He Kyoung Lim,¹† Eu Jin Chung,^2,3† Jin-Cheol Kim,¹ Gyung Ja Choi,¹ Kyoung Soo Jang,¹
Young Ryun Chung,² Kwang Yun Cho,¹ and Seon-Woo Lee³*

Division of Applied Biology, College of Natural Resources and Life Science, Dong-A University,
Busan 604-714, Republic of Korea.

Abstract

A microbial community analysis of forest soil from Jindong Valley, Korea, revealed that the most abundant rRNA genes were related to Acidobacteria, a major taxon with few cultured representatives. To access the microbial genetic resources of this forest soil, metagenomic libraries were constructed in fosmids, with an average DNA insert size of more than 35 kb. We constructed 80,500 clones from Yuseong and 33,200 clones from Jindong Valley forest soils. The double-agar-layer method allowed us to select two antibacterial clones by screening the constructed libraries using Bacillus subtilis as a target organism. Several clones produced purple or brown colonies. One of the selected antibacterial clones, pJEC5, produced purple colonies. Structural analysis of the purified pigments demonstrated that the metagenomic clone produced both the pigment indirubin and its isomer, indigo blue, resulting in purple colonies. In vitro mutational and subclonal analyses revealed that two open reading frames (ORFs) are responsible for the pigment production and antibacterial activity. The ORFs encode an oxygenase-like protein and a putative transcriptional regulator. Mutations of the gene encoding the oxygenase canceled both pigment production and antibacterial activity, whereas a subclone carrying the two ORFs retained pigment production and antibacterial activity. This finding suggests that these forest soil microbial genes are responsible for producing the pigment with antibacterial activity.

Keywords:Acidobacteria, Bacillus subtilis, Escherichia coli, Forest Soil Metagenome Clone, Forest Soil, Organis farming.