AUTHOR=Campelo Ana Belén , López-González María Jesús , Escobedo Susana , Janzen Thomas , Neves Ana Rute , Rodríguez Ana , Martínez Beatriz 

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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.01805

DOI=10.3389/fmicb.2020.01805

ISSN=1664-302X

ABSTRACT=<p>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 <italic>Lactococcus lactis</italic> ABC transporter YsaDCB is shown to constitute, together with TCS-G, a detoxification module that protects <italic>L. lactis</italic> against bacitracin and the bacteriocin Lcn972, both AMPs that inhibit cell wall biosynthesis. Initially, increased expression of <italic>ysaDCB</italic> was detected by RT-qPCR in three <italic>L. lactis</italic> resistant to Lcn972, two of which were also resistant to bacitracin. These mutants shared, among others, single-point mutations in <italic>ysaB</italic> 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 <italic>in trans</italic> of <italic>ysaDCB</italic> in <italic>L. lactis</italic> NZ9000, a strain that lacks a functional detoxification module, enhanced resistance to both AMPs, demonstrating its role as a resistance factor in <italic>L. lactis</italic>. When the three different <italic>ysaB</italic> 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<italic><sub>ysaD</sub></italic> promoter fusions, designed to measure the activation of the detoxification module, revealed that the <italic>ysaB</italic> mutations unlock transcriptional control by TCS-G, resulting in constitutive expression of the <italic>ysaDCB</italic> operon. Finally, deletion of <italic>ysaD</italic> was also performed to get an insight into the function of this gene. <italic>ysaD</italic> encodes a secreted peptide and is part of the <italic>ysaDCB</italic> operon. YsaD appears to modulate signal relay between the ABC transporter and TCS-G, based on the different response of the P<italic><sub>ysaD</sub></italic> 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.</p>