Riboswitch RS as a Molecular Tool in Lactococcus lactis

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Lactococcus lactis is a key bacterium in industrial applications, particularly in dairy production.
• Efficient gene expression control is crucial for microbial adaptation in changing environments.
• Riboswitches are RNA-based regulatory elements known for rapid and precise gene control.

Purpose of the Study:

• To identify and characterize a novel thiamine pyrophosphate (TPP) riboswitch in the Lactococcus lactis transcriptome.
• To validate the function of the identified riboswitch in controlling gene expression.
• To demonstrate the utility of this riboswitch as a molecular genetic tool in L. lactis.

Main Methods:

• RNA sequencing was employed to identify potential riboswitches in the L. lactis MG1363 transcriptome.
• A putative TPP riboswitch upstream of the thiamine transporter gene (thiT) was cloned upstream of the superfolder green fluorescent protein (sfgfp) gene.
• Gene expression was monitored in the presence and absence of thiamine to confirm riboswitch activity.
• The riboswitch was used to restore the mutant phenotype of an L. lactis strain lacking the acmA gene.

Main Results:

• A novel thiamine-responsive TPP riboswitch located upstream of the L. lactis thiT gene was identified and confirmed.
• The riboswitch demonstrated thiamine-dependent regulation of gene expression, with expression levels modulated by thiamine concentration.
• The riboswitch successfully restored the mutant phenotype in an acmA-deficient L. lactis strain, showcasing its practical application.

Conclusions:

• The identified L. lactis thiT TPP riboswitch functions as a natural RNA-based genetic control device.
• This riboswitch provides a tunable system for gradual gene expression downregulation by adjusting thiamine levels.
• The thiamine-responsive riboswitch is a valuable new tool for the L. lactis genetic toolbox, enhancing genetic engineering capabilities.