Isolation of Lactococcus lactis Mutants Simultaneously Resistant to the Cell Wall-Active Bacteriocin Lcn972, Lysozyme, Nisin, and Bacteriophage c2

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits cell wall biosynthesis by binding to lipid II. In this work, two mutants resistant to Lcn972,<jats:named-content content-type="genus-species">Lactococcus lactis</jats:named-content>D1 and D1-20, with high (&gt;320 arbitrary units [AU]/ml) and low (80 AU/ml) susceptibilities, respectively, have been isolated. Resistance to Lcn972 did not impose a burden to growth under laboratory conditions, nor did it substantially alter the physicochemical properties of the cell surface. However, the peptidoglycan of the mutants featured a higher content of muropeptides with tripeptide side chains than the wild-type strain, linking for the first time peptidoglycan remodelling to bacteriocin resistance. Moreover,<jats:named-content content-type="genus-species">L. lactis</jats:named-content>lacking a functional<jats:sc>d,d</jats:sc>-carboxypeptidase DacA (i.e., with a high content of pentapeptide side chain muropeptides) was shown to be more susceptible to Lcn972. Cross-resistance to lysozyme and nisin and enhanced susceptibility to penicillin G and bacitracin was also observed. Intriguingly, the Lcn972-resistant mutants were not infected by the lytic phage c2 and less efficiently infected by phage sk1. Lack of c2 infectivity was linked to a 22.6-kbp chromosomal deletion encompassing the phage receptor protein gene<jats:italic>pip</jats:italic>. The deletion also included maltose metabolic genes and the two-component system (TCS) F. However, a clear correlation between these genes and resistance to Lcn972 could not be clearly established, pointing to the presence of as-yet-unidentified mutations that account for Lcn972 resistance.</jats:p>

The Putative Lactococcal Extracytoplasmic Function Anti-Sigma Factor Llmg2447 Determines Resistance to the Cell Wall-Active Bacteriocin Lcn972

“…Downregulation of genes already identified as playing a role in tolerance to Lcn972 (celB, ptcC) was seen (9). Furthermore, a number of genes puta- tively involved in carbohydrate metabolism were seen as "downregulated" because they are absent from L. lactis D1 as a consequence of the large deletion, encompassing genes llmg0736 to llmg0751, previously identified in this strain (34). Induction of genes of the cell envelope stress response triggered by Lcn972 in L. lactis (llmg2164-llmg2163) and of other genes known to be induced in response to Lcn972 and osmotic shock (busAA, busAB) was observed (28).…”

“…Inhibitory activities were determined by the spot-on-the-lawn test (34). Briefly, melted GM17 agar (supplemented or not supplemented with nisin) was inoculated with overnight lactococcal cultures.…”

“…Remarkably, both mutants had an altered peptidoglycan composition with a high content of muropeptides with tripeptide side chains. Moreover, a large chromosomal deletion was identified, although a clear correlation of this mutation with the loss of susceptibility to Lcn972 could not be established and the genetic basis of the resistance remained elusive (34). In this work, genome-wide transcriptomics was carried out in order to identify the mechanism of L. lactis resistance to Lcn972.…”

“…L. lactis strain D1 was firstly isolated upon adaptation to increasing Lcn972 concentrations, and a derivative of it, L. lactis D1-20, was selected after L. lactis D1 was grown for 200 generations in the absence of Lcn972 (34). Remarkably, both mutants had an altered peptidoglycan composition with a high content of muropeptides with tripeptide side chains.…”

Lytic Infection of Lactococcus lactis by Bacteriophages Tuc2009 and c2 Triggers Alternative Transcriptional Host Responses

“…Notably, L. lactis IL1403, when infected with c2, showed increased transcription of the dltACBD operon, responsible for D-alanyl modification of cell wall components (11). Moreover, modification of peptidoglycan has been linked to reduced susceptibility to 936-species phages in L. lactis MG1363 (58). There have also been suggestions that phage proteins target cell wall biosynthesis and modification in other species.…”