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

Abstract

Background: Lactococcus 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.

Results: Attempts 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.

Conclusions: Inserting 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.

Figure 1

Schematic drawing of the plasmids bearing the Lcn972 gene cluster. The plasmids are based on the pIL252 replicon (4.6 kbp) besides pTLR1_Lcn972, which is based on pTLR1, and pBL1, which is the original plasmid found in the wild-type Lcn972-producing strain L. lactis IPLA972. Black arrow: structural lcn972 gene; grey arrows: immunity genes; white arrow: hypothetical orf4. The different promoters driving expression of the Lcn972 gene cluster are shown in circles. P*₉₇₂: mutated promoter. The size of each plasmid is shown in brackets.

Figure 2

Segregational stability of Lcn972-expressing plasmids in L. lactis NZ9000 grown without erythromycin (Em). All the experiments were started from fresh overnight cultures in GM17Em (except pBL1). Experiments were carried out once except for pIL252 (in A), that was performed three times (only one representative curve is shown), and for pOrf4 (in B), where two independent Em-resistant colonies (−a, −b) were tested. L. lactis 200 g-a is a Em-resistant Lcn972-producing colony isolated from L. lactis pOrf4 after 200 generations.

Figure 3

Segregational stability of the red fluorescent protein mCherry expression plasmid pTRL1 with and without the Lcn972 gene cluster. (A) Pink colony phenotype of pTRL1 to assess the functional expression of the mCherry protein in L. lactis NZ9000 cells. The arrows point to plasmid-free colonies. (B) Stability of the plasmid pTRL1 and pTRL1_Lcn972 in which the Lcn972 gene cluster from pDorf4 was cloned. Cultures were grown in GM17 without erythromycin and the presence of the plasmids was determined by pink/white screening on an average of 390 colonies per sampling time. The experiments were carried out with two independent cultures denoted as (a) and (b).

Figure 4

Integration of the plasmid pOrf4 in the chromosome of L. lactis NZ9000. (A) Results of the PCR strategy to define the region of pOrf4 involved in the molecular reorganization with the chromosome. Primer pairs orf4-F/lcn2, ery5/orf4-R, eryrepF-F/repE-R, and repE-F/repE2-R were used in PCRs A, B, C and D, respectively. (B) Presence of the insertion sequences (black arrows) IS981 and IS1216 as revealed by direct sequencing of the chromosome of L. lactis 200 g and L. lactis 400 g, respectively.

Figure 5

Presence of the Lcn972 gene cluster does not allow direct selection of transformants after plasmid electroporation.L. lactis NZ9000 was electroporated with pDorf4 and colony forming units of the transformation mix right after electroporation (generation 0) and during sub-culturing in GM17 were followed by plating in the presence (GM17Em) or absence (GM17) of erythromycin.