Unveiling the regulatory network controlling natural transformation in lactococci

Abstract

Lactococcus lactis is a lactic acid bacterium of major importance for food fermentation and biotechnological applications. The ability to manipulate its genome quickly and easily through competence for DNA transformation would accelerate its general use as a platform for a variety of applications. Natural transformation in this species requires the activation of the master regulator ComX. However, the growth conditions that lead to spontaneous transformation, as well as the regulators that control ComX production, are unknown. Here, we identified the carbon source, nitrogen supply, and pH as key factors controlling competence development in this species. Notably, we showed that these conditions are sensed by three global regulators (i.e., CcpA, CodY, and CovR), which repress comX transcription directly. Furthermore, our systematic inactivation of known signaling systems suggests that classical pheromone-sensing regulators are not involved. Finally, we discovered that the ComX-degrading MecA-ClpCP machinery plays a predominant role based on the identification of a single amino-acid substitution in the adaptor protein MecA of a highly transformable strain. Contrasting with closely-related streptococci, the master competence regulator in L. lactis is regulated both proximally by general sensors and distantly by the Clp degradation machinery. This study not only highlights the diversity of regulatory networks for competence control in Gram-positive bacteria, but it also paves the way for the use of natural transformation as a tool to manipulate this biotechnologically important bacterium. IMPORTANCELactic acid bacteria (LAB) play important roles in our daily lives as members of our microbiota or as starters of dairy products. Understanding the natural horizontal gene transfer mechanisms that shape their genomes will allow us to better control and understand their evolution over time. The DNA transformation machinery is found in all beneficial LAB species. With the exception of streptococci, however, the conditions of its activation remain unknown. In this study, the physiological conditions that activate competence for DNA transformation in Lactococcus lactis, the most important lactococcal species, were identified. We also unveiled the guardians of the master competence regulator ComX. In this species, it is directly repressed by global carbon and nitrogen regulators (CcpA and CodY) as well as the general stress system CovRS. Additionally, it was discovered that the Clp machinery degrading ComX plays a dominant role in the strict control of competence activation. In Gram-positive cocci, the hierarchical organization of these regulators for controlling competence development in L. lactis is unprecedented.