Functions of a novel signal molecule inducing sporulation in
Streptomyces species
Kentaro TAKADA
Graduate School of Agricultural and Life Sciences,
The University of Tokyo
Research aims
Surugamides A–E are cyclic octapeptides isolated from a marine-derived actinomycete,
Streptomyces sp. JAMM992. We have revealed that other marine Streptomyces isolated fromsea sediment around Japan and several terrestrial Streptomyces produced surugamide A;
Thorson et al. reported that a terrestrial Streptomyces isolated from soil collected at a coalmine fire site in Kentucky also produced surugamide A. The ubiquity of biosynthetic genes
for surugamides led us to speculate that it has some physiological function in the producers.
We set out to identify the biosynthetic gene cluster for surugamide A to generate a mutant
strain that lacks surugamide A productivity and then elucidated the physiological function ofsurugamide A using the mutant strain. Our study on the biosynthetic gene cluster forsurugamide A revealed that surugamides A-E were produced by two NRPS genes, whichwere separated by two other NRPS genes; the central two genes were responsible for thebiosynthesis of an unrelated peptide, termed as surugamide F. The formation of aerial
mycelium was notably delayed in the ΔsurA strain, which was recovered by treatment withsurugamide A in a concentration- dependent manner. In this study, we identified the putative
target protein of surugamide A and revealed that surugamide A promotes aerial mycelium
formation.
Methods
1. Preparation of mutant strains based on the biosynthetic gene cluster of surugamides.
Surugamides A-E are biosynthesized by a combination of two non-ribosomal peptide
synthetases (NRPS) SurA and SurD in the four successive NRPS genes, SurA-D. The aminoacid sequence in surugamide F, biosynthesized by the other NRPS SurB and SurC, is muchdifferent from that in surugamides A-E. To analyze the physiological function ofsurugamides A and F, mutant strains of ΔsurB and ΔsurA, and ΔsurB were generated byin-frame deletion of the A-domain in the first module of each gene.
2. Identification of the protein function to promote aerial mycelium formation.
An enzyme enhanced by surugamide A was isolated by bioassay-guided fractionation

from the culture media of Streptomyces sp. JAMM992. The amino acid sequence at theN-terminus of the enzyme was analyzed by a protein-sequencer. With the sequence in hand,the complete amino acid sequence of the enzyme was obtained from the draft genome
sequence. Recombinant SJP was then expressed in Escherichia coli and purified withNi-Sepharose.
3. Comparison of amino acid sequences in SJP homologs among surugamide-producing
and non-producing Streptomyces.
Genomes derived from several Streptomyces were sequenced using Illumina Hiseq,
followed by read mapping to the sequence of SJP. A phylogenic tree was constructed using
the neighbor-joining method.
Results
With the sequence of the biosynthetic gene cluster for surugamide A, the mutant strain
ΔsurA was prepared by in-frame deletion of the A-domain in the first module of surA.
Sporulation of the mutant strain was clearly delayed, and was recovered by the addition ofSA in a dose-dependent manner. Because SA and SF are biosynthesized by different genes inthe same gene cluster, we hypothesized that SF also plays an important role in the strain.
Therefore, we prepared ΔsurA as well as ΔsurB, and a double mutant, ΔsurAΔsurB. Delayedsporulation similar to that in ΔsurA was observed in both ΔsurB and the double mutant.
Addition of SF also recovered the delay in sporulation. In case of the double mutant,
addition of SA recovered the delay in sporulation partially, whereas addition of both SA andSF recovered the sporulation completely (Figure 1). These results suggest that SA and SF are
implicated in aerial mycelium formation.
Next, we embarked on elucidating the molecular mechanisms to promote aerial myceliumformation. Because SA inhibits cathepsin B activity, we hypothesized that SA affected one ofthe proteases in the strain. After preparation of a crude extract from the surugamide producer,
we checked its enzyme activity in vitro. Eventually we found that SA activated one of theprotease. The crude extract was then separated based on activation of enzyme activity toobtain a purified protease. The amino acid sequence at the N-terminus of the protease wasanalyzed by Edman degradation, and the complete amino acid sequence was obtained fromgenomic data. In order to confirm that SA activated the protease, we prepared a recombinant
protein of the mature protease with His-tag, expressed in E. coli. Finally, we concluded that
activation of the protease promoted aerial mycelium formation. On the other hand, SF did
not activate the recombinant protease, which implied the presence of another moleculartarget for SF.
To the best of our knowledge, this is the first case where aerial mycelium formation waspromoted through protease activation. Therefore, we performed a survey on the activationspecificity for the homologues of the protease from several Streptomyces species. To oursurprise, SA only activated the protease derived from the surugamide-producing
Streptomyces, but not the one from non-producers (Figure 2). Amino acid sequences of these
proteases were obtained from draft genome data. The amino acid sequence of the proteasefrom the producer was obviously different compared to those from non-producers despite
relatively high homology. We concluded that SA activated only specific proteases derived
from surugamide producers.
Conclusion
In this study, we found a novel mechanism wherein surugamides enhance aerial mycelium
formation via activation of one protease by the peptides. Even though a few compounds areknown to control morphological changes by inhibiting extracellular proteases, this is the firstcase where the aerial mycelium formation was promoted through protease activation. We
therefore consider that surugamides are a novel type of signaling molecules.
References
1. Ninomiya A, Katsuyama Y, Kuranaga T, Miyazaki M, Nogi Y, Okada S, Wakimoto T,Ohnishi Y, Matsunaga S*, Takada K*. Biosynthetic Gene Cluster for Surugamide A
Encompasses an Unrelated Decapeptide, Surugamide F. Chem. Biochem. 17, 1709-1712.
(2016)

Figure 1. Recovery of sporulation in the mutants. (upper) addition of SA in ∆surA (middle)
addition of SA in ∆surA ∆surB (lower) addition of SA and SF in ∆surA ∆surBFigure 2. Enzyme activity of proteases after the treatment of SA. (left) proteases fromStreptomyces producing surugamides (right) proteases from Streptomyces not producing
surugamides