The glycopeptide antibiotic gene clusters reported for balhimycin, chloroeremomycin, A40926, A47934 and teicoplanin do not contain Fd or FdR genes (Donadio et al., 2005). Only the biosynthetic gene cluster for the related natural product complestatin contains a single Fd gene (comK) (Chiu et al., 2001). To advance in vitro studies of the cross-linking steps in glycopeptide antibiotic biosynthesis, we present efforts to identify and characterize Fd genes in the balhimycin producer A. balhimycina. The sequencing and annotation

of the entire genome is currently underway. We describe in silico analyses, which reveal 11 different Fd genes in A. balhimycina. Furthermore, we demonstrate the production and KPT-330 concentration purification of two of the newly identified Fds, as well as their ability to participate in electron transfers to OxyB from both A. balhimycina and A. orientalis. The A. balhimycina DSM5908 genome was analyzed using a blast search carried out with six Fd sequences (NP-631171, NP-629284,

NP-631715, NP-625075, NP-628054 and NP-625924) from Streptomyces coelicolor A3(2) (Lamb et al., 2002; Chun et al., 2007), putidaredoxin (P00259) from Pseudomonas putida (Peterson et al., 1990; Pochapsky et al., 1994; Sevrioukova et al., 2003) and the putative ferredoxin comFd (AAK81833) from the complestatin producer Streptomyces lavendulae (Chiu et al., 2001). Eleven putative Fds, named balFd-I to balFd-XI, containing expect (E)-values

<10−6, were identified in the whole genome of A. balhimycina (Table 1). Assignment of the iron–sulfur cluster type was achieved through a blast search of the nonredundant R428 purchase database and a comparison with known Fds. The sequences of the ferredoxins balFd-I to balFd-XI have been deposited in the EMBL database under the accession numbers FN594523-FN594533. The genomic DNA of A. balhimycina DSM5908 was used as a PCR template to amplify the genes coding for the putative [3Fe–4S] ferredoxins balFd-V and balFd-VII. The primers used are shown below (restriction sites underlined): 5′-balFd-V: 5′-TAGACCATATGAAGGTTGTTGTCGACG-3′ 3′-balFd-V: 5′-ATCTACTCGAGGGCCTCTTCGAGGGC-3′ selleckchem 5′-balFd-VII: 5′-TAGACCATATGAAGGTCACCGTGGACG-3′ 3′-balFd-VII: 5′-ATCTACTCGAGCGCGTCCTCGCTCACCG-3 After digestion with NdeI and XhoI, the fragments were cloned between the NdeI/XhoI sites of plasmid pET22b (Novagen) for expression as C-terminal His6-tagged fusion proteins. The nucleotide sequence of each insert was confirmed by sequencing. For production in Escherichia coli Rosetta2(DE3)pLysS (balFd-V) or Origami2(DE3) (balFd-VII), terrific broth medium (400 mL) supplemented with antibiotics and FeSO4 (0.5 mM) was induced with isopropyl-β-d-thiogalactopyranoside (0.2 mM), and shaking at 30 °C for 6 h. Cell pellets in buffer-A (25 mL, 50 mM sodium phosphate pH 7.4, 300 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.