Kimidinomycin, a new antibiotic against Mycobacterium avium complex, produced by Streptomyces sp. KKTA-0263.

During our screening for antibiotics against Mycobacterium avium complex (MAC) with a mass spectrometry network-based indexing approach, a new compound named kimidinomycin was isolated from the culture broth of Streptomyces sp. KKTA-0263 by solvent extraction, HP20 column chromatography, and preparative HPLC. From the structural elucidation, the compound possesses a 38-membered macrolide structure with an N-methylguanidyl group at the terminal side chain. The compound exhibited antimycobacterial activity against M. avium, M. intracellulare, M. smegmatis, and M. bovis BCG with respective MIC values of 12.5, 0.78, 12.5, and 25.0 µg ml-1.

Rifamycin congeners kanglemycins are active against rifampicin-resistant bacteria via a distinct mechanism.

Rifamycin antibiotics (Rifs) target bacterial RNA polymerases (RNAPs) and are widely used to treat infections including tuberculosis. The utility of these compounds is threatened by the increasing incidence of resistance (RifR). As resistance mechanisms found in clinical settings may also occur in natural environments, here we postulated that bacteria could have evolved to produce rifamycin congeners active against clinically relevant resistance phenotypes. We survey soil metagenomes and identify a tailoring enzyme-rich family of gene clusters encoding biosynthesis of rifamycin congeners (kanglemycins, Kangs) with potent in vivo and in vitro activity against the most common clinically relevant RifR mutations. Our structural and mechanistic analyses reveal the basis for Kang inhibition of RifR RNAP. Unlike Rifs, Kangs function through a mechanism that includes interfering with 5'-initiating substrate binding. Our results suggest that examining soil microbiomes for new analogues of clinically used antibiotics may uncover metabolites capable of circumventing clinically important resistance mechanisms.

Fermentation, isolation, purification and characterization of an antitubercular antibiotic from Streptomyces luridus MTCC 4402.

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be a serious public health problem around the world, and it urges the need for development of new antitubercular drugs. An antibiotic producing strain, Streptomyces luridus (MTCC 4402) was earlier isolated from soil by our group. In this work, the phylogenic status was established by 16S rRNA gene sequence analysis. The strain was found to be active against clinically resistant strains. The culture was grown in shake flasks in a medium containing cornsteep liquor, glucose, CaCO(3), soyabean meal and starch. Antibiotic production reached maximum at the end of 72 h. and fermentation profile was obtained. The active compound was extracted into ethyl acetate and was subjected to activity guided purification by column chromatography using silica gel, TLC and HPLC methods. The pure compound eluted at 16.7 min. by gradient elution was subjected to (1)H, (13)C NMR and mass spectral analyses. The acquired data was compared with that of natural products' data base and found to be a known antibiotic, spiramycin. The purified compound was studied for mutagenic, cytotoxicity, antitubercular activities. It was non mutagenic at the concentration of 1000 µg/mL, non cytotoxic and active as antitubercular agent at a concentration of 64 mg/mL and was comparable to rifampicin.

Modification of rifamycin polyketide backbone leads to improved drug activity against rifampicin-resistant Mycobacterium tuberculosis.

Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.

Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin.

Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.

Optimization of culture conditions for production of the anti-tubercular alkaloid hirsutellone A by Trichoderma gelatinosum BCC 7579.

AIMS: This work aimed to optimize the culture conditions for production of a novel and potent anti-tubercular alkaloid, hirsutellone A, by the saprophytic soil fungus Trichoderma gelatinosum BCC 7579. METHODS AND RESULTS: The fungus was initially cultured in shake flasks at 25 degrees C in the potato dextrose broth (PDB) supplemented with various carbon and nitrogen sources and mineral salts to select suitable medium for mycelial growth and hirsutellone A production. Cultivation conditions were further optimized by adjusting initial pH and changing temperature levels to maximize the production of hirsutellone A. The optimal condition that increased the production of hirsutellone A from 19.04 mg l(-1), obtained from basal condition, to 610.55 mg l(-1) and reduced the cultivation time from 40 to 6 days was to cultivate in a shaker at 200 rev min(-1) at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with initial pH of 7. Production of hirsutellone A in larger-scale using a 5-l batch fermenter was also completed yielding 958 mg l(-1) of hirsutellone A within 6 days. CONCLUSIONS: The suitable culture conditions for hirsutellone A production by T. gelatinosum BCC 7579 was the cultivation in 5-l fermenter at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with an initial pH of 7. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of hirsutellone A in a fermenter to obtain a high yield and reduce an incubation period will become very useful in anti-tubercular drug development process in the future.

Enniatin production by the entomopathogenic fungus Verticillium hemipterigenum BCC 1449.

Optimal fermentation conditions for enniatin production using the entomopathogenic fungus Verticillium hemipterigenum BCC 1449 have been investigated. Among various liquid media tested, highest efficiency of enniatin production was achieved by fermentation in yeast extract sucrose. Application of this condition to large-scale fermentation resulted in the isolation of three new analogs, O1, O2 and O3, which are closely related isomers that were characterized as an inseparable mixture, along with seven known enniatins.

Molecular cloning of a D-cycloserine resistance gene from D-cycloserine-producing Streptomyces garyphalus.

A 3.5-kb DNA fragment that confers resistance to D-cycloserine (DCS) was cloned from the chromosomal DNA of a DCS-producing Streptomyces garyphalus into Streptomyces lividans by a shot-gun cloning technique. Nucleotide sequence analysis revealed the existence of four open reading frames (ORFs B, C, D, and E), together with two incomplete ORFs, A and F. By introduction of the cloned fragment into Escherichia coli, the host obtained resistance to DCS. We showed that ORF B, which consists of 903 bp, is a DCS resistance gene. The hydropathy plot analysis of a protein deduced from ORF B revealed that the protein carries membrane-integral domains spanning the membrane 10 times, which suggests that the DCS-resistance determinant may be a factor associated with DCS transport.

Deciphering tuberactinomycin biosynthesis: isolation, sequencing, and annotation of the viomycin biosynthetic gene cluster.

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.

Isolation and characterization of 27-O-demethylrifamycin SV methyltransferase provides new insights into the post-PKS modification steps during the biosynthesis of the antitubercular drug rifamycin B by Amycolatopsis mediterranei S699.

The gene rif orf14 in the rifamycin biosynthetic gene cluster of Amycolatopsis mediterranei S699, producer of the antitubercular drug rifamycin B, encodes a protein of 272 amino acids identified as an AdoMet: 27-O-demethylrifamycin SV methyltransferase. Frameshift inactivation of rif orf14 generated a mutant of A. mediterranei S699 that produces no rifamycin B, but accumulates 27-O-demethylrifamycin SV (DMRSV) as the major new metabolite, together with a small quantity of 27-O-demethyl-25-O-desacetylrifamycin SV (DMDARSV). Heterologous expression of rif orf14 in Escherichia coli yielded a 33.8-kDa polyhistidine-tagged polypeptide, which efficiently catalyzes the methylation of DMRSV to rifamycin SV, but not that of DMDARSV or rifamycin W. 27-O-Demethylrifamycin S was methylated poorly, if at all, by the enzyme to produce rifamycin S. The purified enzyme does not require a divalent cation for catalytic activity. While Ca(2+) or Mg(2+) inhibits the enzyme activity slightly, Zn(2+), Ni(2+), and Co(2+) are strongly inhibitory. The K(m) values for DMRSV and S-adenosyl-L-methionine (AdoMet) are 18.0 and 19.3 microM, respectively, and the K(cat) is 87s(-1). The results indicate that DMRSV is a direct precursor of rifamycin SV and that acetylation of the C-25 hydroxyl group must precede the methylation reaction. They also suggest that rifamycin S is not the precursor of rifamycin SV in rifamycin B biosynthesis, but rather an oxidative shunt-product.