BACKGROUND
rna regulators are important players in control of gene expression in bacteria and often mediate a response to changes in the environment .
some regulatory rnas, designated riboswitches, are part of the mrna they regulate.
riboswitches are sequences in the 5' end of mrnas that change conformation upon binding of a ligand, affecting transcription or translation of the down-stream gene .
other rna regulators bind to proteins and regulate their function, whereas the largest group of small rnas , act by base pairing with target rnas.
base pairing srnas generally fall into two groups: cis-acting srnas that have capacity for extensive base pairing, and trans-encoded srnas with a more limited potential for base pairing with their target rna.
trans-acting srnas regulate the translation and/or the stability of their target rnas and each often regulate more than one target.
for the most part, these srnas affect target genes in a negative fashion by binding to the region surrounding the start codon and ribosome binding site, but can act through base pairing in a region far upstream, and occasionally affect translation positively .
many of the trans-encoded srnas require the rna chaperone hfq for function.
hfq promotes rna-rna interactions between the srna and its target mrna and the protein may additionally stabilize the srna in vivo.
rna regulators furthermore control pathogenesis in bacteria such as staphylococcus aureus  <cit> , salmonella typhimurium  <cit> , vibrio cholerae  <cit> , group a streptococcus  <cit> , pseudomonas aeruginosa  <cit> , clamydia trachomatis  <cit> , and clostridium perfringens  <cit> .
francisella tularensis is a gram-negative bacterium that causes the disease tularaemia in humans and in a large number of animals and is one of the most virulent bacterial pathogens known.
the ability of f. tularensis to invade and replicate in host cells, particularly in immune cells such as macrophages, is critical for its capacity to cause disease.
once inside host cells, f. tularensis resides transiently inside a phagosome that matures into a late endosomal stage and the bacterium then escapes to replicate in the cytoplasm.
the mechanism by which the bacterium escapes the phagosome is not well understood, but genes encoded in a pathogenicity island  are required for this step in the infectious cycle.
several regulatory proteins regulate virulence gene expression in f. tularensis by activating transcription of the fpi.
these comprise the proteins mgla  <cit> , sspa  <cit> , prma  <cit> , and fevr   <cit> .
f. tularensis unlike many other bacteria encodes only one alternative sigma factor  <cit> , no complete two component regulator pairs  <cit> , and very little information exists concerning control of gene expression apart from the above mentioned regulation of the fpi.
we have recently studied the role of hfq in the physiology and virulence of f. tularensis  <cit> .
transcriptional analyses revealed that hfq - directly or indirectly - regulates the expression of numerous genes in this pathogen.
functional studies showed that hfq is required for stress resistance as well as for full virulence in both a fully virulent strain and in the attenuated live vaccine strain .
since hfq normally performs its function by promoting srna-mrna interactions, these results strongly suggest that srnas are expressed in this organism and are involved in diverse functions.
here, we have initiated the identification of the srnas expressed by f. tularensis lvs.
we have assessed and identified the srna species that are expressed at high levels and found the commonly known  <dig> s rna, 5s rrna, and the transfer messenger rna .
additionally, we identified two novel srna species by cdna cloning and have characterized these.
studies of srna mutant strains suggested potential targets for regulation.
as an alternative approach to identify srnas in f. tularensis, we performed a bioinformatic prediction of srnas.
interestingly, this analysis found both the experimentally determined srna loci and additionally identified a number of other putative srna-containing intergenic regions.
RESULTS
identification of highly expressed small rna species of francisella
to identify srnas in f. tularensis we first assessed the highly expressed srnas by polyacrylamide gel electrophoresis followed by ethidium bromide staining .
this approach has been used successfully to find novel srnas in s. aureus  <cit> .
we isolated total rna from f. tularensis lvs after growth in normal complex broth in exponential growth phase  or in stationary phase , after oxidative stress  or after growth at high osmolarity .
we chose to assess the rna profile under different conditions because srna expression in other bacteria was found to be constitutive for some srnas , but induced under specific conditions  for most trans-encoded srnas  <cit> .
however, no major differences were observed in the pattern of rna species that were visible after page .
four bands ranging in size from ~ <dig> nt to ~ <dig> nt were visible after staining .
to determine the origin of these four rna species, we eluted the rnas from the gel and performed linker ligation, cdna synthesis, and pcr amplification of each eluted rna .
sequence analysis of cloned pcr fragments identified the rnas, although in all cases we also found contaminating rrna and/or trna sequences.
ten clones corresponding to rna # <dig>  the largest rna, were sequenced and seven of these identified the rna as the transfer messenger rna .
this rna is also designated ssra and is  <dig> nt in size.
tmrnas are found in all bacteria and are highly expressed.
the tmrna has properties of both a trna and a mrna.
it participates in trans-translation, which is a reaction that transfers the translational complex to the tmrna and ultimately leads to degradation of the polypeptide .
based on the size of rna # <dig>  we presumed it was the 5s rrna .
this was confirmed after cdna sequencing.
next, rna # <dig> was identified as the  <dig> s rna component of the signal recognition particle .
ftl_r <dig> was found in one of ten clones sequenced, whereas the other nine clones were the 5s rrna.
this is not very surprising considering the very similar size of these two transcripts and the high expression of the 5s rna.
despite several attempts, we did not obtain any cdna clones of rna # <dig>  the rna with an apparent size of ~ <dig> nt.
cloning and identification of additional srnas
as our analysis of highly expressed rna did not result in the identification of novel small rna transcripts, we proceeded to construct a limited cdna library of low molecular weight rnas.
for this, we used the same approach as described for the highly expressed srnas.
total rna from bacteria grown under the different conditions described above, but depleted of ribosomal rnas , was separated on a 8% denaturing polyacrylamide gel and rnas of approximately 100- <dig> nt were eluted from the gel and processed the same way as for the highly expressed rnas.
the gel was divided into smaller areas before eluting the rnas and ten clones from each area  were sequenced in order to identify new srna species.
by this method, we identified two different rnas, one of which was identified twice from the same gel piece, whereas the remaining clones contained rrna, trna or other contaminating sequences.
to further confirm that the two identified candidates are authentic srnas, we performed northern blotting analysis  using 32p-labeled oligonucleotides as probes.
as can be seen in figure  <dig>  a strong band was observed for each of the srna candidates with sizes of approximately ~ <dig> nt and ~ <dig> nt, respectively.
we refer to these srnas as ftra and ftrb for francisella tularensis srna a and b. in addition to the major bands, two bands of larger size were seen for ftra and one with a lower size for ftrb, suggesting that processing of the transcripts might be occurring.
srna mapping, localization, and structure analysis
to map the ends of the rnas we performed 5'- and 3'-race .
this revealed that ftra is  <dig> nt and ftrb is  <dig> nt in length.
the size found for ftrb is in agreement with the strong bands seen in the northern blot, whereas it seems to correspond to one of the larger, but less intense, bands seen in the northern blot of ftra.
a coordinate of the 5' end of srna .
b coordinate of the 3' end of srna .
c the orientation of the srna and its flanking genes .
">" designates a gene encoded on the coding strand and "<" designates a gene encoded on the non-coding strand.
the ftra gene is located between ftl_ <dig> and ftl_ <dig>  and is encoded on the coding strand.
the flanking genes encode a transposase and a conserved hypothetical protein.
ftrb is encoded on the coding strand between ftl_ <dig> and ftl_ <dig>  encoding two hypothetical proteins.
the predicted secondary structures of ftra and ftrb, using the quikfold program http://dinamelt.bioinfo.rpi.edu/quikfold.php <cit> , are shown in figure  <dig>  both ftra and ftrb are predicted to be highly structured and contain several stem-loops.
to determine whether ftra and ftrb are similar to known bacterial srnas, we performed blast to search the sequences of the non-coding rna database http://ncrnadb.trna.ibch.poznan.pl/blast.html.
no bacterial rnas in the database showed sequence similarity to the two f. tularensis srnas.
sequences were also queried against the rfam database http://rfam.sanger.ac.uk/, which did not result in any matches.
furthermore, a blastn search of the genes encoding either srna against the ncbi total sequence database showed that the genes are only found in francisella and not in any other bacterial genome.
this strongly indicates that the two rnas are novel bacterial srnas.
characterization of ftra or ftrb mutants
to study the role of the srnas, we proceeded by creating mutant strains carrying a chromosomal deletion of either the ftra or ftrb gene.
sequencing and northern blotting were respectively used to confirm deletion of these loci and the absence of their corresponding transcripts .
neither mutant strain exhibited any growth defects or increased sensitivity to several stress conditions , showing that deleting either ftra or ftrb does not affect the ability of f. tularensis to survive a number of stresses.
likewise, we did not observe any change in expression of either ftra or ftrb after exposure to oxidative or osmolarity stress .
we next tested if either gene plays a role in f. tularensis intracellular multiplication.
as shown in figure  <dig>  both mutant strains multiplied intracellularly in j <dig> murine macrophage-like cells in a manner indistinguishable from the wild-type strain, indicating that neither ftra nor ftrb is required for multiplication in macrophages in vitro.
to examine if ftra or ftrb is required for virulence of f. tularensis, we assessed the ability of each mutant strain to induce disease in the mouse model of infection.
no major difference in survival was observed between mice infected with wild-type lvs or with either of the mutant strains, showing that neither gene is required for virulence .
in addition, after infecting mice with a 1: <dig> mixture of mutant and wild-type bacteria we recovered equal numbers of wild-type and mutant bacteria from the spleen .
this further demonstrates that deleting ftra or frtb does not affect f. tularensis pathogenicity in the mouse model.
it is important to recall that most srnas affect gene expression negatively.
deleting a specific srna gene and thus alleviating the repressive effect may not be of major consequence for the bacterium.
our results from in vitro characterization of the mutant strains therefore do not rule out the possibility that ftra or ftrb may indeed control functions related to growth, stress resistance or virulence.
identification of potential targets
most characterized srnas control expression of their target genes by base-pairing with a target mrna.
in addition to affecting translation, base-pairing between a srna and its target frequently leads to changed stability of both srna and mrna.
therefore, to experimentally identify potential targets for regulation by ftra and ftrb, we compared the transcriptomes of lvs wild-type bacteria grown in regular broth to that of either the ftra or the ftrb mutant.
this way we identified several potential targets for ftra and ftrb .
none of the potential targets identified by microarray analysis are located in the same genomic region as the srnas, indicating that the srnas are trans-acting.
the analysis identified four genes for which the transcript was found at a higher level in the ftra mutant than in lvs.
these are ftl_ <dig> , ftl_ <dig> , ftl_ <dig> , and ftl_ <dig> .
interestingly, pyrf transcripts were also found at higher levels in a hfq mutant  <cit> .
thus, deleting either hfq or ftra affects pyrf mrna levels in the same manner, in agreement with a regulatory mechanism in which hfq-mediated ftra-pyrf mrna base-pairing leads to degradation of the target mrna.
this, however, needs to be demonstrated experimentally.
the mrna levels of five genes were changed in the ftrb mutant compared to wild-type bacteria: two were found in higher levels  and three at lower levels  in the ftrb mutant.
of these, ftl_ <dig>  was also found to be down-regulated in the f. tularensis hfq mutant  <cit> .
this finding could be explained by a mechanism in which rna duplex formation leads to stabilization of the mrna.
to confirm the results obtained by microarray analysis, we chose two genes with changed expression in either mutant strain and performed quantitative rt-pcr analysis.
for each mutant strain, this analysis confirmed the change in expression for both genes .
presently we do not know if ftra and ftrb regulate these targets directly as it is also possible that the observed changes in mrna level are due to indirect effects of deleting the srna.
a genes included had ≥ <dig>  average fold-change in mrna level  and for which each of the five spots for the gene on the microarray had fold-changes ≥ <dig>
b change in mrna level in mutant strain compared to wild-type lvs
identification of putative srnas using bioinformatics analysis
while our cloning based screen proved effective in identifying previously unknown srnas, the relatively small number of clones sequenced meant that only abundant small transcripts could be found using this approach.
in an effort to identify putative srnas in f. tularensis that may have been missed in our physical screen, we used the bioinformatic tool sipht  <cit>  to conduct a genome-wide bioinfomatic screen for candidate srnas in f. tularensis lvs.
sipht identifies candidate intergenic loci based on the co-localization of intergenic conservation and rho-independent terminators.
it then annotates these candidates for several features, including the similarity of their primary sequence and predicted secondary structure to intergenic sequences in other bacterial replicons, their proximity to the putative binding sites of various transcription factors, and their homology to previously confirmed or predicted srnas and cis-regulatory rna elements.
a total of  <dig> candidate loci were identified by sipht, including three intergenic regions  each containing two overlapping candidate loci, leading to a total of  <dig> igrs .
importantly, both the ftra and ftrb srna were identified in the bioinformatic prediction  and candidate  <dig> ), suggesting that other candidate loci identified by sipht may represent real non-coding rnas.
one of these candidates was annotated by sipht as sharing sequence homology with sprb, a previously identified but uncharacterized srna in staphylococcus aureus  <cit> , while another was found in a similar genomic location based on its flanking genes to p <dig>  an srna of unknown function in pseudomonas aeruginosa.
for fourteen of the loci identified, including ftra and ftrb, conservation of secondary structure was predicted by qrna  <cit> .
putative sigma- <dig> promoters were identified within  <dig> nucleotides upstream of the predicted terminators of  <dig> candidates, including two upstream of ftra, one of which in a position consistent with the larger band seen by northern analysis and the results of the 5'-race.
a srna candidates, 5' and 3' ends predicted using sipht  <cit> .
b the orientation of the srna and its flanking genes .
">" designates a gene encoded on the coding strand and "<" designates a gene encoded on the non-coding strand.
numbers outside of the "|" characters denote the distance between the boundaries of the predicted locus and its flanking genes.
c potential orfs are indicated by their length in amino acids.
* denotes an orf staring with an alternative start codon.
for sequence of each rna see additional file  <dig>
neither tmrna nor  <dig> s rna were identified by sipht.
further analysis revealed that these loci were missed because they were not associated with predicted rho-independent terminators.
this may reflect a true paucity of rho-independent terminators in f. tularensis or limitations in the ability of the three terminator predicting algorithms used by sipht to identify terminators in an at rich genomes such as that of f. tularensis.
the fact that these two srnas were not identified by sipht suggests that other f. tularensis srnas were likely missed in our screen.
several characteristics of francisella likely hinder predictions of srnas based on the proximity of intergenic sequence conservation and rho-independent terminators.
first, no genome sequences for close relatives of f. tularensis are available for blast comparisons and the relatively low gc content  of the f. tularensis genome leads to spurious hits in blast searches that are more likely due to low sequence complexity than to actual sequence conservation.
second, as described above, it is also likely that existing terminator prediction programs, most of which had been trained on e. coli, are less effective in identifying terminators in at-rich species such as francisella.
indeed, in a kingdom-wide search for putative srnas in over  <dig> different strains, all francisella sp.
were among the  <dig> strains with the lowest density of putative intergenic rho-independent terminators.
however, while these factors likely contribute to both the decreased sensitivity and specificity of our srna predictions, the fact that both ftra and ftrb were identified in our in silico screen suggests that the predictive algorithm used by sipht is effective in identifying novel srnas even in species such as francisella that are at-rich and have relatively few sequenced relatives.
recently, a number of small orfs encoding small peptides , previously non-annotated, have been identified in other bacterial species  <cit> .
we therefore examined whether the predicted srna loci in f. tularensis contain orfs encoding small peptides .
a few loci could encode small peptides of 24- <dig> amino acids, but most loci contained none or only very short orfs, further supporting the hypothesis that the majority of the loci encode putative rnas.
CONCLUSIONS
here, we have confirmed the expression in f. tularensis of the well-known non-coding rnas tmrna and  <dig> s rna after direct purification and cloning of the cdnas.
two additional srnas expressed by f. tularensis were identified from a cdna library prepared from low molecular weight rna.
the two putative srnas , which are encoded in intergenic regions, were confirmed to be genuine srnas by northern blot analysis and the transcripts were characterized with respect to transcription endpoints.
the srnas do not show sequence similarity to any known srnas and their dna sequences are only found in francisella species, indicating that these are novel srnas.
deletion of either the ftra or ftrb gene had no effect on bacterial survival during normal growth or stress, and did not affect the capacity of francisella to induce disease in mice.
this suggests that these srnas affect functions that are not required under the conditions tested or that additional srnas with redundant functions may exist.
specifically, in silico analysis identified conserved sequence between ftrb and srna candidate # <dig>  suggesting they may be functional paralogues.
subsequently, microarray analysis allowed us to identify a number of potential targets of ftra and ftrb regulation.
further experiments will be needed to verify if these represent real targets.
finally, in silico prediction of putative srna loci identified a number of francisella igrs, including the two harboring ftra and ftrb.
future work will determine which of these igrs indeed encode authentic srnas.

 rna regulators are important players in control of gene expression in bacteria and often mediate a response to changes in the environment. some regulatory rnas, designated riboswitches, are part of the mrna they regulate. riboswitches are sequences in the 5' end of mrnas that change conformation upon binding of a ligand, affecting transcription or translation of the down-stream gene. other rna regulators bind to proteins and regulate their function, whereas the largest group of small rnas, act by base pairing with target rnas. base pairing srnas generally fall into two groups: cis-acting srnas that have capacity for extensive base pairing, and trans-encoded srnas with a more limited potential for base pairing with their target rna. trans-acting srnas regulate the translation and/or the stability of their target rnas and each often regulate more than one target. for the most part, these srnas affect target genes in a negative fashion by binding to the region surrounding the start codon and ribosome binding site, but can act through base pairing in a region far upstream, and occasionally affect translation positively. many of the trans-encoded srnas require the rna chaperone hfq for function. hfq promotes rna-rna interactions between the srna and its target mrna and the protein may additionally stabilize the srna in vivo. rna regulators furthermore control pathogenesis in bacteria such as staphylococcus aureus, salmonella typhimurium, vibrio cholerae, group a streptococcus, pseudomonas aeruginosa, clamydia trachomatis, and clostridium perfringens. francisella tularensis is a gram-negative bacterium that causes the disease tularaemia in humans and in a large number of animals and is one of the most virulent bacterial pathogens known. the ability of f. tularensis to invade and replicate in host cells, particularly in immune cells such as macrophages, is critical for its capacity to cause disease. once inside host cells, f. tularensis resides transiently inside a phagosome that matures into a late endosomal stage and the bacterium then escapes to replicate in the cytoplasm. the mechanism by which the bacterium escapes the phagosome is not well understood, but genes encoded in a pathogenicity island are required for this step in the infectious cycle. several regulatory proteins regulate virulence gene expression in f. tularensis by activating transcription of the fpi. these comprise the proteins mgla, sspa, prma, and fevr. f. tularensis unlike many other bacteria encodes only one alternative sigma factor, no complete two component regulator pairs, and very little information exists concerning control of gene expression apart from the above mentioned regulation of the fpi. we have recently studied the role of hfq in the physiology and virulence of f. tularensis. transcriptional analyses revealed that hfq - directly or indirectly - regulates the expression of numerous genes in this pathogen. functional studies showed that hfq is required for stress resistance as well as for full virulence in both a fully virulent strain and in the attenuated live vaccine strain. since hfq normally performs its function by promoting srna-mrna interactions, these results strongly suggest that srnas are expressed in this organism and are involved in diverse functions. here, we have initiated the identification of the srnas expressed by f. tularensis lvs. we have assessed and identified the srna species that are expressed at high levels and found the commonly known s rna, 5s rrna, and the transfer messenger rna. additionally, we identified two novel srna species by cdna cloning and have characterized these. studies of srna mutant strains suggested potential targets for regulation. as an alternative approach to identify srnas in f. tularensis, we performed a bioinformatic prediction of srnas. interestingly, this analysis found both the experimentally determined srna loci and additionally identified a number of other putative srna-containing intergenic regions.  identification of highly expressed small rna species of francisella to identify srnas in f. tularensis we first assessed the highly expressed srnas by polyacrylamide gel electrophoresis followed by ethidium bromide staining. this approach has been used successfully to find novel srnas in s. aureus. we isolated total rna from f. tularensis lvs after growth in normal complex broth in exponential growth phase or in stationary phase, after oxidative stress or after growth at high osmolarity. we chose to assess the rna profile under different conditions because srna expression in other bacteria was found to be constitutive for some srnas, but induced under specific conditions for most trans-encoded srnas. however, no major differences were observed in the pattern of rna species that were visible after page. four bands ranging in size from ~ nt to ~ nt were visible after staining. to determine the origin of these four rna species, we eluted the rnas from the gel and performed linker ligation, cdna synthesis, and pcr amplification of each eluted rna. sequence analysis of cloned pcr fragments identified the rnas, although in all cases we also found contaminating rrna and/or trna sequences. ten clones corresponding to rna # the largest rna, were sequenced and seven of these identified the rna as the transfer messenger rna. this rna is also designated ssra and is nt in size. tmrnas are found in all bacteria and are highly expressed. the tmrna has properties of both a trna and a mrna. it participates in trans-translation, which is a reaction that transfers the translational complex to the tmrna and ultimately leads to degradation of the polypeptide. based on the size of rna # we presumed it was the 5s rrna. this was confirmed after cdna sequencing. next, rna # was identified as the s rna component of the signal recognition particle. ftl_r was found in one of ten clones sequenced, whereas the other nine clones were the 5s rrna. this is not very surprising considering the very similar size of these two transcripts and the high expression of the 5s rna. despite several attempts, we did not obtain any cdna clones of rna # the rna with an apparent size of ~ nt. cloning and identification of additional srnas as our analysis of highly expressed rna did not result in the identification of novel small rna transcripts, we proceeded to construct a limited cdna library of low molecular weight rnas. for this, we used the same approach as described for the highly expressed srnas. total rna from bacteria grown under the different conditions described above, but depleted of ribosomal rnas, was separated on a 8% denaturing polyacrylamide gel and rnas of approximately 100- nt were eluted from the gel and processed the same way as for the highly expressed rnas. the gel was divided into smaller areas before eluting the rnas and ten clones from each area were sequenced in order to identify new srna species. by this method, we identified two different rnas, one of which was identified twice from the same gel piece, whereas the remaining clones contained rrna, trna or other contaminating sequences. to further confirm that the two identified candidates are authentic srnas, we performed northern blotting analysis using 32p-labeled oligonucleotides as probes. as can be seen in figure a strong band was observed for each of the srna candidates with sizes of approximately ~ nt and ~ nt, respectively. we refer to these srnas as ftra and ftrb for francisella tularensis srna a and b. in addition to the major bands, two bands of larger size were seen for ftra and one with a lower size for ftrb, suggesting that processing of the transcripts might be occurring. srna mapping, localization, and structure analysis to map the ends of the rnas we performed 5'- and 3'-race. this revealed that ftra is nt and ftrb is nt in length. the size found for ftrb is in agreement with the strong bands seen in the northern blot, whereas it seems to correspond to one of the larger, but less intense, bands seen in the northern blot of ftra. a coordinate of the 5' end of srna. b coordinate of the 3' end of srna. c the orientation of the srna and its flanking genes. ">" designates a gene encoded on the coding strand and "<" designates a gene encoded on the non-coding strand. the ftra gene is located between ftl_ and ftl_ and is encoded on the coding strand. the flanking genes encode a transposase and a conserved hypothetical protein. ftrb is encoded on the coding strand between ftl_ and ftl_ encoding two hypothetical proteins. the predicted secondary structures of ftra and ftrb, using the quikfold program http://dinamelt.bioinfo.rpi.edu/quikfold.php, are shown in figure both ftra and ftrb are predicted to be highly structured and contain several stem-loops. to determine whether ftra and ftrb are similar to known bacterial srnas, we performed blast to search the sequences of the non-coding rna database http://ncrnadb.trna.ibch.poznan.pl/blast.html. no bacterial rnas in the database showed sequence similarity to the two f. tularensis srnas. sequences were also queried against the rfam database http://rfam.sanger.ac.uk/, which did not result in any matches. furthermore, a blastn search of the genes encoding either srna against the ncbi total sequence database showed that the genes are only found in francisella and not in any other bacterial genome. this strongly indicates that the two rnas are novel bacterial srnas. characterization of ftra or ftrb mutants to study the role of the srnas, we proceeded by creating mutant strains carrying a chromosomal deletion of either the ftra or ftrb gene. sequencing and northern blotting were respectively used to confirm deletion of these loci and the absence of their corresponding transcripts. neither mutant strain exhibited any growth defects or increased sensitivity to several stress conditions, showing that deleting either ftra or ftrb does not affect the ability of f. tularensis to survive a number of stresses. likewise, we did not observe any change in expression of either ftra or ftrb after exposure to oxidative or osmolarity stress. we next tested if either gene plays a role in f. tularensis intracellular multiplication. as shown in figure both mutant strains multiplied intracellularly in j murine macrophage-like cells in a manner indistinguishable from the wild-type strain, indicating that neither ftra nor ftrb is required for multiplication in macrophages in vitro. to examine if ftra or ftrb is required for virulence of f. tularensis, we assessed the ability of each mutant strain to induce disease in the mouse model of infection. no major difference in survival was observed between mice infected with wild-type lvs or with either of the mutant strains, showing that neither gene is required for virulence. in addition, after infecting mice with a 1: mixture of mutant and wild-type bacteria we recovered equal numbers of wild-type and mutant bacteria from the spleen. this further demonstrates that deleting ftra or frtb does not affect f. tularensis pathogenicity in the mouse model. it is important to recall that most srnas affect gene expression negatively. deleting a specific srna gene and thus alleviating the repressive effect may not be of major consequence for the bacterium. our results from in vitro characterization of the mutant strains therefore do not rule out the possibility that ftra or ftrb may indeed control functions related to growth, stress resistance or virulence. identification of potential targets most characterized srnas control expression of their target genes by base-pairing with a target mrna. in addition to affecting translation, base-pairing between a srna and its target frequently leads to changed stability of both srna and mrna. therefore, to experimentally identify potential targets for regulation by ftra and ftrb, we compared the transcriptomes of lvs wild-type bacteria grown in regular broth to that of either the ftra or the ftrb mutant. this way we identified several potential targets for ftra and ftrb. none of the potential targets identified by microarray analysis are located in the same genomic region as the srnas, indicating that the srnas are trans-acting. the analysis identified four genes for which the transcript was found at a higher level in the ftra mutant than in lvs. these are ftl_, ftl_, ftl_, and ftl_. interestingly, pyrf transcripts were also found at higher levels in a hfq mutant. thus, deleting either hfq or ftra affects pyrf mrna levels in the same manner, in agreement with a regulatory mechanism in which hfq-mediated ftra-pyrf mrna base-pairing leads to degradation of the target mrna. this, however, needs to be demonstrated experimentally. the mrna levels of five genes were changed in the ftrb mutant compared to wild-type bacteria: two were found in higher levels and three at lower levels in the ftrb mutant. of these, ftl_ was also found to be down-regulated in the f. tularensis hfq mutant. this finding could be explained by a mechanism in which rna duplex formation leads to stabilization of the mrna. to confirm the results obtained by microarray analysis, we chose two genes with changed expression in either mutant strain and performed quantitative rt-pcr analysis. for each mutant strain, this analysis confirmed the change in expression for both genes. presently we do not know if ftra and ftrb regulate these targets directly as it is also possible that the observed changes in mrna level are due to indirect effects of deleting the srna. a genes included had ≥ average fold-change in mrna level and for which each of the five spots for the gene on the microarray had fold-changes ≥ b change in mrna level in mutant strain compared to wild-type lvs identification of putative srnas using bioinformatics analysis while our cloning based screen proved effective in identifying previously unknown srnas, the relatively small number of clones sequenced meant that only abundant small transcripts could be found using this approach. in an effort to identify putative srnas in f. tularensis that may have been missed in our physical screen, we used the bioinformatic tool sipht to conduct a genome-wide bioinfomatic screen for candidate srnas in f. tularensis lvs. sipht identifies candidate intergenic loci based on the co-localization of intergenic conservation and rho-independent terminators. it then annotates these candidates for several features, including the similarity of their primary sequence and predicted secondary structure to intergenic sequences in other bacterial replicons, their proximity to the putative binding sites of various transcription factors, and their homology to previously confirmed or predicted srnas and cis-regulatory rna elements. a total of candidate loci were identified by sipht, including three intergenic regions each containing two overlapping candidate loci, leading to a total of igrs. importantly, both the ftra and ftrb srna were identified in the bioinformatic prediction and candidate ), suggesting that other candidate loci identified by sipht may represent real non-coding rnas. one of these candidates was annotated by sipht as sharing sequence homology with sprb, a previously identified but uncharacterized srna in staphylococcus aureus, while another was found in a similar genomic location based on its flanking genes to p an srna of unknown function in pseudomonas aeruginosa. for fourteen of the loci identified, including ftra and ftrb, conservation of secondary structure was predicted by qrna. putative sigma- promoters were identified within nucleotides upstream of the predicted terminators of candidates, including two upstream of ftra, one of which in a position consistent with the larger band seen by northern analysis and the results of the 5'-race. a srna candidates, 5' and 3' ends predicted using sipht. b the orientation of the srna and its flanking genes. ">" designates a gene encoded on the coding strand and "<" designates a gene encoded on the non-coding strand. numbers outside of the "|" characters denote the distance between the boundaries of the predicted locus and its flanking genes. c potential orfs are indicated by their length in amino acids. * denotes an orf staring with an alternative start codon. for sequence of each rna see additional file neither tmrna nor s rna were identified by sipht. further analysis revealed that these loci were missed because they were not associated with predicted rho-independent terminators. this may reflect a true paucity of rho-independent terminators in f. tularensis or limitations in the ability of the three terminator predicting algorithms used by sipht to identify terminators in an at rich genomes such as that of f. tularensis. the fact that these two srnas were not identified by sipht suggests that other f. tularensis srnas were likely missed in our screen. several characteristics of francisella likely hinder predictions of srnas based on the proximity of intergenic sequence conservation and rho-independent terminators. first, no genome sequences for close relatives of f. tularensis are available for blast comparisons and the relatively low gc content of the f. tularensis genome leads to spurious hits in blast searches that are more likely due to low sequence complexity than to actual sequence conservation. second, as described above, it is also likely that existing terminator prediction programs, most of which had been trained on e. coli, are less effective in identifying terminators in at-rich species such as francisella. indeed, in a kingdom-wide search for putative srnas in over different strains, all francisella sp. were among the strains with the lowest density of putative intergenic rho-independent terminators. however, while these factors likely contribute to both the decreased sensitivity and specificity of our srna predictions, the fact that both ftra and ftrb were identified in our in silico screen suggests that the predictive algorithm used by sipht is effective in identifying novel srnas even in species such as francisella that are at-rich and have relatively few sequenced relatives. recently, a number of small orfs encoding small peptides, previously non-annotated, have been identified in other bacterial species. we therefore examined whether the predicted srna loci in f. tularensis contain orfs encoding small peptides. a few loci could encode small peptides of 24- amino acids, but most loci contained none or only very short orfs, further supporting the hypothesis that the majority of the loci encode putative rnas.  here, we have confirmed the expression in f. tularensis of the well-known non-coding rnas tmrna and s rna after direct purification and cloning of the cdnas. two additional srnas expressed by f. tularensis were identified from a cdna library prepared from low molecular weight rna. the two putative srnas, which are encoded in intergenic regions, were confirmed to be genuine srnas by northern blot analysis and the transcripts were characterized with respect to transcription endpoints. the srnas do not show sequence similarity to any known srnas and their dna sequences are only found in francisella species, indicating that these are novel srnas. deletion of either the ftra or ftrb gene had no effect on bacterial survival during normal growth or stress, and did not affect the capacity of francisella to induce disease in mice. this suggests that these srnas affect functions that are not required under the conditions tested or that additional srnas with redundant functions may exist. specifically, in silico analysis identified conserved sequence between ftrb and srna candidate # suggesting they may be functional paralogues. subsequently, microarray analysis allowed us to identify a number of potential targets of ftra and ftrb regulation. further experiments will be needed to verify if these represent real targets. finally, in silico prediction of putative srna loci identified a number of francisella igrs, including the two harboring ftra and ftrb. future work will determine which of these igrs indeed encode authentic srnas. 
francisella tularensis is a highly pathogenic gram-negative bacterium that causes the disease tularaemia in humans and animals. our findings suggest that f. tularensis encodes a significant number of non-coding regulatory rnas, including members of well conserved families of structural and housekeeping rnas and other poorly conserved transcripts that may have evolved more recently to help f. tularensis deal with the unique and diverse set of environments with which it must contend. deletion of these srnas did not, however, significantly alter f. tularensis growth under various stress conditions in vitro, its replication in murine cells, or its ability to induce disease in a mouse model of f. tularensis infection. we have used a combination of experimental assays and in silico prediction to identify srnas in f. tularensis strain lvs. regulation of bacterial gene expression by small rnas have proved to be important for many biological processes. we have also discovered two abundant putative srnas that share no sequence similarity or conserved genomic context with any previously annotated regulatory transcripts.
 regulation of bacterial gene expression by small rnas have proved to be important for many biological processes. francisella tularensis is a highly pathogenic gram-negative bacterium that causes the disease tularaemia in humans and animals. relatively little is known about the regulatory networks existing in this organism that allows it to survive in a wide array of environments and no srna regulators have been identified so far.  we have used a combination of experimental assays and in silico prediction to identify srnas in f. tularensis strain lvs. using a cdna cloning and sequencing approach we have shown that f. tularensis expresses homologues of several srnas that are well-conserved among diverse bacteria. we have also discovered two abundant putative srnas that share no sequence similarity or conserved genomic context with any previously annotated regulatory transcripts. deletion of either of these two loci led to significant changes in the expression of several mrnas that likely include the cognate target of these srnas. deletion of these srnas did not, however, significantly alter f. tularensis growth under various stress conditions in vitro, its replication in murine cells, or its ability to induce disease in a mouse model of f. tularensis infection. we also conducted a genome-wide in silico search for intergenic loci that suggests f. tularensis encodes several other srnas in addition to the srnas found in our experimental screen.  our findings suggest that f. tularensis encodes a significant number of non-coding regulatory rnas, including members of well conserved families of structural and housekeeping rnas and other poorly conserved transcripts that may have evolved more recently to help f. tularensis deal with the unique and diverse set of environments with which it must contend. 