ABSTRACTInducible gene expression systems are invaluable tools for the functional characterization of genes and in the construction of protein overexpression hosts. Controllable expression is especially important for the study of essential and toxic genes or genes where the level of expression tightly influences their cellular effect. Here, we implemented the well-characterized tetracycline-inducible expression system in two industrially important lactic acid bacteria,Lactococcus lactisandStreptococcus thermophilus. Using a fluorescent reporter gene, we show that optimization of the repression level is necessary for efficient induction using anhydrotetracycline in both organisms. Random mutagenesis in the ribosome binding site of the tetracycline repressor TetR inLactococcus lactisindicated that altering the expression levels of TetR was necessary for efficient inducible expression of the reporter gene. Through this approach, we achieved plasmid-based, inducer-responsive, and tight gene expression inLactococcus lactis. We then verified the functionality of the optimized inducible expression system inStreptococcus thermophilusfollowing its chromosomal integration using a markerless mutagenesis approach and a novel DNA fragment assembly tool presented herein. This inducible expression system holds several advantages over other described systems in lactic acid bacteria, although more efficient techniques for genetic engineering are still needed to realize these advantages in industrially relevant species, such asS. thermophilus. Our work expands the molecular toolbox of these bacteria, which can accelerate future physiological studies.IMPORTANCELactococcus lactisandStreptococcus thermophilusare two industrially important lactic acid bacteria globally used in dairy fermentations and, therefore, are of considerable commercial interest to the food industry. Moreover, due to their general history of safe usage, these microorganisms are increasingly being explored as hosts for the production of heterologous proteins and various chemicals. Development of molecular tools in the form of inducible expression systems and mutagenesis techniques facilitates their in-depth physiological characterization as well as their exploitation in biotechnological applications.