The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, viomycin, from Streptomyces sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives.It was recently estimated that between the years 1998 and 2030 there will be 225 million new cases of tuberculosis (TB) and 79 million TB-related deaths (40). These numbers are astonishing when one considers that treatments for this disease, in the forms of vaccines or chemotherapy, have been available for more than 50 years (29). Mycobacterium tuberculosis, the causative agent of TB, is notoriously slowly growing and during infection can persist in a latent form in many individuals. These attributes contribute to the reasons why typical chemotherapy regimens for TB last 6 to 9 months (6) and why TB is so persistent. This prolonged treatment presents significant hurdles in the development of new antibiotics and in retaining the efficacies of the antibiotics used at present. Side effects and toxicity from a particular compound can be magnified when a patient takes a drug for this length of time, and there are increased incidences of poor adherence to the chemotherapy regimen by unmonitored patients, resulting in the development of multidrug-resistant (MDR) TB infections. These facts, together with alarming interactions between human immunodeficiency virus and TB infections that can result in increased numbers of infected individuals and MDR TB (30), make it of paramount importance to develop new chemotherapy agents or introduce modifications to the agents available at present to reduce their toxicities and increase their activities against MDR TB.The tuberactinomycins (TUBs; this abbreviation refers to the antibiotic family as a whole) (Fig. 1) are used specifically for the treatment of MDR TB (14). The importance of TUBs is reflected by some members being included on the World Health Organization's Model List of Essential M...

Among all types of RNA, tRNA is unique given that it possesses the largest assortment and abundance of modified nucleosides. The methylation at N 1 of adenosine 58 is a conserved modification, occurring in bacterial, archaeal, and eukaryotic tRNAs. In the yeast Saccharomyces cerevisiae, the tRNA 1-methyladenosine 58 (m 1 A58) methyltransferase (Mtase) is a two-subunit enzyme encoded by the essential genes TRM6 (GCD10) and TRM61 (GCD14). While the significance of many tRNA modifications is poorly understood, methylation of A58 is known to be critical for maintaining the stability of initiator tRNA Met in yeast. Furthermore, all retroviruses utilize m 1 A58-containing tRNAs to prime reverse transcription, and it has been shown that the presence of m 1 A58 in human tRNA 3 Lys is needed for accurate termination of plus-strand strong-stop DNA synthesis during HIV-1 replication. In this study we have identified the human homologs of the yeast m

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A synthetic genetic array was used to identify lethal and slow-growth phenotypes produced when a mutation in TRM6, which encodes a tRNA modification enzyme subunit, was combined with the deletion of any non-essential gene in Saccharomyces cerevisiae. We found that deletion of the REX1 gene resulted in a slow-growth phenotype in the trm6-504 strain. Previously, REX1 was shown to be involved in processing the 3′ ends of 5S rRNA and the dimeric tRNAArg-tRNAAsp. In this study, we have discovered a requirement for Rex1p in processing the 3′ end of tRNAiMet precursors and show that precursor tRNAiMet accumulates in a trm6-504 rex1Δ strain. Loss of Rex1p results in polyadenylation of its substrates, including tRNAiMet, suggesting that defects in 3′ end processing can activate the nuclear surveillance pathway. Finally, purified Rex1p displays Mg2+-dependent ribonuclease activity in vitro, and the enzyme is inactivated by mutation of two highly conserved amino acids.