Lactococcus lactis are Gram-positive, immobile, facultatively anaerobic, non-spore-forming lactic acid bacteria that have become an important model organism due to their industrial role as primary components of dairy starter cultures and their impact on public health due to their probiotic properties [1]. In order to maintain and improve these properties it is important to understand how these microorganisms adapt to environmental changes, especially when subjected to stress conditions caused by temperature changes, nutrient availability or other stress inducing factors.
Small regulatory RNAs (sRNAs) are amongst the most abundant gene-regulating RNAs, serving as key post-transcriptional regulators essential for bacterial adaptation to environmental changes [2]. These sRNAs regulate gene expression by base pairing with target mRNAs influencing its translation or by directly interacting with proteins and affecting their activity [3]. Gaining deeper insight into the role of regulatory RNAs in controlling stress responses and metabolic processes in Lactococcus lactis could potentially lead to innovative practical industrial applications [4]. In the Department of Biological DNA Modification, it was shown that transcription of several small regulatory RNAs is observed in a region of the L. lactis genome, which we have named the sLLM1238 system.
The aim of this study is to determine the mechanism of action of L. lactis sLLM1238 sRNAs. Firstly, we determined different forms of sRNAs by Northern blotting. Afterwards, to facilitate overexpression of sRNAs, we created a vector with a sLLM1238 gene and an empty one for control and electroporated them into L. lactis. In order to see expression changes of genes possibly regulated by sLLM1238, we performed total RNA sequencing analysis of L. lactis electroporated with these vectors. Furthermore, we analysed how L. lactis with and without overexpression of sRNAs responds when grown on agar plates with different concentrations of lysozyme. In addition, we extracted total RNA samples from L. lactis subjected to lysozyme in order to observe sRNA expression changes.
The obtained results will help to understand the mechanisms of action of sRNAs in L. lactis and their role in gene regulation as well as response to environment changes. This work was supported by funding from Research Council of Lithuania [MIP-22-11 to GV].