Holomycin, a member of the pyrrothine class of antibiotics, displayed broad-spectrum antibacterial activity, inhibiting a variety of gram-positive and gram-negative bacteria, with the exception of Enterobacter cloacae, Morganella morganii, and Pseudomonas aeruginosa. The antibiotic lacked activity against the eukaryotic microorganisms Saccharomyces cerevisiae and Candida kefyr. Holomycin exhibited a bacteriostatic response against Escherichia coli that was associated with rapid inhibition of RNA synthesis in whole cells. Inhibition of RNA synthesis could have been a secondary consequence of inhibiting tRNA aminoacylation, thereby inducing the stringent response. However, the levels of inhibition of RNA synthesis by holomycin were similar in a stringent and relaxed pair of E. coli strains that were isogenic except for the deletion of the relA gene. This suggests that inhibition of RNA synthesis by holomycin could reflect direct inhibition of DNA-dependent RNA polymerase. Examination of the effects of holomycin on the kinetics of the appearance of ␤-galactosidase in induced E. coli cells was also consistent with inhibition of RNA polymerase at the level of RNA chain elongation. However, holomycin only weakly inhibited E. coli RNA polymerase in assays using synthetic poly(dA-dT) and plasmid templates. Furthermore, inhibition of RNA polymerase was observed only at holomycin concentrations in excess of those required to inhibit the growth of E. coli. It is possible that holomycin is a prodrug, requiring conversion in the cell to an active species that inhibits RNA polymerase.Thiolutin and holomycin (see Fig. 1) are members of the pyrrothine class of naturally occurring antibiotics that are characterized by the possession of a unique pyrrolinonodithiole nucleus (2). Although these antimicrobial agents were originally discovered more than 40 years ago (6, 13, 23, 26), relatively little is known about their mode of action. Limited studies with thiolutin suggest that the pyrrothines may act as inhibitors of DNA-dependent RNA polymerase. Thus, thiolutin preferentially inhibits RNA synthesis in both Saccharomyces cerevisiae (10) and Escherichia coli (14) and is reported to be a potent inhibitor of partially purified RNA polymerases from S. cerevisiae (10, 25). Furthermore, by monitoring the effects of thiolutin on the induction of ␤-galactosidase in E. coli, Khachatourians and Tipper (14) concluded that the antibiotic interfered with RNA chain elongation rather than with initiation of transcription.However, there are several observations that cast doubt upon the hypothesis that thiolutin, and therefore the pyrrothines as a class, prevents microbial growth by interfering with the elongation of RNA transcripts catalyzed by RNA polymerase. For instance, Sivasubramanian and Jayaraman (24) were unable to reproduce the observations made by Khachatourians and Tipper (14) concerning the effects of thiolutin on ␤-galactosidase induction in E. coli. Thus, these authors (24) concluded that thiolutin inhibits initiation of RNA tran...

A collection of rifampin-resistant mutants of Staphylococcus aureus with characterized RNA polymerase ␤-subunit (rpoB) gene mutations was cross-screened against a number of other RNA polymerase inhibitors to correlate susceptibility with specific rpoB genotypes. The rpoB mutants were cross-resistant to streptolydigin and sorangicin A. In contrast, thiolutin, holomycin, corallopyronin A, and ripostatin A retained activity against the rpoB mutants. The second group of inhibitors may be of interest as drug development candidates.Bacterial DNA-dependent RNA polymerase is an attractive drug target because RNA chain elongation is essential for bacterial growth (6, 16). Among those antibiotics discovered in the last 50 years, there are several known, or suspected, inhibitors of bacterial DNA-dependent RNA polymerase that have not been developed and could be candidates for new drugs. These agents include thiolutin (18), holomycin (B. Oliva, A. O'Neill, J. M. Wilson, P. J. O'Hanlon and I. Chopra, submitted for publication), streptolydigin (6, 16), the ripostatins, corallopyronins and sorangicins (10-12, 23) (Fig. 1). However, bacterial resistance has already developed to the rifamycins, the only class of RNA polymerase inhibitor that is in use clinically (21). Therefore, before considering whether other RNA polymerase inhibitors might be developed, it is important to establish whether resistance to rifamycins, such as rifampin, also confers cross-resistance to the other agents. Some attempts to address this issue have been made (9,12,13,15,24). However, the data are incomplete and the genetic basis of resistance to rifamycins in those strains used for cross-screening has rarely been determined. Furthermore, some data are contradictory; e.g., cross-resistance between rifampin and streptolydigin has been observed by some authors (13) but not by others (9,15).To assist the evaluation of these older agents we crossscreened them against a collection of rifampin-resistant mutants of Staphylococcus aureus, generated in an isogenic background, with defined RNA polymerase ␤-subunit (rpoB) gene mutations. These S. aureus strains, which provide a model for rpoB mutations occurring in naturally occurring isolates of staphylococci and other organisms (1,7,8,15,22,28,29), have allowed us to correlate susceptibility with specific rpoB genotypes.The antibiotics used here were either purchased from Sigma (rifampin and streptolydigin) or were gifts from H. Reichenbach, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany (corallopyronin A, ripostatin A, and sorangicin A); P. O'Hanlon, SmithKline Beecham Pharmaceuticals, Harlow, United Kingdom (holomycin and thiolutin); and Pharmacia & Upjohn (rifabutin). Spontaneous rifampin-resistant mutants of S. aureus 8325-4 (20) were isolated by plating approximately 10 8 CFU onto Iso-Sensitest agar (Oxoid, Basingstoke, United Kingdom) containing 0.032 g of rifampin/ml (four times the MIC). A number of rifampin-resistant mutants were picked at random, and their MICs of rifampin were d...

Tetracycline analogs fell into two classes on the basis of their mode of action. Tetracycline, chlortetracycline, minocycline, doxycycline, and 6-demethyl-6-deoxytetracycline inhibited cell-free translation directed by either Escherichia coli or Bacillus subtilis extracts. A second class of analogs tested, including chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, failed to inhibit protein synthesis in vitro or were very poor inhibitors. Tetracyclines of the second class, however, rapidly inhibited the in vivo incorporation of precursors into DNA and RNA as well as protein. The class 2 compounds therefore have a mode of action that is entirely distinct from the class 1 compounds, such as tetracycline that are used clinically. Although tetracyclines of the second class entered the cytoplasm, the ability of these analogs to inhibit macromolecular synthesis suggests that the cytoplasmic membrane is their primary site of action. The interaction of class 1 and class 2 tetracyclines with ribosomes was studied by examining their effects on the chemical reactivity of bases in 16S rRNA to dimethyl sulfate. Class 1 analogs affected the reactivity of bases to dimethyl sulfate. The response with class 2 tetracyclines varied, with some analogs affecting reactivity and others (chelocardin and 4-epi-anhydrotetracycline) not.The tetracyclines are a group of broad-spectrum antibiotics which are generally considered to prevent bacterial growth by inhibiting protein synthesis. This results from binding of antibiotic to a single site in the 30S ribosomal subunit which prevents attachment of aminoacyl tRNA to the ribosomal acceptor site (3). In order to reach the ribosome, these antibiotics must traverse the hydrophobic lipid bilayer of the bacterial cytoplasmic membrane (3). At physiological pH tetracyclines can exist as an equilibrium mixture of two free base forms: a low-energy, lipophilic nonionized species and a high-energy, hydrophilic zwitterionic structure (7). A solvent-dependent equilibrium between the two forms has been demonstrated with oxytetracycline free base and is supported by X-ray analyses of tetracyclines crystallized from aqueous and nonaqueous solvents (7,15). Both forms are believed to be important for the antibacterial activity of tetracyclines, the low-energy, lipophilic conformational form (Fig. 1A) for uptake across the cytoplasmic membrane and the hydrophilic, zwitterionic structure (Fig. 1B) for binding to the ribosome (7).Chelocardin (14) is a naturally occurring anhydrotetracycline derivative with a modified A ring (Fig. 2). In contrast to the solvent-dependent equilibrium of the two tetracycline species mentioned above, chelocardin apparently exists in the same conformation in both polar and nonpolar solvents, as evidenced by circular dichroism measurements (6). We believe this is related to the planarity of the BCD rings in chelocardin and that a lipophilic form, perhaps related to that of tetracycline, is the preferred species. Thes...

The modes of action of atypical tetracyclines that do not directly inhibit bacterial protein synthesis were investigated. The analogs tested, chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, were bactericidal and caused the lysis of Escherichia coli accompanied by the release of the cytoplasmic enzyme 1-galactosidase into the supernatant. Examination by electron microscopy demonstrated that cells exposed to these analogs underwent marked morphological alterations that included the formation of numerous ghosts and the appearance of cellular debris in the culture medium.Although atypical tetracyclines promoted lysis in intact organisms, they did not cause lysis of E. coli spheroplasts, indicating that the analogs do not directly destroy the cytoplasmic membrane. These agents may promote cell lysis and death by interfering with the membrane's electrochemical gradient, which in turn leads to stimulation of autolytic enzyme activity and cellular lysis. The results support recently published data which indicate that tetracyclines are divisible into two classes on the basis of their modes of action.

The PL assay is unsuitable for detection of membrane-damaging agents in S. aureus. The other assays, including the BG assay, detect membrane damage. The OD assay should be sufficient for most purposes since it is effective, rapid and cheap to perform. Studies requiring maximum sensitivity and discrimination should employ the ATP assay.

The stringent response in Escherichia coli and many other organisms is regulated by the nucleotides ppGpp and pppGpp. We show here for the first time that at least six staphylococcal species also synthesize ppGpp and pppGpp upon induction of the stringent response by mupirocin. Spots corresponding to ppGpp and pppGpp on thin-layer chromatograms suggest that pppGpp is the principal regulatory nucleotide synthesized by staphylococci in response to mupirocin, rather than ppGpp as in E. coli.Bacteria adapt to amino acid or carbon source insufficiency by a complex series of regulatory events known as the stringent response (reviewed in reference 4). In Escherichia coli, for example, amino acid starvation leads to an accumulation of uncharged cognate tRNA. When the ratio of charged to uncharged tRNA falls below a critical threshold (24), occupation of the vacant mRNA codon at the ribosomal A site by uncharged cognate tRNA leads to stalling of peptide chain elongation and synthesis of the nucleotides pppGpp and ppGpp from GTP and ATP in an idling reaction involving RelA (2, 9, 13). Intracellular concentrations of (p)ppGpp are controlled by the relA gene, encoding the ribosome-dependent (p)ppGpp synthetase I, or stringent factor (19), and the spoT gene, encoding the ribosome-independent ppGpp 3Ј-pyrophosphohydrolase/ppGpp synthetase II (14,33).Induction of the stringent response rapidly reduces the synthesis of mRNA because of inhibition of RNA polymerase by ppGpp (28,31). This coincides with the down-regulation of a wide range of energetically demanding cellular processes, including the synthesis of stable RNA, DNA, protein, and peptidoglycan. Close coordination between transcription and translation can be maintained through the effects of the stringent response on the regulation of individual ribosomal proteins; elongation factors G, Tu, and Ts; certain aminoacyl tRNA synthetases; and stationary-phase-specific sigma factor ( s ) (11). Ultimately, changes such as these enhance survival in a nutrient-poor environment.The antibiotic mupirocin (30) produces cellular effects similar to those of isoleucine starvation by preventing the charging of tRNA Ile due to inhibition of isoleucyl tRNA synthetase in E. coli (16,17), Staphylococcus aureus (5), and other organisms, thereby inhibiting protein synthesis. In the presence of mupirocin, RNA synthesis in E. coli and S. aureus is also inhibited as a consequence of the stringent response; however, this inhibition can be reversed by chloramphenicol (15), which in E. coli, binds close to the binding site for the terminal CCA of aminoacyl tRNA in the peptidyl transferase A site (23) and reduces the amount of RelA-dependent (p)ppGpp synthesis (8, 10). A stringent response mediated by (p)ppGpp has been detected in some gram-positive and gram-negative bacteria and higher organisms after amino acid starvation or induction by a variety of antibiotics (7,18,26). However, in halobacteria (25) and streptococci (21), stringency is not necessarily coupled with (p)ppGpp production. Thus, in S...

Aims: The aim of this study was to analyse the antimycotic properties of Melaleuca alternifolia essential oil (tea tree oil, TTO) and its principal components and to compare them with the activity of 5-fluorocytosine and amphotericin B. Methods and Results:The screening for the antimycotic activity was performed by serial twofold dilutions in Roswell Park Memorial Institute medium with the inclusion of Tween-80 (0AE5%). TTO and terpinen-4-olo were the most active compounds. Conclusions: The majority of the organisms were sensitive to the essential oil, with TTO and terpinen-4-olo being the most active oils showing antifungal activity at minimum inhibitory concentration values lower than other drugs. Significance and Impact of the study: This study provides a sample large enough to determine the antifungal properties of TTO and terpinen-4-olo and suggests further studies for a possible therapeutic use.

Clofazimine is a potent anti-staphylococcal agent. It appears to be a membrane-disrupting agent and does not inhibit RNA polymerase.