A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis

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.

Plasmid Complement of Lactococcus lactis NCDO712 Reveals a Novel Pilus Gene Cluster

“…The plasmids often specify traits of industrial importance such as the ability to grow on lactose, to utilize milk protein or stress resistance [9,10]. Other important plasmid-encoded functions include bacteriocin production [11,12] and resistance [13,14], metal ion resistance [15], antibiotic resistance [16,17] and bacteriophage resistance [18,19]. A recent publication described a CRISPR-Cas system that was encoded by a lactococcal plasmid, although it was concluded not to be functional [20].…”

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

“…Mohedano and coworkers (32) adapted the broad-host-range vector pNZ8048 to study gene expression in Lactobacillus and showed that the mCherry construct could be used to study complex promoter induction mechanisms, such as bacteriocin production by Lactobacillus acidophilus. The role that lactococcin 972 (a bacteriocin produced by Lactococcus lactis) plays in the stabilization of gene expression in L. lactis was studied by monitoring expression of the mCherry gene (33). The adhesion of L. plantarum and Lactobacillus fermentum strains to the intestinal tract of zebrafish was studied by transferring plasmid pRCR12, containing the mCherry gene, into the bacterial cells (34).…”

Antimicrobial Peptides Produced by Bacteria: The Bacteriocins

“…Production of these proteins will be a function of the concentration of nisin added to the cell culture (Mierau and Kleerebezem 2005). The immunity determinants have also been exploited as food-grade markers in cloning vectors that make use of bacteriocins as selecting agents and, more recently, the introduction of full bacteriocin operons into plasmids has demonstrated to prevent plasmid segregation under non-selecting conditions (Takala and Saris 2002;Campelo et al 2014). The development of these plasmids helps to reduce the use of antibiotics in biotechnological processes with genetically modified bacteria.…”