Fig 1.
Structures and biosynthetic gene clusters of natural lincosamides.
The resistance genes are marked in grey, the genes homologous in both gene clusters are black. The genes highlighted by red line exhibit inactivation pattern enabling their assignment to condensation reaction, i.e. formation of amide bond (in red oval in the structures).
Table 1.
Abbreviations of specific compounds and proteins.
Table 2.
Comparative analysis of lincomycin and celesticetin biosynthetic gene clusters.
Fig 2.
The lincomycin biosynthetic pathway.
Gray background highlights the condensation step solved herein. Red—proteins with already proved functions, A—adenylation domain, CP—carrier protein, PPL—4-propyl-L-proline, MTL—methylthiolincosamide.
Fig 3.
The biological activity assay of S. lincolnensis inactivation mutants.
The inhibition zones correspond to the antibiotic production. WT—wild type strain (positive control); ΔT, ΔN, N-ΔID, N-ΔCP indicate the respective disrupted lincomycin biosynthetic gene or its part; NC—not complemented; PPL or MTL—complemented by the respective intermediate of lincomycin biosynthesis. For each sample, at least two independent cultivations were evaluated in triplicates as described in Material and methods. A typical result is shown.
Fig 4.
The biological activity assay of S. lincolnensis inactivation mutants.
The inhibition zones correspond to the antibiotic production. WT—wild type strain (positive control); ΔC, ΔD, ΔE, ΔF, ΔV, ΔIH, ΔQ—indicate the respective disrupted lincomycin biosynthetic gene; NC—not complemented; PPL, MTL or PPL+MTL—indicate complementation by the respective intermediate of lincomycin biosynthesis or their combination. For each sample, at least two independent cultivations were evaluated in triplicates as described in Material and methods. A typical result is shown.
Fig 5.
Evidence of two translation starts in LmbN coding gene.
(A) Western blot analysis of LmbN forms produced by S. lincolnensis, and heterologously in S. coelicolor. 1 and 7: standards of MW (His8-tagged LmbN, His8-tagged LmbN-ID, His8-tagged LmbN-CP); 2: S. lincolnensis ΔlmbN mutant; 3: S. lincolnensis WT; 4: S. coelicolor M145 WT; 5: S. coelicolor M145 containing mutant form of lmbN with artificial stop codon introduced immediately downstream of the translation start 1; 6: S. coelicolor M145 containing native lmbN gene. (B) The detailed scheme of internal translation start (start 2) in lmbN. The red colour corresponds to CP domain coding sequence; the green colour corresponds to amino sugar isomerase domain (ID) coding sequence. RBS—ribosome binding site. Start 1—regular translation start producing full length LmbN.
Table 3.
MS analysis of LmbN-CP.