EMBOSS Palindrome Proteasome inhibitor 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 AZD4547 supplier 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 mafosfamide 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.