Chromosomal instability in Streptomyces avermitilis: major deletion in the central region and stable circularized chromosome

Abstract

Background: The chromosome of Streptomyces has been shown to be unstable, frequently undergoing gross chromosomal rearrangements. However, the mechanisms underlying this phenomenon remain unclear, with previous studies focused on two chromosomal ends as targets for rearrangements. Here we investigated chromosomal instability of Streptomyces avermitilis, an important producer of avermectins, and characterized four gross chromosomal rearrangement events, including a major deletion in the central region. The present findings provide a valuable contribution to the mechanistic study of genetic instability in Streptomyces.

Results: Thirty randomly-selected "bald" mutants derived from the wild-type strain all contained gross chromosomal rearrangements of various types. One of the bald mutants, SA1-8, had the same linear chromosomal structure as the high avermectin-producing mutant 76-9. Chromosomes of both strains displayed at least three independent chromosomal rearrangements, including chromosomal arm replacement to form new 88-kb terminal inverted repeats (TIRs), and two major deletions. One of the deletions eliminated the 36-kb central region of the chromosome, but surprisingly did not affect viability of the cells. The other deletion (74-kb) was internal to the right chromosomal arm. The chromosome of another bald mutant, SA1-6, was circularized with deletions at both ends. No obvious homology was found in all fusion sequences. Generational stability analysis showed that the chromosomal structure of SA1-8 and SA1-6 was stable.

Conclusions: Various chromosomal rearrangements, including chromosomal arm replacement, interstitial deletions and chromosomal circularization, occurred in S. avermitilis by non-homologous recombination. The finding of an inner deletion involving in the central region of S. avermitilis chromosome suggests that the entire Streptomyces chromosome may be the target for rearrangements, which are not limited, as previously reported, to the two chromosomal ends.

Figure 1

Gross chromosomal rearrangements in spontaneous bald mutants from S. avermitilis wild-type (WT) strain ATCC31267. (A) AseI restriction map of wild-type chromosome. (B and C) AseI restriction patterns of genomic DNA of bald mutants (SA). (D) Similar AseI profiles of 76-9 and SA1-8. PFGE conditions for separating large fragments were: (B and D) 1.2% agarose, 4.5 V/cm, 20-130 s pulses, 36 h; 4.5 V/cm, 60-90 s pulses, 2 h; 4.5 V/cm, 5-10 s pulses, 8 h; conditions for separating small fragments were: (C) 1.5% agarose, 6 V/cm, 5-10 s pulses, 24 h. Fragments D and E overlapped because of their extremely similar migration; overlap was also found for fragments G1/G2, O/P/N, and S/T. SAP1: 94.3-kb linear plasmid. Solid arrows: missing fragments; Open arrows: potential missing fragments; Triangles: new bands.

Figure 2

PFGE analysis of the chromosomes of S. avermitilis strains. (A) PFGE of intact chromosome treated with Proteinase K (PK) and SDS. (B) PFGE analysis of AseI digested chromosome with PK and SDS treatment, showing that fragment NA2 is a new end bound to terminal protein. PFGE conditions for (A) were: 1% agarose, 3 V/cm, 180 s pulses, 20 h. Conditions for SA1-8 and wild-type in (B) were the same as for Fig 1B and 1C, respectively. "+" represents DNA sample treated with PK; "-'' represents DNA sample treated with SDS.

Figure 3

Southern hybridization analysis of chromosomal rearrangements in SA1-8 (A, B) and schematic representation of the chromosomes of wild-type strain and mutant SA1-8, showing three independent rearrangements (C). Total DNAs were in gel digested with AseI, and probed by aveC, Dr, D600, NA3, G2-152, and G1-139, respectively (A, B). Probe aveC was in the ave gene cluster of fragment A. Distance between probe and extreme right end of chromosome was 600-kb for D600, 196-bp for Dr. Probes G2-152 and G1-139 were located on fragments G2 and G1, respectively. PFGE conditions were the same as for Fig. 1B. (C) Open bar: simplified chromosome map with fragment designations and sizes in kilobases; Vertical lines: AseI sites; Horizontal lines: probes; Diagonal lines: internal regions not displayed; Thick arrows: 88-kb TIRs; Solid circles: terminal proteins; Black bars: inner deletion regions.

Figure 4

Analysis of recombination point in fragment NA1. (A) Restriction maps of fragments involved in the recombination event in NA1. The 1.84-kb PstI junction fragment resulted from fusion in opposite orientation of partially deleted 6.4-kb and 7.0-kb PstI fragments from left and right chromosomal arms, termed A6.4 and D7.0 respectively. (B) Hybridization analysis of the PstI fusion fragment. (C) Inverse PCR to obtain the left unknown sequence of 1.84-kb PstI junction fragment. (D) The fusion sequence in NA1 joins the partial region of fragment A6.4 and D7.0 at a 5-bp overlapping sequence. Bold and non-bold fonts represent nucleotide sequences from fragment A6.4 and D7.0, respectively. Dashed lines represent deleted regions. Ps: PstI. Primers 113 and 114 were used in inverse PCR. Primers 118 and 113 were used in PCR for amplifying fusion sequence.

Figure 5

Analysis of fusion sequence in fragment NA2. (A) Location of chromosomal deletion ends and fusion junction. Left and right deletion termini were characterized by stepwise PCR mapping. Deleted and fused regions are indicated by dashed and shaded lines, respectively. Kp, KpnI. (B) Southern analysis of fusion fragment with probe N2, which was prepared using primers 236 and 239. (C) Junction sequence, showing no obvious homology between the original sequences.

Figure 6

Analysis of fusion sequence in fragment NA3. (A) Location of chromosomal deletion ends and fusion junction. Ba, BamHI. (B) Southern analysis of junction fragment with probe N3, which was prepared using primers 248 and 272. (C) Junction sequence in NA3. The 3-bp overlapping sequence is boxed.

Figure 7

Characterization of circular chromosome in SA1-6. (A) Schematic representation of the chromosomes of wild-type strain and mutant SA1-6, showing deletions at both ends. (B) Location of chromosomal deletion ends and fusion junction. Bg, BglII. (C) Southern analysis of fusion fragment with probe N4, which was prepared using primers 405 and 406. (D) Junction sequence, showing no obvious homology between the original sequences.