Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic

Abstract

Lantibiotics are ribosomally synthesized peptides that undergo posttranslational modifications to their mature, antimicrobial form. They are characterized by the unique amino acids lanthionine and methyllanthionine, introduced by means of dehydration of Ser/Thr residues followed by reaction of the resulting dehydro amino acids with cysteines to form thioether linkages. Two-component lantibiotics use two peptides that are each posttranslationally modified to yield two functionally distinct products that act in synergy to provide bactericidal activity. By using genetic data instead of isolation, a two-component lantibiotic, haloduracin, was identified in the genome of the Gram-positive alkaliphilic bacterium Bacillus halodurans C-125. We show that heterologously expressed and purified precursor peptides HalA1 and HalA2 are processed by the purified modification enzymes HalM1 and HalM2 in an in vitro reconstitution of the biosynthesis of a two-component lantibiotic. The activity of each HalM enzyme is substrate-specific, and the assay products exhibit antimicrobial activity after removal of their leader sequences at an engineered Factor Xa cleavage site, indicating that correct thioether formation has occurred. Haloduracin's biological activity depends on the presence of both modified peptides. The structures of the two mature haloduracin peptides Halalpha and Halbeta were investigated, indicating that they have similarities as well as some distinct differences compared with other two-component lantibiotics.

Fig. 1.

Sequence alignment of two-component lantibiotics. (A) Sequence alignment of the structural peptide of HalA1 with that of the α/A1 prepeptides from plantaracin W (PlwAα, AAG02567), staphylococcin C55 (SacAα, BAB78438), lacticin 3147 (LtnA1, O87236), BHT, (BhtA1, AAZ76603), and Smb (SmbA1, BAD72776). Ser/Thr residues that are dehydrated are shown in blue, Ser/Thr residues that are not dehydrated are underlined, and Ser converted to D-Ala are shown in green. The superscript a signifies that the reported mass (18) indicates seven of eight Ser/Thr residues are dehydrated, and possibly also one or two Ser to D-Ala conversions based on precedent in Ltnα (35) and the presence of an homolog of the dehydrogenase LtnJ involved in this process (38). (B) Sequence alignment of the structural region of HalA2 with that of the β/A2 prepeptides from plantaracin W (PlwAβ, AAG02566), BHT (BhtA2, AAZ76602), Smb (SmbA2, BAD72777), lacticin 3147 (LtnA2, O87237), staphylococcin C55 (SacAβ, BAB78439), and the two peptides of cytolysin (CylLA_L, AAK67264; CylLA_S, AAK67265). For cytolysin, the sites of the seven dehydrations in CylL_L (based on its molecular weight in ref. 39) are not known. For cases where additional proteolysis takes place (PlwAβ and CylL-AS), the N terminus of the mature products is denoted with a red arrow.

Fig. 2.

MALDI-TOF mass spectrum of HalA1 and HalA2 incubated with HalM1 and HalM2 (solid line). HalA1, calculated 9,671.86 Da (M + H − 3H₂O) and observed 9,674 Da; HalA2, calculated 8,892.01 Da (M + H − 7H₂O) and observed 8,892 Da. For comparison, the spectrum of HalA1 and HalA2 that were not subjected to HalM1 and HalM2 is presented in the dashed line. HalA1, calculated 9,725.86 Da (M + H) and observed 9,726 Da; HalA2, calculated 9,018.01 Da (M + H) and observed 9,021 Da.

Fig. 3.

Structure determination of Halα and Halβ. (A) MALDI-TOF mass spectrum of Halα and Halβ treated with 1 mM TCEP and 10 mM iodoacetamide (solid line). Halα, calculated 3,162.58 Da (M + H + 2 IAA adducts) and observed 3,164 Da; Halβ, calculated 2,332.83 Da (M + H) and observed 2,334 Da. A spectrum of Halα and Halβ that were not treated with iodoacetamide is shown in dashed line. Halα, calculated 3,046.58 Da (M + H) and observed 3,046 Da; Halβ, calculated 2,332.83 Da (M + H) and observed 2,332 Da. (B) Electrospray ionization (ESI)-FTMS/MS spectrum of Halβ. Fragment ions are indicated.

Fig. 4.

Bioassay with the indicator strain L. lactis CNRZ 117. Lane 1, haloduracin isolated from B. halodurans C-125; lane 2, HalA1-Xa and HalA2-Xa treated with HalM1, HalM2, and Factor Xa to generate Halα and Halβ; lane 3, HalA1-Xa treated with HalM1 and factor Xa to generate Halα; lane 4, HalA2-Xa treated with HalM2 and factor Xa to generate Halβ; lane 5, HalA1-Xa and HalA2-Xa treated with HalM1 and HalM2 only (no factor Xa); lane 6, HalA1-Xa and HalA2-Xa treated with Factor Xa only (no HalM1 or HalM2).

Fig. 5.

Proposed structures for the Halα and Halβ peptides of the two-component lantibiotic haloduracin. Ser/Thr residues in the Hal peptides that are predicted to remain unmodified are shown in green.