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.Actinomycete bacteria, especially those belonging to the family Streptomycetaceae, are well-known producers of secondary metabolites with diverse biological activities. Representatives of the genus Streptomyces produce a variety of antibiotics with antibacterial, antifungal, and antitumor activities. The majority of antibiotic-producing streptomycetes have been isolated from terrestrial environments, while antibiotic-producing streptomycetes from the marine sources remain largely unexplored. Therefore, studies of streptomycetes from the marine environment are important for unraveling their potential for antibiotic production. In addition, such studies might reveal the means by which antibiotic biosynthesis and resistance genes are spread in nature.It is widely acknowledged that plasmids play an important role in genetic exchange between bacterial species. Conjugative plasmids are quite common in Streptomyces strains (13), and a number of these mobile genetic elements have been characterized in detail. The characterized mobile genetic elements include both circular plasmids, such as pIJ101 from Streptomyces lividans (14) and SCP2 from Streptomyces coelicolor (2, 35), and linear plasmids, such as SLP2 from S. lividans (6) and SCP1 from S. coelicolor (38, 39). The presence of a linear plasmid in Streptomyces was first reported in 1979, and the plasmid was the 17-kb pSLA2 plasmid of Streptomyces rochei (11). SCP1 of S. coelicolor was discovered in the early 1970s (38, 39), but because of its large size (356 kb), isolation of this plasmid with conventional techniques was not possible and therefore it was not recognized as a linear plasmid until pulsed-field gel electrophoresis (PFGE) was invented. Later, SCP1 was shown to harbor a complete set of genes for biosynthesis of the antibiotic methylenomycin (21; K. F. Chater, C. J. Bruton, S. J. O''Rouke, and A. W. Wietzorrek, 5 July 2001, Patent Cooperation Treaty international application WO/2001/048228), while another linear plasmid, found in S. rochei, has been shown to contain genes for biosynthesis of both lankamycin and lankacidin (16, 19, 28, 36). Other examples of plasmids include pPZG103 carrying oxytetracycline biosynthesis genes acquired from the chromosome of Streptomyces rimosus (10) and pKSL from Streptomyces lasaliensis, which might be involved in the production of lasalocid and/or echinomycin (17, 20).Linear plasmids can be transferred between Streptomyces strains by means of conjugation, and SCP1 is an example of a conjugative linear plasmid as it is easily transferred from an SCP1⁺ strain to an SCP1⁻ strain (39). Interspecific transfer to S. lividans and Streptomyces parvulus has also been reported for this plasmid, and it was demonstrated that the recipient strains had acquired the ability to produce and be resistant to methylenomycin (12, 21). Transfer of intact linear plasmids containing mercury resistance genes from two Streptomyces strains isolated from the marine environment to S. lividans, conferring mercury resistance to the initially mercury-sensitive recipient, has been reported by Ravel et al. (32). It has also been shown that interspecific transfer of linear plasmids is possible in sterile amended soil microcosms, suggesting that mercury resistance might be spread by plasmid transfer in polluted environments (31).We report here isolation and screening of several thousand actinobacterial strains from the Trondheim fjord (Norway), which resulted in identification of producers of both known and potentially new polyene macrolides with antifungal activity. The ability to produce the polyene macrolide candicidin D was found to be widespread among the Trondheim fjord Streptomyces isolates. We also report that the candicidin biosynthesis genes (can) are present on a linear plasmid identified in one of these isolates, suggesting that the can genes might be spread by means of conjugation.