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.

FIG. 1.

Putative nisin resistance mechanisms. The putative nisin resistance pathway in L. lactis can be divided into four putative mechanisms. For each mechanism, the genes (putatively) involved, based on DNA microarray results, are indicated below between parentheses or in the text (Table 4 details their respective gene expressions). Dark squares, involvement of either the cell membrane or cell wall composition in the resistance mechanism. Mechanism A, cell wall changes, preventing nisin from reaching lipid II in the cytoplasmic membrane, by thickening the cell wall (pbp2A), by becoming more densely packed (galE and pbp2A) and by becoming less negatively charged (dltD). Mechanism B, the L. lactis Nis^r strain changes the local acidity on the outside of the membrane nisin by changing the expression of genes encoded by the arc operon. This results in an elevated pH, possibly promoting the degradation of the nisin molecule or forcing it to bind to the cell wall. Mechanism C, reduced levels of proteins encoded by the fab operon, which is involved in the saturation of the fatty acid chains in membrane phospholipids, might make the membrane more fluid, preventing nisin insertion into the membrane. Mechanism D, ABC transporters might be involved in transporting nisin out of the membrane, preventing nisin-lipid II binding.