1 Faculty of Science, SDU2 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU3 University of Southern Denmark4 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU
During the past decade, small non-coding regulatory RNAs (sRNAs) have been identified in several bacterial species. In many cases, the sRNAs are only transiently transcribed in response to a particular stress or growth condition, reflecting the fact that many sRNAs regulate genes that are important for stress tolerance and virulence. One of the principle mechanisms used by bacteria to transiently express genes is through the use of alternative sigma factors. Under non-inducing conditions, alternative sigma factors are repressed by a complex regulatory network. However, in response to (a) particular signal(s) (low temperature, high osmolarity, amino acid stress etc.) the alternative sigma factor is de-repressed and is subsequently free to activate the expression of a specific regulon. L. monocytogenes encodes four alternative sigma factors, where only sigma B (SigB) has been extensively characterized. We thought it likely that L. monocytogenes might encode sRNAs that were specifically regulated by alternative sigma factors. To approach this issue, we are in the process of developing a bioinformatics tool that allows us to predict candidate sRNA genes that are likely to be regulated by an alternative sigma factor. Using this approach we have successfully identified a novel sRNA of ~75 nucleotides in L. monocytogenes that is specifically regulated by SigB. This sRNA, which we have termed SigB1 is expressed from the 3'-UTR of a large operon. SigB1 is expressed in a SigB dependant manner only in response to known SigB associated stresses such as salt- and ethanol-stress or entrance into stationary phase. Using transcriptional promoter-lacZ reporter assays, we have confirmed that SigB1 is not the result of an RNA-processing event. Interestingly, SigB1 does not contain any obvious Hfq binding sites and so far we haven't been able to detect any difference in the expression of SigB1 between a wt strain and a Dhfq strain. Finally, we have constructed an in-frame deletion of SigB1 and are currently in the process of analysing this mutant.