Chapter 14 : Unstable Linear Chromosomes: the Case of Streptomyces

Invertron stracture of the Streptomyces linear chromosomal DNA. The physical (Ase I) and genetic map of the S. ambofaciens DSM40697 chromosome typifies the invertron structure of the Streptomyces chromosomes. The 8-Mb chromosomal DNA is linear and possesses TIRs associated with proteins. The oriC locus is approximately opposite the ends of the DNA. Most gene localizations are from Leblond et al. ( 72 ) or unpublished data; the transcriptional orientation of the rrn loci are from Berger et al. ( 10 ), and the localization of hasL/R are from Fischer et al. ( 39 ). The unstable and deletable region, including Ase I fragments F, D, G, E, and J, is localized at the ends of the DNA and is indicated by dark shading.

Different levels of genetic instability in Streptomyces. Hypervariability in S. ambofaciens DSM40697 is shown; no preponderant phenotype can be seen in the progeny of a pigment-defective colony generated from the WT strain ( 69 ).

Mutacor states. Mutator states are revealed by the appearance of pigment-defective papillae on the typical grey pigment of the WT colony ( 80a ). The WT strain grown on solid medium for 14 days gives rise to a minority of fully pigmented (Pig+) colonies (≤30%) and to a majority of papillae-harboring colonies (≥70%) as well as fully pigment-defective colonies (about 1%; not symbolized). The colonies can also be sectored. When the number of papillae per colony was plotted against the number of colonies, the distribution differed from the theoretical Poisson distribution. Mutators at the extremity of the distribution were defined as colonies harboring more than 20 papillae per colony.

Chromosomal deletions on the linear chromosomal DNA of Streptomyces. The different locations of the chromosomal deletions are symbolized. The WT chromosome is represented under the racket frame model ( 101 ), where the extremities of the DNA consisting of large inverted repeats (TIRs) are associated. The length of the TIRs is symbolized by that of the parallel terminal regions. The solid circles represent the terminal proteins covalently associated with the DNA ends. (A) The TIRs and subterminal regions can be deleted, leading to circularization of the chromosome. (B) DNA amplification takes place from amplifiable loci called AUDs localized in the unstable region. Amplification is frequently associated with an adjacent deletion including all sequence separating the AUD from the chromosomal end. Thus, an unknown structure (question mark) replaces the bacterial telomere. (C) Internal deletions in one chromosomal arm were observed in S. ambofaciens, leading to the shortening of the TIRs. (D) Deletions were also observed associated with sister chromosomal exchanges, leading to the increase of the TIR length. (E) Loss of DNA extremities and replacement by an unknown structure (question mark) were also described in Streptomyces.

Hypothetical flow chart of the cascade of molecular events involved in genetic instability. Structural instability would proceed by two different recombination pathways: intramolecular and intermolecular interactions (sister chromosome exchange). Both recombination events could take place at the chromosomal replication in the terminus region. A primary event (terminal protein loss, DNA degradation, or collapse of the replication fork) would lead to the loss of a DNA extremity. The creation of this reactive extremity would result in either circularization of the chromosome (A) or DNA end fusion between the two newly replicated chromatids (B). This latter form would enter an equivalent to the breakage-fusion-bridge cycle described in eukaryotes by McClintock ( 81 ). The fusion of DNA ends would be a good explanation for the unknown nature of chromosomes that have lost one DNA extremity and kept the other one intact ( Fig. 4 ). DNA amplification could take place either at the end of the replicated chromatids by a Young and Cullum mechanism ( 120 ) or on the circularized or fused molecules. Secondly, interchromosomal interactions could explain the variation of the TIR length. If the recombination event (homologous or illegitimate) takes place between two regions specific to each chromosomal arm (C, event 1), then it results in the exchange of chromosomal arms, with extension of the TIR. On the other hand, recombination between two regions, one in the TIR and the other one specific to a chromosomal arm (D, event 2), would result, on the right replicon, in the shortening of the TIR accompanied by an internal deletion. The reciprocal could remain undetectable, since no deletion would result from this event, but a duplication of an internal TIR sequence is produced (arrows). When segregated, this structure might also trigger an instability cascade by homologous recombination that could be initiated between the large duplicated areas.

Evolutionary implications of genetic instability. Interactions between linear plasmids and chromosomes leading to the generation of hybrid chromosomes were recently reported in Streptomyces ( 91 ). This mechanism results in acquisition of new terminal information on the chromosomal DNA. This exchange could result from homologous or illegitimate recombination pathways. The hybrids could be unstable and undergo recombinational exchanges between newly replicated sister chromosomes, as reported in S. ambofaciens. This event would result in the homogeneity of the terminal sequence and thus introduce a dramatic change in the terminal sequences of this species. The symbols are as for other figures for chromosomal DNA. Plasmid DNA (open box) possesses an invertron structure with TIRs (hatched arrows) and terminal proteins (shaded circles). Broken lines symbolize the recombination event.