BACKGROUND
multiple studies demonstrate that non coding rnas ) possess regulatory roles in the bacterial stress response
<cit> .
bacterial srna regulators typically range from  <dig> –  <dig> nts and are often transcribed from intergenic regions , although open reading frames may also encode srnas
<cit> .
most srnas act as regulators at the post-transcriptional level by base-pairing with target mrnas; these srna-mrna binding regions are often short and imperfect and may require an additional rna chaperone, which in most cases is the hfq protein
<cit> .
this imperfect binding allows each srna molecule to control multiple targets
<cit> , where either the translation of the target mrna is upregulated, or more commonly inhibited.
many srna regulators are upregulated when bacteria sense environmental stress: these include oxidative stress
<cit> , low ph environment
<cit> , nutrient deprivation
<cit>  and glucose-phosphate stress
<cit> .
despite overwhelming evidence that srnas play a role when bacteria experience physiological stress, no systematic study has been undertaken to ascertain the impact or levels of srna production in bacteria when antibiotics are present.
naturally susceptible pathogens can develop drug resistance when treated with antibiotics
<cit> .
genetically acquired antibiotic resistance in pathogenic bacteria, via spontaneous / random mutations and horizontal gene transfer, is a significant issue in the treatment of infectious diseases
<cit> .
intrinsic regulatory networks such as those mediated by the transcriptional regulators mara, soxs and rama are also implicated in the development of antibiotic resistance particularly since these systems control the influx / efflux of antibiotics
<cit> .
thus far studies that have focused on the intrinsic antibiotic resistome are limited to gene and protein networks mediated by these gene operons or other transcription factors
<cit> .
hence the role of the newly uncovered class of regulatory molecules such as srnas in controlling or contributing to the antimicrobial resistance phenotype is largely unknown.
some evidence for the role of srnas in mediating antimicrobial resistance already exists: for example, the expression of bacterial outer membrane proteins, ompf and ompc, involved in antibiotic import, is controlled by the srnas micf and micc respectively
<cit> .
additionally, the overexpression of another srna  was recently found to induce multidrug resistance in escherichia coli via the mdtef efflux pump
<cit> .
nevertheless, whether the functional role of micf, micc and dsra is indeed part of the bacteria’s intrinsic stress response to antibiotic challenge remains unknown.
tigecycline is a member of the glycylcycline group of antibiotics, and was registered in the eu in april 2006
<cit> .
this bacteriostatic antibiotic acts as a protein synthesis inhibitor by binding to the 30s ribosomal subunit
<cit> .
tigecycline is active against a broad range of bacteria, with only few naturally resistant exceptions, namely, proteus spp., morganella morganii, providencia spp., and pseudomonas aeruginosa.
specifically, tigecycline is effective against multidrug resistant bacteria such as staphylococcus aureus , vancomycin-resistant enterococcus , extended-spectrum beta-lactamase -expressing enterobacteriaceae, and carbapenem-resistant strains
<cit> .
reports of resistance to tigecycline have been rare in naturally susceptible pathogens, however in resistant variants efflux pump overexpression has contributed to tigecycline resistance
<cit> .
salmonella, a member of enterobacteriaceae, encodes both the rama transcriptional factor and the acrab efflux pump, which when overexpressed confers tigecycline resistance
<cit> .
additionally, salmonella represents a model bacterium for srna mining
<cit>  and genome manipulation
<cit> , making it an ideal system for our study, but more importantly represents a paradigm for other members of enterobacteriaceae.
hence in this study we used a cloning strategy to determine the srna population after tigecycline exposure in salmonella enterica serovar typhimurium, and also whether the absence of these srnas would render the cells less adaptable to tigecycline challenge.
RESULTS
cdna library construction and analysis
a cdna library was constructed from the cells that were challenged by half the minimal inhibitory concentration  of tigecycline  at od600 =  <dig> .
approximately ~ <dig> clones were obtained; from these  <dig> random candidates were sequenced and analysed.
the nature of the cdna library construction procedure  allowed us to obtain the sequences in an orientation specific manner.
the cdna sequences were mapped to the s. typhimurium sl <dig> genome  using blast .
of the mapped sequences, 31% encoded trnas; 6% and 9% matched to rrnas and protein coding sequences, respectively; 4% partially overlapped with open reading frames , and 50% aligned to igrs.
of all the igr readings, 90% were located between the 16s and 23s rrna encoding genes .
analyses of the cdna sequences encoding partial orfs indicated which genes were expressed in the presence of tigecycline.
as stated above, 9% of the sequences identified matched to rrnas, in addition to a further sequence which was found to overlap the 30s ribosomal protein and another mapped to elongation factor tu.
this is perhaps not surprising, given that the specific target for tigecycline is the ribosome
<cit> .
on the other hand, sequences overlapping known stress-response genes were also captured in the cdna library, e.g.
dinf and a gene encoding a putative outer membrane protein .
the dinf gene is a member of the sos response family and encodes an efflux pump which belongs to the multidrug and toxic compound extrusion  family
<cit> , and sl1344_ <dig>  encoding a putative outer membrane protein homologous to ycfr in e. coli, which influences biofilm formation through stress response and surface hydrophobicity
<cit> .
the expression of these genes supports our hypothesis that challenge at half the mic of tigecycline triggers a stress response.
of note, the cdna library also contained sequences of different lengths that mapped to open reading frames, which we postulate to be a result of mrna degradation, rather than a representation of bona fide srna regulators.
meanwhile, 4% of all sequences that partially overlap orfs, all do so at the 5’ end of the orfs.
this suggests that these sequences might be 5’ untranslated regions, or encode riboswitches and/or control the expression of the downstream genes.
northern blot verification
northern blot analysis was performed on rna extracted from sl <dig> that were either unchallenged or challenged with half the mic of tigecycline.
since most srnas are produced from igrs
<cit> , only sequences from these regions  were selected for further validation by northern blot analysis.
as 90% of the igr sequences are located between 16s and 23s rrna coding sequences, most of which are identical, there were  <dig> unique igr sequences  that were assayed, of which four  were found to consistently show elevated expression with tigecycline challenge .
the remaining srna candidates were either not detectable by northern blots, or did not show differential levels of transcription.
correspondingly all further analyses focused on these four srnas.
the relative fold increase in srna expression was determined by northern blots in challenged versus unchallenged cells.
upon tigecycline exposure, the expression levels of syj <dig>  syj <dig>  syj <dig> and syj <dig> , relative to unchallenged cells, were increased to  <dig>   <dig>   <dig> and  <dig> fold, respectively .
we also tested the level of the four srnas in cells challenged with half the mic of tetracycline .
as expected, all of the four srnas were also found to be upregulated compared to the control sample .
this is possibly due to the fact that tigecycline and tetracycline are related compounds, and they may as well trigger stress response pathways that share a common set of regulatory molecules.
of note and as shown in figure
4a, the level of 5s rna was not affected by the presence of half the mic of tigecycline or tetracycline .
bioinformatic analysis
all four srna sequences were searched against s. typhimurium sl <dig> using ncbi blast.
the syj <dig> encoding sequence is located between the 16s  and 23s rrna  coding loci on the sense strand ).
blast analysis uncovered two additional identical copies in the genome sequence of sl <dig> .
similar to syj <dig>  syj <dig> is also encoded from the igr between the 16s and 23s rrna coding sequences, but from a different genetic locus ).
the sequence encoding syj <dig> has an identical copy  and additionally five other paralogs with 93% - 99% identity on the sl <dig> chromosome.
the encoding sequence of syj <dig> is flanked by entc downstream , and fepb upstream  ).
it also has a paralog that shares 90% identity, starting at position  <dig> on the s. typhimurium sl <dig> genome and located between pntb  and an un-annotated gene .
syj <dig> was previously identified by vogel et al.
in e. coli as sroa
<cit> , encoded by a sequence downstream of yabn  and upstream of tbpa   and
5a).
syj <dig>  syj <dig>  and syj <dig> are all highly conserved within the different members of enterobacteriaceae, although the coding sequences of syj <dig>  syj <dig> and syj <dig> are also found in other families of bacteria , in plants  and in animals .
in contrast, syj <dig> is only found in salmonella, enterobacter, photorhabdus and citrobacter.
syj <dig> , syj <dig>  syj <dig> and syj <dig> in other species and relevance to other drug classes
we proceeded to determine whether the increased expression of these srnas would be salmonella specific or drug-class specific.
hence, we assessed the levels of our srna candidates  in other members of enterobacteriaceae  when challenged with sub-inhibitory levels of tigecycline .
additionally, in order to determine whether these srnas are upregulated solely as a result of tigecycline challenge or are generally upregulated as a result of sub-inhibitory antibiotic challenge, s. typhimurium sl <dig> was challenged with either half the mic of ampicillin  or ciprofloxacin .
as shown in figure
3b, none of the four tested srnas were upregulated in response to ciprofloxacin exposure, whilst three  were found to be upregulated in the presence of ampicillin.
interestingly, e. coli did not upregulate the expression of the three candidate srnas  in response to challenge at half the mic of tigecycline.
however, syj <dig> exhibited an elevated level of expression in k. pneumoniae in the presence of tigecycline .
of note, although the syj <dig>  coding sequence is present in k. pneumoniae, two transcripts were detected after hybridisation.
however it was the larger rna species that appeared upregulated in rna derived from klebsiella cells challenged with half the mic of tigecycline.
hence we surmise that this larger rna transcript, consistent with the larger intergenic region in k. pneumoniae, is where the syj <dig> homolog coding sequence is located.
from these results we show that the upregulation of srnas identified in this study are neither species nor drug specific in the presence of unrelated classes of antibiotics.
5’ rapid amplifed cdna ends  of syj <dig>
to determine the transcriptional start site  of syj <dig> , we performed 5’ race analysis.
as shown in figure
<dig>  the 5’ race result reveals that the tss of syj <dig> and tbpa lies  <dig> bases upstream of the start codon of tbpa, consistent with previous findings
<cit> .
quantitative real time pcr
syj <dig> : the upregulation of syj <dig> in s. typhimurium challenged by half the mic of tigecycline or tetracycline was quantified with qpcr.
as shown in figure
<dig>  compared to the control, cells challenged by tigecycline or tetracycline produced ~ <dig> fold more syj <dig>  interestingly, the transcription level of the downstream gene, tbpa, was hardly affected by the presence of the antibiotics.
this suggests that syj <dig>  but not the tbpa gene product, is upregulated as a result of tigecycline or tetracycline challenge.
dinf  and ycfr : as mentioned previously, the rna transcripts of these two stress responsive genes were picked up in the srna cloning and is suggestive that half the mic of tigecycline does induce a stress response in s. typhimurium.
in order to confirm this, we performed a qpcr on s. typhimurium challenged by half the mic of tigecycline or tetracycline, and compared the transcriptional levels of dinf and ycfr to the control.
as shown in figure
<dig>  the transcriptional level of dinf increased to  <dig>  and  <dig>  fold when the cells were challenged by half the mic of tigecycline and tetracycline, respectively; the level of ycfr increased to  <dig> and  <dig> fold when the cells were challenged by half the mic of tigecycline and tetracycline, respectively.
survival rate assays
survival rate assays were performed to investigate whether the deletion of syj <dig>  would highlight any phenotypic deficiencies when challenged with tigecycline.
our initial tests showed that the mics of the mutant  and the wild type strains  were identical to tigecycline .
we then performed growth curves in rdm, where both sl <dig> and yj <dig> exhibited similar growth rates, as determined by od <dig> readings, even in the presence of tigecycline .
to determine whether syj <dig> confers an advantage to bacterial survival in the presence of tigecycline challenge, the survival frequencies were determined for the wild type sl <dig> and yj <dig> in the presence of  <dig> ×,  <dig> ×, 4 × and 8 × mic of tigecycline.
both sl <dig> and yj <dig> failed to form any colonies on  <dig> ×, 4 × and 8 × mic plates after overnight incubation at 37°c.
the survival rates for sl <dig> and yj <dig> at 1 × the mic were ~ <dig>  × 10- <dig> and  <dig>  × 10- <dig> respectively .
despite this modest decrease, statistical analysis on four biological replicate experiments supports that the reduced survival rate observed in yj <dig> is indeed significant .
the survival rate was restored upon complementation where yj <dig>  yielded a survival frequency close but higher than sl <dig> , and as expected the plasmid control yj <dig>  had a similar survival rate to yj <dig> .
this reduction in the survival rate of yj <dig> compared to the one of yj <dig> was also found to be statistically significant .
overall, it suggests that the absence of syj <dig> could confer a subtle but reduced survival rate in the presence of tigecycline.
discussion
small rnas are regulatory molecules that enhance a bacterium’s adaptability in a constantly changing environment
<cit> .
as regulatory molecules, srnas have several advantages over their protein counterparts.
firstly, srnas consist of a short nucleotide sequence which does not require translation into a peptide sequence.
this ensures that the response from srna mediated regulators would be much more rapid than protein mediated factors
<cit> .
accordingly, modelling studies suggest that due to the rapid kinetics associated with srna production, the downstream regulon response is correspondingly prompt when compared to protein based factors, a valuable trait in constantly evolving environments
<cit> .
moreover, base pairing flexibility presumably allows rapid evolution of srnas
<cit> .
finally, srna-mrna interaction generally lacks specificity and often imperfect binding occurs ensuring that more than one target mrna is affected, thereby expanding the repertoire of the srna regulators
<cit> .
at antibiotic concentrations at or higher than the mic, cells are likely to halt cellular replication and / or stop growing, or result in the accumulation of advantageous genomic mutations
<cit> , which may permanently alter the transcriptional profiles of bacteria
<cit> .
hence we surmised that the srnas upregulated in the cells under these conditions may not be a direct result of antibiotic stress response but possibly due to genetic mutations or global perturbations.
therefore, a cdna library was constructed from the cells that were challenged by half the mic of tigecycline at mid-log phase.
in support of our hypothesis, our screen identified genes involved in the stress response when the bacterial cells were challenged with half the mic of tigecycline.
these include a sos response gene, dinf, encoding a mate family efflux pump, and a gene homologous to ycfr in e. coli, encoding a putative outer membrane protein.
qpcr confirms the upregulation of the two genes when s. typhimurium is challenged with half the mic of tigecycline or tetracycline .
our finding of four srnas , syj <dig>  syj <dig> and syj118) that are upregulated in the presence of tigecycline or tetracycline provides the first direct evidence that srnas are differentially expressed upon antibiotic exposure.
it is known that tetracycline triggers mrna accumulation in bacteria
<cit> .
however, this is unlikely to be the case as increased transcription was not noted for e.g.
tbpa , and the gene encoding the 5s rna .
two of the four srnas  we describe in this study are novel.
additionally, our work shows that these four srnas are not species specific as both syj <dig> and syj <dig> are upregulated in k. pneumoniae when challenged with half the mic of tigecycline, or drug specific as syj <dig>  syj <dig> and syj <dig> are upregulated as a result of ampicillin challenge .
both syj <dig>  previously identified as styr- <dig> in salmonella <cit> , and syj <dig>  a novel srna discovered in this study, are located between 16s and 23s rrna coding sequences .
both tigecycline and tetracycline target the 30s ribosomal subunit in bacterial cells.
this might trigger over-production of the 16s-23s rrna molecules, which also includes syj <dig> and syj <dig>  this may raise the possibility that syj <dig> and syj <dig> are “by-products” rather than bona fide srna regulators.
however, in support of syj <dig> and syj <dig> being classed as srnas, not all 16s-23s rrna intergenic regions identified in our screen were upregulated in the presence of tigecycline when assessed by northern blots .
furthermore, only syj <dig>  not syj <dig>  was upregulated in k. pneumoniae when challenged with tigecycline .
taken together these data lead to the observation that these different inter 16s-23s rrna regions  may have alternative functions independent of rrna processing, which could be regulatory srna.
in this work we have used the 5s rna as a loading control for northern blot assays.
given that it is a ribosomal rna we wondered whether the 5s rna levels would be affected by either tigecycline or tetracycline exposure.
as shown in figure
4a, the 5s rna expression levels were unaltered when the cells were challenged with half the mic of tigecycline or tetracycline, and therefore it is a suitable loading control for the northern blot assays.
the four srnas  that were upregulated as a response to tigecycline challenge in s. typhimurium were also upregulated in tetracycline challenged cells .
this is not surprising since both tigecycline and tetracycline target the 30s ribosomal subunit.
it is possible that the similar mechanisms of action of tetracycline and tigecycline trigger comparable stress-responsive pathways, which possibly include syj <dig>  syj <dig>  syj <dig> and syj <dig>
syj <dig> has not been previously described and thus is also a novel srna discovered in this study.
its conservation among several species and its upregulation in s. typhimurium upon challenge with tigecycline and tetracycline,  suggest that syj <dig>  combined with its conservation across different species, may represent a common denominator in the response to tigecycline / tetracycline exposure.
interestingly, none of the four srnas were found upregulated when s. typhimurium was exposed to ciprofloxacin, or when e. coli was challenged with tigecycline .
when challenged with tigecycline, both s. typhimurium and k. pneumoniae upregulated two srnas, namely syj <dig> and syj <dig> .
despite encoding these sequences, no upregulation was noted in e. coli cells exposed to tigecycline compared to the unexposed controls .
this suggests two possibilities: the first, where the tigecycline stress response involving srnas in e. coli is different from that in k. pneumoniae and s. typhimurium, and the second, where the srnas  may be linked to regulatory networks contributing to tigecycline resistance, i.e.
rama, only found in s. typhimurium and k.pneumoniae but not in e. coli <cit> .
however targetrna
<cit>  predictions for syj <dig> for cognate mrna binding partners, using default parameters, yields four mrna sequences .
of note, pspb and pspa which are involved in stress-response and the virulence attributes of several bacterial species
<cit>  are potential targets of syj <dig>  syj20-mediated control of the psp operon may explain the reduced fitness of the sroa  deleted salmonella strain in a mouse infection model
<cit> .
the sequence of syj <dig>  was applied as the input for targetrna  prediction with default parameters.
a recent study undertaken to map srna profiles in sl <dig> using massive parallel sequencing technology identified  <dig> srnas.
notably, syj <dig> and syj <dig> were not identified in this large scale study which suggests that firstly, these srnas are produced as a result of conditional exposure e.g.
tigecycline and secondly that our small scale screen is able to uncover novel srnas
<cit> .
the encoding sequences of three srnas  identified in this screen have more than one paralog within s. typhimurium’s genome, making it difficult to pinpoint their exact roles in the bacterial response against antibiotic challenge through genetic analysis.
due to this reason, only syj <dig> and its associated phenotype were investigated further.
syj <dig>  also known as sroa
<cit> , is encoded immediately upstream of the tbpayabkyabj operon  and contains a thi-box sequence required as a riboswitch for the modulation of the tbpayabkyabj operon .
the deletion of the chromosomal sequence of syj <dig> would have very likely removed the tss of the downstream gene tbpa .
however, tbpa transcript levels remained unaltered upon tigecycline / tetracycline exposure .
therefore the polar effect of the syj <dig> deletion is considered to be minimal.
when survival rate assays were performed a subtle but reproducible deficiency  as reflected by a reduction in the viability in the Δsyj <dig> strain  compared to the wild type strain   was observed.
this deficiency was alleviated when a plasmid encoding allele of syj <dig> was transformed in yj <dig> , where the vector only control  did not .
this subtle change of phenotype is not entirely surprising, as it has been observed that srna deletions usually have little, if any, effect
<cit> .
in fact, syj <dig>  or sroa, has been linked to other phenotypes such as reduced fitness by a Δsroa s. typhimurium strain  during competitive infection with the wild type strain in mice
<cit> .
however it is not evident from the work whether the reduction in competitiveness of the Δsroa s. typhimurium strain is due to altered tbpa expression.
previous work suggests that syj <dig>  may function as a riboswitch for the tbpayabkyabj  operon
<cit>  in e. coli and that this regulatory role does not require hfq
<cit> .
in our studies, we can show that the wild type strain s. typhimurium  produces syj <dig>  in the presence of sub-inhibitory concentration of ciprofloxacin  whilst the Δhfq strain
<cit>  produced less .
this suggests that syj <dig>  apart from its putative riboswitch role, can act as a trans-regulatory srna, as hfq is typically required for functionality and stability by trans-encoded srnas
<cit> .
this is further supported by the two facts that a) the mild defect due to the chromosomal deletion of syj <dig> in sl <dig> can be complemented by the plasmid-coding allele , which cannot be attributed to its role as riboswitch, since the rna transcripts of syj <dig>  and tbpa  are on separate strands, and b) syj <dig> was upregulated in s. typhimurium challenged with half the mic of tigecycline or tetracycline, where the transcriptional level of tbpa remained the same .
the transcript size of syj <dig>  as detected by northern blot analysis, is approximately  <dig> nts which is consistent with the size reported in e. coli
<cit> .
as has been suggested previously, it is possible that syj <dig> is generated by transcription attenuation of tbpayabkyabj <cit> ; and the released short syj <dig>  functions as a srna by regulating alternative targets in trans in the cell.
CONCLUSIONS
our work shows that srnas upregulated in response to tigecycline exposure can also be produced in a non drug or species specific manner.
the deletion of the srna, syj <dig>  confers a subtle survival disadvantage in the presence of tigecycline, possibly due to its role as a trans-regulatory srna after tigecycline exposure.
our results although preliminary, suggest that srna levels can be altered upon antibiotic exposure and presumably provide an initial survival advantage under antibiotic challenge.
however, ongoing analyses are required to dissect the regulatory impact of srna upregulation and its contribution to antibiotic resistance in bacteria.

 multiple studies demonstrate that non coding rnas ) possess regulatory roles in the bacterial stress response. bacterial srna regulators typically range from – nts and are often transcribed from intergenic regions, although open reading frames may also encode srnas. most srnas act as regulators at the post-transcriptional level by base-pairing with target mrnas; these srna-mrna binding regions are often short and imperfect and may require an additional rna chaperone, which in most cases is the hfq protein. this imperfect binding allows each srna molecule to control multiple targets, where either the translation of the target mrna is upregulated, or more commonly inhibited. many srna regulators are upregulated when bacteria sense environmental stress: these include oxidative stress, low ph environment, nutrient deprivation and glucose-phosphate stress. despite overwhelming evidence that srnas play a role when bacteria experience physiological stress, no systematic study has been undertaken to ascertain the impact or levels of srna production in bacteria when antibiotics are present. naturally susceptible pathogens can develop drug resistance when treated with antibiotics. genetically acquired antibiotic resistance in pathogenic bacteria, via spontaneous / random mutations and horizontal gene transfer, is a significant issue in the treatment of infectious diseases. intrinsic regulatory networks such as those mediated by the transcriptional regulators mara, soxs and rama are also implicated in the development of antibiotic resistance particularly since these systems control the influx / efflux of antibiotics. thus far studies that have focused on the intrinsic antibiotic resistome are limited to gene and protein networks mediated by these gene operons or other transcription factors. hence the role of the newly uncovered class of regulatory molecules such as srnas in controlling or contributing to the antimicrobial resistance phenotype is largely unknown. some evidence for the role of srnas in mediating antimicrobial resistance already exists: for example, the expression of bacterial outer membrane proteins, ompf and ompc, involved in antibiotic import, is controlled by the srnas micf and micc respectively. additionally, the overexpression of another srna was recently found to induce multidrug resistance in escherichia coli via the mdtef efflux pump. nevertheless, whether the functional role of micf, micc and dsra is indeed part of the bacteria’s intrinsic stress response to antibiotic challenge remains unknown. tigecycline is a member of the glycylcycline group of antibiotics, and was registered in the eu in april 2006. this bacteriostatic antibiotic acts as a protein synthesis inhibitor by binding to the 30s ribosomal subunit. tigecycline is active against a broad range of bacteria, with only few naturally resistant exceptions, namely, proteus spp., morganella morganii, providencia spp., and pseudomonas aeruginosa. specifically, tigecycline is effective against multidrug resistant bacteria such as staphylococcus aureus, vancomycin-resistant enterococcus, extended-spectrum beta-lactamase -expressing enterobacteriaceae, and carbapenem-resistant strains. reports of resistance to tigecycline have been rare in naturally susceptible pathogens, however in resistant variants efflux pump overexpression has contributed to tigecycline resistance. salmonella, a member of enterobacteriaceae, encodes both the rama transcriptional factor and the acrab efflux pump, which when overexpressed confers tigecycline resistance. additionally, salmonella represents a model bacterium for srna mining and genome manipulation, making it an ideal system for our study, but more importantly represents a paradigm for other members of enterobacteriaceae. hence in this study we used a cloning strategy to determine the srna population after tigecycline exposure in salmonella enterica serovar typhimurium, and also whether the absence of these srnas would render the cells less adaptable to tigecycline challenge.  cdna library construction and analysis a cdna library was constructed from the cells that were challenged by half the minimal inhibitory concentration of tigecycline at od600 = . approximately ~ clones were obtained; from these random candidates were sequenced and analysed. the nature of the cdna library construction procedure allowed us to obtain the sequences in an orientation specific manner. the cdna sequences were mapped to the s. typhimurium sl genome using blast. of the mapped sequences, 31% encoded trnas; 6% and 9% matched to rrnas and protein coding sequences, respectively; 4% partially overlapped with open reading frames, and 50% aligned to igrs. of all the igr readings, 90% were located between the 16s and 23s rrna encoding genes. analyses of the cdna sequences encoding partial orfs indicated which genes were expressed in the presence of tigecycline. as stated above, 9% of the sequences identified matched to rrnas, in addition to a further sequence which was found to overlap the 30s ribosomal protein and another mapped to elongation factor tu. this is perhaps not surprising, given that the specific target for tigecycline is the ribosome. on the other hand, sequences overlapping known stress-response genes were also captured in the cdna library, e.g. dinf and a gene encoding a putative outer membrane protein. the dinf gene is a member of the sos response family and encodes an efflux pump which belongs to the multidrug and toxic compound extrusion family, and sl1344_ encoding a putative outer membrane protein homologous to ycfr in e. coli, which influences biofilm formation through stress response and surface hydrophobicity. the expression of these genes supports our hypothesis that challenge at half the mic of tigecycline triggers a stress response. of note, the cdna library also contained sequences of different lengths that mapped to open reading frames, which we postulate to be a result of mrna degradation, rather than a representation of bona fide srna regulators. meanwhile, 4% of all sequences that partially overlap orfs, all do so at the 5’ end of the orfs. this suggests that these sequences might be 5’ untranslated regions, or encode riboswitches and/or control the expression of the downstream genes. northern blot verification northern blot analysis was performed on rna extracted from sl that were either unchallenged or challenged with half the mic of tigecycline. since most srnas are produced from igrs, only sequences from these regions were selected for further validation by northern blot analysis. as 90% of the igr sequences are located between 16s and 23s rrna coding sequences, most of which are identical, there were unique igr sequences that were assayed, of which four were found to consistently show elevated expression with tigecycline challenge. the remaining srna candidates were either not detectable by northern blots, or did not show differential levels of transcription. correspondingly all further analyses focused on these four srnas. the relative fold increase in srna expression was determined by northern blots in challenged versus unchallenged cells. upon tigecycline exposure, the expression levels of syj syj syj and syj, relative to unchallenged cells, were increased to and fold, respectively. we also tested the level of the four srnas in cells challenged with half the mic of tetracycline. as expected, all of the four srnas were also found to be upregulated compared to the control sample. this is possibly due to the fact that tigecycline and tetracycline are related compounds, and they may as well trigger stress response pathways that share a common set of regulatory molecules. of note and as shown in figure 4a, the level of 5s rna was not affected by the presence of half the mic of tigecycline or tetracycline. bioinformatic analysis all four srna sequences were searched against s. typhimurium sl using ncbi blast. the syj encoding sequence is located between the 16s and 23s rrna coding loci on the sense strand ). blast analysis uncovered two additional identical copies in the genome sequence of sl. similar to syj syj is also encoded from the igr between the 16s and 23s rrna coding sequences, but from a different genetic locus ). the sequence encoding syj has an identical copy and additionally five other paralogs with 93% - 99% identity on the sl chromosome. the encoding sequence of syj is flanked by entc downstream, and fepb upstream ). it also has a paralog that shares 90% identity, starting at position on the s. typhimurium sl genome and located between pntb and an un-annotated gene. syj was previously identified by vogel et al. in e. coli as sroa, encoded by a sequence downstream of yabn and upstream of tbpa and 5a). syj syj and syj are all highly conserved within the different members of enterobacteriaceae, although the coding sequences of syj syj and syj are also found in other families of bacteria, in plants and in animals. in contrast, syj is only found in salmonella, enterobacter, photorhabdus and citrobacter. syj, syj syj and syj in other species and relevance to other drug classes we proceeded to determine whether the increased expression of these srnas would be salmonella specific or drug-class specific. hence, we assessed the levels of our srna candidates in other members of enterobacteriaceae when challenged with sub-inhibitory levels of tigecycline. additionally, in order to determine whether these srnas are upregulated solely as a result of tigecycline challenge or are generally upregulated as a result of sub-inhibitory antibiotic challenge, s. typhimurium sl was challenged with either half the mic of ampicillin or ciprofloxacin. as shown in figure 3b, none of the four tested srnas were upregulated in response to ciprofloxacin exposure, whilst three were found to be upregulated in the presence of ampicillin. interestingly, e. coli did not upregulate the expression of the three candidate srnas in response to challenge at half the mic of tigecycline. however, syj exhibited an elevated level of expression in k. pneumoniae in the presence of tigecycline. of note, although the syj coding sequence is present in k. pneumoniae, two transcripts were detected after hybridisation. however it was the larger rna species that appeared upregulated in rna derived from klebsiella cells challenged with half the mic of tigecycline. hence we surmise that this larger rna transcript, consistent with the larger intergenic region in k. pneumoniae, is where the syj homolog coding sequence is located. from these results we show that the upregulation of srnas identified in this study are neither species nor drug specific in the presence of unrelated classes of antibiotics. 5’ rapid amplifed cdna ends of syj to determine the transcriptional start site of syj, we performed 5’ race analysis. as shown in figure the 5’ race result reveals that the tss of syj and tbpa lies bases upstream of the start codon of tbpa, consistent with previous findings. quantitative real time pcr syj: the upregulation of syj in s. typhimurium challenged by half the mic of tigecycline or tetracycline was quantified with qpcr. as shown in figure compared to the control, cells challenged by tigecycline or tetracycline produced ~ fold more syj interestingly, the transcription level of the downstream gene, tbpa, was hardly affected by the presence of the antibiotics. this suggests that syj but not the tbpa gene product, is upregulated as a result of tigecycline or tetracycline challenge. dinf and ycfr: as mentioned previously, the rna transcripts of these two stress responsive genes were picked up in the srna cloning and is suggestive that half the mic of tigecycline does induce a stress response in s. typhimurium. in order to confirm this, we performed a qpcr on s. typhimurium challenged by half the mic of tigecycline or tetracycline, and compared the transcriptional levels of dinf and ycfr to the control. as shown in figure the transcriptional level of dinf increased to and fold when the cells were challenged by half the mic of tigecycline and tetracycline, respectively; the level of ycfr increased to and fold when the cells were challenged by half the mic of tigecycline and tetracycline, respectively. survival rate assays survival rate assays were performed to investigate whether the deletion of syj would highlight any phenotypic deficiencies when challenged with tigecycline. our initial tests showed that the mics of the mutant and the wild type strains were identical to tigecycline. we then performed growth curves in rdm, where both sl and yj exhibited similar growth rates, as determined by od readings, even in the presence of tigecycline. to determine whether syj confers an advantage to bacterial survival in the presence of tigecycline challenge, the survival frequencies were determined for the wild type sl and yj in the presence of ×, ×, 4 × and 8 × mic of tigecycline. both sl and yj failed to form any colonies on ×, 4 × and 8 × mic plates after overnight incubation at 37°c. the survival rates for sl and yj at 1 × the mic were ~  × 10- and  × 10- respectively. despite this modest decrease, statistical analysis on four biological replicate experiments supports that the reduced survival rate observed in yj is indeed significant. the survival rate was restored upon complementation where yj yielded a survival frequency close but higher than sl, and as expected the plasmid control yj had a similar survival rate to yj. this reduction in the survival rate of yj compared to the one of yj was also found to be statistically significant. overall, it suggests that the absence of syj could confer a subtle but reduced survival rate in the presence of tigecycline. discussion small rnas are regulatory molecules that enhance a bacterium’s adaptability in a constantly changing environment. as regulatory molecules, srnas have several advantages over their protein counterparts. firstly, srnas consist of a short nucleotide sequence which does not require translation into a peptide sequence. this ensures that the response from srna mediated regulators would be much more rapid than protein mediated factors. accordingly, modelling studies suggest that due to the rapid kinetics associated with srna production, the downstream regulon response is correspondingly prompt when compared to protein based factors, a valuable trait in constantly evolving environments. moreover, base pairing flexibility presumably allows rapid evolution of srnas. finally, srna-mrna interaction generally lacks specificity and often imperfect binding occurs ensuring that more than one target mrna is affected, thereby expanding the repertoire of the srna regulators. at antibiotic concentrations at or higher than the mic, cells are likely to halt cellular replication and / or stop growing, or result in the accumulation of advantageous genomic mutations, which may permanently alter the transcriptional profiles of bacteria. hence we surmised that the srnas upregulated in the cells under these conditions may not be a direct result of antibiotic stress response but possibly due to genetic mutations or global perturbations. therefore, a cdna library was constructed from the cells that were challenged by half the mic of tigecycline at mid-log phase. in support of our hypothesis, our screen identified genes involved in the stress response when the bacterial cells were challenged with half the mic of tigecycline. these include a sos response gene, dinf, encoding a mate family efflux pump, and a gene homologous to ycfr in e. coli, encoding a putative outer membrane protein. qpcr confirms the upregulation of the two genes when s. typhimurium is challenged with half the mic of tigecycline or tetracycline. our finding of four srnas, syj syj and syj118) that are upregulated in the presence of tigecycline or tetracycline provides the first direct evidence that srnas are differentially expressed upon antibiotic exposure. it is known that tetracycline triggers mrna accumulation in bacteria. however, this is unlikely to be the case as increased transcription was not noted for e.g. tbpa, and the gene encoding the 5s rna. two of the four srnas we describe in this study are novel. additionally, our work shows that these four srnas are not species specific as both syj and syj are upregulated in k. pneumoniae when challenged with half the mic of tigecycline, or drug specific as syj syj and syj are upregulated as a result of ampicillin challenge. both syj previously identified as styr- in salmonella, and syj a novel srna discovered in this study, are located between 16s and 23s rrna coding sequences. both tigecycline and tetracycline target the 30s ribosomal subunit in bacterial cells. this might trigger over-production of the 16s-23s rrna molecules, which also includes syj and syj this may raise the possibility that syj and syj are “by-products” rather than bona fide srna regulators. however, in support of syj and syj being classed as srnas, not all 16s-23s rrna intergenic regions identified in our screen were upregulated in the presence of tigecycline when assessed by northern blots. furthermore, only syj not syj was upregulated in k. pneumoniae when challenged with tigecycline. taken together these data lead to the observation that these different inter 16s-23s rrna regions may have alternative functions independent of rrna processing, which could be regulatory srna. in this work we have used the 5s rna as a loading control for northern blot assays. given that it is a ribosomal rna we wondered whether the 5s rna levels would be affected by either tigecycline or tetracycline exposure. as shown in figure 4a, the 5s rna expression levels were unaltered when the cells were challenged with half the mic of tigecycline or tetracycline, and therefore it is a suitable loading control for the northern blot assays. the four srnas that were upregulated as a response to tigecycline challenge in s. typhimurium were also upregulated in tetracycline challenged cells. this is not surprising since both tigecycline and tetracycline target the 30s ribosomal subunit. it is possible that the similar mechanisms of action of tetracycline and tigecycline trigger comparable stress-responsive pathways, which possibly include syj syj syj and syj syj has not been previously described and thus is also a novel srna discovered in this study. its conservation among several species and its upregulation in s. typhimurium upon challenge with tigecycline and tetracycline, suggest that syj combined with its conservation across different species, may represent a common denominator in the response to tigecycline / tetracycline exposure. interestingly, none of the four srnas were found upregulated when s. typhimurium was exposed to ciprofloxacin, or when e. coli was challenged with tigecycline. when challenged with tigecycline, both s. typhimurium and k. pneumoniae upregulated two srnas, namely syj and syj. despite encoding these sequences, no upregulation was noted in e. coli cells exposed to tigecycline compared to the unexposed controls. this suggests two possibilities: the first, where the tigecycline stress response involving srnas in e. coli is different from that in k. pneumoniae and s. typhimurium, and the second, where the srnas may be linked to regulatory networks contributing to tigecycline resistance, i.e. rama, only found in s. typhimurium and k.pneumoniae but not in e. coli. however targetrna predictions for syj for cognate mrna binding partners, using default parameters, yields four mrna sequences. of note, pspb and pspa which are involved in stress-response and the virulence attributes of several bacterial species are potential targets of syj syj20-mediated control of the psp operon may explain the reduced fitness of the sroa deleted salmonella strain in a mouse infection model. the sequence of syj was applied as the input for targetrna prediction with default parameters. a recent study undertaken to map srna profiles in sl using massive parallel sequencing technology identified srnas. notably, syj and syj were not identified in this large scale study which suggests that firstly, these srnas are produced as a result of conditional exposure e.g. tigecycline and secondly that our small scale screen is able to uncover novel srnas. the encoding sequences of three srnas identified in this screen have more than one paralog within s. typhimurium’s genome, making it difficult to pinpoint their exact roles in the bacterial response against antibiotic challenge through genetic analysis. due to this reason, only syj and its associated phenotype were investigated further. syj also known as sroa, is encoded immediately upstream of the tbpayabkyabj operon and contains a thi-box sequence required as a riboswitch for the modulation of the tbpayabkyabj operon. the deletion of the chromosomal sequence of syj would have very likely removed the tss of the downstream gene tbpa. however, tbpa transcript levels remained unaltered upon tigecycline / tetracycline exposure. therefore the polar effect of the syj deletion is considered to be minimal. when survival rate assays were performed a subtle but reproducible deficiency as reflected by a reduction in the viability in the Δsyj strain compared to the wild type strain was observed. this deficiency was alleviated when a plasmid encoding allele of syj was transformed in yj, where the vector only control did not. this subtle change of phenotype is not entirely surprising, as it has been observed that srna deletions usually have little, if any, effect. in fact, syj or sroa, has been linked to other phenotypes such as reduced fitness by a Δsroa s. typhimurium strain during competitive infection with the wild type strain in mice. however it is not evident from the work whether the reduction in competitiveness of the Δsroa s. typhimurium strain is due to altered tbpa expression. previous work suggests that syj may function as a riboswitch for the tbpayabkyabj operon in e. coli and that this regulatory role does not require hfq. in our studies, we can show that the wild type strain s. typhimurium produces syj in the presence of sub-inhibitory concentration of ciprofloxacin whilst the Δhfq strain produced less. this suggests that syj apart from its putative riboswitch role, can act as a trans-regulatory srna, as hfq is typically required for functionality and stability by trans-encoded srnas. this is further supported by the two facts that a) the mild defect due to the chromosomal deletion of syj in sl can be complemented by the plasmid-coding allele, which cannot be attributed to its role as riboswitch, since the rna transcripts of syj and tbpa are on separate strands, and b) syj was upregulated in s. typhimurium challenged with half the mic of tigecycline or tetracycline, where the transcriptional level of tbpa remained the same. the transcript size of syj as detected by northern blot analysis, is approximately nts which is consistent with the size reported in e. coli. as has been suggested previously, it is possible that syj is generated by transcription attenuation of tbpayabkyabj; and the released short syj functions as a srna by regulating alternative targets in trans in the cell.  our work shows that srnas upregulated in response to tigecycline exposure can also be produced in a non drug or species specific manner. the deletion of the srna, syj confers a subtle survival disadvantage in the presence of tigecycline, possibly due to its role as a trans-regulatory srna after tigecycline exposure. our results although preliminary, suggest that srna levels can be altered upon antibiotic exposure and presumably provide an initial survival advantage under antibiotic challenge. however, ongoing analyses are required to dissect the regulatory impact of srna upregulation and its contribution to antibiotic resistance in bacteria. 
the upregulation of some of these srnas were also observed when s. typhimurium was challenged by ampicillin; or when klebsiella pneumoniae was challenged by tigecycline. small rnas are overexpressed as a result of antibiotic exposure in s. typhimurium where the same molecules are upregulated in a related species or after exposure to different antibiotics. genome analysis found that syj and syj mapped between the 16s and 23s rrna encoding genes. syj is encoded upstream of the tbpayabkyabj operon and is classed as a riboswitch, whilst its role in antibiotic stress-response appears independent of its riboswitch function. despite mounting evidence for the importance of srnas in stress response, their role upon antibiotic exposure remains unknown. in this study, we sought to determine firstly, whether differential expression of srnas occurs upon antibiotic exposure and secondly, whether these srnas could be attributed to microbial tolerance to antibiotics. bacteria employ complex transcriptional networks involving multiple genes in response to stress, which is not limited to gene and protein networks but now includes small rnas. these findings imply that the srna mediated response is a component of the bacterial response to antibiotic challenge. a small scale srna cloning strategy of salmonella enterica serovar typhimurium sl challenged with half the minimal inhibitory concentration of tigecycline identified four srnas which were reproducibly upregulated in the presence of either tigecycline or tetracycline.
 bacteria employ complex transcriptional networks involving multiple genes in response to stress, which is not limited to gene and protein networks but now includes small rnas. these regulatory rna molecules are increasingly shown to be able to initiate regulatory cascades and modulate the expression of multiple genes that are involved in or required for survival under environmental challenge. despite mounting evidence for the importance of srnas in stress response, their role upon antibiotic exposure remains unknown. in this study, we sought to determine firstly, whether differential expression of srnas occurs upon antibiotic exposure and secondly, whether these srnas could be attributed to microbial tolerance to antibiotics.  a small scale srna cloning strategy of salmonella enterica serovar typhimurium sl challenged with half the minimal inhibitory concentration of tigecycline identified four srnas which were reproducibly upregulated in the presence of either tigecycline or tetracycline. the coding sequences of the four srnas were found to be conserved across a number of species. genome analysis found that syj and syj mapped between the 16s and 23s rrna encoding genes. syj is encoded upstream of the tbpayabkyabj operon and is classed as a riboswitch, whilst its role in antibiotic stress-response appears independent of its riboswitch function. syj is encoded between genes that are involved in enterobactin transport and metabolism. additionally we find that the genetic deletion of syj rendered a reduced viability phenotype in the presence of tigecycline, which was recovered when complemented. the upregulation of some of these srnas were also observed when s. typhimurium was challenged by ampicillin; or when klebsiella pneumoniae was challenged by tigecycline.  small rnas are overexpressed as a result of antibiotic exposure in s. typhimurium where the same molecules are upregulated in a related species or after exposure to different antibiotics. syj a riboswitch, appears to possess a trans-regulatory srna role in antibiotic tolerance. these findings imply that the srna mediated response is a component of the bacterial response to antibiotic challenge. 