Table 1.
Whole-genome comparison between Actinomadura sp. 32–07 and closely related Actinomadura species identified by TrueBac™.
Fig 1.
Maximum likelihood tree generated from nearly complete 16S rRNA gene sequences showing the relationship between Actinomadura sp. 32–07 and the type strains of species with validly published names in the genus Actinomadura.
Thermomonospora curvata DSM 43183T was used as an outgroup. The tree with the highest log likelihood is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (only values above 40% are shown). The tree is drawn to scale, with branch lengths measured in number of substitutions per site (bar = 0.01 substitutions per site). Nodes marked with a full circle were also recovered in the neighbour-joining and maximum parsimony trees. T’s at the end of strain names indicate type strains.
Fig 2.
Photographic, light microscopy and electron microscopy images of Actinomadura sp. 32–07.
After 21 days of growth on ISP medium 2, colonies are characterised by four major lobes, which often split in two minor sectors, and a central hollow pit (A, no magnification). Spore chains appear as long, flexuous series of subtle oval swelling stemming from a thinner, smooth hypha. Endospores inside the swellings can be viewed by light microscopy after staining with malachite green and counterstaining with safranin O (B, 100X magnification) or by closer inspection with electron microscopy (C, 8,500X magnification). Free spores of Actinomadura sp. 32–07 appear spherical and smooth, measuring 200–300 nm in diameter (D, 20,000X magnification).
Table 2.
Main morphological, cultural and physiological traits that distinguish Actinomadura sp. 32–07 from A. macra NBRC 14102T.
Table 3.
Summary of the main chemotaxonomic markers of Actinomadura sp. 32–07.
Fig 3.
Selected results of the antiSMASH analysis on the genome of Actinomadura sp. 32–07.
(A) A total of 27 regions were identified that contained one or more predicted biosynthetic clusters. (B) Of all the results, only region 11 (124,153 bp) contained a gene cluster coding for NRPS modules whose arrangement and domain composition agreed with the chemical structure of zelkovamycin (zelA–C). The core biosynthetic clusters in the region are highlighted in red and ORFs are numbered according to their position in the genome. Where available, the modules and domains coded by the ORFs of interest and their predicted substrate specificity are shown. (C) Schematic of the organization of the putative zelkovamycin synthetase according to the structure of zelkovamycin. The similarities between the expected residue specificity of some A domains and the respective software prediction are illustrated, along with the domains missing from the software prediction.
Fig 4.
(A) Comparison between the linear structures of zelkovamycin and argyrins, (B) the NRPS systems behind their production and (C) the core biosynthetic genes coding for them.
Genes, modules and amino acid residues of each compound are color-coded. The structure of the argyrin cluster and argyrin synthetase are adapted from Pogorevc et al. [60].
Fig 5.
Detection, purification and antibiotic activity of zelkovamycin from Actinomadura sp. 32–07.
(A) Extracted ion chromatograms of commercial zelkovamycin (top) and a representative example of an extracted sample (bottom). Zelkovamycin can be detected as a monodisperse peak eluted at 6.14 min. (B) Zelkovamycin extraction steps from the inoculum medium, the fermented medium and the harvested mycelium. The compound is present in both culture media and can be successfully extracted upon resin elution with 100% methanol. (C) Antibiotic sensitivity testing on S. aureus SH1000 using the most concentrated zelkovamycin extracts (E3) from each fraction. The lower concentration of antibiotic in the extracts obtained from the seed culture and the mycelium is reflected by their significantly lower, albeit detectable, inhibitory activity. FT: flow-through; W: water wash; E1–3: 20%, 50% and 100% MeOH elution, respectively; Z: zelkovamycin standard (1 mg/mL); S: seed culture extract; F: fermented broth extract; M: mycelium extract; C: 100% MeOH control.