wellesley.edu/targetRNA/) prediction with default parameters. A recent study undertaken AZD1080 to map sRNA profiles in SL1344 using massive parallel sequencing technology identified 140 sRNAs. Notably, sYJ5 and sYJ75 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 [34]. The encoding sequences of three sRNAs (sYJ5, sYJ75 and sYJ118) 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 sYJ20 and its associated phenotype
were investigated further. sYJ20, also known as SroA [5], is encoded immediately upstream of the tbpAyabKyabJ operon (homologous to thiBPQ in E. coli) and contains a THI-box sequence required as a riboswitch for the modulation of the tbpAyabKyabJ operon (Figure 5). The 3-MA cost deletion of the chromosomal sequence of sYJ20 would have very likely removed the TSS of the downstream gene tbpA (Figure 5). However, tbpA transcript levels remained unaltered upon tigecycline / tetracycline exposure (Figure 6). Therefore the polar effect of the sYJ20 deletion is considered to be minimal. When survival rate assays were performed a subtle but reproducible deficiency (P < 0.05) as reflected click here by a reduction in the viability in the ΔsYJ20 strain (YJ104) compared to the wild type strain (SL1344) (Figure 7) was observed. This deficiency was alleviated when a plasmid encoding allele of sYJ20 was transformed in YJ104 (i.e. YJ107), where the vector only control (i.e. YJ110) did not (Figure 7). This subtle change of phenotype is not entirely surprising, as it has been observed that sRNA deletions usually have little,
if any, effect [45]. In fact, sYJ20, or SroA, has been linked to other phenotypes such as reduced fitness by a ΔsroA S. Typhimurium strain (sroA encodes sYJ20) during competitive infection with the wild type strain in mice [44]. However it is not evident Ixazomib manufacturer from the work whether the reduction in competitiveness of the ΔsroA S. Typhimurium strain is due to altered tbpA expression. Previous work suggests that sYJ20 (SroA) may function as a riboswitch for the tbpAyabKyabJ (thiBPQ) operon [5] in E. coli and that this regulatory role does not require Hfq [46]. In our studies, we can show that the wild type strain S. Typhimurium (SL1344) produces sYJ20 (transcript size around 100 nts) in the presence of sub-inhibitory concentration of ciprofloxacin (0.0078 μg/ml) whilst the Δhfq strain [7] produced less (Figure 4B). This suggests that sYJ20, 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 [47].
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