Production of a class II two-component lantibiotic of Streptococcus pneumoniae using the class I nisin synthetic machinery and leader sequence

Abstract

Recent studies showed that the nisin modification machinery can successfully dehydrate serines and threonines and introduce lanthionine rings in small peptides that are fused to the nisin leader sequence. This opens up exciting possibilities to produce and engineer larger antimicrobial peptides in vivo. Here we demonstrate the exploitation of the class I nisin production machinery to generate, modify, and secrete biologically active, previously not-yet-isolated and -characterized class II two-component lantibiotics that have no sequence homology to nisin. The nisin synthesis machinery, composed of the modification enzymes NisB and NisC and the transporter NisT, was used to modify and secrete a putative two-component lantibiotic of Streptococcus pneumoniae. This was achieved by genetically fusing the propeptide-encoding sequences of the spr1765 (pneA1) and spr1766 (pneA2) genes to the nisin leader-encoding sequence. The chimeric prepeptides were secreted out of Lactococcus lactis, purified by cation exchange fast protein liquid chromatography, and further characterized. Mass spectrometry analyses demonstrated the presence and partial localization of multiple dehydrated serines and/or threonines and (methyl)lanthionines in both peptides. Moreover, after cleavage of the leader peptide from the prepeptides, both modified propeptides displayed antimicrobial activity against Micrococcus flavus. These results demonstrate that the nisin synthetase machinery can be successfully used to modify and produce otherwise difficult to obtain antimicrobially active lantibiotics.

FIG. 1.

Putative pneumococcin A1 and A2 gene cluster. Organization of the chromosomally located biosynthetic gene cluster of pneumococcin A1 and A2 genes (spr1765 and spr1766, respectively) in S. pneumoniae. ORFs are represented by thick gray arrows, and their SPR gene identification numbers are shown in the gray arrows. The putative promoters are represented by thin and bent black arrows.

FIG. 2.

Amino acid sequence alignment of nisin with pneumococcins A1 and A2. The cleavage site in the peptide sequence is highlighted in gray. Identical amino acid residues for all three sequences are indicated by an asterisk, conserved residues by a colon, and semiconserved amino acids by a point.

FIG. 3.

Tris-Tricine gel illustrating trypsin-treated and non-trypsin-treated purified chimeric peptides and nisin. Lane M, marker; lane 1, prePneA1; lane 1A, trypsin-treated PneA1; lane 2, prePneA2; lane 2A, trypsin-treated PneA2; lane 3, prenisin; lane 3A, trypin-treated prenisin.

FIG. 4.

Modified PneA1 contains thioether bridges. The SAAASSKVCISAAVSGIGGLVSYNNDCLG fragment dehydrated fourfold, threefold, and twofold (solid line) is treated with CDAP (dotted line) to detect the presence of modifiable cysteines (yielding peptide plus 25 Da) and nonmodifiable thioether linkage-forming cysteines. The 2,643.6-Da peak which is dehydrated fourfold shows a mild single CDAP addition. The 2,661.9-Da (threefold-dehydrated) peak shows single and double CDAP additions, as indicated by black arrows. The 2,680.5-Da (twofold-dehydrated) peak shows clear single and double CDAP additions; 1 CDAP, 2,705.3 Da (plus 25 Da), and 2 CDAP, 2,729.8 Da (plus 50 Da). The fivefold-dehydration peak is becoming visible after CDAP addition, indicating that this peptide has two thioether rings.