Streptomyces spp. From the Marine Sponge Antho dichotoma: Analyses of Secondary Metabolite Biosynthesis Gene Clusters and Some of Their Products.

Actinomycete bacteria from marine environments represent a potential source for new antibiotics and anti-tumor drugs. Ten strains belonging to the genus Streptomyces isolated from the marine sponge Antho dichotoma collected at the bottom of the Trondheim fjord (Norway) were screened for antibiotic activity. Since only few isolates proved to be bioactive in the conditions tested, we decided to gain an insight into their biosynthetic potential using genome sequencing and analysis. Draft genomes were analyzed for the presence of secondary metabolite biosynthesis gene clusters (BGCs) using antiSMASH software. BGCs specifying both known and potentially novel secondary metabolites were identified, suggesting that these isolates might be sources for new bioactive compounds. The results of this analysis also implied horizontal transfer of several gene clusters between the studied isolates, which was especially evident for the lantibiotic- and thiopeptide-encoding BGCs. The latter implies the significance of particular secondary metabolites for the adaptation of Streptomyces to the spatially enclosed marine environments such as marine sponges. Two bioactive isolates, one showing activity against both yeast and Bacillus subtilis, and one only against yeast were analyzed in details, leading to the identification of cycloheximide, linearmycins, and echinomycins that are presumably responsible for the observed bioactivities.

Actinoalloteichus fjordicus sp. nov. isolated from marine sponges: phenotypic, chemotaxonomic and genomic characterisation.

Two actinobacterial strains, ADI 127-17T and GBA 129-24, isolated from marine sponges Antho dichotoma and Geodia barretti, respectively, collected at the Trondheim fjord in Norway, were the subjects of a polyphasic study. According to their 16S rRNA gene sequences, the new isolates were preliminarily classified as belonging to the genus Actinoalloteichus. Both strains formed a distinct branch, closely related to the type strains of Actinoalloteichus hoggarensis and Actinoalloteichus hymeniacidonis, within the evolutionary radiation of the genus Actinoalloteichus in the 16S rRNA gene-based phylogenetic tree. Isolates ADI 127-17T and GBA 129-24 exhibited morphological, chemotaxonomic and genotypic features distinguishable from their close phylogenetic neighbours. Digital DNA: DNA hybridization and ANI values between strains ADI 127-17T and GBA 129-24 were 97.6 and 99.7%, respectively, whereas the corresponding values between both tested strains and type strains of their closely related phylogenetic neighbours, A. hoggarensis and A. hymeniacidonis, were well below the threshold for delineation of prokaryotic species. Therefore, strains ADI 127-17T (= DSM 46855T) and GBA 129-24 (= DSM 46856) are concluded to represent a novel species of the genus Actinoalloteichus for which the name of Actinoalloteichus fjordicus sp. nov. (type strain ADI 127-17T = DSM 46855T = CECT 9355T) is proposed. The complete genome sequences of the new strains were obtained and compared to that of A. hymeniacidonis DSM 45092T and A. hoggarensis DSM 45943T to unravel unique genome features and biosynthetic potential of the new isolates.

Genomics of sponge-associated Streptomyces spp. closely related to Streptomyces albus J1074: insights into marine adaptation and secondary metabolite biosynthesis potential.

A total of 74 actinomycete isolates were cultivated from two marine sponges, Geodia barretti and Phakellia ventilabrum collected at the same spot at the bottom of the Trondheim fjord (Norway). Phylogenetic analyses of sponge-associated actinomycetes based on the 16S rRNA gene sequences demonstrated the presence of species belonging to the genera Streptomyces, Nocardiopsis, Rhodococcus, Pseudonocardia and Micromonospora. Most isolates required sea water for growth, suggesting them being adapted to the marine environment. Phylogenetic analysis of Streptomyces spp. revealed two isolates that originated from different sponges and had 99.7% identity in their 16S rRNA gene sequences, indicating that they represent very closely related strains. Sequencing, annotation, and analyses of the genomes of these Streptomyces isolates demonstrated that they are sister organisms closely related to terrestrial Streptomyces albus J1074. Unlike S. albus J1074, the two sponge streptomycetes grew and differentiated faster on the medium containing sea water. Comparative genomics revealed several genes presumably responsible for partial marine adaptation of these isolates. Genome mining targeted to secondary metabolite biosynthesis gene clusters identified several of those, which were not present in S. albus J1074, and likely to have been retained from a common ancestor, or acquired from other actinomycetes. Certain genes and gene clusters were shown to be differentially acquired or lost, supporting the hypothesis of divergent evolution of the two Streptomyces species in different sponge hosts.

Production of a new thiopeptide antibiotic, TP-1161, by a marine Nocardiopsis species.

Twenty-seven marine sediment- and sponge-derived actinomycetes with a preference for or dependence on seawater for growth were classified at the genus level using molecular taxonomy. Their potential to produce bioactive secondary metabolites was analyzed by PCR screening for genes involved in polyketide and nonribosomal peptide antibiotic synthesis. Using microwell cultures, conditions for the production of antibacterial and antifungal compounds were identified for 15 of the 27 isolates subjected to this screening. Nine of the 15 active extracts were also active against multiresistant gram-positive bacterial and/or fungal indicator organisms, including vancomycin-resistant Enterococcus faecium and multidrug-resistant Candida albicans. Activity-guided fractionation of fermentation extracts of isolate TFS65-07, showing strong antibacterial activity and classified as a Nocardiopsis species, allowed the identification and purification of the active compound. Structure elucidation revealed this compound to be a new thiopeptide antibiotic with a rare aminoacetone moiety. The in vitro antibacterial activity of this thiopeptide, designated TP-1161, against a panel of bacterial strains was determined.

Biosynthesis of macrolactam BE-14106 involves two distinct PKS systems and amino acid processing enzymes for generation of the aminoacyl starter unit.

BE-14106 is a macrocyclic lactam with an acyl side chain previously identified in a marine-derived Streptomyces sp. The gene cluster for BE-14106 biosynthesis was cloned from a Streptomyces strain newly isolated from marine sediments collected in the Trondheimsfjord (Norway). Bioinformatics and experimental analyses of the genes in the cluster suggested an unusual mechanism for assembly of the molecule. Biosynthesis of the aminoacyl starter apparently involves the concerted action of a distinct polyketide synthase (PKS) system and several enzymes that activate and process an amino acid. The resulting starter unit is loaded onto a second PKS complex, which completes the synthesis of the macrolactam ring. Gene inactivation experiments, enzyme assays with heterologously expressed proteins, and feeding studies supported the proposed model for the biosynthesis and provided new insights into the assembly of macrolactams with acyl side chain.

Candicidin biosynthesis gene cluster is widely distributed among Streptomyces spp. isolated from the sediments and the neuston layer of the Trondheim fjord, Norway.

A large number of Streptomyces bacteria with antifungal activity isolated from samples collected in the Trondheim fjord (Norway) were found to produce polyene compounds. Investigation of polyene-containing extracts revealed that most of the isolates produced the same compound, which had an atomic mass and UV spectrum corresponding to those of candicidin D. The morphological diversity of these isolates prompted us to speculate about the involvement of a mobile genetic element in dissemination of the candicidin biosynthesis gene cluster (can). Eight candicidin-producing isolates were analyzed by performing a 16S rRNA gene-based taxonomic analysis, pulsed-field gel electrophoresis, PCR, and Southern blot hybridization with can-specific probes. These analyses revealed that most of the isolates were related, although they were morphologically diverse, and that all of them contained can genes. The majority of the isolates studied contained large plasmids, and two can-specific probes hybridized to a 250-kb plasmid in one isolate. Incubation of the latter isolate at a high temperature resulted in loss of the can genes and candicidin production, while mating of the "cured" strain with a plasmid-containing donor restored candicidin production. The latter result suggested that the 250-kb plasmid contains the complete can gene cluster and could be responsible for conjugative transfer of this cluster to other streptomycetes.

Actinomycetes from sediments in the Trondheim fjord, Norway: diversity and biological activity.

The marine environment represents a largely untapped source for isolation of new microorganisms with potential to produce biologically active secondary metabolites. Among such microorganisms, Gram-positive actinomycete bacteria are of special interest, since they are known to produce chemically diverse compounds with a wide range of biological activities. We have set out to isolate and characterize actinomycete bacteria from the sediments in one of the largest Norwegian fjords, the Trondheim fjord, with respect to diversity and antibiotic-producing potential. Approximately 3,200 actinomycete bacteria were isolated using four different agar media from the sediment samples collected at different locations and depths (4.5 to 450 m). Grouping of the isolates first according to the morphology followed by characterization of isolates chosen as group representatives by molecular taxonomy revealed that Micromonospora was the dominating actinomycete genus isolated from the sediments. The deep water sediments contained a higher relative amount of Micromonospora compared to the shallow water samples. Nine percent of the isolates clearly required sea water for normal growth, suggesting that these strains represent obligate marine organisms. Extensive screening of the extracts from all collected isolates for antibacterial and antifungal activities revealed strong antibiotic-producing potential among them. The latter implies that actinomycetes from marine sediments in Norwegian fjords can be potential sources for the discovery of novel anti-infective agents.

Hydrophobicity development, alkane oxidation, and crude-oil emulsification in a Rhodococcus species.

The relationship between the phenomena alkane oxidation, extreme hydrophobicity of the cell surface, and crude-oil emulsification in Rhodococcus sp. strain 094 was investigated. Compounds that induce the emulsifying ability simultaneously induced the cytochrome P450-containing alkane oxidizing system and the transition from low to high cell-surface hydrophobicity. Exposed to inducers of crude-oil emulsification, the cells developed a strong hydrophobic character during exponential growth, which was rapidly lost when entering stationary phase. The loss in hydrophobicity coincided in time with the crude-oil emulsification, indicating that the components responsible for the formation of cell-surface hydrophobicity act as excellent emulsion stabilisers only after release from the cells. Rhodococcus sp. strain 094 possessed three distinct levels of cell-surface hydrophobicity. One level of low hydrophobicity was characteristic of cells in late stationary phase and was independent of growth substrate. A second and more hydrophobic level was observed for cells in exponential phase grown on water-soluble substrates, while a third level, characterised by extreme cell hydrophobicity, was observed for cells in exponential phase cultivated on hydrophobic substrates such as hexadecane. The production of the oil-emulsifying agents seems to require external sources of nitrogen and phosphate.