Figure 1.
Structures of the two GE2270 congeners described in this study.
Figure 2.
Phylogenetic tree based on 16S rRNA gene sequences of selected actinomycetes.
Neighbor-joining cladogram constructed in MEGA5 [44]; alignment of the sequences was done with CLUSTALW, bootstrap values (in percent) are calculated from 1000 resamplings and shown at the respective nodes for values >50%, the scale bar represents 0.02% sequence divergence. The tree is rooted to 16S rRNA from Bacillus cereus ATCC 14579.
Figure 3.
Insert of cosmid 2F7 and construction of cosmids derived from 2F7.
A) Insert of 2F7 and 2F7cat comprising the GE2270 biosynthetic gene cluster (pbt), the tufR gene coding for the GE2270-resistant EF-Tu, 25 adjacent ribosomal genes and rpoC coding for RNA polymerase β′-subunit (see Table S2). B) Introduction of the inducible tcp830 promoter in front of the regulatory gene pbtR resulting in cosmid pbtKA01. C) For the construction of pbtKA02 the tcp830 promoter was introduced in front of pbtG1 under loss of pbtR. D) Replacement of 22 genes encoding ribosomal proteins with an apramycin resistance cassette (aac(3)IV) resulted in cosmid pbtCK01. E) pbtCK02 was constructed by introduction of the constitutive promoter ermE* and associated replacement of the ribosomal genes rpsL, rpsG and fusA with a hygromycin resistance cassette (hyg). F–H) Introduction of the inducible tcp830 promoter at three distinct positions in each case followed by subsequent removal of the employed aac(3)IV cassette. F) tcp830 introduced in front of the regulatory gene pbtR resulting in cosmids pbtCK03. G) tcp830 introduced in front of pbtG1 under loss of pbtR resulting in cosmid pbtCK04 and H) tcp830 introduced in front of the structural gene pbtA resulting in cosmid pbtCK05. I) negative control cosmid pbtCK08 was constructed by replacement of the pbt biosynthetic genes of cosmid 2F7cat by an apramycin resistance cassette (aac(3)IV). For each construct, the efficiency of conjugal transfer into Streptomyces coelicolor M1146 is expressed as number of exconjugants per 1 million spores.
Figure 4.
HPLC-MS analysis of a methanolic extract of S. coelicolor M1146(pbtCK05).
Data confirms the presence of GE2270A; GE2270B1 is identified as the main product.
Figure 5.
GE2270A production over time in S. coelicolor M1146(pbtCK01).
Cultivation was performed in SM13 medium in 300-square deepwell plates [28].
Figure 6.
GE2270 production of S. coelicolor M1146 strains containing different constructs.
A) GE2270A production of S. coelicolor M1146 strains containing different constructs of the GE2270 biosynthetic gene cluster (see Figure 3). The amount of GE2270A was determined after 8 days of cultivation in 24-square deepwell plates [28]. B) GE2270B1 production in S. coelicolor M1146(pbtCK05). Values are mean ± SEM from triplicated cultivation of three individual exconjugants each.
Figure 7.
Agar diffusion test to determine the resistance of Streptomyces coelicolor strains against GE2270A.
GE2270A was applied in amounts of 0.4 µg to 12 µg; 20 µg kanamycin (Kan) were applied as positive control.