Hence we surmised that the sRNAs upregulated in the cells under t

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 (Figure

KU-57788 price 6). Our finding of four sRNAs (sYJ20 (SroA), sYJ5, sYJ75 and sYJ118) that are upregulated in the presence of tigecycline learn more 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 [38]. However, this is unlikely to be the case as increased transcription was not noted for e.g. tbpA (open reading frame lying downstream

click here of sYJ20, Figure 6), and the gene encoding the 5S RNA (Figure 4A). Two of the four sRNAs (sYJ5 and sYJ75) we describe in this study are novel. Additionally, our work shows that these four sRNAs are not species specific as both sYJ20 and sYJ118 are upregulated in K. pneumoniae when challenged with half the MIC of tigecycline, or drug specific as sYJ5, sYJ75 and sYJ118 are upregulated as a result of ampicillin challenge (Figure 3B). Both sYJ118, previously identified as StyR-44 in Salmonella[39], and sYJ5, a novel sRNA discovered in this study, are located between 16S and 23S rRNA coding sequences (Figure 2C). 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 sYJ5 and sYJ118. This may raise the possibility that sYJ5 and sYJ118 are “by-products” rather than bona fide sRNA regulators. However, in support of sYJ5 and sYJ118 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 (data not shown). Furthermore, only sYJ118, not sYJ5, was upregulated in K. pneumoniae when challenged with tigecycline (Figure 3B).

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