EMBOSS Palindrome buy ICG-001 analysis (http://emboss.bioinformatics.nl/) was used to locate palindromic sequences upstream of desE and desF. blastn analysis identified sequences with similarity to the DmdR-binding consensus from Streptomyces coelicolor A3(2) (Flores & Martín, 2004). The S. tropica des mutant was grown in A1 media (10 g L−1 potato starch,
4 g L−1 yeast extract, 2 g L−1 peptone, 28 g L−1 Instant Ocean) with 36 μM FeSO4 to mid-log phase, and 300 μL of cells was spread on iron-limited media plates. To test siderophore uptake, triplicate filter discs with 10 μg DFO E or yersiniabactin (EMC Microcollections GmbH, Germany), 130 μg FeSO4 or ultrapure water were placed on overlays. To probe the function of the putative
siderophore biosynthetic loci in S. tropica CNB-440 and S. arenicola CNS-205, we inactivated each by insertional inactivation of critical structural genes. In both species, the des mutants grew poorly in iron-limited media, the growth of which was visible after 2 months. When supplemented with FeSO4, the des mutant growth improved with cultures growing within 2 weeks, compared to wild-type cultures that grew densely and reached stationary phase within 1 week in iron-replete media. In contrast, mutagenesis of sid2-4 did not alter the growth or phenotype of the cells in either iron-limited or iron-sufficient conditions. CAS assays determined the presence of iron chelators in wild-type and mutant cultures. Mutagenesis of the des cluster, but not sid2-4, abolished CAS activity in S. tropica CNB-440 in the both the total culture and extracted supernatant. Similarly, disruption Pifithrin-�� concentration of des, but not sid2, resulted in the loss of CAS activity in the S. arenicola CNS-205 total culture and supernatant. These observations suggest that the primary growth-essential iron chelator in Salinispora laboratory cultures is a siderophore associated with the des locus. To confirm the lack of activity from sid2–sid4, we used RT-PCR to determine the conditions under which the putative siderophore biosynthetic loci were transcribed
(Fig. 2). The des gene cluster was transcribed in both PIK-5 species under iron limitation and repressed under iron-replete conditions. Interestingly, the sid2 transcript was detected in iron-limited S. tropica CNB-440, whereas it was only identified in iron-replete S. arenicola CNS-205. Transcript was detected under iron limitation from most genes within the S. tropica CNB-440 sid2 gene cluster (Table 1), except for a methyltransferase (stro2056), FAD-dependent monooxygenase (stro2658), hypothetical protein (stro2659) and putative transporters (stro2651-2). Despite the detection of sid2 transcript in both strains, no chemotypic differences were detected by the analytical HPLC in the extracts of mutants compared with the wild-type. The sid3 and sid4 gene clusters were not transcribed under iron-limited or iron-replete conditions.