Dataset Information

Mutations Selected After Exposure to Bacteriocin Lcn972 Activate a Bce-Like Bacitracin Resistance Module in Lactococcus lactis.

ABSTRACT: Resistance against antimicrobial peptides (AMPs) is often mediated by detoxification modules that rely on sensing the AMP through a BceAB-like ATP-binding cassette (ABC) transporter that subsequently activates a cognate two-component system (TCS) to mount the cell response. Here, the Lactococcus lactis ABC transporter YsaDCB is shown to constitute, together with TCS-G, a detoxification module that protects L. lactis against bacitracin and the bacteriocin Lcn972, both AMPs that inhibit cell wall biosynthesis. Initially, increased expression of ysaDCB was detected by RT-qPCR in three L. lactis resistant to Lcn972, two of which were also resistant to bacitracin. These mutants shared, among others, single-point mutations in ysaB coding for the putative Bce-like permease. These results led us to investigate the function of YsaDCB ABC-transporter and study the impact of these mutations. Expression in trans of ysaDCB in L. lactis NZ9000, a strain that lacks a functional detoxification module, enhanced resistance to both AMPs, demonstrating its role as a resistance factor in L. lactis. When the three different ysaB alleles from the mutants were expressed, all of them outperformed the wild-type transporter in resistance against Lcn972 but not against bacitracin, suggesting a distinct mode of protection against each AMP. Moreover, P ysaD promoter fusions, designed to measure the activation of the detoxification module, revealed that the ysaB mutations unlock transcriptional control by TCS-G, resulting in constitutive expression of the ysaDCB operon. Finally, deletion of ysaD was also performed to get an insight into the function of this gene. ysaD encodes a secreted peptide and is part of the ysaDCB operon. YsaD appears to modulate signal relay between the ABC transporter and TCS-G, based on the different response of the P ysaD promoter fusions when it is not present. Altogether, the results underscore the unique features of this lactococcal detoxification module that warrant further research to advance in our overall understanding of these important resistance factors in bacteria.

SUBMITTER: Campelo AB

PROVIDER: S-EPMC7438565 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Mutations Selected After Exposure to Bacteriocin Lcn972 Activate a Bce-Like Bacitracin Resistance Module in Lactococcus lactis.

Campelo Ana Belén AB López-González María Jesús MJ Escobedo Susana S Janzen Thomas T Neves Ana Rute AR Rodríguez Ana A Martínez Beatriz B

Frontiers in microbiology 20200813

Resistance against antimicrobial peptides (AMPs) is often mediated by detoxification modules that rely on sensing the AMP through a BceAB-like ATP-binding cassette (ABC) transporter that subsequently activates a cognate two-component system (TCS) to mount the cell response. Here, the Lactococcus lactis ABC transporter YsaDCB is shown to constitute, together with TCS-G, a detoxification module that protects L. lactis against bacitracin and the bacteriocin Lcn972, both AMPs that inhi ...[more]

PMID: 32903467

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Project description: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.

Dataset Information

Mutations Selected After Exposure to Bacteriocin Lcn972 Activate a Bce-Like Bacitracin Resistance Module in Lactococcus lactis.

Publications

Mutations Selected After Exposure to Bacteriocin Lcn972 Activate a Bce-Like Bacitracin Resistance Module in <i>Lactococcus lactis</i>.

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