The SapB morphogen is a lantibiotic-like peptide derived from the product of the developmental gene ramS in Streptomyces coelicolor

Abstract

SapB is a morphogenetic peptide that is important for aerial mycelium formation by the filamentous bacterium Streptomyces coelicolor. Production of SapB commences during aerial mycelium formation and depends on most of the genes known to be required for the morphogenesis of aerial hyphae. Furthermore, the application of purified SapB to mutants blocked in morphogenesis restores their capacity to form aerial hyphae. Here, we present evidence that SapB is a lantibiotic-like peptide that is derived by posttranslational modification from the product of a gene (ramS) in the four-gene ram operon, which is under the control of the regulatory gene ramR. We show that the product of another gene in the operon (ramC) contains a region that is similar to enzymes involved in the biosynthesis of lantibiotics, suggesting that it might be involved in the posttranslational processing of RamS. We conclude that SapB is derived from RamS through proteolytic cleavage and the introduction of four dehydroalanine residues and two lanthionine bridges. We provide an example of a morphogenetic role for an antibiotic-like molecule.

Fig. 1.

The ram gene cluster and sequence analysis of ramC.(A) The arrangement of the five genes in the ram cluster of S. coelicolor.(B) Amino acid alignment of the C terminus of RamC of S. coelicolor with the C termini of the lantibiotic modification enzymes CinM and MrsM, from Streptomyces cinnamoneus and Bacillus spp., respectively. Dashed lines indicate conserved sequences commonly found in Lan cyclases.

Fig. 2.

Alignment of RamS of S. coelicolor with its ortholog AmfS of S. griseus. Also shown is the amino acid sequence of SapB obtained by Edman degradation after reduction with ethanethiol under alkaline conditions (20).

Fig. 3.

Proposed primary (A) and covalent (B) structure of an internal SapB fragment generated by acid hydrolysis. Lan is the first and only S containing residue, whereas Dha (residue 4) is distinguishable by the double bond between the α and β carbons.

Fig. 4.

MALDI-TOF tandem MS analysis of SapB (A) and the proposed covalent structure of SapB (B). The cleavage site indicated by the pink arrow in the covalent structure (B) would yield a 1,913.8-Da fragmentation product, corresponding to the minor peak indicated by the pink arrow in A. Other fragmentation products are similarly labeled in both the chromatogram and covalent structure.

Fig. 5.

Proposed primary structure of SapB. The deduced bridging pattern yields two cyclic structures of eight residues, most of which are hydrophobic. The loops are flanked and hinged by less hydrophobic amino acids.

Fig. 6.

Molecular model of SapB. (A) Schematic representation of the model. The C- and N-terminal macrocyclic loops are colored magenta and cyan, respectively, and the thioether (Lan bridge) sulfurs are colored yellow. (B) Spacefilling representation of the model. The view is rotated by 180° with respect to A, as indicated by the C and N termini. The basic N-terminal group and Arg-4 side chain are colored blue, and the acidic C-terminal group and Asp-12 side chain are colored red.