Analysis of fusion junctions of circularized chromosomes in Streptomyces griseus

Abstract

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.

FIG. 1

Restriction and cosmid maps at both chromosomal ends of S. griseus 2247 and deleted regions in two afsA-negative mutants. All the AflII (Af), AseI (As), SpeI (Sp), and SspI (Ss) sites in the terminal 600-kb regions are shown. Cosmids 17E10 and 16C1, which filled in the gap that remained at the right side of cosmid F2D2, are newly isolated. The deleted regions in afsA-negative mutants are indicated by dashed lines.

FIG. 2

Location of the chromosomal deletion ends and the fusion junction in mutant 404-23. The right and left deletion ends of the 404-23 chromosome were narrowed by stepwise physical mapping of the deletion end cosmids 6E12 (A) and F2D2 (B) and finally located by comparison with the junction fragment (C). The deleted and fused regions are shown by dashed and shaded lines, respectively. Ba, BamHI; Ps, PstI; Ec, EcoRI; Kp, KpnI; SI, SacI; Ec*, EcoRI sites derived from the cosmid vector Supercos1.

FIG. 3

Southern hybridization analysis of the chromosomal deletion ends and the fusion junction in mutant 404-23. The 2247 and 404-23 DNAs were digested with BamHI, separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with the following probes: the end PstI fragment of cosmid 6E12 (A), the 9.5-kb KpnI fragment of cosmid F2D2 (B), and the 7.2-kb BamHI fusion fragment (C).

FIG. 4

Nucleotide sequences of the fusion junction (J) in mutant 404-23 and the corresponding right (R) and left (L) deletion ends in the parent strain, 2247. The fusion junction is located between nt 203 and 204. Putative ORFs and the corresponding protein sequences (in the one-letter code) are shown together with an RBS and a stop codon (*). The numbering for the nucleotide sequence is shown on the right.

FIG. 5

Locations of the chromosomal deletion ends and the fusion junction in mutant N2. The right and left deletion ends and the fusion junction were determined by stepwise physical mapping of the deletion end cosmids 6C8 (A) and F1F12 (B) and by comparison with the fusion fragment (C). SII, SacII; Sa, SalI. Other abbreviations are the same as for Fig. 2.

FIG. 6

Southern hybridization analysis of the chromosomal deletion ends and the fusion junction in mutant N2. The SacI digests of the 2247 and N2 DNAs were hybridized with the following probes: the 2.6-kb SacI fragment of cosmid 6C8 (A), the 1.9-kb EcoRI-SacI fragment of F1F12 (B), and the 2.3-kb SacI fusion fragment (C).

FIG. 7

Nucleotide sequences of the fusion junction (J) in mutant N2 and the right (R) and left (L) deletion ends in the parent strain, 2247. The fusion junction is composed of 6-bp nucleotides with the sequence TCCCAC, shown by a box. A putative ORF is carried on the complementary strand near the left deletion end, and the corresponding protein sequence is shown, in the one-letter code.