Identification of tirandamycins as specific inhibitors of the futalosine pathway

To identify compounds that specifically inhibit the futalosine pathway, we employed a previously developed screening method.⁸ For the initial screening, we used the paper disk method and employed two closely related Bacillus strains, Bacillus subtilis strain 168 and Bacillus halodurans C-125, as the test organisms. By genome sequencing, these two strains had been shown to possess a high degree of similarity.⁹ However, B. subtilis strain 168 and B. halodurans C-125 use the classical pathway and the futalosine pathway, respectively, for the biosynthesis of MK. We therefore proposed that a compound inhibiting the biosynthesis of MK in the futalosine pathway may specifically repress the growth of B. halodurans C-125 only. To test this, we first screened candidate compounds for their ability to specifically inhibit B. halodurans C-125 using a paper disk assay. We tested ~3200 culture broths (2800 actinomycetes broths and 400 fungi broths). Of these, 18 culture broths were found to specifically inhibit the growth of B. halodurans C-125 (0.6%). In particular, the growth of B. halodurans C-125 was clearly inhibited in the presence of sample no. SF2910, but this inhibition was reversed by adding MK (0.1 mg ml⁻¹), even in the presence of sample no. SF2910. This result suggested that sample no. SF2910 contained a compound that specifically inhibited the futalosine pathway. Therefore, we next investigated active components in sample no. SF2910.

The MIC value of 1 against B. halodurans C-125 was calculated to be 1 μg ml⁻¹ by measuring the OD₆₀₀ of liquid cultures containing various concentrations of 1, and no growth inhibition for B. subtilis strain 168 was observed up to 100 μg ml⁻¹ of 1. Tirandamycins A and B, originally isolated from Streptomyces tirandis in 1971, are antibiotics that exhibit antimicrobial activity against a number of bacteria with an MIC range of 1–10 μg ml⁻¹.^{12, 13} In vitro experiments have revealed that tirandamycins inhibit the chain initiation and elongation steps of RNA polymerase transcription.¹⁴ Among tirandamycin-sensitive bacteria, some use a canonical MK biosynthetic pathway (Bacillus megaterium, Clostridium pasteurianum, Streptococcus pyogenes and Bacteroides fragilis) and others use a futalosine pathway (Streptomyces prasinus). Therefore, tirandamycins are suggested to target both transcription and the futalosine pathway.

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