Genome-Guided Investigation Provides New Insights into Secondary Metabolites of Streptomyces parvulus SX6 from Aegiceras corniculatum.

Whole-genome sequencing and genome mining are recently considered an efficient approach to shine more light on the underlying secondary metabolites of Streptomyces. The present study unearths the biosynthetic potential of endophytic SX6 as a promising source of biologically active substances and plant-derived compounds for the first time. Out of 38 isolates associated with Aegiceras corniculatum (L.) Blanco, Streptomyces parvulus SX6 was highly active against Pseudomonas aeruginosa ATCC® 9027™ and methicillin-resistant Staphylococcus epidermidis (MRSE) ATCC® 35984™. Additionally, S. parvulus SX6 culture extract showed strong cytotoxicity against Hep3B, MCF-7, and A549 cell lines at a concentration of 30 µg/ml, but not in non-cancerous HEK-293 cells. The genome contained 7.69 Mb in size with an average G + C content of 72.8% and consisted of 6,779 protein-coding genes. AntiSMASH analysis resulted in the identification of 29 biosynthetic gene clusters (BGCs) for secondary metabolites. Among them, 4 BGCs showed low similarity (28-67% of genes show similarity) to actinomycin, streptovaricin, and polyoxypeptin gene clusters, possibly attributed to antibacterial and anticancer activities observed. In addition, the complete biosynthetic pathways of plant-derived compounds, including daidzein and genistein were identified using genome mining and HPLC-DAD-MS analysis. These findings portray an exciting avenue for future characterization of promising secondary metabolites from mangrove endophytic S. parvulus.

Uncovering the cytochrome P450-catalyzed methylenedioxy bridge formation in streptovaricins biosynthesis.

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.

Involvement of the beta subunit of RNA polymerase in resistance to streptolydigin and streptovaricin in the producer organisms Streptomyces lydicus and Streptomyces spectabilis.

Streptomyces lydicus NRRL2433 and S. spectabilis NRRL2494 produce two inhibitors of bacterial RNA polymerase: the 3-acyltetramic acid streptolydigin and the naphthalenic ansamycin streptovaricin, respectively. Both strains are highly resistant to their own antibiotics. Independent expression of the S. lydicus and S. spectabilis rpoB and rpoC genes, encoding the beta- and beta'-subunits of RNA polymerase, respectively, in S. albus showed that resistance is mediated by rpoB, with no effect of rpoC. Within the beta-subunit, resistance was confined to an amino acid region harboring the "rif region." Comparison of the beta-subunit amino acid sequences of this region from the producer strains and those of other streptomycetes and site-directed mutagenesis of specific differential residues located in it (L485 and D486 in S. lydicus and N474 and S475 in S. spectabilis) showed their involvement in streptolydigin and streptovaricin resistance. Other amino acids located close to the "Stl pocket" in the S. lydicus beta-subunit (L555, F593, and M594) were also found to exert influence on streptolydigin resistance.

Effects of streptovaricins and their degradation products on RNA-directed DNA polymerase of Rauscher leukemia virus.

The activities of streptovaricin complexes, streptovaricins, streptovals, and streptovarinic degradation products were elevated against RNA-directed DNA polymerases of Rauscher leukemia virus, DNA-dependent DNA polymerase of bacterial and mammalian cells, and DNA-dependent RNA polymerases of mammalian origin. The activities of streptovaricins were also listed for comparison purposes. The effects of streptovaricin complexes on viral DNA polymerases varied significantly from lot to lot, and streptovaricin complex lot 7 was the most active. All the streptovals and streptovaricin degradation products except varicinal A showed a marked improvement (twofold to tenfold) in activity against the viral enzyme over the parent streptovaricins. None of these compounds, however, displayed any significant effect on either the DNA polymerase of L1210 leukemia cells and Escherichia coli or the RNA polymerase of isolated nuclei of mouse liver. As a result of tests in these systems, some specific inhibitors of RNA-directed DNA polymerases of Rauscher leukemia virus were selected.

Effects of streptovaricins and their degradation products on infectivity of Rauscher leukemia virus.

The virucidal effects of streptovaricin (Sv) A, SvC, SvD, streptoval (Sval) C, Sval Fc, and streptovarone were evaluated by incubation of the drug with Rauscher leukemia virus (RLV) at 37 degrees C for 60 minutes prior to dillution and addition to cells (in vitro assay) or before ip injection into animals (in vivo assay). The in vitro and in vivo assays were plaque formation and splenomegaly, respectively. A dose-related effect was observed with all six compounds with the in vitro assay. On an equimolar basis, the Sv degradation products, i.e., Sval C, Sval Fc, and streptovarone were most inhibitory, followed by SvD; SvA and SvC were least active. At 0.0625 mumoles, the three Sv degradation products inactivated over 90% of the RLV. Similar results were obtained through the in vivo assay. At 0.06 mumoles, streptovarone, Sval C, and SvD showed 78,62, and 29% inhibition of splenomegaly, respectively; SvA and SvC were essentially inactive. A direct relationship was observed between inhibition on RNA-directed DNA polymrase of RLV by these compounds and their virucidal effects. No drug given at the time of injection, however, showed any significant effect on virus infective processes in vitro or in vivo. The reason for the lack of therapeutic effects of these compounds is discussed.