Transcriptome Analysis Reveals Mechanisms by Which Lactococcus lactis Acquires Nisin Resistance

Abstract

Nisin, a posttranslationally modified antimicrobial peptide produced by Lactococcus lactis, is widely used as a food preservative. Yet, the mechanisms leading to the development of nisin resistance in bacteria are poorly understood. We used whole-genome DNA microarrays of L. lactis IL1403 to identify the factors underlying acquired nisin resistance mechanisms. The transcriptomes of L. lactis IL1403 and L. lactis IL1403 Nis r , which reached a 75-fold higher nisin resistance level, were compared. Differential expression was observed in genes encoding proteins that are involved in cell wall biosynthesis, energy metabolism, fatty acid and phospholipid metabolism, regulatory functions, and metal and/or peptide transport and binding. These results were further substantiated by showing that several knockout and overexpression mutants of these genes had strongly altered nisin resistance levels and that some knockout strains could no longer become resistant to the same level of nisin as that of the wild-type strain. The acquired nisin resistance mechanism in L. lactis is complex, involving various different mechanisms. The four major mechanisms are (i) preventing nisin from reaching the cytoplasmic membrane, (ii) reducing the acidity of the extracellular medium, thereby stimulating the binding of nisin to the cell wall, (iii) preventing the insertion of nisin into the membrane, and (iv) possibly transporting nisin across the membrane or extruding nisin out of the membrane.Nisin, a lanthionine-containing peptide produced by certain strains of Lactococcus lactis (24), is widely used in the food industry as a safe and natural preservative (11) because of its antimicrobial activity against a broad range of gram-positive bacteria, including Listeria monocytogenes. Nisin disrupts the cytoplasmic membrane of a bacterial cell through pore formation, which leads to the release of small cytoplasmic compounds, depolarization of the membrane potential, and, ultimately, cell death (4, 5). The lantibiotic uses lipid II as a docking molecule in the target membrane for efficient pore formation. By the binding of nisin to lipid II, bacterial cell wall synthesis is also inhibited (4, 5), which provides another mechanism of cell killing (59).The efficiency of nisin as an antimicrobial agent could be seriously compromised by the occurrence of nisin resistance in spoilage or pathogenic bacteria. The generation of nonstable nisin-resistant (Nis r ) strains of L. lactis under laboratory conditions is achieved relatively easily by stepwise exposure of cells to increasing concentrations of nisin (20,39,42). Since nisin sensitivity and the lipid II content in the cytoplasmic membrane are not directly correlated (29), nisin resistance is expected to be achieved through another mechanism(s).Nisin resistance in bacteria has been associated with an altered fatty acid composition of phospholipids (39,42) or an altered phospholipid composition of the cytoplasmic membrane (41). Lipoteichoic acid (LTA) was shown to play an important role in the nisin re...

SpxB Regulates O-Acetylation-dependent Resistance of Lactococcus lactis Peptidoglycan to Hydrolysis

“…In Group A streptococcus, a TCS was shown to be induced by human saliva, which could also cause cell wall damage because it is rich in lysozyme (49). A link between spxB and the cell wall stress response is supported by recent transcriptome results on L. lactis strain IL1403 that had acquired resistance to the cell wall-targeted antimicrobial peptide nisin: in this strain, spxB (yneH), oatA (yvhB), and cesSR (kinD llrD) were among the up-regulated genes (50).…”

Lantibiotic Resistance

“…Additionally, this study showed that wild-type strains of Staphylococcus bearing additional copies of the dlt operon contained larger amounts of Dalanylated teichoic acids and hence repelled cationic proteins more effectively and were less sensitive to gallidermin (45). It was also notable that spontaneously nisin-resistant mutants of L. lactis IL1403 expressed the dlt and gal operons at higher levels (51). The relevance of the gal operon, associated with the Leloir pathway (52), is that galE encodes a UDP-glucose 4-epimerase that is responsible for the synthesis of ␣-galactose, which is transported across the membrane to become a substituent of LTA.…”

“…This observation led to further studies of L. lactis MG1363, which established that L. lactis MG1363 ⌬galAMK was twice as sensitive to nisin as the wild type, suggesting that ␣-galactose incorporation has an effect on LTA structure and thus is important in nisin resistance. In addition, the LTA of the resistant strain contained twice as much ␣-galactose as the wild type, which may indicate a more densely packed LTA, ultimately making the cell wall barrier less negatively charged due to the action of DltA (51). Nisin-resistant cells also appeared to have more lipoteichoic acid than nisin-sensitive cells.…”

“…This strain was also slightly more resistant to mersacidin but was sensitive to a variety of beta-lactam antibiotics. Similarly, expression of the gene encoding PBP2A (a class B PBP) was higher in a nisinresistant mutant of L. lactis IL1403 than in the corresponding wild-type strain, leading to speculation that resistance was provided by a thicker and more densely packed cell wall (51).…”

Stress Physiology of Lactic Acid Bacteria

“…lactis ⌬cesR mutant was two times more sensitive to nisin, although this peptide did not activate the cesFSR promoter (441). Nevertheless, a nisin-resistant strain obtained through a nisin adaptation process overexpressed cesFSR and its regulon (383). The cesFSR system is also induced by vancomycin, bacitracin, and the bacteriocins plantaricin C and Lcn972 (441).…”