Physical Map of the Linear Chromosome of Streptomyces hygroscopicus 10-22 Deduced by Analysis of Overlapping Large Chromosomal Deletions

Abstract

The chromosomal DNA of Streptomyces hygroscopicus 10-22, a derivative of strain 5102-6, was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestions with AseI gave 11 fragments with a total length of ca. 7.36 Mb. The AseI sites were mapped by analysis of overlapping chromosomal deletions in different mutants and confirmed by Southern hybridizations using partially digested genome fragments and linking cosmids as probes. PFGE analysis of DNA with and without proteinase K treatment, together with the hybridization results, suggested a linear organization with terminal proteins and large terminal inverted repeats. Some deletion mutants had circular chromosomes.Streptomyces species are gram-positive, mycelium-forming soil bacteria with a high GϩC content (70 to 74%) in their DNA (5). They display a complex life cycle which involves the production of a large number of secondary metabolites, many of which are antibiotics. Pulsed-field gel electrophoresis (PFGE) allows the physical mapping of large bacterial chromosomes, and the physical maps of several Streptomyces chromosomes have been reported (11,16,17,21). Different approaches were used for mapping bacterial chromosomes (8). For example, hybridization of "linking clones" to Southern blots of restriction fragments separated by PFGE was used to establish the physical map of Streptomyces coelicolor A3(2) (11), Streptomyces lividans 66 (16), Streptomyces griseus (17), Streptomyces rimosus (21), and Saccharopolyspora erythraea (26). Physical maps were constructed for Mycoplasma mobile (2) and Pseudomonas aeruginosa (27) by employing one-and two-dimensional PFGE. A high-resolution physical map was established by ordering a set of cosmids in S. coelicolor A3(2) (25).One of the outstanding features of Streptomyces is its frequent chromosomal deletions (30, 31), which could occur spontaneously or be induced by exposure to physical or chemical factors, such as UV radiation (32) and antibiotics (33). This phenomenon is believed to be related to the linearity of the chromosomes, and this genetic instability has not been shown to occur in circular chromosomes (7,20).Streptomyces hygroscopicus 10-22 (37) can produce at least three useful antifungal antibiotics, one of which is used extensively and the others of which are used on a small scale in Chinese agriculture. Antibiotic 5102-I, an aminoglycoside similar to validamycin, is highly effective against Pellicularia sasakii, which causes rice sheath blight disease, Pellicularia filamentosa, which causes seedling blight of cotton, and Helminthosporium sigmoideum, which causes sclerotial disease of rice. Antibiotic II is a polypeptide useful for protection against leaf spot of corn caused by Cochlioobolus heterostrophus. Antibiotic III, whose structure is unknown, is useful for the control of cotton wilt caused by Fusarium oxysporum. Extensive efforts have therefore been devoted to identifying the compounds (35, 36), developing a gene cloning system (22), and improving th...

Streptomyces coelicolor A3(2) Lacks a Genomic Island Present in the Chromosome of Streptomyces lividans 66

“…General growth media and growth conditions for E. coli and Streptomyces strains and standard methods for handling E. coli and Streptomyces in vivo and in vitro, such as preparation of plasmid and chromosome DNAs, construction of a Streptomyces genomic library in E. coli, transformation, and Southern hybridization, were as described previously (23,32), unless stated otherwise. DNA samples were prepared, digested, and separated by PFGE as described previously (31). DNA sequencing was performed using ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kits from Perkin-Elmer Applied Biosystems.…”

Regulation of the biosynthesis of thiopeptide antibiotic cyclothiazomycin by the transcriptional regulator SHJG8833 in Streptomyces hygroscopicus 5008

“…Interestingly, three putative regulatory genes were identified, with two at the end of, and one flanking, the cluster. Since S. hygroscopicus 10-22 is much more difficult to manipulate genetically than S. hygroscopicus 5008 (Pang et al, 2002;Qin et al, 1994), here we used strain 5008 as a model to investigate the roles of these three regulatory genes in the biosynthesis of cyclothiazomycin. We proved that the cluster is functional in S. hygroscopicus 5008 and we showed that one of the regulatory genes, SHJG8833, is essential for cyclothiazomycin biosynthesis.…”

Chromosomal Circularization inStreptomyces griseusby Nonhomologous Recombination of Deletion Ends

“…Filamentous soil bacteria, Streptomyces, are unusual in carrying an 8-9 Mb linear chromosome: [1][2][3][4][5][6][7] the genome project for Streptomyces coelicolor A3(2), a model strain for Streptomyces genetics, has been recently completed. 8) Streptomyces species are also known to frequently have linear plasmids, which range from 12 to 1700 kb in size.…”