47NISR Research GRANT
2010 Young Investigator Research Grant

Clarification of new regulatory network for antibiotic
production
Shigeru KITANIInternational Center for Biotechnology, Osaka University

Research aims
Gram-positive bacteria of the genus Streptomyces are industrially important microorganisms, producing more than
70% of commercially important antibiotics. The productionof these compounds is often regulated by diffusible signaling molecules called autoregulators. One well-studied family of autoregulators consists of the g -butyrolactones, which
are active at nanomolar concentrations and which elicit antibiotic production by modulating the DNA-binding activityof cognate receptor proteins, and hence have been referred
to as bacterial hormones. Although 60% of Streptomyces
strains may utilize g -butyrolactone autoregulators, little is
known about signaling molecules used by other members of
this genus.
Streptomyces avermitilis produces a family of polyketide
macrocyclic lactones known as avermectins, which havepotent antiparasitic and broad-spectrum activities against
nematodes and arthropod parasites, together with two otherfamilies of polyketide antibiotics, filipins and oligomycins.
While the biosynthetic pathways of the three antibiotics
have been predicted and partially characterized, the regulatory mechanism of antibiotics production in S. avermitilis
has so far remained unclear. Moreover, nothing is known
about the autoregulator itself that might control antibiotic
production in S. avermitilis.
In this study, I purified a signaling molecule from Streptomyces avermitilis, and determined its structure, including
stereochemistry. In addition, I investigated the functionalrole of avaR3, one of the clustered-receptor genes, to know
its involvement in antibiotic production in S. avermitilis.

amounts of each antibiotic were quantified with HPLC system using authentic standards.
Results
1. Identification of avenolide, an S. avermitilis autoregulator belonging to a new class of Streptomyces
hormone1)
Genes involved in autoregulator biosynthesis and those
encoding autoregulator receptor proteins are frequently
clustered at a same locus in various Streptomyces species.
The genes sav3702-3706 of S. avermitilis include threegenes (avaR1, avaR2, and avaR3) encoding homologues ofg -butyrolactone-autoregulator receptor proteins and the aco
gene that encode an acyl-CoA oxidase. To determine
whether the aco gene is important in antibiotic production,an aco gene knockout mutant was evaluated for antibioticproduction. The level of avermectin production in the aco

Methods
Two thousands liters of wild-type culture broth were prepared to isolate the ligand (avenolide) of AvaR1, which wasexpressed in E. coli and purified as a single protein. Twobioassays were used to detect avenolide activity: a DNA
dissociation activity on the rAvaR1-DNA complex in thegel-shift experiment, and an avermectin inducing activity
with S. avermitilis aco mutant, which lacks ability to produce both avenolide and avermectin. NMR (1H: 500 MHz,13
C: 125 MHz) spectra were obtained on JEOL JNM-ECP
500 FT NMR SYSTEM. The wild-type avaR3 gene was replaced with the disrupted allele (DavaR3) by homologous
recombination for construction of the DavaR3 strain. TheFig. 1 (A) A chemical structure of avenolide. (B) Effect of avenolide onavermectin production. (C) Avermectin production with increasing concentrations of avenolide.

48mutant was markedly reduced to about 6% of the wild-typelevel. The defect of the mutant in the avermectin production
was restored completely by the addition of an ethyl acetate(AcOEt)-extract of the wild-type culture broth. Interestingly, an AcOEt-extract of the aco mutant was unable to induce avermectin production, which suggests that signaling
molecule(s) eliciting avermectin production is present in
the wild-type strain, but not in the aco mutant. Furthermore, the AcOEt-extract of the wild-type culture clearly
prevented AvaR1 from forming the AvaR1-DNA complex,
whereas the AcOEt-extract of the aco mutant showed almost no activity. These results implied that the factor inhibiting the DNA-binding activity of AvaR1 seems to besynthesized by enzymatic function of Aco and the factor is
responsible for inducing avermectin production as a signaling molecule. Thus, I purified the factor from wild-type
culture broth by several procedures including a silica gelcolumn chromatography and HPLC with C18 reverse-phasecolumn, and determined the structure of signaling moleculetermed “avenolide”, which triggered avermectin production
at a minimum effective concentration of 4 nM (Fig. 1).
2. Functional analysis of AvaR3, an autoregulator-receptor homologue2)
avaR3, one of the clustered-receptor genes, encodes aprotein containing an extra stretch of amino acid residues
that has not been found in the family of g -butyrolactoneautoregulator receptors. In order to investigate the in vivorole of avaR3 in S. avermitilis, an avaR3-deletion mutant
was constructed. In liquid cultivation, significantly decreased production of avermectins was observed in theavaR3 mutant with delayed expression of avermectinbiosynthetic genes, suggesting that AvaR3 positively controls the avermectin biosynthetic genes. Moreover, the disruption caused an increased production of filipin without
any changes in the transcriptional profile of the filipinbiosynthetic genes, suggesting that filipin production is indirectly controlled by AvaR3. AvaR3 is also involved inboth antibiotic productions on solid medium. Furthermore,the avaR3 mutant showed more fragmented mycelia with aremarkable decrease in the mean diameter of the pellet, indicating that AvaR3 plays an important role in the mycelial
aggregation in liquid cultivation.
Conclusion
In this study, I identified avenolide, a novel signaling
molecule controlling avermectin production in S. avermitilis, and showed functional roles of the avaR3 gene encoding an autoregulator-receptor homologue in the secondary
metabolism of this bacterium. The identification of avenolide as a new Streptomyces autoregulator should allow theconstruction of high level producers of antibiotics, including avermectin, as well as facilitating genetic understandingof regulatory mechanism for antibiotic production. On theother hand, the completed Streptomyces genome sequences
have demonstrated that the putative autoregulator receptor
genes are frequently located far from the antibiotic biosynthetic gene clusters on the genomes. Thus, it is expectedthat further understanding of the regulatory mechanism by
AvaR3, whose gene is located far away from the avermectinbiosynthetic gene cluster, will lead to higher yields in antibiotic production.
The results obtained in this study suggest that we coulddiscover clinically-useful natural compounds by activating
the numerous silent natural product gene clusters prevalentin actinomycete genomes.
References1)

2)

Kitani, S., Miyamoto, K. T., Takamatsu, S., Herawati, E., Iguchi, H.,
Nishitomi, K., Uchida, M., Nagamitsu, T., Omura, S., Ikeda, H., andNihira, T. (2011) Avenolide, a Streptomyces hormone controlling antibiotic production in Streptomyces avermitilis. Proc. Natl. Acad. Sci.
U.S.A. 108: 16410–16415.
Miyamoto, K. T., Kitani, S., Komatsu, M., Ikeda, H., and Nihira, T.
(2011) The autoregulator receptor homologue AvaR3 plays a regulatory role in antibiotic production, mycelial aggregation and colony
development of Streptomyces avermitilis. Microbiology 157: 2266–2275.