Hunting for cultivable Micromonospora strains in soils of the Atacama Desert.

Innovative procedures were used to selectively isolate small numbers of Micromonospora strains from extreme hyper-arid and high altitude Atacama Desert soils. Micromonosporae were recognised on isolation plates by their ability to produce filamentous microcolonies that were strongly attached to the agar. Most of the isolates formed characteristic orange colonies that lacked aerial hyphae and turned black on spore formation, whereas those from the high altitude soil were dry, blue-green and covered by white aerial hyphae. The isolates were assigned to seven multi- and eleven single-membered groups based on BOX-PCR profiles. Representatives of the groups were assigned to either multi-membered clades that also contained marker strains or formed distinct phyletic lines in the Micromonospora 16S rRNA gene tree; many of the isolates were considered to be putatively novel species of Micromonospora. Most of the isolates from the high altitude soils showed activity against wild type strains of Bacillus subtilis and Pseudomonas fluorescens while those from the rhizosphere of Parastrephia quadrangulares and from the Lomas Bayas hyper-arid soil showed resistance to UV radiation.

Structure-guided functional characterization of enediyne self-sacrifice resistance proteins, CalU16 and CalU19.

Calicheamicin γ1I (1) is an enediyne antitumor compound produced by Micromonospora echinospora spp. calichensis, and its biosynthetic gene cluster has been previously reported. Despite extensive analysis and biochemical study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach and biochemical characterization, two proteins encoded by genes from the 1 biosynthetic gene cluster assigned as "unknowns", CalU16 and CalU19, were characterized. Structure analysis revealed that they possess the STeroidogenic Acute Regulatory protein related lipid Transfer (START) domain known mainly to bind and transport lipids and previously identified as the structural signature of the enediyne self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were cleaved by 1 in vitro. Through site-directed mutagenesis and mass spectrometry, we identified the site of cleavage in each protein and characterized their function in conferring resistance against 1. This report emphasizes the importance of structural genomics as a powerful tool for the functional annotation of unknown proteins.

Micromonospora tulbaghiae sp. nov., isolated from the leaves of wild garlic, Tulbaghia violacea.

A novel actinomycete, strain TVU1(T), was isolated from leaves of the indigenous South African plant Tulbaghia violacea. Applying a polyphasic approach, the isolate was identified as a member of the genus Micromonospora. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain TVU1(T) was most closely related to Micromonospora echinospora DSM 43816(T). However, phylogenetic analysis based on gyrB gene sequences showed that strain TVU1(T) was most closely related to the type strains of Micromonospora aurantiaca and Micromonospora chalcea. DNA-DNA relatedness values between strain TVU1(T) and the type strains of M. echinospora, M. aurantiaca and M. chalcea were 7.6+/-4.5, 45.9+/-2.0 and 60.9+/-4.5 %, respectively. Strain TVU1(T) could be distinguished from the type strains of all three of these species by several physiological characteristics, such as colony colour, NaCl tolerance, growth temperature range and sole carbon source utilization pattern. Strain TVU1(T) (=DSM 45142(T)=NRRL B-24576(T)) therefore represents a novel species for which the name Micromonospora tulbaghiae sp. nov. is proposed.

TLN-05220, TLN-05223, new Echinosporamicin-type antibiotics, and proposed revision of the structure of bravomicins(*).

The deposited strain of the hazimicin producer, Micromonospora echinospora ssp. challisensis NRRL 12255 has considerable biosynthetic capabilities as revealed by genome scanning. Among these is a locus containing both type I and type II PKS genes. The presumed products of this locus, TLN-05220 (1) and TLN-05223 (2), bear a core backbone composed of six fused rings starting with a 2-pyridone moiety. The structures were confirmed by conventional spectral analyses including MS, and 1D and 2D NMR experiments. Comparison of both the 1H and 13C NMR data of the newly isolated compound with those of echinosporamicin and bravomicin A led us to propose a revision of the structure of the latter to include a 2-pyridone instead of the pyran originally postulated. Both compounds (1 and 2) possessed strong antibacterial activity against a series of gram-positive pathogens including several strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci (VRE), and cytotoxic activities against several human tumor cell lines. The TLN compounds are the first of this group with reported anticancer activity.

Modulation of glycogen and trehalose levels in Micromonospora echinospora (ATCC 15837).

The growth of Micromonospora echinospora was studied in high and low C/N ratio medium using both batch and continuous culture. Asparagine was consumed rapidly in batch cultures where it served as both a nitrogen and carbon source. Glucose consumption was low suggesting that asparagine functions as the major carbon source under these conditions. The effect of nutrient limitation on the accumulation of storage carbohydrate in batch culture revealed an intimate association between nitrogen limitation and the accumulation of carbonaceous reserves. This study revealed that glycogen constituted the major carbohydrate reserve associated with the onset of sporulation. Intracellular trehalose levels were found to be relatively low and may have been affected by the availability of carbon. Continuous culture studies revealed a correlation between glycogen accumulation and increasing growth rate. It was also found that elevated cellular ATP levels correlated with the increase in glycogen, and reduced glycolytic activity. At the higher growth rates cellular ATP levels were elevated and coincided with reduced activity of the key glycolytic enzyme, phosphofructokinase, suggesting that glycogen can act as a convenient energy reservoir when excess carbon flux dictates.

4'-N-methyl-5'-hydroxystaurosporine and 5'-hydroxystaurosporine, new indolocarbazole alkaloids from a marine Micromonospora sp. strain.

Two new indolocarbazole alkaloids, 4'-N-methyl-5'-hydroxystaurosporine (2) and 5'-hydroxystaurosporine (3), were isolated together with the known staurosporine (1) from the culture broth of a marine Micromonospora sp. (strain L-31-CLCO-002). The fermentation, structural data and cytotoxic activities of these compounds against various tumor cell lines are given.

Detection of antitumor and antimicrobial activities in marine organism associated actinomycetes isolated from the Taiwan Strait, China.

The purpose of this work was to screen the actinomycetes having antitumor or antimicrobial activity, which were isolated from the surface, epidermis and intestines of sea plants and animals collected from the Taiwan Strait, China. Antitumor activity was studied by the MTT assay and DNA target activity was studied by the biochemical induction assay while antimicrobial activity was determined by observing bacterial and fungal growth inhibition. 20. 6% of marine actinomycete cultures displayed cytotoxic activity on P388 cells at dilutions at and below 1:320 and 18.6% on KB cells. 2. 96% of marine actinomycete cultures displayed inducing activity. Among all marine actinomycetes isolated, the genus Micromonospora has the highest positive rate of inducing activity. However, most antimicrobial activity was found in the genus Streptomyces. These results indicate that marine organism associated actinomycetes could be a promising source for antitumor and antimicrobial bioactive agents.

Arisostatins A and B, new members of tetrocarcin class of antibiotics from Micromonospora sp. TP-A0316. I. Taxonomy, fermentation, isolation and biological properties.

Arisostatins A and B, new members of tetrocarcin class of antibiotics were isolated from the culture broth of an actinomycete strain. The producing strain, TP-A0316, was identified as Micromonospora sp. Arisostatins were obtained from the culture fluid by solvent extraction and chromatographic purification. They showed antibiotic activity against Gram-positive bacteria and antitumor activity.

Antifungal anthracycline antibiotics, spartanamicins A and B from Micromonospora spp.

Spartanamicins A and B, two antifungal antibiotics, were produced by a culture of Micromonospora spp. strain No. MSU-43097 (ATCC 53803), isolated from a potted soil containing asparagus (Asparagus officinalis L.) plants. The antibiotics were isolated from the mycelial cake using organic solvents. The structures of spartanamicins A and B were determined by spectral and chemical means. Spartanamicin B is more active as an antifungal compound than it's analogue, A. The minimum inhibitory concentration for spartanamicin B on Candida albicans and Aspergillus, Cladosporium, Cryptococcus, Rhodotorula and Staphylococcus spp. ranged from 0.2 to 1 microgram/ml. It was not active against Staphylococcus aureus, Escherichia coli and Citrobacter spp. but some strains of S. aureus were sensitive.

Conditions for protoplasting, regenerating, and transforming the calicheamicin producer, Micromonospora echinospora.

We report methods to generate protoplasts, to regenerate mycelia, and to transform Micromonospora echinospora. This actinomycete produces the unusual antitumor antibiotics, the calicheamicins. These protocols may be applied to other actinomycetes that have been difficult to transform. These methods also may facilitate the cloning of calicheamicin biosynthetic genes by genetic complementation.

Calicheamicins, a novel family of antitumor antibiotics: taxonomy, fermentation and biological properties.

A novel family of antitumor antibiotics, the calicheamicins, were isolated from the fermentation broth of Micromonospora echinospora subsp. calichensis. These antibiotics exhibited significant activity against Gram-positive and Gram-negative bacteria in vitro. Calicheamicin gamma 1I demonstrated antitumor activity against P388 leukemia and B16 melanoma in vivo.

Temporally regulated tandem promoters in Micromonospora echinospora.

A collection of promoters from the Micromonospora echinospora strain that produces the calichemicin antitumor antibiotics was identified by the use of the promoter-probe vector pIJ486 in Streptomyces lividans. A 0.4-kilobase-pair Micromonospora DNA fragment was found to contain multiple tandem promoters which were characterized by S1 nuclease protection, Northern blotting, and DNA sequence determination. Analysis of RNA isolated from timed Micromonospora cultures revealed two classes of promoters within the 0.4-kilobase-pair fragment. The P2 promoter was maximally active during the exponential phase. In contrast, the P1 promoter cluster, consisting of three closely spaced start sites located 80 base pairs upstream of P2, was maximally active during the stationary phase. Because P1 was strongly induced in synchrony with calichemicin drug production, P1 is of potential utility in expressing cloned genes specifically during the stationary phase.