Abstract
BackgroundLactococcus lactis is widely used as a dairy starter and has been extensively studied. Based on the acquired knowledge on its physiology and metabolism, new applications have been envisaged and there is an increasing interest of using L. lactis as a cell factory. Plasmids constitute the main toolbox for L. lactis genetic engineering and most rely on antibiotic resistant markers for plasmid selection and maintenance. In this work, we have assessed the ability of the bacteriocin Lactococcin 972 (Lcn972) gene cluster to behave as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics. Lcn972 is a non-lantibiotic bacteriocin encoded by the 11-kbp plasmid pBL1 with a potent antimicrobial activity against Lactococcus.ResultsAttempts to clone the full lcn972 operon with its own promoter (P972), the structural gene lcn972 and the immunity genes orf2-orf3 in the unstable plasmid pIL252 failed and only plasmids with a mutated promoter were recovered. Alternatively, cloning under other constitutive promoters was approached and achieved, but bacteriocin production levels were lower than those provided by pBL1. Segregational stability studies revealed that the recombinant plasmids that yielded high bacteriocin titers were maintained for at least 200 generations without antibiotic selection. In the case of expression vectors such as pTRL1, the Lcn972 gene cluster also contributed to plasmid maintenance without compromising the production of the fluorescent mCherry protein. Furthermore, unstable Lcn972 recombinant plasmids became integrated into the chromosome through the activity of insertion sequences, supporting the notion that Lcn972 does apply a strong selective pressure against susceptible cells. Despite of it, the Lcn972 gene cluster was not enough to avoid the use of antibiotics to select plasmid-bearing cells right after transformation.ConclusionsInserting the Lcn972 cluster into segregational unstable plasmids prevents their lost by segregation and probable could be applied as an alternative to the use of antibiotics to support safer and more sustainable biotechnological applications of genetically engineered L. lactis.
Highlights
Lactococcus lactis is widely used as a dairy starter and has been extensively studied
The use of antimicrobial peptides produced by bacteria as selecting agents and their cognate immunity genes as selectable marker genes have been explored as food-grade cloning strategies
These results supported the notion that the Lactococcin 972 (Lcn972) gene cluster might be involved in plasmid maintenance by post-segregational killing, i.e. eliminating those cells which have lost the plasmid during division due to the strong toxicity of the bacteriocin
Summary
Lactococcus lactis is widely used as a dairy starter and has been extensively studied. Lactococcus lactis is widely used as starter in the manufacture of cheese It belongs to the diverse group of lactic acid bacteria which are characterized by the production of large quantities of lactic acid as a consequence of the fermentative metabolism of carbohydrates. The deep knowledge of L. lactis physiology along with the advent of high-throughput technologies (−omics) has To address these challenges, several genetic tools have been devised that expand from replicative cloning plasmids to chromosomal integration strategies, (inducible-) gene expression platforms and synthetic promoters, among others [1,5]. Lacticin 481 and lacticin 3147 conjugative plasmids could be transferred in mating experiments by selection of transconjugants with the corresponding bacteriocin [14,15] These examples support the potential of bacteriocin-based approaches to develop food-grade genetic tools
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