Alternatively, the yet-unknown compound(s) in the soil might have been converted to

tryptophan or anthranilate by the bacterial activity. Because the soil extracts did not induce the andA promoter (Nishiyama et al., 2010), such compounds might be resistant to extraction and/or poorly soluble, like lignin or humic substances. It is unlikely that levels of these inducers would be very high, and this could explain the relatively low levels of expression in soil compared with those in the in vitro cultures (Fig. 3). The andA-mutant cells failed to proliferate at the initial stage of incubation in the soil (see Fig. 5, days 7 to day 15). These data clearly indicated that the anthranilate dioxygenase genes play pivotal roles Cyclopamine cell line in the proliferation of ATCC 17616 cells in the soil. It is puzzling that loss of anthranilate dioxygenase alone abolished the growth in soil. When wild-type and andA-mutant cells were grown in the M9 minimal medium supplemented with succinate and soil extracts,

the turbidities of the culture reached a higher level than when grown in the M9 minimal medium supplemented with succinate alone, indicating that energy sources are present in the soil and that andA mutant can utilize them. Although further investigations are needed, the important role of the anthranilate dioxygenase might be to remove anthranilate, which might accumulate and have adverse effects on cells growing in the soil. As anthranilate is known to be a precursor of the signal molecules mediating intercellular see more communication for some Gram-negative bacteria (Bredenbruch et al., 2005; Farrow & Pesci, 2007; Oglesby et al., 2008), the accumulation of anthranilate might confuse the quorum sensing. We thank Dr Paul B.

Rainey for providing the IVET plasmid pUIC3. We thank Dr T. Ohmori for providing the soil sample collected from the Ehime Agricultural Experiment Station. This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. “
“The model organism Streptomyces coelicolor A3(2) harbors a 356-kb linear plasmid, SCP1. We report here development of a recombinational cloning method for Niclosamide deleting large segment from one telomere of SCP1 followed by replacing with the telomere of pSLA2 and sequentially inserting with the overlapping cosmids in vivo. The procedure depends on homologous recombination coupled with cleavage at telomere termini by telomere terminal protein. Using this procedure, we cloned the 81-kb avermectin and the 76-kb spinosad biosynthetic gene clusters into SCP1. Heterologous expression of avermectin production in S. coelicolor was detected. These results demonstrate the utility of SCP1 for cloning large DNA segments such as antibiotic biosynthetic gene clusters.