54NISR Research GRANT
2005 Research Grant

Antibiotic Production in Streptomyces Induced in Co-culturing System
Yasuhiro IGARASHI
Department of Engineering, Toyama Prefectural University

Research aims
Pure culture is the fundamental basis in modern microbiology. Except for a part of symbiotic bacteria, it is widely
accepted that microorganisms should be purely cultured
both for scientific and industrial purposes and the contamination should be eliminated. Actinomycetes have a great
ability of producing diverse bioactive compounds, many ofwhich are industrially produced as pharmaceuticals andagrochemicals. In the use of actinomycetes, it is normal toculture them purely as same as other microorganisms. However, it was unexpectedly found in our laboratory that acontaminated bacterium induced the pigment production by
Streptomyces lividans on the agar plate. We initiated this research to investigate the potential utility of the bacterium in
the induction of antibiotic production by actinomycetes.
Methods
The bacterium used for the co-culture with Streptomyces
was isolated as a contaminant from the culture plate ofStreptomyces lividans. It is a Gram-positive bacillus, andwas identified as Tsukamurella pulmonis TP-B0596 basedon the 16S rDNA analysis. The actinomycete strains usedin this study were isolated from the soil and plant samples
in our laboratory, and identified as Streptomyces based on
the taxonomic study. The seed and production fermentation
was carried out in V-22 and A-3M medium, respectively1).
The medium composition is as follows: V-22; soluble starch
1%, glucose 0.5%, NZ-case 0.3%, yeast extract 0.2%, tryptone 0.1%, K2HPO4 0.1%, MgSO4 · 7H2O 0.05%, CaCO3
0.3% (pH 7) and A-3M; soluble starch 2%, glucose 0.5%,glycerol 2%, Pharmamedia 1.5%, yeast extract 0.3%, Diaion HP-20 1% (pH 7). The HPLC analysis was performedon an Agilent HP-1100 system with a photodiode array detector.
Results
The co-culture of actinomycetes and T. pulmonis was
carried out in the following manner. Actinomycete strains
were cultured in V-22 medium for 4 days and T. pulmonisfor 1 day, separately. To the A-3M medium were transferred
3% (v/v) of the seed actinomycete culture and 0.3% (v/v) of
the seed T. pulmonis culture, and the production fermentation was carried out for 6 days at 30°C on a rotary shaker.
The whole broth was extracted with 1-butanol, the organic
layer was concentrated in vacuo, and the extract was sub-jected to antimicrobial assay. Similarly, the extract was prepared from the pure-cultured broth of actinomycetes andsubjected to the bioassay. Among the 122 Streptomyces
strains examined, 8 strains were identified to show antibacterial activity when co-cultured with T. pulmonis but notwhen purely cultured. In addition, production of secondarymetabolites was detected in the co-cultured extract by theHPLC analysis, that were not found in the pure culture extract. Then, we isolated the secondary metabolites producedin the co-culture and analyzed their structures. Spectroscopic analysis using NMR and MS revealed that in S. albogriseolus S430 was induced the production of antibiotic
A33853 and in S. panayensis TT1712 the production ofaclacynomycins. Furthermore, two new compounds wereisolated from the co-cultured broth: antibiotic TPU-0043 isthe polyene macrolide from S. sp. TP-A06252) and alchivemycin is the antimicrobial macrocyclic compound from S.
sp. S5223), both of which are produced in the co-culture
with T. pulmonis.
Conclusion
We found an interesting and unusual phenomenon thatthe co-culture of Streptomyces with a bacterium T. pulmonis
induced the production of secondary metabolites whichwere not produced in a pure culture of Streptomyces. Although some metabolites produced in the co-culturing sys-55

tem are known compounds, two new compounds werefound from this novel fermentation method. One of the newcompounds is antibiotic TPU-0043, a stereoisomer of thepentaene macrolide chainin, and the other is alchivemycin
which possesses the unprecedented heterocyclic structure
revealed by X-ray crystallography. It is noteworthy that thesecondary metabolism which is dormant under the normal
condition is activated by ‘contamination’ and the new
bioactive compounds are produced.
Recently, the frequency of finding a new compound fromactinomycetes is decreasing4). However, an innovative fermentation technology such as the co-culturing system
shown in this study may provide a new insight into thescreening program from actinomycetes. The secondary metabolism is not induced by other bacteria, e.g. Bacillus subtilis, Escherichia coli, except for T. pulmonis. Furtheranalysis on the mechanism of the co-culture and secondary

metabolism induction in rare actinomycetes is under investigation.
References
1) Sasaki, T., Igarashi, Y., Saito, N. and Furumai, T. 2001. Cedarmycin
A and B, new antimicrobial antibiotics from Streptomyces sp. TPA0456. J. Antibiot. 54: 567–572.
2) Igarashi, Y., In, Y., Ishida, T., Fujita, T., Yamakawa, T., Onaka, H.
and Furumai, T. 2005. Absolute configuration of TPU-0043, a pentaene macrolide from Streptomyces sp. J. Antibiot. 58: 523–525.
3) Igarashi, Y., Ishida, T., In, Y., Kan, Y., Fujita, T., Azumi, M.,
Yoahida, R. and Furumai, T. 2005. Structure determination of microbial secondary metabolites with unusual cyclic structures. Book ofAbstracts of 9th International Conference of the Chemistry of Antibiotics and other Bioactive Compounds. (Archachon, France)
4) Berdy, J. 2005. Bioactive microbial metabolites. J. Antibiot. 58:
1–26.