-encoded srnas, mrna leader sequences and sense srnas on the chromosome. between 8% and 22% more srnas than expected from an equal distribution of srnas were found on this replicon. in contrast, 9% more cis-encoded antisense rnas than expected from an equal distribution were identified on megaplasmid psymb. srna candidate regions were almost evenly distributed over the whole genome. thus, clustering was only observed at the gene or intergenic region level. about half of all antisense srna candidates overlap with the 3'-end of the coding region and the 3'-utr. 18% overlap with the 5'-utr and 26% are located on the opposite strand inside the orf. the majority of sense srna candidates are located within the coding region of the associated gene or overlap the 3'-utr and the orf. transcription units in repeats and transposable elements a number of recent studies revealed non-coding transcripts within transposable elements or repeat regions. hence, the transcriptional activity of these regions in the s. meliloti genome was analyzed. this suggested repeat regions from two groups showing transcriptional activity. the first group comprises repeat regions, to nt in length, with transcriptional activity. these are associated with transposase genes, either in antisense, in sense orientation or as leader transcript. an example for this group is transposon trm that occurs in several copies on the chromosome as well as on the megaplasmids. it possesses a type antisense srna of nt which overlaps the 5'-end of the transposase gene. antisense srnas with similar features were found in trm and trm the partial transposases trm20c and trm5n are associated with a type antisense srna. furthermore, trm trm sma and sma possess sense srnas within the transposase-encoding region or overlapping its 3'-end. interestingly, a putative mrna leader was identified upstream of sma which presumably contains two distinct mrna leader-like transcription start sites. the second group comprises repetitive extragenic palindromic elements, repeats and three regions which include both rep elements and repeats in s. meliloti with transcriptional activity. identification and expression profiling of srnas by oligonucleotide microarray and chip hybridizations as a complementary approach to the deep sequencing strategy, oligonucleotide microarray and affymetrix symbiosis chip hybridizations were carried out using arrays that contain probes representing coding regions and intergenic regions. rna for these experiments was obtained from identical stress conditions and growth phases as previously applied for deep sequencing. in the oligonucleotide microarray hybridizations, signals derived from fractions composed of small and long rnas were compared. this strategy allowed for identifying sequence regions predominantly present in the small rna fraction excluding sequence regions that were also represented in the long rna fraction at a considerable level. applying a cut-off of ≥ to the ratio of signals derived from the small rna to signals from the long rna fraction srna candidate regions were identified. among these were the s rna, the inca srnas, and a number of trnas confirming the applicability of this strategy. according to the standard procedure of classification, candidates were classified as putative trans-encoded srnas. of these were mapped to intergenic regions oriented in antisense to neighboring genes but not overlapping the 5'- or 3'-utrs and thus classified as type trans-encoded srna candidates, whereas were classified as type sharing the same orientation as at least one of the two neighboring genes. the remaining candidates fell into the groups of cis-encoded antisense srnas and mrna leader transcripts. in support of the oligonucleotide microarray-based analysis, affymetrix symbiosis chip hybridizations were carried out using the small rna fractions from the experiments described above. signals were classified as small non-coding rna candidates when exhibiting the following characteristics: a signal intensity at least two fold higher than the background, and a distance of less than nt between two probes with a positive signal. candidates listed in were classified following the standard procedure. comparison of the microarray with the affymetrix symbiosis chip data revealed trans-encoded srnas, cis-encoded antisense srnas, and mrna leader candidates identified by both approaches. the candidates from the oligonucleotide microarray hybridizations were clustered on the basis of their expression profiles under the eight growth conditions tested. information on expression patterns of trans-encoded srnas with unambiguous 5'- and 3'-ends identified by deep sequencing could be retrieved from the microarray data. expression patterns could also be deduced for cis-encoded antisense srnas and mrna leader sequences that are also shared by both data sets. only of these trans-encoded srna candidates appeared to be generally expressed in all conditions. the other candidates from this srna class were detected only in a subset of conditions with the largest cluster comprising candidates only found in a single condition. clustering of the expression patterns of the putative cis-encoded antisense srnas shared by both data sets revealed candidates expressed in all conditions, whereas candidates showed expression in two to seven conditions, and candidates in only one condition. from the putative mrna leader transcripts identified by both approaches, were predominantly detected in the small rna fraction in two to five conditions, whereas were identified in only one condition. none of these leader transcripts was detected in all conditions. validation of selected candidates prediction of peptide-encoding srnas since a short transcript may have dual functions as regulatory srna and mrna, srna candidate sequences were screened for coding regions of at least nt preceded by a putative ribosome binding site. results of this analysis are summarized in. among the trans-encoded srnas, six candidates carry such a coding region, of which three are preceded by a predicted rbs. the class of antisense srnas includes candidates with a coding region, of which one possesses a putative rbs. in case of sense srnas, candidates were found that may encode a peptide and of these also carried an upstream rbs motif. different types of coding regions were found, orfs that overlap the corresponding gene or are located within the coding region of this gene. the reading frame of most of the predicted srna orfs differs from the reading frame of the corresponding gene, but share the same reading frame and hence the same stop codon with this gene. a similar situation was found in the class of mrna leader transcripts. here, candidates include an orf, of these with a potential rbs. these orfs do not include those starting with the start codon of the corresponding gene because these lack an inframe stop codon on the srna sequence. only orfs were included that map to the 5'-utr or overlap the coding region of the corresponding gene in a different reading frame. analysis of srna candidates by 5'-race and northern hybridizations srna candidates selected from different classes were further analyzed by 5'-race and northern hybridizations. race experiments confirmed the 5'-ends derived from the sequencing data of the trans-encoded srnas smelb smela smelb smelc smelb and smelb the cis-encoded antisense srna smela the mrna leader transcript smela and the intragenic sense srna smelb northern hybridizations were carried out for six candidates using total rna obtained from the exponential growth phase in complex medium, after shifting the culture to higher or lower temperature, and after adding salt to the culture. hybridizations of the trans-encoded srnas smelb smelc smelb and smelb showed signals in all conditions with just small variations in signal strength. in contrast, smela and smela060/smela showed differential expression. smela was only detected at a very low level in complex medium and in the same medium at decreased temperature. however, it was strongly detected after the shift from 30°c to 40°c and at a medium level in gms medium. smela and smela encode two srnas of the same length that differ by just one nucleotide. these srnas were detected only after the temperature shift to 40°c and after adding mm nacl to a culture in gms medium. microarray hybridization data is available for smela060/smela represented by oligonucleotide probes on the array. these results are in good agreement with the results of the northern hybridization, with the highest ratios obtained under heat shock and salt shock conditions, and values slightly above the threshold for srna detection in the other conditions. this indicates that expression patterns can be retrieved from the microarray data. the srna lengths of smelb and smelb estimated from the northern hybridizations are shorter than those derived from the sequencing data. this might be explained by incomplete denaturation of these srnas or by degradation processes during rna isolation. in case of smelb a weak band at the expected size was detected that may correspond to the full length contig derived from the sequencing data. northern hybridization of smel resulted in two close bands which may have originated from alternative 3'-ends found by cdna sequencing. two dominant bands were also detected for smela and smela060/smela the former are also indicated by the sequence coverage whereas no hint on the origin of the additional band could be found for smela060/smela interestingly, a structure composed of three stem-loops with identical sequence motifs in the loops was predicted for smela. shorter transcripts suggested by northern hybridizations and cdna sequencing were predicted to lack one or two of the stem-loop substructures. stable structures corresponding to predicted structural domains of the full-length transcripts were also predicted for shorter transcripts associated to the srnas smelc smelb and smelb. this is not the case for the putative mrna leader transcript smela the structure predicted for the short 5'-transcript differs from that of the full-length rna. z-scores computed for the srna candidates significantly differ from zero indicating that these srnas form a well-defined secondary structure. predicted structures and z-scores of the srnas analyzed by northern and 5'-race are shown in figures and bioinformatics analysis of srna candidates blast homology search blast homology searches for the transcripts from sequencing were carried out at different cut-offs. we report here on results for an e-value cut-off at 7e- which is the most stringent cut-off for which all our transcripts match against their origin genome. the match covering at least 70% of the transcript length was an additional requirement. data for this setting is shown in table under these criteria, we obtained about hits in alpha-proteobacteria, and about in other bacteria. hits were classified as "known" if there was annotation information associated with the matched region. as expected, the class of sense transcripts comprised the highest percentage of "known" matches, followed by mrna leaders. the low percentage for antisense and trans-encoded transcripts can be explained by the fact that only few transcripts of this type are annotated in the databases. data for other blast cut-offs are given in. results of the blast search with srna trancripts against sequence databases with an e-value cut-off 7e- and the requirement that the hit covers at least 70% of the query are shown. hits are classified as known if annotation information is associated with the database sequence. hit counts are given in taxonomic order. hits to the taxonomic group do not include the hits to the subgroup which is listed above it. for the alpha-proteobacteria, the percentage of "known" versus "unknown" hits for each class of transcript is listed. y, known; n, unknown. structural analysis we investigated whether our transcripts appear to have a well-defined secondary structure. rather than looking at individual minimum free energy foldings, we computed accumulated boltzmann probabilities of all foldings of the rna which exhibit the same abstract shape. we determined the dominant shape for each sequence and computed z-scores against a background distribution taken from the s. meliloti genome. the result is shown in figure which shows that classes of trans-encoded, sense and antisense trancripts tend to have positive z-scores, centered around + this implies that for these classes a well-defined secondary structure may be associated with their function. leader transcripts showed no bias. standard rfam homology search from the non-coding srnas from s. meliloti already known in rfam, belonging to families, our approach confirmed by direct positional matching, although with corrections in their precise position. these were also returned by standard rfam search with regular cut-offs. beyond these known transcripts, candidates were classified as - heretofore unknown - members of different rna families, excluding three families of hiv sequences attracting hits, which were considered false positives. of the remaining families included known members from s. meliloti. the only "new" families resulting from this search were rf and rf, the latter of which appears biologically implausible. hence, members of only one new family were identified applying a standard rfam search. refined rfam homology search we performed a simple but complete evaluation of the family models presently available in rfam, comparing scores obtained from the covariance models to scores from plain sequence models generated from the same sequence alignments. we observed that most of the covariance models strongly rely on sequence similarity. they practically behave as hmm sequence models, with the encoded structure only contributing to the score in a negligible way. a diverged sequence with a conserved structure will rarely pass the model thresholds, explaining the lack of generalization. as known srnas are scarce in the class of alpha-proteobacteria, we sought a way to extract more information from rfam models. we designed a pipeline using rnasifter that combines independent evidence of structural conservation with an rfam search below the suggested cut-offs. by this procedure, we obtained families and families above cut-offs of *t and *t, respectively, where t is the default cut-off of the family. these candidates require individual analysis; an encouraging observation about the refined search strategy is the correct classification of smela as a group ii intron, which was neither observed with the standard rfam search nor applying a blast search. the list of new families is given in. discussion identification of srna candidates in s. meliloti here we report on the first comprehensive screen for srnas in an alpha-proteobacterium applying deep sequencing and microarray hybridizations. the symbiotic nitrogen-fixing soil bacterium s. meliloti was analyzed as representative of the rhizobiales. our study suggested a total of srna candidates that were classified as trans-encoded srnas, cis-encoded antisense srnas, mrna leader transcripts, and sense srnas overlapping coding regions. these results are in good agreement with a number of recent studies that reported genome-wide screens for prokaryotic srnas by cdna sequencing and hybridization to tiling microarrays. the first studies of this type were reported for e. coli, but similar studies were also published for other gram-negative bacteria and gram-positive bacteria. in the group of alpha-proteobacteria, only one global srna screening study was published so far, which was a tiling microarray-based srna screen in c. crescentus that resulted in novel srna candidates. for alpha-proteobacteria, only one pilot study applying pyrosequencing of cdna derived from total rna depleted for rrna sequences of s. meliloti was reported. but this approach aimed at identifying transcripts of new protein-encoding genes and delivered just reads derived from non-rrna sequences that indicated new orfs. depending on the methods and strategies of data analysis applied, and the selection criteria for srna candidates, these studies suggested up to several hundred srna candidates in different bacteria. this indicates that not only mirna genes in eukaryotes but also srna genes in bacteria represent a significant proportion of the genome, although only a small proportion of these candidates was further validated. srna discovery by deep sequencing for discovery of srnas in s. meliloti we have combined two deep sequencing technologies. sequencing of cdnas turned out to be the most valuable data resource. sequence coverage defined 5'- and 3'-ends of the srna candidates which are of great advantage for both structural and comparative analyses. moreover, enrichment of primary 5'-ends of transcripts allowed for identifying transcriptional start sites of srna candidates with several of these validated by 5'-race. data from enrichment of processed 5'-ends was ambiguous with a very high proportion of 5'-ends present in both the primary and the processed transcript-enriched samples. this effect may have been caused by a pyrophosphatase activity which eliminates the pyrophosphate from the 5'-end of primary transcripts making these transcripts accessible for the sequencing procedure followed in the processed transcript-enriched sample. pyrophosphatase activity was recently described in several bacteria, such as the nudix enzymes rpph in e. coli and bdrpph in bdellovibrio bacteriovorus. nevertheless, a number of srna candidate regions displayed alternative 5'-ends that may have been caused by posttranscriptional processing of the transcripts. processing of transcripts at the 5'-end is a well characterized mechanism, an example of which is the srna gady in e. coli. we also found a number of srnas with transcription start sites varying by only one or two nucleotides. these were probably generated by the activity of a single promoter with a diffuse transcription start site, as was previously described for the sral srna. alternatively, transcripts with more than one distinct 5'-end may be products of alternative promoters. alternative 3'-ends observed in this study might be generated by endo- or exonucleolytic cleavage of the full length transcript. examples of srnas processed by such mechanisms are micx in vibrio cholerae and arcz in e. coli and salmonella. the former is processed by rnase e into two transcripts with different 3'-ends whereas the primary and 5'-processed transcripts of the latter undergo 3'-exonucleolytic degradation. another possible mechanism that results in alternative 3'-ends is the presence of two or more termination sites. for instance, gcvb of e. coli possesses two termination sites, resulting in two distinct transcript sizes. illumina/solexa sequencing of cdna derived from total rna revealed only few additional trans-encoded srna candidates. the deep sequencing data from this study suggests an average length of the srna candidates of about nt. this is in good agreement with results from several other studies that deduced a typical size range of srnas of to nt. filtering and classification of srna candidates the stringency of criteria for selection of srna candidate regions based on cdna sequencing data has a great effect on the number of candidates derived from this approach. in this study, we have based the sequence data analyses on strict criteria that defined seed regions of to nt in length with a coverage of at least reads per nucleotide. selection criteria were relaxed to a minimum of five reads per nucleotide to complete the contigs. definition of a lower coverage would have resulted in a higher number of candidate regions with an increasing number of false positives from transcriptional background and mrna degradation. classification of srna candidates in putative trans-encoded srnas, cis-encoded antisense srnas, mrna leader transcripts, and sense srnas overlapping coding regions is ambiguous. the main difficulty is the definition of a gene region including 5'- and 3'-utr, since promoter and terminator predictions are not well established in s. meliloti. therefore, the classification was based on the estimated minimal length of 5'- and 3'-utrs which might have resulted in misclassification of a number of candidates. moreover, the majority of sense srnas completely or partially overlapping coding regions probably are stable mrna degradation products. several previous studies provided also evidences for the presence of antisense srna and mrna leader transcripts in addition to the intergenic trans-encoded srnas. cis-encoded antisense srna transcripts appear to be very dominant in synechocystis sp. pcc georg et al. suggested that about 10% of all genes in this organism are influenced by antisense rnas. recently, evidence for antisense rnas that may affect about 3% of the protein-encoding genes was obtained by parallel sequencing in vibrio cholerae. such a high proportion of antisense transcripts as in synechocystis sp. pcc was not observed in our study in s. meliloti applying deep sequencing. only about 2% of the protein-encoding genes seem to be partly transcribed in antisense direction. in contrast, our microarray experiments suggested about 11% of genes with overlapping antisense transcripts. such discrepancy may be organism-specific, but probably is more influenced by employing different discovery strategies. in this study, a multi-tiered approach to srna identification was taken applying different complementary identification strategies. illumina/solexa sequencing was carried out for cdna derived from total rna which resulted in a proportion of 87% of reads mapping to rrna genes or repeat regions. usage of total rna and the inability to retrieve strand information from this sequencing data did not allow for identifying srnas associated to coding regions, but identified intergenic srna genes and revealed information on transcription units of protein-encoding genes. in contrast, the sequencing approach including size fractionation and primary 5'-end enrichment provided rich information on short transcripts encoded in intergenic regions as well as antisense srnas and leader transcripts associated to coding regions. a microarray approach cannot provide exact information on 5'- and 3'-ends of srnas and is limited by the genome coverage with oligonucleotide probes. the sm14koli microarray used in this study contained probes distributed on both strands of intergenic regions at irregular distances. coding regions were just represented by a single probe. the strategy of comparing short rna to long rna fractions was therefore tailored to predominantly identify intergenic srna genes. in comparison to the high number of microarray hybridizations covering different conditions, only a low number of hybridizations of short rna to the affymetrix symbiosis chip were performed resulting in only few reliable srna candidates. because of the different strengths of the applied strategies, a reasonable comparison of identified candidates is only possible for the class of trans-encoded srnas. about 40% of the trans-encoded srnas identified by the sequencing approach were also identified by the illumina/solexa sequencing and/or the microarray approaches. only candidates were identified by all approaches. it has to be noted that these numbers change with relaxing the strict criteria applied in analysis of the deep sequencing data. classification and determination of srna ends is more reliable based on the sequencing-derived dataset. because of the short illumina/solexa reads as well as distribution and length of microarray probes, the definition of the regions is less accurate based on these approaches. this explains discrepancies in classification of a few srna regions and mapping of some srna regions identified by one method to multiple regions identified by one or both of the other approaches. deep sequencing data confirmed previously reported srnas in addition to s rna, tmrna, trnas and rrnas, our deep sequencing approach confirmed previously published srna candidates. the tmrna, the two inca antisense transcripts, and two rose-like elements were found in studies dedicated to one or few srnas, whereas the other candidates resulted from global screening approaches. overlap, percent overlap of the candidates from the cited studies or database compared to the region covered by a srna candidate in this study. n, strand was not or not undoubtedly determined. id; rna id; type, rna type; st., strand; ov., overlap; id, rna id corresponding to the given reference; north., experimentally validated by northern blot, given are the references for the northern hybridizations. rna id: *, solexa candidate. rna type: trans-encoded srna; cis-encoded antisense srna; mrna leader; sense srna; open reading frame. the presumed location of the srnas predicted by ulvé et al., derived from the location of the dot blot probes. thus an overlap analysis to our candidates could not be performed. among the mrna leader transcripts were two candidates that corresponded to the predicted rose-like elements upstream of ibpa and smb encoding heat shock proteins in s. meliloti. it has to be noted that the ibpa rose element was represented by only eight reads in the sequencing data and thus marginally failed our filter for candidate selection. maclellan et al. characterized the cis-encoded antisense srna gene inca situated within the repabc operon on each megaplasmid. inca is located within the small intergenic region between repb and repc in opposite orientation to these coding regions. high sequence coverage of both inca srnas was also evident in the sequencing data. the s. meliloti tmrna was identified because of its homology to sra from bradyrhizobium japonicum. northern blot analysis and mapping of both 5'- and 3'-ends identified two pieces, a nt mrna-like domain and a nt trna-like domain, both highly stable, whereas the premature form was unstable. both transcripts were also identified in our study with a high sequence coverage. the s. meliloti strain used in this study harbours three classes of group ii introns, rmint, smb21477/smb, and sma. transcript smela probably corresponding to the intron lariat rna was identified by the sequencing approach. our sequencing data supported srna candidates that resulted from the previous genome-wide screens of which were confirmed by northern hybridizations. from the trans-encoded srnas identified in our study, have previously been predicted. this high percentage of re-discovery of known srna candidates increases the confidence in the new putative srnas. in the classes of antisense srnas, mrna leader transcripts, and sense srnas, only few known candidates were re-identified. these were out of antisense srnas, out of mrna leader transcripts, and out of sense srnas. hence, our approach resulted in a high number of new candidates. furthermore, additional information on 5'- and 3'-ends as well as expression patterns under several growth and stress conditions could be derived for a number of previously described candidates from the sequencing and microarray data. in several cases our srna regions differed in orientation from, only partially overlapped, or were located inside the regions predicted in other studies. our approach also confirmed rfam annotated transcripts of s. meliloti. putative srna functions in s. meliloti putative functions of srnas trans-encoded and cis-encoded antisense srnas predominantly seem to act via base pairing mechanisms. the former usually are located in intergenic regions and share only poor and incomplete complementarity with target sequences, which complicates computational target predictions. in contrast, the latter share extended regions of complementarity to an overlapping sense encoded gene. thus, in most cases antisense srnas modulate expression of the overlapping sense gene on transcriptional or posttranscriptional level, although trans targets may also be affected. the majority of antisense srnas identified in this study are complementary to the 3'-utr of the corresponding gene. it is well established that this type of antisense rnas is involved in posttranscriptional regulation of the steady-state amount. thus, either mrna stabilization by masking of potential ribonuclease recognition sites or mrna degradation by formation of a ribonuclease sensitive rna duplex are probably mediated by the antisense srnas mapping to the 3'-utr. only cis-encoded antisense srnas were located in small intergenic regions in antisense to both neighboring genes which presumably form an operon structure. this type of srna may mediate transcription termination inside the operon structure resulting in down-regulation of the downstream gene expression. microarray hybridizations supported a number of srna candidates identified by the deep sequencing approach and provided further information on expression patterns in different growth phases and stress conditions. a group of trans-encoded srna candidates, antisense srnas, and mrna leader candidates were found only in one of the eight conditions tested. this indicates a specific expression pattern and provides hints to the biological context of their function. among these were the antisense srna smela found under oxidative stress as well as smelc and smelb found under salt stress conditions. of the mrna leader candidates were only found in response to heat stress. the majority of cis-encoded antisense srnas have been described in plasmids, phages and transposons. such srnas probably inhibiting translation of the transposase are encoded by tn and tn. transposon-encoded antisense rnas are quite stable which may explain the high sequence coverage of the majority of the transposable element-related srna candidates found in our study. another group of repeats that were represented in the small rna fraction analyzed in this study were rep elements. although these elements are widely distributed within the genome, their functions remain obscure. it has been shown that their presence can affect the level of gene expression. it was speculated that these elements play a role in gene transcription termination. another proposed role is in relation to chromosomal structure, because both dna gyrase and dna polymerase i can bind to rep elements. functional classification via database search an important observation from this study is that searching rfam family models with over srna candidates only re-discovered those sequences from s. meliloti already known and stored in rfam. there was not a single case of an unclassified transcript that was predicted to be a member of a certain family, except for the ire family and some apparent false positives. this is in strong contrast to the state of the art with protein-coding genes. it can be explained in part by the fact that identification of srnas in bacteria is still at its outset, and in part by the fact that current rfam models are very strongly based on sequence similarity rather than conserved structure. alternative ways to construct structural family models have been recently suggested, but an implementation is not yet available. our refined rfam search strategy yielded family associations which are still awaiting validation. the blast search suggested a number of homologous regions, mainly in the rhizobiales but also in other alpha-proteobacteria, indicating that srna candidates derived from the comprehensive experimental screen in s. meliloti may support the computational prediction of srnas in related bacteria.  genome-wide experimental screening for srnas several genome-wide screens for srnas applying parallel sequencing of cdnas and hybridizations of tiling microarrays have recently been published. apart from a single study that used tiling oligonucleotide chips to discover srnas in c. crescentus, our study is the first comprehensive approach to discover srnas in an alpha-proteobacterium. therefore, this data set will be a valuable resource for comparative studies in related bacteria. identification of homologous srnas and assignment to rna families assigning srna candidates to functional rna families with present bioinformatics tools is a difficult task. a recent study shows that even human expertise in describing structural patterns does not take us much beyond a simple blast search. the study argues for an effort in developing curated databases of aligned secondary structures, as a prerequisite for increasing the power of comparative analysis. another perspective is that the use of structural models from locomotif, which include the thermodynamics of rna folding and can handle simple cases of pseudoknots, may generalize better than the combinatorial pattern descriptions via rnamotif used in the cited study. perspectives for functional analyses global experimental screening strategies have delivered a high number of srna candidates in all bacteria studied so far. in contrast to these well-developed discovery strategies, approaches to identify targets of srnas in high throughput are still in the infancy. experimental approaches mainly rely on transcript or protein profiling in srna mutant or over-expression strains, and in vitro analysis of srna-mrna interaction. in contrast to computational predictions of eukaryotic microrna targets, predictions of bacterial srna targets are complicated by poor and incomplete complementarity. a first small step towards unravelling the functions of the srnas in s. meliloti is provided by the expression patterns of a subset of srnas derived from this study. 
enrichment for rnas with primary 5'-ends prior to sequencing of cdnas suggested transcriptional start sites corresponding to predicted srna regions. searching rfam family models with over srna candidates, re-discovered only those sequences from s. meliloti already known and stored in rfam, whereas blast searches suggested a number of homologs in related alpha-proteobacteria. the screening data suggests that in s. meliloti about 3% of the genes encode trans-encoded srnas and about 2% antisense transcripts. expression patterns derived from microarray hybridizations provided further information on conditions of expression of a number of srna candidates. this study reports on a comprehensive screen for srnas in the symbiotic nitrogen-fixing alpha-proteobacterium sinorhizobium meliloti applying deep sequencing of cdnas and microarray hybridizations. thus, this first comprehensive screen for srnas applying deep sequencing in an alpha-proteobacterium shows that srnas also occur in high number in this group of bacteria. a total of srna candidates that were classified as trans-encoded srnas, cis-encoded antisense srnas, mrna leader transcripts, and sense srnas overlapping coding regions were identified in a size range of to nucleotides. small untranslated rnas are widespread regulators of gene expression in bacteria. among these were transcripts corresponding to previously reported srna candidates. the consensus σ promoter motif cttgac-n17-ctatat was found upstream of srna candidates. furthermore, genbank, embl, ddbj, pdb, and rfam databases were searched for homologs of the srna candidates identified in this study.
 small untranslated rnas are widespread regulators of gene expression in bacteria. this study reports on a comprehensive screen for srnas in the symbiotic nitrogen-fixing alpha-proteobacterium sinorhizobium meliloti applying deep sequencing of cdnas and microarray hybridizations.  a total of srna candidates that were classified as trans-encoded srnas, cis-encoded antisense srnas, mrna leader transcripts, and sense srnas overlapping coding regions were identified in a size range of to nucleotides. among these were transcripts corresponding to previously reported srna candidates. enrichment for rnas with primary 5'-ends prior to sequencing of cdnas suggested transcriptional start sites corresponding to predicted srna regions. the consensus σ promoter motif cttgac-n17-ctatat was found upstream of srna candidates. expression patterns derived from microarray hybridizations provided further information on conditions of expression of a number of srna candidates. furthermore, genbank, embl, ddbj, pdb, and rfam databases were searched for homologs of the srna candidates identified in this study. searching rfam family models with over srna candidates, re-discovered only those sequences from s. meliloti already known and stored in rfam, whereas blast searches suggested a number of homologs in related alpha-proteobacteria.  the screening data suggests that in s. meliloti about 3% of the genes encode trans-encoded srnas and about 2% antisense transcripts. thus, this first comprehensive screen for srnas applying deep sequencing in an alpha-proteobacterium shows that srnas also occur in high number in this group of bacteria. 