ABSTRACT
We have comprehensively analyzed the linear chromosomes of Streptomyces griseus mutants constructed and kept in our laboratory. During this study, macrorestriction analysis of AseI and DraI fragments of mutant 402-2 suggested a large chromosomal inversion. The junctions of chromosomal inversion were cloned and sequenced and compared with the corresponding target sequences in the parent strain 2247. Consequently, a transposon-involved mechanism was revealed. Namely, a transposon originally located at the left target site was replicatively transposed to the right target site in an inverted direction, which generated a second copy and at the same time caused a 2.5-Mb chromosomal inversion. The involved transposon named TnSGR was grouped into a new subfamily of the resolvase-encoding Tn3 family transposons based on its gene organization. At the end, terminal diversity of S. griseus chromosomes is discussed by comparing the sequences of strains 2247 and IFO13350.
Subject(s)
Chromosome Inversion , Chromosomes, Bacterial , Retroelements , Streptomyces griseus/genetics , Transposon Resolvases/genetics , Base Sequence , DNA, Bacterial/genetics , Molecular Sequence Data , Mutation , Restriction Mapping , Sequence Analysis, DNAABSTRACT
A filamentous soil bacterium, Streptomyces griseus 2247, carries a 7. 8-Mb linear chromosome. We previously showed by macrorestriction analysis that mutagenic treatments easily caused deletions at both ends of its linear chromosome and changed the chromosome to a circular form. In this study, we confirmed chromosomal circularization by cloning and sequencing the junction fragments from two deletion mutants, 404-23 and N2. The junction sequences were compared with the corresponding right and left deletion end sequences in the parent strain, 2247. No homology and a 6-bp microhomology were found between the two deletion ends of the 404-23 and N2 mutants, respectively, which indicate that the chromosomal circularization was caused by illegitimate recombination without concomitant amplification. The circularized chromosomes were stably maintained in both mutants. Therefore, the chromosomal circularization might have occurred to prevent lethal deletions, which otherwise would progress into the indispensable central regions of the chromosome.
Subject(s)
Chromosomes, Bacterial/genetics , Streptomyces griseus/genetics , Amino Acid Sequence , Base Sequence , Chromosome Deletion , Cosmids , DNA, Bacterial/genetics , DNA, Circular/genetics , Gene Library , Molecular Sequence Data , Plasmids , Restriction Mapping/methods , Sequence Alignment , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA- mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome.
Subject(s)
Chromosomes, Bacterial/genetics , Gene Deletion , Genes, Bacterial , Streptomyces griseus/genetics , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/biosynthesis , Chromosomes, Bacterial/ultrastructure , Cosmids , Restriction Mapping , Streptomyces griseus/metabolismABSTRACT
The genes coding for the protease inhibitors, SSI and API-2c', have been analyzed by comparing DNA macrorestriction patterns of Streptomyces albogriseolus S-3253 and S. griseoincarnatus KTo-250 with those of inhibitor-deficient mutants. The mutants were found to suffer from chromosomal deletions rather than plasmid loss which resulted in the loss of the relevant genes. Hybridization experiments indicated that the ssi homologs in S. lividans and S. coelicolor A3(2) are located near the end of the linear chromosome.
Subject(s)
Protease Inhibitors/metabolism , Streptomyces/chemistry , Streptomyces/genetics , Subtilisins/antagonists & inhibitors , Subtilisins/genetics , DNA, Bacterial/analysis , Electrophoresis, Gel, Pulsed-Field , Gene Deletion , Genes, Bacterial/genetics , Plasmids/genetics , Restriction MappingABSTRACT
The chromosomal DNA of Streptomyces griseus 2247 (a derivative of strain IFO3237) was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestion with AseI and DraI gave 15 and 9 fragments, respectively, the total sizes of which were 7.8 Mb. All the AseI and DraI fragments were aligned on a linear chromosome map by using linking plasmids and cosmids. PFGE analysis of the intact chromosome also showed a linear DNA band of about 8 Mb. Detailed physical maps of both terminal regions were constructed; they revealed the presence of a 24-kb terminal inverted repeat on each end. PFGE analysis with and without proteinase K treatment suggested that each end of the chromosome carries a protein molecule.
