Riboswitch RSthiT as a Molecular Tool in Lactococcus lactis.

ABSTRACT

Previous RNA sequencing has allowed the identification of 129 long 5' untranslated regions (UTRs) in the Lactococcus lactis MG1363 transcriptome. These sequences potentially harbor cis-acting riboswitches. One of the identified extended 5' UTRs is a putative thiamine pyrophosphate (TPP) riboswitch. It is located immediately upstream of the thiamine transporter gene thiT (llmg_0334). To confirm this assumption, the 5'-UTR sequence was placed upstream of the gene encoding the superfolder green fluorescent protein (sfGFP), sfgfp, allowing the examination of the expression of sfGFP in the presence or absence of thiamine in the medium. The results show that this sequence indeed represents a thiamine-responsive TPP riboswitch. This RNA-based genetic control device was used to successfully restore the mutant phenotype of an L. lactis strain lacking the major autolysin gene, acmA. The L. lactis thiT TPP riboswitch (RSthiT) is a useful molecular genetic tool enabling the gradual downregulation of the expression of genes under its control by adjusting the thiamine concentration. IMPORTANCE The capacity of microbes with biotechnological importance to adapt to and survive under quickly changing industrial conditions depends on their ability to adequately control gene expression. Riboswitches are important RNA-based elements involved in rapid and precise gene regulation. Here, we present the identification of a natural thiamine-responsive riboswitch of Lactococcus lactis, a bacterium used worldwide in the production of dairy products. We used it to restore a genetic defect in an L. lactis mutant and show that it is a valuable addition to the ever-expanding L. lactis genetic toolbox.