Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan.

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemia salina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells. Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemiasalina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Anti-parasitic activities of specific bacterial extracellular products of Streptomyces griseus SDX-4 against Ichthyophthirius multifiliis.

The ciliate Ichthyophthirius multifiliis is one of the most pathogenic parasites of fish maintained in captivity. In this study, effects of bacterial extracellular products of Streptomyces griseus SDX-4 against I. multifiliis were determined. The fermentation liquor of S. griseus was extracted successively in a separating funnel with petroleum ether, ethyl acetate, and n-butanol. In vitro assays revealed that the n-butanol extracts (NBu-E) and ethyl acetate extracts (Eto-E) of S. griseus were observed to be more effective against theronts than the other extracts with median effective concentration (EC50) values of 0.86 and 12.5 mg L(-1), respectively, and significantly reduced the survival of the tomonts and the total number of theronts released by the tomonts (P<0.05). All encysted tomonts were killed when the concentration of NBu-E was 30.0 mg L(-1). Results of in vivo test demonstrated that the number of I. multifiliis trophonts on the grass carp treated with NBu-E was markedly lower compared to the control group at 11 days after exposed to theronts (P<0.05). In the control group, 100% mortality was observed owing to heavy I. multifiliis infection at 11 days after the exposure. On the other hand, only 9.5% mortality owing to parasite infection was recorded in the groups treated with the NBu-E (30 mg L(-1)). The median lethal dose (LD50) of NBu-E for grass carp was 152.4 mg L(-1). Our results indicate that n-butanol extract of S. griseus will be useful in aquaculture for controlling I. multifiliis infections.

Diversity analysis of streptomycetes and associated phosphotranspherase genes in soil.

An attempt was made to verify the observation that Streptomyces griseus was prevalent in soil based on isolation work. A genus-specific PCR was developed for Streptomyces based on the housekeeping gene atpD and used to investigate species diversity within selected soils. The presence of S. griseus was investigated to determine coexistence of resistance-only streptomycin phosphotransferase (strA) in the same soil as streptomycin producers. Two additional PCR-based assays were developed; one specific for strA in association with production, the other for more diverse strA and other related phosphotranferases. Both the S. griseus atpD and strA genes were below the PCR detection limit in all soils examined. A number of more diverse phosphotransferase genes were amplified, a minority of which may be associated with streptomycin production. We conclude that neither streptomycin producers nor S. griseus are prevalent in the fresh or chitin and starch-amended soils examined (less than 0.1% of soil actinobacteria). One of the soil sites had received plantomycin (active ingredient: streptomycin) and diversity studies suggested that this altered the streptomycete populations present in the soil.

Multilocus sequence analysis of Streptomyces griseus isolates delineating intraspecific diversity in terms of both taxonomy and biosynthetic potential.

Systematics can provide a fundamental framework for understanding the relationships and diversification of organisms. Multilocus sequence analysis (MLSA) has shown great promise for an elaborate taxonomic grouping of streptomycete diversity. To evaluate the practical significance of MLSA as a valuable systematic tool for streptomycetes, we examined six endophytic Streptomyces griseus isolates and two S. griseus reference strains possessing obvious antagonistic activities and identical 16S rRNA gene sequences, using both housekeeping genes and secondary metabolic genes. All the eight strains contained PKS-I and NRPS genes, but not PKS-II genes, and showed similar diversity in both the MLSA phylogeny based on five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) and fingerprinting of KS-AT genes. We also inferred a phylogeny based on concatenated amino acid sequences of representative KS-AT genes from the strains, which displayed a topology correlated well with those of housekeeping-gene MLSA and KS-AT fingerprinting. The good congruence observed between phylogenies based on the different datasets verified that the MLSA scheme provided robust resolution at intraspecific level and could predict the overall diversity of secondary metabolic potential within a Streptomyces species, despite somewhat of a discrepancy with antimicrobial data. It is therefore feasible to apply MLSA to dissecting natural diversity of streptomycetes for a better understanding of their evolution and ecology, as well as for facilitating their bioprospecting.

Taxonomic evaluation of the Streptomyces griseus clade using multilocus sequence analysis and DNA-DNA hybridization, with proposal to combine 29 species and three subspecies as 11 genomic species.

Streptomyces griseus and related species form the biggest but least well-defined clade in the whole Streptomyces 16S rRNA gene tree. Multilocus sequence analysis (MLSA) has shown promising potential for refining Streptomyces systematics. In this investigation, strains of 18 additional S. griseus clade species were analysed and data from a previous pilot study were integrated in a larger MLSA phylogeny. The results demonstrated that MLSA of five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) is better than the previous six-gene scheme, as it provides equally good resolution and stability and is more cost-effective; MLSA using three or four of the genes also shows good resolution and robustness for differentiating most of the strains and is therefore of value for everyday use. MLSA is more suitable for discriminating strains that show >99 % 16S rRNA gene sequence similarity. DNA-DNA hybridization (DDH) between strains with representative MLSA distances revealed a strong correlation between the data of MLSA and DDH. The 70 % DDH value for current species definition corresponds to a five-gene MLSA distance of 0.007, which could be considered as the species cut-off for the S. griseus clade. It is concluded that the MLSA procedure can be a practical, reliable and robust alternative to DDH for the identification and classification of streptomycetes at the species and intraspecies levels. Based on the data from MLSA and DDH, as well as cultural and morphological characteristics, 18 species and three subspecies of the S. griseus clade are considered to be later heterotypic synonyms of 11 genomic species: Streptomyces griseinus and Streptomyces mediolani as synonyms of Streptomyces albovinaceus; Streptomyces praecox as a synonym of Streptomyces anulatus; Streptomyces olivoviridis as a synonym of Streptomyces atroolivaceus; Streptomyces griseobrunneus as a synonym of Streptomyces bacillaris; Streptomyces cavourensis subsp. washingtonensis as a synonym of Streptomyces cyaneofuscatus; Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies and Streptomyces flavofuscus as synonyms of Streptomyces fimicarius; Streptomyces flavogriseus as a synonym of Streptomyces flavovirens; Streptomyces erumpens, 'Streptomyces ornatus' and Streptomyces setonii as synonyms of Streptomyces griseus; Streptomyces graminofaciens as a synonym of Streptomyces halstedii; Streptomyces alboviridis, Streptomyces griseus subsp. alpha, Streptomyces griseus subsp. cretosus and Streptomyces luridiscabiei as synonyms of Streptomyces microflavus; and Streptomyces californicus and Streptomyces floridae as synonyms of Streptomyces puniceus.

Taxonomic study of a chromomycin-producing strain and reclassification of Streptomyces cavourensis subsp. washingtonensis as a later synonym of Streptomyces griseus.

A chromomycin-producing actinomycete, strain AP19, was isolated from a sample of faeces collected from Foping national nature reserve in China. Chemotaxonomic and morphological properties indicated that the novel isolate was a member of the genus Streptomyces. Phylogenetic analyses based on an almost complete 16S rRNA gene sequence of the strain and on the 120-nt nucleotide variable gamma-region of this molecule revealed that it was closely related to Streptomyces griseus ISP 5236(T) and Streptomyces cavourensis subsp. washingtonensis ATCC 27732(T). DNA-DNA relatedness values among these strains were above 70 %. Streptomyces cavourensis subsp. washingtonensis could be readily distinguished from Streptomyces cavourensis ATCC 14889(T) by differing BOX-PCR fingerprinting patterns, relatively low 16S rRNA gene sequence similarity and a low DNA-DNA relatedness value. It is proposed, therefore, that Streptomyces cavourensis subsp. washingtonensis is a later synonym of Streptomyces griseus.

Genetic and phenotypic evidence for Streptomyces griseus ecovars isolated from a beach and dune sand system.

This study was designed to determine the biogeography of six alkaliphilic Streptomyces strains which had been isolated from four locations within a 60 m transect across a beach and dune sand system. The six strains shared >99% 16S rRNA gene similarities with one another and with representative strains of Streptomyces griseus. Infraspecific diversity amongst the strains was investigated by multilocus sequence typing (MLST) in combination with carbon utilisation phenotypic testing. The results show that each of the strains is genotypically and phenotypically distinct. Furthermore, the MLST and carbon utilisation profiles were congruent thereby providing preliminary evidence which suggests that the observed infraspecific diversity is consistent with ecological selection. The results also demonstrate that infraspecific diversity can be observed over small spatial scales. These findings support the hypothesis that the six isolates are ecovars of Streptomyces griseus. The implications of these findings for prokaryotic biogeography and bioprospecting are discussed.

Streptomyces griseus 45H, a producer of the extracellular autoregulator protein factor C, is a member of the species Streptomyces albidoflavus.

Streptomyces griseus strain 45H, isolated in 1960 during a mutagenesis programme on the industrial streptomycin producer S. griseus 52-1, encodes an extracellular, pleiotropic autoregulatory signalling protein, factor C, which stimulates sporulation of S. griseus 52-1 in submerged culture. The facC gene, which codes for factor C, is present in very few streptomycetes and is not present in S. griseus 52-1. Based on 16S rRNA gene sequencing and other molecular data, S. griseus 45H, the factor C producer, is here shown to be related to the original laboratory strain of Streptomyces flavofungini, which was being studied in the same laboratory in 1960, and to Streptomyces albidoflavus. Southern blotting revealed that three out of four independently isolated strains of S. albidoflavus possess facC. Both the original strain of S. flavofungini and S. griseus 45H are therefore identified as members of the species Streptomyces albidoflavus, and we propose that S. griseus 45H should be renamed Streptomyces albidoflavus 45H.

A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: use of multilocus sequence analysis for streptomycete systematics.

Streptomycetes are a complex group of actinomycetes that produce diverse bioactive metabolites of commercial significance. Systematics can provide a useful framework for identifying species that may produce novel metabolites. However, previously proposed approaches to the systematics of Streptomyces have suffered from either poor interlaboratory comparability or insufficient resolution. In particular, the Streptomyces griseus 16S rRNA gene clade is the most challenging and least defined group within the genus Streptomyces in terms of phylogeny. Here we report the results of a multilocus sequence analysis scheme developed to address the phylogeny of this clade. Sequence fragments of six housekeeping genes, atpD, gyrB, recA, rpoB, trpB and 16S rRNA, were obtained for 53 reference strains that represent 45 valid species and subspecies. Analysis of each individual locus confirmed the suitability of loci and the congruence of single-gene trees for concatenation. Concatenated trees of three, four, five and all six genes were constructed, and the stability of the topology and discriminatory power of each tree were analysed. It can be concluded from the results that phylogenetic analysis based on multilocus sequences is more accurate and robust for species delineation within Streptomyces. A multilocus phylogeny of six genes proved to be optimal for elucidating the interspecies relationships within the S. griseus 16S rRNA gene clade. Our multilocus sequence analysis scheme provides a valuable tool that can be applied to other Streptomyces clades for refining the systematic framework of this genus.

Elloxazinones A and B, New Aminophenoxazinones from Streptomyces griseus Acta 2871.

Two new aminophenoxazinone compounds with antitumor activity, elloxazinone A and B, were isolated from the culture filtrate of Streptomyces griseus Acta 2871. Their chemical structures were determined by mass spectrometry, NMR spectroscopy and X-ray analysis. Elloxazinones A and B showed a moderate inhibition of the proliferation of human cells from gastric adenocarcinoma in vitro but a strong inhibition of hepatocellular carcinoma cells whereas elloxazinone B strongly inhibited the proliferation of human breast carcinoma cells.

Structure determination of germacrane-type sesquiterpene alcohols from an endophyte Streptomyces griseus subsp.

Three germacrane-type sesquiterpene alcohols were isolated from an endophyte of mangrove plant Kandelia candel. Their structures were characterized as 1(10)E,5E-germacradiene-11-ol (1), 1(10)E,5E-germacradiene-3,11-diol (2), 1(10)E,5E-germacradiene-2,11-diol (3) based on the extensive NMR studies. Among them, 2 and 3 are identified as new compounds.

Classification of Streptomyces griseus (Krainsky 1914) Waksman and Henrici 1948 and related species and the transfer of 'Microstreptospora cinerea' to the genus Streptomyces as Streptomyces yanii sp. nov.

A soil actinomycete, strain 80-133(T), with the non-validly published name 'Microstreptospora cinerea', was the subject of a polyphasic study designed to clarify its taxonomic status. Comparative 16S rRNA gene sequence studies indicated that the organism belonged to the genus Streptomyces, a result in line with previous chemotaxonomic and morphological data. The strain belonged to the Streptomyces griseus clade, but could be distinguished from representatives of species assigned to this taxon by using DNA-DNA relatedness and phenotypic data. In light of these findings, it is proposed that the organism should be recognized as a novel species of the genus Streptomyces. The name proposed for this taxon is Streptomyces yanii sp. nov., with isolate 80-133(T) (=AS 4.1146(T)=JCM 3331(T)) as the type strain. It was also shown that representative strains of Streptomyces argenteolus, Streptomyces caviscabies, S. griseus and Streptomyces setonii belong to the same genomic species and have key phenotypic properties in common. It is proposed that S. caviscabies and S. setonii should be considered as later heterotypic synonyms of S. griseus and that S. argenteolus AS 4.1693(T) should also be assigned to this taxon.

Reclassification of Streptomyces nigrifaciens as a later synonym of Streptomyces flavovirens; Streptomyces citreofluorescens, Streptomyces chrysomallus subsp. chrysomallus and Streptomyces fluorescens as later synonyms of Streptomyces anulatus; Streptomyces chibaensis as a later synonym of Streptomyces corchorusii; Streptomyces flaviscleroticus as a later synonym of Streptomyces minutiscleroticus; and Streptomyces lipmanii, Streptomyces griseus subsp. alpha, Streptomyces griseus subsp. cretosus and Streptomyces willmorei as later synonyms of Streptomyces microflavus.

A DNA-DNA hybridization survey was performed on 13 Streptomyces species and two subspecies, dispersed over five genotypically defined clusters as delineated by Lanoot et al. [Syst Appl Microbiol 27 (2004), 84-92]. Within each of the latter clusters, strains shared DNA-DNA relatedness values above 70 %. On the basis of published recommendations, the following eight Streptomyces species with validly published names are considered as later synonyms: Streptomyces nigrifaciens as a synonym of Streptomyces flavovirens; Streptomyces citreofluorescens, Streptomyces chrysomallus subsp. chrysomallus and Streptomyces fluorescens as synonyms of Streptomyces anulatus; Streptomyces chibaensis as a synonym of Streptomyces corchorusii; Streptomyces flaviscleroticus as a synonym of Streptomyces minutiscleroticus; and Streptomyces lipmanii, Streptomyces griseus subsp. alpha, Streptomyces griseus subsp. cretosus and Streptomyces willmorei as synonyms of Streptomyces microflavus. Emended descriptions are proposed.

Molecular analysis of sporulation in Streptomyces griseus.

Previous evidence suggested that orf1590 from Streptomyces griseus has the potential to encode two polypeptide products from temporally regulated nested open frames (orfs) and that the longer polypeptide may be a DNA-binding protein. We have developed a hypothetical model of the role of orf1590 in sporulation of S. griseus and have begun to test this model by determining the nucleotide sequence of the orf1590 counterpart from Streptomyces coelicolor. The conservation of the helix-turn-helix domain and the two potential translation start codons is consistent with our model. Continued analysis of bald mutants of S. griseus has indicated that several prematurely synthesize sporulation septa and spore walls. One of these nonsporulating strains appears to be a bldA mutant of S. griseus. Complementation analysis suggests that at least three genetic loci are involved in the correct timing of deposition of sporulation septa and wall thickening.

New antibiotics 4181-A and B from Streptomyces griseus; taxonomy, fermentation, isolation and characterization.

The new antibiotics 4181-A and B were isolated from the fermentation broth of Streptomyces griseus, a soil isolate. Their molecular formulae were determined as C29H21NO9 and C28H19NO9, respectively. The UV, IR and NMR spectra suggest that they possess a quinone moiety in their structures. They were found to have antibacterial, antifungal and antitumor activity.

Enzyme-linked immunosorbent assay (ELISA) as a means of taxonomic analysis of Streptomyces and related organisms.

Fourteen Streptomyces strains from various numerical taxonomic classes and representatives of three other genera of actinomycetes were studied using an indirect enzyme-linked immunosorbent assay (IND-ELISA) to determine their serological relationships. The IND-ELISA results agreed with those from previous numerical taxonomic analyses and Ouchterlony double-diffusion studies. The IND-ELISA method is quicker, more quantitative and less subjective than Ouchterlony assays and thus should be useful in Streptomyces taxonomy. The results indicated that Frankia sp. CpI1 was related to Streptomyces.

[Correlations in the utilization of carbon sources in the range of griseus species of the genus Streptomyces]. / Korreliatsii v ispol'zovanii istochnikov ugleroda u vidov serii griseua roda Streptomyces.

The author proposes a new scheme for arranging the data about the assimilation of carbon sources in tables; this is illustrated with the Griseus species belonging to the Streptomyces genus and with the information about them presented by Bergey [2]. In the scheme, carbon sources in the tables are arranged in the order of their diminishing availability for streptomycetes, and the lists of species within the morphological groups are constructed according to the property of a carbon source not being assimilated. Such an arrangement of data in the tables is very clear and convenient to use; it allows one to make optimal schemes for determining the assimilation of carbon sources by actinomycetes, and makes it easy to divide any large number of cultures into groups according to a similar assimilation of carbon sources. The scheme has been used to analyse the data available in science and to establish correlations in the assimilation of carbon sources by species of the Griseus series belonging to the Streptomyces genus.

C-19393 S2 and H2, new carbapenem antibiotics. I. Taxonomy of the producing strain, fermentation and antibacterial properties.

C-19393 S2 and H2 are new carbapenem antibiotics produced by a streptomycete. The producing strain was taxonomically studied and named Streptomyces griseus subsp. cryophilus. Cobaltous compounds were necessary for production of the antibiotics. C-19393 S2 and H2 showed a broad spectrum of antibacterial activities with C-19393 H2 being 8 approximately 120 times more active than C-19393 S2. They also exhibited beta-lactamase-inhibiting activities and acted synergistically with ampicillin and cefotiam against clinical isolates resistant to beta-lactam antibiotics.