BACKGROUND
in bacterial genomes the non-coding rnas  identified were the ribosomal rnas  and transfer rnas , which are important components of the protein synthesis machinery .
in addition, cis-encoded antisense rnas  and trans-encoded small rnas  were also identified.
analyses of asrnas and srnas targets have shown that these ncrnas could alter the translation process or mrna stability by target base pairing .
moreover, srnas may have multiple targets  <cit> .
non-coding rna elements present in many bacterial genomes add a further complexity to the comprehension of bacterial gene regulation  <cit> .
recently, several ncrnas with different genomic origins, lengths, functions, and gene regulation mechanisms have been identified .
there are evidences that ncrnas may regulate important processes, such as pathogenesis, iron metabolism, and quorum sensing  <cit> .
novel ncrnas are difficult to detect by conventional biochemical screenings  <cit> .
as an alternative, in silico approaches associated to a functional analysis validation have proved to be effective in the identification of ncrnas .
in at-rich genomes, the ncrna genes show a relatively higher gc-content  <cit>  and therefore, compositional-based analyses that scan for local gc-content have had success in detecting ncrnas.
the algorithm single genome ncrna search   <cit>  is a whole-genome eukaryote predictor that uses these features.
given that most functional rnas rely on a stable secondary structure, prediction of the minimum free energy of a transcript is also used as a means of detecting ncrna genes  <cit> .
target prediction is the main step in understanding bacterial srna function.
recently, computational target prediction methods had their performance improved by inclusion of rna accessibility and conservation information .
interacting rna  and rnaplex are reliable srna target prediction software  <cit> .
intarna uses the energy score of the interaction, which is calculated as the sum of the free energy of hybridization and the free energy required for making the interaction sites accessible  <cit> .
rnaplex  <cit>  is a refinement of the rnahybrid software and uses a simplified algorithm to reduce the time needed to localize putative hybridization sites, mainly by neglecting intramolecular interactions and by using a slightly simplified energy model.
the rnaplex tool is combined with the rnaup tool and can find high-confidence targets, with only a slight loss of sensitivity.
moreover, rnaplex also uses an energy score of the interaction of srna and putative targets to predict molecule interactions.
mycoplasmas are bacteria of the class mollicutes characterized by small genomes and low gc content.
mycoplasma hyopneumoniae, mycoplasma flocculare and mycoplasma hyorhinis are important species that have been identified in the porcine respiratory system .
m. hyopneumoniae is the etiological agent of porcine enzootic pneumonia  <cit> , while m. hyorhinis can also cause swine polyserositis and arthritis  <cit> .
although m. flocculare is widespread in swine herds, it has so far been recognized as a commensal species  <cit> .
currently, the genome sequences of several mycoplasma species are available, allowing a comparative analysis of the gene content among different species.
however, information related to regulatory elements is very limited in these organisms.
furthermore, the mycoplasma species present a low number of proteins involved in transcriptional regulation  <cit> .
these evidences suggest the presence of alternative transcriptional regulatory mechanisms in mycoplasmas.
it was previously shown that most genes from the genomes of m. hyopneumoniae, m. flocculare and m. hyorhinis are expressed at some basal level and that the majority of the genes are co-transcribed  <cit> .
therefore, a global determination of the genomic functional elements is a prerequisite to expand our knowledge regarding transcriptional small rna regulation in swine respiratory mycoplasmas.in the current study, we have analyzed and predicted trans-encoded small rnas from m. hyopneumoniae, m. flocculare and m. hyorhinis genomes.
moreover, we have analyzed rna-rna interaction and accessed target genes for the srna candidates.
some predicted m. hyopneumoniae srnas were also experimentally investigated by a reverse transcription amplification approach in three different culture conditions.
RESULTS
global screening for small rnas
knowledge related to the presence and role of small rnas in mycoplasma remains limited, therefore a genome-wide screen for srnas was conducted using in silico prediction approaches.
only srnas present in the intergenic regions  were searched as the input file, since all regions marked as coding sequence  and known ncrnas, such as rrna, trna, rnase p and others, were masked.
these igrs have a gc content of 21 % for m. hyopneumoniae, 23 % for m. flocculare and 21 % for m. hyorhinis representing, respectively,  <dig>   <dig> and 14 % of the total genome.
the segments with a high gc cumulative, comparable to the known ncrnas, were considered srna candidates.
the analysis of the m. hyopneumoniae genome using the sigrs software allowed the identification of  <dig> previously known ncrnas and  <dig> predicted putative novel srnas.
however, the sigrs outputs associate with distinct regions, defined some of them as unique ncrna candidates.
therefore, a fragmentation algorithm  was applied, resulting in  <dig> previously known ncrnas  and  <dig> putative novel srnas, named srna_ <dig> to srna_ <dig> .
the sequences of these predicted srnas including their information related to genome location, length, gc content, and free energy of the secondary structure of the srna are supplied in additional file 2: table s <dig>  the rnaspace software recovered 87 % of the sigrs/fraps predictions supporting the previous sigrs/fraps results for the srna genes.
m. hyopneumoniae srnas have an average length of 128 bp, ranging from  <dig> to 424 bp and  <dig>  % of gc content.
furthermore, the minimum fold energy  among the putative srnas varies from − <dig>  kcal/mol to − <dig>  kcal/mol .
the srna sequences were analyzed by blast search to locate homologous sequences in the genomes of the other m. hyopneumoniae strains available.
remarkably,  <dig> predicted novel srnas  were present in all m. hyopneumoniae genome strains , while only the srna_ <dig> was exclusive to the m. hyopneumoniae  <dig> genome .
interestingly, only the srna_ <dig> was identified in the genomes of all pathogenic strains and was absent in the genome of the non-pathogenic m. hyopneumoniae j strain.
moreover, srna_ <dig> was found only in the genome of the two pathogenic strains  <dig> and  <dig> isolated from brazilian swine herds.
in order to evaluate if some of the predicted srna genes were transcribed, a stem-loop-rt-pcr analysis was used to investigate the presence of srna transcripts in three different culture conditions.
figure 1b illustrates the primers design for the stem-loop-rt-pcr approach.
primer pair positions were based on a full-length srna prediction, for both strands using specific primers for each srna tested.
the low gc content of the m. hyopneumoniae genome and the mandatory full-length primers position with high specificity resulted in  <dig> srnas subject to experimental analysis.
it was possible to show that all the  <dig> predicted and experimentally analyzed srnas were detected as transcribed in the three different growth conditions, except for srna_ <dig> and srna_ <dig>  which were not transcribed in the standard condition or heat shock condition, respectively .fig.
<dig> schematic representation of srna position at m. hyopneumoniae  <dig> genome and primers design.
a overview of the location of srna_ <dig> in the m. hyopneumoniae  <dig> genome region from  <dig>  to  <dig>  base pair.
white arrows represent the genes position and the srna_ <dig> is represented by a gray box.
representation from artemis.
b primers location for the rt-pcr stem-loop reaction.
slim arrows indicate the amplicon orientation.
black portions represent amplification and gray portions represent the primers location.
the amplification from specific forward primer to universal reverse primer is the entire srna length
the possible presence of sequences homologous to the novel srnas predicted for m. hyopneumoniae in other mycoplasma species, also found in the swine respiratory tract, was analyzed by blast search.
sequences homologous to the novel srnas of m. hyopneumoniae were not found in the genomes of m. flocculare and m. hyorhinis, supporting the notion that these novel srnas are species-specific.
therefore, the genomes of m. flocculare and m. hyorhinis were screened for the presence of srnas applying the same methodology used for m. hyopneumoniae.
using sigrs plus fraps, it was possible to predict  <dig> novel srnas for m. flocculare, which were named srna_f <dig> to srna_f <dig>  and  <dig> novel srnas for m. hyorhinis, which were named srna_r <dig> to srna_r <dig> .
rnaspace predictor was able to identify approximately 50 % of the sigrs/fraps predictions, for both genomes.
furthermore, the main profile of the new predicted srnas was similar to that found for the srnas predicted in m. hyopneumoniae.
the srnas of m. flocculare have an average length of 167 bp, ranging from  <dig> to 402 bp and  <dig>  % of gc content , while the minimum fold energy  among the putative srnas varies from − <dig>  kcal/mol to − <dig>  kcal/mol .
the srnas of m. hyorhinis have an average length of 85 bp, ranging from  <dig> to 209 bp with  <dig>  % of gc content and the minimum fold energy  varied from − <dig>  kcal/mol to − <dig>  kcal/mol .
a homologous sequence search was carried out for each novel putative m. flocculare and m. hyorhinis srna in the available genomes of other m. flocculare, m. hyorhinis and m. hyopneumoniae strains.
all predicted srnas were present only in the corresponding genomes of m. flocculare  or m. hyorhinis , as shown in additional file 4: table s <dig> and additional file 5: table s <dig>
small rna targets prediction
the importance of srnas as a distinct class of gene regulators in bacteria is well established, as many diverse processes have been shown to be controlled by srnas in different species  <cit> .
the interaction of srnas with different targets is an important mechanism to control the complex regulatory networks in bacterial cells.
therefore, to analyze the interaction of srnas to a multitude of different target mrnas and their role in gene regulation, the binding region of the srna:target duplex needs to be investigated.
a computational analysis of the potential targets for the srnas identified using rnaplex and intarna predicted  <dig> targets for the srnas of m. hyopneumoniae ,  <dig> targets for the srnas of m. flocculare  and  <dig> targets for the srnas of m. hyorhinis .
an interaction was deemed functional only if respecting both criteria free energy smaller or equal to a threshold of −13 kcal/mol and equally predicted by the two software.
detailed information  related to the srnas/targets interactions is available in each additional file.
the in silico predictions identified interaction with target genes for  <dig> novel m. hyopneumoniae srnas.
the majority of the srnas showed multiple target genes varying from  <dig> to  <dig> , however most of them  interacted with up to seven target genes .
considering the whole genome of m. hyopneumoniae,  <dig> genes out of  <dig> showed a predicted interaction with at least one novel srna, representing 21 % of the m. hyopneumoniae genes used as input .
in general, the genes that showed such predicted interaction were also found to pair with multiple srnas, although some genes interact with only a single srna.
from the  <dig> genes,  <dig>  were found as single target, meaning that the mrnas encoded by these genes could potentially interact with only one of the predicted novel srnas .
the products of these genes are mainly related to basal cell metabolism, transport system and lipoproteins; many products are related to protein synthesis while most are related with unknown products .
a detailed analysis of the data  revealed that approximately 25 % of the target genes were predicted as capable to interact with up to three srnas.
nevertheless, some targets are ubiquitous: for example the gene aaz <dig> , which encodes the exonuclease protein, showed interactions with six predicted srnas.
another gene with multiple srna interactions is aaz <dig>  gene, which codes for the prolipoprotein p <dig>  which may interact with three different srnas.
the three novel srnas without target prediction were srna_ <dig>  srna_ <dig> and srna_ <dig>  detailed analysis of the interaction predictions revealed that these srnas have putative target genes found by the two algorithms, however the energy interaction in the intarna software was higher than −13 kcal/mol.
the srna_ <dig>  experimentally analyzed, has two putative target genes: mhp7448_ <dig>  encoding glycine cleavage system h protein, and the mhp7448_ <dig>  encoding a hypothetical protein, with energy interactions around −12 kcal/mol for both of them.
in turn, the srna_ <dig> and srna_ <dig> interact with targets with energy around −10 kcal/mol as: the mhp7448_ <dig>  and mhp7448_ <dig>  as targets for srna_27; and mhp7448_ <dig> , mhp7448_ <dig>  and mhp7448_ <dig>  as targets for srna_ <dig> .
similarly to the m. hyopneumoniae results, rnaplex and intarna predicted target gene interactions for  <dig> novel m. flocculare srnas and for all novel m. hyorhinis srnas.
most of the srnas were predicted to bind to multiple targets and the number of targets for each srna ranged from one to nine for m. flocculare and from one to  <dig> for m. hyorhinis .
as shown in additional file 4: table s <dig> and additional file 5: table s <dig>  the majority of the predicted novel srnas from both genomes were able to interact with a restricted number of target genes.
the srnas with the higher interaction number were the srna_f <dig> and the srna_r <dig>  with  <dig> and  <dig> target interactions, respectively.
data analyses, as presented in additional file 9: table s <dig> and additional file 10: table s <dig>  showed that  <dig> of the encoded mrnas of m. flocculare and  <dig> of the encoded mrnas of m. hyorhinis interact with at least one novel srna, representing, respectively,  <dig> and 6 % of all the genes used as input.
as expected, the different products of the target genes have different metabolic functions as observed in the m. hyopneumoniae analysis.
some of the genes encode for enzymes involved in cell metabolism, cell division proteins, transcription regulation, adhesins, while a high number of the gene products are classified as unknown or hypothetical proteins.
the m. flocculare srnas without target prediction were srna_f <dig> and srna_f <dig>  detailed analysis of the interaction predictions shows these srnas with putative target genes found by the two algorithms, however the energy interaction in the intarna software was higher than −13 kcal/mol.
focusing in these in silico interactions, the srna_f <dig> was able to bind to genes rpsk , trxa , and mf <dig> .
furthermore, srna_f <dig> could interact with mf <dig> and mf <dig>  and also with mf <dig> .
the interaction energies for both srnas were around −10 kcal/mol.
discussions
small bacterial rnas generally act by base pairing with mrnas, regulating many aspects of bacterial physiology leading to positive or negative regulation of target protein synthesis.
to search for srnas in the genomes of m. hyopneumoniae, m. flocculare and m. hyorhinis, we combined in silico prediction approaches with transcription analysis .
the in silico prediction of novel trans-acting srnas in the three mycoplasma genomes was performed by applying two different methods, ncrna search  and rnaspace, allowing the in silico identification of srnas following the combination of the results generated by both algorithms.
using this approach,  <dig>   <dig> and  <dig> putative novel srnas were predicted in the genomes of m. hyopneumoniae, m. flocculare and m. hyorhinis, respectively.
the average length of these putative srnas in the genomes of the three mycoplasma species ranged from  <dig> to 167 bp.
previously, the availability of transcriptome maps of m. hyopneumoniae, m. flocculare and m. hyorhinis allowed the in silico identification of  <dig>   <dig> and  <dig> putative novel ncrnas, respectively, with lengths ranging from  <dig> to  <dig> nucleotides  <cit> .
however, this approach was unable to identify the srnas among the ncrnas  <cit> .
although in escherichia coli the majority of the new srnas, varying in length from  <dig> to  <dig> nucleotides, have been identified through an in silico prediction  <cit> , the application of this proposed methodology to other bacterial species has had only limited success as it requires reliable species-specific consensus promoter and terminator sequences.
in vibrio cholerae,  <dig> candidates for novel srnas were predicted by relying only on putative terminators and regions of sequence conservation in intergenic regions  <cit> .
previous studies on the m. pneumoniae genome demonstrated the presence of  <dig> ncrnas, the majority of which were classified as asrnas, and probably only  <dig> are srnas  <cit> .
among other bacteria, the number of srnas is variable as  <dig> trans-encoded srnas found in the listeria monocytogenes genome .
taken together, it is possible to suggest that the number and size of the srnas predicted in the genomes of m. hyopneumoniae, m. flocculare and m. hyorhinis are variable as those found in other bacterial species.
aiming to validate the in silico srna prediction, some m. hyopneumoniae srnas were amplified.
nineteen out of  <dig> predicted srnas were tested, corresponding to all candidates that could be full-length amplified by stem-loop rt specific primers.
the experimental data were able to validate the in silico approach as transcripts were detected from all tested srnas.
interestingly, two of the srnas  showed differential expression dependent on the growth conditions tested.
the srna_ <dig> was transcribed in both stress conditions tested and the presence of the srna_ <dig> transcript was detected only in normal culture and oxidative stress conditions.
these results support the notion that the expression of regulatory rnas  changes in response to external stimuli and therefore contributes to an adaptive expression program.
moreover, different studies have also established the indispensable nature of bacterial srnas in cell adaptation, immediate responses to changing environments, survival, and pathogenesis  <cit> .
no homologs of the  <dig> m. hyopneumoniae srnas were found in the genomes of the other swine respiratory tract mycoplasma species.
the same result was observed when srnas from m. flocculare and m. hyorhinis were analyzed.
these results demonstrate that the srnas of these three swine respiratory tract mycoplasmas are species-specific.
we have looked for any difference that could be associated to the non-conservation among species.
however, the genome composition, including gc content within the coding and non-coding regions is very similar in mycoplasmas species.
moreover, the species-specific srnas were identified in the genomes of the different strains of each species analyzed.
previous studies have demonstrated that, although some srnas are conserved in closely related species, a similar species-specific srna pattern appears to be found in other bacterial species  <cit> .
the number of regulatory rnas predicted in m. hyopneumoniae, m. flocculare and m. hyorhinis indicates that many diverse processes could be controlled by bacterial srnas.
in order to predict targets for the novel srnas, two algorithms were used.
a genome-wide cog analysis of srna–mrna target interactions demonstrated the absence of correlation to specific classes of genes, suggesting that srnas might be used to control general processes within the mycoplasma cell as found in other bacteria  <cit> .
moreover, we observed that the ortholog target genes predicted for a mycoplasma species could interact with another species; so, target genes could be considered as not species-specific.
similarly to data previously described for other bacteria, some m. hyopneumoniae, m. flocculare and m. hyorhinis srnas have a predicted interaction with single targets  while others show a multitude of targets , possibly acting as global regulators.
taken together, our results indicate that the majority of the predicted mrna targets are encoded hypothetical proteins or genes involved in the cellular general metabolism.
previous analyses described the presence of differential transcription in the m. hyopneumoniae transcriptome .
therefore, the genes up or down-regulated in the above studies were correlated with the genes predicted as interacting with srnas.
among all differentially expressed genes ,  <dig>  were predicted as target genes for at least one novel srna.
the gene mhp7448_ <dig> that encode the p <dig> prolipoprotein, which can interact with three different srnas  have been shown to be differentially expressed in heat chock condition  <cit> .
p <dig> is an immunodominant surface lipoprotein of m. hyopneumoniae used in the serological diagnosis of infections  <cit> .
moreover, the gene mhp7448_ <dig> that encodes a putative mgte transporter and is also responsive to heat chock stress  <cit>  showed putative interaction with three different srnas .
mgte is a magnesium transporter protein expressed in a number of gram-negative and gram-positive bacteria that can modulate bacteria virulence and cytotoxicity  <cit> .
homologous sequences for the small rnas were searched in the genomes of other m. hyopneumoniae strains available.
interestingly, only one srna  was exclusive of the genomes of pathogenic strains of m. hyopneumoniae .
the srna_ <dig> interacts in silico with two mrnas encoding hypothetical products and one mrna, which encodes a protoporphirogen oxidase that has methylase activity.
so, further analysis will be required to characterize the hypothetical proteins and identify a possible function for this exclusive srna of m. hyopneumoniae.
CONCLUSIONS
in the current study, we have analyzed and predicted trans-encoded small rnas from the genomes of m. hyopneumoniae, m. flocculare and m. hyorhinis.
moreover, we have analyzed rna-rna interaction and accessed putative target genes for the srna candidates.
some predicted m. hyopneumoniae srnas were also experimentally investigated by a reverse transcription amplification approach, in three different culture conditions.
in conclusion, we were able to identify  <dig>   <dig> and  <dig> novel srnas in m. hyopneumoniae, m. flocculare and m. hyorhinis, respectively.
the number of srnas is similar to the one predicted in other bacterial species.
all m. hyopneumoniae srnas tested were transcribed in at least one condition; however, the differential expression profile of two srnas in response to oxidative stress and heat shock stress suggests that its expression is influenced by environmental signals.
target genes for the novel srna candidates were accessed showing that many srnas can interact with different targets, and that different srnas could regulate the same mrnas.
in this context, complex global regulatory networks might be implicated in mycoplasma.

 in bacterial genomes the non-coding rnas identified were the ribosomal rnas and transfer rnas, which are important components of the protein synthesis machinery. in addition, cis-encoded antisense rnas and trans-encoded small rnas were also identified. analyses of asrnas and srnas targets have shown that these ncrnas could alter the translation process or mrna stability by target base pairing. moreover, srnas may have multiple targets. non-coding rna elements present in many bacterial genomes add a further complexity to the comprehension of bacterial gene regulation. recently, several ncrnas with different genomic origins, lengths, functions, and gene regulation mechanisms have been identified. there are evidences that ncrnas may regulate important processes, such as pathogenesis, iron metabolism, and quorum sensing. novel ncrnas are difficult to detect by conventional biochemical screenings. as an alternative, in silico approaches associated to a functional analysis validation have proved to be effective in the identification of ncrnas. in at-rich genomes, the ncrna genes show a relatively higher gc-content and therefore, compositional-based analyses that scan for local gc-content have had success in detecting ncrnas. the algorithm single genome ncrna search is a whole-genome eukaryote predictor that uses these features. given that most functional rnas rely on a stable secondary structure, prediction of the minimum free energy of a transcript is also used as a means of detecting ncrna genes. target prediction is the main step in understanding bacterial srna function. recently, computational target prediction methods had their performance improved by inclusion of rna accessibility and conservation information. interacting rna and rnaplex are reliable srna target prediction software. intarna uses the energy score of the interaction, which is calculated as the sum of the free energy of hybridization and the free energy required for making the interaction sites accessible. rnaplex is a refinement of the rnahybrid software and uses a simplified algorithm to reduce the time needed to localize putative hybridization sites, mainly by neglecting intramolecular interactions and by using a slightly simplified energy model. the rnaplex tool is combined with the rnaup tool and can find high-confidence targets, with only a slight loss of sensitivity. moreover, rnaplex also uses an energy score of the interaction of srna and putative targets to predict molecule interactions. mycoplasmas are bacteria of the class mollicutes characterized by small genomes and low gc content. mycoplasma hyopneumoniae, mycoplasma flocculare and mycoplasma hyorhinis are important species that have been identified in the porcine respiratory system. m. hyopneumoniae is the etiological agent of porcine enzootic pneumonia, while m. hyorhinis can also cause swine polyserositis and arthritis. although m. flocculare is widespread in swine herds, it has so far been recognized as a commensal species. currently, the genome sequences of several mycoplasma species are available, allowing a comparative analysis of the gene content among different species. however, information related to regulatory elements is very limited in these organisms. furthermore, the mycoplasma species present a low number of proteins involved in transcriptional regulation. these evidences suggest the presence of alternative transcriptional regulatory mechanisms in mycoplasmas. it was previously shown that most genes from the genomes of m. hyopneumoniae, m. flocculare and m. hyorhinis are expressed at some basal level and that the majority of the genes are co-transcribed. therefore, a global determination of the genomic functional elements is a prerequisite to expand our knowledge regarding transcriptional small rna regulation in swine respiratory mycoplasmas.in the current study, we have analyzed and predicted trans-encoded small rnas from m. hyopneumoniae, m. flocculare and m. hyorhinis genomes. moreover, we have analyzed rna-rna interaction and accessed target genes for the srna candidates. some predicted m. hyopneumoniae srnas were also experimentally investigated by a reverse transcription amplification approach in three different culture conditions.  global screening for small rnas knowledge related to the presence and role of small rnas in mycoplasma remains limited, therefore a genome-wide screen for srnas was conducted using in silico prediction approaches. only srnas present in the intergenic regions were searched as the input file, since all regions marked as coding sequence and known ncrnas, such as rrna, trna, rnase p and others, were masked. these igrs have a gc content of 21 % for m. hyopneumoniae, 23 % for m. flocculare and 21 % for m. hyorhinis representing, respectively, and 14 % of the total genome. the segments with a high gc cumulative, comparable to the known ncrnas, were considered srna candidates. the analysis of the m. hyopneumoniae genome using the sigrs software allowed the identification of previously known ncrnas and predicted putative novel srnas. however, the sigrs outputs associate with distinct regions, defined some of them as unique ncrna candidates. therefore, a fragmentation algorithm was applied, resulting in previously known ncrnas and putative novel srnas, named srna_ to srna_. the sequences of these predicted srnas including their information related to genome location, length, gc content, and free energy of the secondary structure of the srna are supplied in additional file 2: table s the rnaspace software recovered 87 % of the sigrs/fraps predictions supporting the previous sigrs/fraps results for the srna genes. m. hyopneumoniae srnas have an average length of 128 bp, ranging from to 424 bp and  % of gc content. furthermore, the minimum fold energy among the putative srnas varies from −  kcal/mol to −  kcal/mol. the srna sequences were analyzed by blast search to locate homologous sequences in the genomes of the other m. hyopneumoniae strains available. remarkably, predicted novel srnas were present in all m. hyopneumoniae genome strains, while only the srna_ was exclusive to the m. hyopneumoniae genome. interestingly, only the srna_ was identified in the genomes of all pathogenic strains and was absent in the genome of the non-pathogenic m. hyopneumoniae j strain. moreover, srna_ was found only in the genome of the two pathogenic strains and isolated from brazilian swine herds. in order to evaluate if some of the predicted srna genes were transcribed, a stem-loop-rt-pcr analysis was used to investigate the presence of srna transcripts in three different culture conditions. figure 1b illustrates the primers design for the stem-loop-rt-pcr approach. primer pair positions were based on a full-length srna prediction, for both strands using specific primers for each srna tested. the low gc content of the m. hyopneumoniae genome and the mandatory full-length primers position with high specificity resulted in srnas subject to experimental analysis. it was possible to show that all the predicted and experimentally analyzed srnas were detected as transcribed in the three different growth conditions, except for srna_ and srna_ which were not transcribed in the standard condition or heat shock condition, respectively.fig. schematic representation of srna position at m. hyopneumoniae genome and primers design. a overview of the location of srna_ in the m. hyopneumoniae genome region from to base pair. white arrows represent the genes position and the srna_ is represented by a gray box. representation from artemis. b primers location for the rt-pcr stem-loop reaction. slim arrows indicate the amplicon orientation. black portions represent amplification and gray portions represent the primers location. the amplification from specific forward primer to universal reverse primer is the entire srna length the possible presence of sequences homologous to the novel srnas predicted for m. hyopneumoniae in other mycoplasma species, also found in the swine respiratory tract, was analyzed by blast search. sequences homologous to the novel srnas of m. hyopneumoniae were not found in the genomes of m. flocculare and m. hyorhinis, supporting the notion that these novel srnas are species-specific. therefore, the genomes of m. flocculare and m. hyorhinis were screened for the presence of srnas applying the same methodology used for m. hyopneumoniae. using sigrs plus fraps, it was possible to predict novel srnas for m. flocculare, which were named srna_f to srna_f and novel srnas for m. hyorhinis, which were named srna_r to srna_r. rnaspace predictor was able to identify approximately 50 % of the sigrs/fraps predictions, for both genomes. furthermore, the main profile of the new predicted srnas was similar to that found for the srnas predicted in m. hyopneumoniae. the srnas of m. flocculare have an average length of 167 bp, ranging from to 402 bp and  % of gc content, while the minimum fold energy among the putative srnas varies from −  kcal/mol to −  kcal/mol. the srnas of m. hyorhinis have an average length of 85 bp, ranging from to 209 bp with  % of gc content and the minimum fold energy varied from −  kcal/mol to −  kcal/mol. a homologous sequence search was carried out for each novel putative m. flocculare and m. hyorhinis srna in the available genomes of other m. flocculare, m. hyorhinis and m. hyopneumoniae strains. all predicted srnas were present only in the corresponding genomes of m. flocculare or m. hyorhinis, as shown in additional file 4: table s and additional file 5: table s small rna targets prediction the importance of srnas as a distinct class of gene regulators in bacteria is well established, as many diverse processes have been shown to be controlled by srnas in different species. the interaction of srnas with different targets is an important mechanism to control the complex regulatory networks in bacterial cells. therefore, to analyze the interaction of srnas to a multitude of different target mrnas and their role in gene regulation, the binding region of the srna:target duplex needs to be investigated. a computational analysis of the potential targets for the srnas identified using rnaplex and intarna predicted targets for the srnas of m. hyopneumoniae, targets for the srnas of m. flocculare and targets for the srnas of m. hyorhinis. an interaction was deemed functional only if respecting both criteria free energy smaller or equal to a threshold of −13 kcal/mol and equally predicted by the two software. detailed information related to the srnas/targets interactions is available in each additional file. the in silico predictions identified interaction with target genes for novel m. hyopneumoniae srnas. the majority of the srnas showed multiple target genes varying from to, however most of them interacted with up to seven target genes. considering the whole genome of m. hyopneumoniae, genes out of showed a predicted interaction with at least one novel srna, representing 21 % of the m. hyopneumoniae genes used as input. in general, the genes that showed such predicted interaction were also found to pair with multiple srnas, although some genes interact with only a single srna. from the genes, were found as single target, meaning that the mrnas encoded by these genes could potentially interact with only one of the predicted novel srnas. the products of these genes are mainly related to basal cell metabolism, transport system and lipoproteins; many products are related to protein synthesis while most are related with unknown products. a detailed analysis of the data revealed that approximately 25 % of the target genes were predicted as capable to interact with up to three srnas. nevertheless, some targets are ubiquitous: for example the gene aaz, which encodes the exonuclease protein, showed interactions with six predicted srnas. another gene with multiple srna interactions is aaz gene, which codes for the prolipoprotein p which may interact with three different srnas. the three novel srnas without target prediction were srna_ srna_ and srna_ detailed analysis of the interaction predictions revealed that these srnas have putative target genes found by the two algorithms, however the energy interaction in the intarna software was higher than −13 kcal/mol. the srna_ experimentally analyzed, has two putative target genes: mhp7448_ encoding glycine cleavage system h protein, and the mhp7448_ encoding a hypothetical protein, with energy interactions around −12 kcal/mol for both of them. in turn, the srna_ and srna_ interact with targets with energy around −10 kcal/mol as: the mhp7448_ and mhp7448_ as targets for srna_27; and mhp7448_, mhp7448_ and mhp7448_ as targets for srna_. similarly to the m. hyopneumoniae results, rnaplex and intarna predicted target gene interactions for novel m. flocculare srnas and for all novel m. hyorhinis srnas. most of the srnas were predicted to bind to multiple targets and the number of targets for each srna ranged from one to nine for m. flocculare and from one to for m. hyorhinis. as shown in additional file 4: table s and additional file 5: table s the majority of the predicted novel srnas from both genomes were able to interact with a restricted number of target genes. the srnas with the higher interaction number were the srna_f and the srna_r with and target interactions, respectively. data analyses, as presented in additional file 9: table s and additional file 10: table s showed that of the encoded mrnas of m. flocculare and of the encoded mrnas of m. hyorhinis interact with at least one novel srna, representing, respectively, and 6 % of all the genes used as input. as expected, the different products of the target genes have different metabolic functions as observed in the m. hyopneumoniae analysis. some of the genes encode for enzymes involved in cell metabolism, cell division proteins, transcription regulation, adhesins, while a high number of the gene products are classified as unknown or hypothetical proteins. the m. flocculare srnas without target prediction were srna_f and srna_f detailed analysis of the interaction predictions shows these srnas with putative target genes found by the two algorithms, however the energy interaction in the intarna software was higher than −13 kcal/mol. focusing in these in silico interactions, the srna_f was able to bind to genes rpsk, trxa, and mf. furthermore, srna_f could interact with mf and mf and also with mf. the interaction energies for both srnas were around −10 kcal/mol. discussions small bacterial rnas generally act by base pairing with mrnas, regulating many aspects of bacterial physiology leading to positive or negative regulation of target protein synthesis. to search for srnas in the genomes of m. hyopneumoniae, m. flocculare and m. hyorhinis, we combined in silico prediction approaches with transcription analysis. the in silico prediction of novel trans-acting srnas in the three mycoplasma genomes was performed by applying two different methods, ncrna search and rnaspace, allowing the in silico identification of srnas following the combination of the results generated by both algorithms. using this approach, and putative novel srnas were predicted in the genomes of m. hyopneumoniae, m. flocculare and m. hyorhinis, respectively. the average length of these putative srnas in the genomes of the three mycoplasma species ranged from to 167 bp. previously, the availability of transcriptome maps of m. hyopneumoniae, m. flocculare and m. hyorhinis allowed the in silico identification of and putative novel ncrnas, respectively, with lengths ranging from to nucleotides. however, this approach was unable to identify the srnas among the ncrnas. although in escherichia coli the majority of the new srnas, varying in length from to nucleotides, have been identified through an in silico prediction, the application of this proposed methodology to other bacterial species has had only limited success as it requires reliable species-specific consensus promoter and terminator sequences. in vibrio cholerae, candidates for novel srnas were predicted by relying only on putative terminators and regions of sequence conservation in intergenic regions. previous studies on the m. pneumoniae genome demonstrated the presence of ncrnas, the majority of which were classified as asrnas, and probably only are srnas. among other bacteria, the number of srnas is variable as trans-encoded srnas found in the listeria monocytogenes genome. taken together, it is possible to suggest that the number and size of the srnas predicted in the genomes of m. hyopneumoniae, m. flocculare and m. hyorhinis are variable as those found in other bacterial species. aiming to validate the in silico srna prediction, some m. hyopneumoniae srnas were amplified. nineteen out of predicted srnas were tested, corresponding to all candidates that could be full-length amplified by stem-loop rt specific primers. the experimental data were able to validate the in silico approach as transcripts were detected from all tested srnas. interestingly, two of the srnas showed differential expression dependent on the growth conditions tested. the srna_ was transcribed in both stress conditions tested and the presence of the srna_ transcript was detected only in normal culture and oxidative stress conditions. these results support the notion that the expression of regulatory rnas changes in response to external stimuli and therefore contributes to an adaptive expression program. moreover, different studies have also established the indispensable nature of bacterial srnas in cell adaptation, immediate responses to changing environments, survival, and pathogenesis. no homologs of the m. hyopneumoniae srnas were found in the genomes of the other swine respiratory tract mycoplasma species. the same result was observed when srnas from m. flocculare and m. hyorhinis were analyzed. these results demonstrate that the srnas of these three swine respiratory tract mycoplasmas are species-specific. we have looked for any difference that could be associated to the non-conservation among species. however, the genome composition, including gc content within the coding and non-coding regions is very similar in mycoplasmas species. moreover, the species-specific srnas were identified in the genomes of the different strains of each species analyzed. previous studies have demonstrated that, although some srnas are conserved in closely related species, a similar species-specific srna pattern appears to be found in other bacterial species. the number of regulatory rnas predicted in m. hyopneumoniae, m. flocculare and m. hyorhinis indicates that many diverse processes could be controlled by bacterial srnas. in order to predict targets for the novel srnas, two algorithms were used. a genome-wide cog analysis of srna–mrna target interactions demonstrated the absence of correlation to specific classes of genes, suggesting that srnas might be used to control general processes within the mycoplasma cell as found in other bacteria. moreover, we observed that the ortholog target genes predicted for a mycoplasma species could interact with another species; so, target genes could be considered as not species-specific. similarly to data previously described for other bacteria, some m. hyopneumoniae, m. flocculare and m. hyorhinis srnas have a predicted interaction with single targets while others show a multitude of targets, possibly acting as global regulators. taken together, our results indicate that the majority of the predicted mrna targets are encoded hypothetical proteins or genes involved in the cellular general metabolism. previous analyses described the presence of differential transcription in the m. hyopneumoniae transcriptome. therefore, the genes up or down-regulated in the above studies were correlated with the genes predicted as interacting with srnas. among all differentially expressed genes, were predicted as target genes for at least one novel srna. the gene mhp7448_ that encode the p prolipoprotein, which can interact with three different srnas have been shown to be differentially expressed in heat chock condition. p is an immunodominant surface lipoprotein of m. hyopneumoniae used in the serological diagnosis of infections. moreover, the gene mhp7448_ that encodes a putative mgte transporter and is also responsive to heat chock stress showed putative interaction with three different srnas. mgte is a magnesium transporter protein expressed in a number of gram-negative and gram-positive bacteria that can modulate bacteria virulence and cytotoxicity. homologous sequences for the small rnas were searched in the genomes of other m. hyopneumoniae strains available. interestingly, only one srna was exclusive of the genomes of pathogenic strains of m. hyopneumoniae. the srna_ interacts in silico with two mrnas encoding hypothetical products and one mrna, which encodes a protoporphirogen oxidase that has methylase activity. so, further analysis will be required to characterize the hypothetical proteins and identify a possible function for this exclusive srna of m. hyopneumoniae.  in the current study, we have analyzed and predicted trans-encoded small rnas from the genomes of m. hyopneumoniae, m. flocculare and m. hyorhinis. moreover, we have analyzed rna-rna interaction and accessed putative target genes for the srna candidates. some predicted m. hyopneumoniae srnas were also experimentally investigated by a reverse transcription amplification approach, in three different culture conditions. in conclusion, we were able to identify and novel srnas in m. hyopneumoniae, m. flocculare and m. hyorhinis, respectively. the number of srnas is similar to the one predicted in other bacterial species. all m. hyopneumoniae srnas tested were transcribed in at least one condition; however, the differential expression profile of two srnas in response to oxidative stress and heat shock stress suggests that its expression is influenced by environmental signals. target genes for the novel srna candidates were accessed showing that many srnas can interact with different targets, and that different srnas could regulate the same mrnas. in this context, complex global regulatory networks might be implicated in mycoplasma. 
we performed the identification of trans-encoded small rnas from the genomes of mycoplama hyopneumoniae, mycoplasma flocculare and mycoplasma hyorhinis, which are mycoplasma species that have been identified in the porcine respiratory system. mycoplasma hyopneumoniaemycoplasma floccularemycoplasma hyorhinissmall rnasporcine respiratory system6th solbio international conference soibio 2016riviera maya, mexico 22- april http://www.soibio.org/en/icmexico2016issue-copyright-statement© the author 2016 furthermore, the expression profile of some m. hyopneumoniae srnas was determined by a reverse transcription amplification approach, in three different culture conditions. a detailed investigation revealed a differential expression profile for two m. hyopneumoniae srnas in response to oxidative and heat shock stress conditions, suggesting that their expression is influenced by environmental signals. bacterial non-coding rnas act by base-pairing as regulatory elements in crucial biological processes. moreover, we analyzed srna-mrna hybrids and accessed putative target genes for the novel srna candidates. a total of and putative srnas were predicted in m. hyopneumoniae, m. flocculare and m. hyorhinis, respectively. the majority of the srnas showed interaction with multiple target genes, some of which could be linked to pathogenesis and cell homeostasis activity.
 bacterial non-coding rnas act by base-pairing as regulatory elements in crucial biological processes. we performed the identification of trans-encoded small rnas from the genomes of mycoplama hyopneumoniae, mycoplasma flocculare and mycoplasma hyorhinis, which are mycoplasma species that have been identified in the porcine respiratory system.  a total of and putative srnas were predicted in m. hyopneumoniae, m. flocculare and m. hyorhinis, respectively. a comparative genomic analysis revealed the presence of species or lineage specific srna candidates. furthermore, the expression profile of some m. hyopneumoniae srnas was determined by a reverse transcription amplification approach, in three different culture conditions. all tested srnas were transcribed in at least one condition. a detailed investigation revealed a differential expression profile for two m. hyopneumoniae srnas in response to oxidative and heat shock stress conditions, suggesting that their expression is influenced by environmental signals. moreover, we analyzed srna-mrna hybrids and accessed putative target genes for the novel srna candidates. the majority of the srnas showed interaction with multiple target genes, some of which could be linked to pathogenesis and cell homeostasis activity.  this study contributes to our knowledge of mycoplasma srnas and their response to environmental changes. furthermore, the mrna target prediction provides a perspective for the characterization and comprehension of the function of the srna regulatory mechanisms. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mycoplasma hyopneumoniaemycoplasma floccularemycoplasma hyorhinissmall rnasporcine respiratory system6th solbio international conference soibio 2016riviera maya, mexico 22- april http://www.soibio.org/en/icmexico2016issue-copyright-statement© the author 2016 