Isolation and Characterization of Transposon-Insertional Mutants from Paenibacillus polymyxa E681 Altering the Biosynthesis of Indole-3-Acetic Acid Quyet Tien Phi Æ Sang-Ho Oh Æ Yu-Mi Park Æ Seung-Hwan Park Æ Choong-Min Ryu Æ Sa-Youl Ghim Received: 20 November 2007 / Accepted: 28 December 2007 / Published online: 19 February 2008 Ó Springer Science+Business Media, LLC 2008 Abstract We screened a mini-Tn10 insertional mutant library of the spore-forming bacterium Paenibacillus polymyxa E681 with variable indole-3-acetic acid (IAA) productivity. Four mutants, of which two showed a decrease in IAA production and the other two showed an increase in IAA production, were finally selected. Further analyses demonstrated different levels of IAA intermedi- ates from culture supernatant of wild-type strain and mutants. In addition, mutants showed different promotions on the early growth of 10-day-old maize in terms of the increase in shoot and root weights. DNA fragments flank- ing the transposon insertion in four mutants were cloned and sequenced. The target sites of insertion were gene gpr1, disrupted at two sites, 49 bp downstream of the spo0F gene, and relA/spoT homologue, which codes for GPR1/FUN34/YaaH family protein, stage 0 sporulation protein F, and RelA/SpoT domain protein, respectively. This evidence suggests that there may be a number of genes involved in the regulation of IAA biosynthesis of P. polymyxa. Introduction Indole-3-acetic acid (IAA), a major plant growth hormone of the auxin class, has been found to be produced by numerous organisms including of plants, bacteria, fungi, and algae. The identification of IAA intermediates dem- onstrated five different pathways of IAA biosynthesis in bacteria [15]. Many reports have described the factors involved in the level of IAA biosynthesis, which are both genetic and environmental factors [15]. Related to the effects of genetic elements, the location of IAA biosyn- thetic genes, mode of expression, transcriptional regulator RpoS, and two-component system GacS/GacA have been shown to affect the level of IAA production in some bac- teria [8, 16, 19]. By transposon-insertional mutagenesis, several genes and proteins from bacteria have been repor- ted to be relevant to the level of IAA biosynthesis, mainly involved in transcriptional regulators, transport system proteins, outer membrane proteins, and cytochrome c bio- genesis genes [11, 23]. Paenibacillus polymyxa, a Gram-positive bacterium, has been proved to dependently synthesize IAA from the main precursor tryptophan (Trp) [10]. However, studies on the identification and characterization of the key genes and proteins related to this process in the bacterium have not been conducted. P. polymyxa E681 used in this experiment was isolated from the rhizosphere of winter barley grown in Korea [18]. This strain is known to form endospores, suppress plant diseases, produce antimicrobial compounds, secrete diverse degrading enzymes, and produce phyto- hormones [18]. The bacterium was previously reported to produce IAA dependent on exogenous Trp via only the indole-3-pyruvic acid (IPA) pathway. In addition, the enzyme indole- 3-pyruvate decarboxylase (IPDC), the key enzyme in the IPA pathway, from the bacterium was Q. T. Phi Á S.-H. Oh Á Y.-M. Park Á S.-H. Park Department of Microbiology, Kyungpook National University, Daegu 702-701, Korea S.-H. Park Á C.-M. Ryu Genome Research Center, KRIBB, Daejon 305-600, Korea S.-Y. Ghim (&) Department of Microbiology, College of Natural Sciences, Kyungpook National University, 1370 Sankyug-Dong, Buk-Gu, Daegu, Korea e-mail: ghimsa@mail.knu.ac.kr 123 Curr Microbiol (2008) 56:524–530 DOI 10.1007/s00284-008-9118-8