tetracycline and kalafungin

Many microorganisms produce molecules having antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbours. Given that these molecules are often toxic to the producer, mechanisms must exist to ensure that the assembly of the export apparatus accompanies or precedes biosynthesis. Streptomyces coelicolor produces the polyketide antibiotic actinorhodin in a multistep pathway involving enzymes encoded by genes that are clustered together. Embedded within the cluster are genes for actinorhodin export, two of which, actR and actA resemble the classic tetR and tetA repressor/efflux pump-encoding gene pairs that confer resistance to tetracycline. Like TetR, which represses tetA, ActR is a repressor of actA. We have identified several molecules that can relieve repression by ActR. Importantly (S)-DNPA (an intermediate in the actinorhodin biosynthetic pathway) and kalafungin (a molecule related to the intermediate dihydrokalafungin), are especially potent ActR ligands. This suggests that along with the mature antibiotic(s), intermediates in the biosynthetic pathway might activate expression of the export genes thereby coupling export to biosynthesis. We suggest that this could be a common feature in the production of many bioactive natural products.

Topics: Anthraquinones; Anti-Bacterial Agents; Bacterial Proteins; Biological Transport; Biosensing Techniques; DNA, Bacterial; Gene Expression Regulation, Bacterial; Ligands; Multigene Family; Mutation; Naphthalenes; Naphthoquinones; Pyrans; Repressor Proteins; Streptomyces coelicolor; Tetracycline

We have developed a high capacity screen for compounds that inhibit the 3C protease of human rhinovirus-1b. The assay uses a recombinant strain of Escherichia coli expressing both the protease and a tetracycline resistance-conferring protein modified to contain the minimal protease cleavage site. Cultures growing in microtiter plates containing tetracycline are treated with potential inhibitors and simultaneously monitored for change in growth over time using an oxygen probe. Most of the cultures, not containing an inhibitor of the 3C protease, show reduced growth due to cleavage of the essential gene product; normal growth is seen only in the infrequent culture that contains an inhibitor. In the present example, we have used the tetA gene of plasmid pACYC184 as the modified gene. The system has been validated using inhibitors of protease 3C, and has been used to identify three new inhibitors of the enzyme, active in the micromolar range.

Topics: 3C Viral Proteases; Amino Acid Sequence; Antiporters; Arabinose; Autoanalysis; Bacterial Proteins; Citrinin; Cysteine Endopeptidases; Escherichia coli; Gene Expression; Molecular Sequence Data; Naphthoquinones; Oligopeptides; Plasmids; Protease Inhibitors; Pyrones; Recombinant Fusion Proteins; Tetracycline; Tetracycline Resistance; Viral Proteins