DNA rearrangements at the extremities of the Streptomyces ambofaciens linear chromosome: Evidence for developmental control

Biochimie 82 (2000) 29−34 © 2000 Société française de biochimie et biologie moléculaire/Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400003485/FLA

DNA rearrangements at the extremities of the Streptomyces ambofaciens linear chromosome: Evidence for developmental control Annie Dary*, Patricia Martin, Thomas Wenner, Bernard Decaris, Pierre Leblond Laboratoire de Génétique et Microbiologie associé à l’Institut National de la Recherche Agronomique (unité 952), Faculté des Sciences de l’Université Henri-Poincaré, Nancy 1, BP239, 54506 Vandoeuvre-lès-Nancy, France (Received 18 August 1999; accepted 5 October 1999) Abstract — In Streptomyces, a genomic instability results from frequent recombination events which occur at the ends of the linear chromosomal DNA. These events are believed to be responsible for the variability observed in these regions among Streptomyces species and strains. In order to identify functions able to control this type of genome plasticity, mutators as well as mutants produced at different stages of development have been characterized in S. ambofaciens. Their characterization suggests the existence of a relationship between genomic instability and colony development. © 2000 Société française de biochimie et biologie moléculaire/ Éditions scientifiques et médicales Elsevier SAS Streptomyces ambofaciens / DNA rearrangements / genomic instability / genome plasticity

1. Introduction Recombination plays a key role in the cell, particularly through DNA damage repair, and can promote stability or variability of the DNA sequence. Recombination frequencies are not randomly distributed along the chromosome, rather, specific chromosomal loci or regions undergo frequent recombination events. Such is the case of the Escherichia coli terminus of replication or of the telomeric and subtelomeric regions of the prokaryotic and eukaryotic chromosomes [1, 2]. The subtelomeric regions, i.e., sequences associated with the telomere, of most organisms are dynamic. In eukaryotes or prokaryotes, homologous or illegitimate recombination events are responsible for the variability observed between species or strains in telomeric and subtelomeric regions [2, 3]. The ends of the Streptomyces linear chromosomal DNA do not seem to constitute an exception to this rule since they are prone to frequent recombination events which generate a high level of genome plasticity. This plasticity consists of deletions of several hundred kilobases associated, in some cases, with the tandem reiteration of adjacent DNA sequences [4]. 2. Genomic instability in Streptomyces Chromosomal DNA of Streptomyces is a linear molecule of about 8 Mb ending with terminal inverted repeats * Correspondence and reprints

(TIRs) covalently bound to terminal proteins. This DNA molecule contains a centrally located bidirectional origin of replication which is believed to be active. Two chromosomal arms, approximately similar in size, are served by this replication origin [5, 6]. Finally, as has been proposed for the Streptomyces linear plasmid SCP1, the TIRs might be synaptically associated to form a ‘racket frame’ structure [7]. The linear conformation of the Streptomyces chromosomal DNA allows for the differentiation between telomeric (the extreme ends) and subtelomeric (the sequences located near telomeres) regions. Although Streptomyces chromosomes and linear plasmids show a high degree of conservation in the first 166–168 bp terminal fragments, beyond this terminal homology, sequences do not cross-hybridize [8]. TIRs of variable size are found among the Streptomyces genus [8-12]. As in other organisms harboring a linear chromosomal DNA, homologous and illegitimate recombination events are exceptionally frequent within the telomeric and subtelomeric regions and are responsible for the high degree of variability observed in these regions among Streptomyces. These recombination events potentially modify not only the size and sequence of the TIRs but also the conformation of the chromosomal DNA. Several chromosomal structures produced by this genomic instability have been characterized. Some mutants harbor a deletion which is internal to one chromosomal arm (figure 1A) and results from a recombination event between a DNA sequence located inside the TIR and one located outside. All of these mutants exhibit

30 reduced TIRs. Mutants with a circular chromosomal DNA have also been observed. Such a circularization results from the loss of both extremities after a recombination event between sequences located on both arms, either inside or outside of the TIRs [13, 14]. The identification of endpoint deletions in several mutants indicates that recombination events seem to occur randomly but within defined regions, i.e., the chromosomal DNA extremities [13-15]. Circularization of the chromosome does not stabilize the structure since spontaneously or artificially circularized chromosomes are even more unstable than linear ones [14, 16, 17]. Mutant chromosomes with TIRs larger than those found in WT organisms have also been described (figure 1B). These result from the deletion of one chromosomal end and its replacement by a sequence identical to the end of the undeleted chromosomal arm. Fischer et al. [12] showed that such structures were generated by homologous recombination between two copies of a duplicated DNA sequence (the has genes), each located on one chromosomal arm. The simplest hypothesis to explain the replacement of one extremity by the other and the subsequent increase in TIR size is to consider that such a structure is generated by an ectopic crossover between the two copies of the has gene. Replacement of one chromosomal extremity by another has also been observed in S. rimosus. A recombination event between the linear plasmid pPZG101 and the chromosomal DNA [18] leads to a hybrid chromosome which remains linear but has lost its invertron structure. Fischer et al. [12] have proposed that this hybrid chromosome might return to its invertron conformation through an interchromosomal exchange similar to that postulated for the mutants having extended TIRs. Finally, some mutants have lost one chromosomal extremity while retaining a linear structure, a deletion that is associated with a RecA-dependent amplification of an adjacent DNA sequence in some mutant strains [13, 14, 19]. The exact chromosome structure of these mutants remains to be elucidated. Although prone to frequent rearrangements, the Streptomyces subtelomeric regions are not silent but rather contain numerous genes which are actively expressed [20]. Most of them encode proteins involved in secondary metabolism, i.e., a subset of genes required for morphological and biochemical differentiation of Streptomyces. Indeed, Streptomyces are Gram-positive soil bacteria whose complex life cycle has led to a highly differentiated organism. On solid medium, a spore germinates to form a vegetative mycelium, later giving rise to an aerial mycelium which forms apical spores [21]. Thus, genomic rearrangements which occur in the terminal chromosomal regions generate mutants displaying various phenotypes. The presence of a potentially active bidirectional origin at the chromosome center implies that the extremities constitute regions of replication completion. Conse-

Dary et al. quently, the replication forks can be slowed in these regions. The arrest of replication forks is associated with generation of double strand breaks (DSBs) [22]. DSBs constitute lethal lesions, and all organisms possess at least one of the two types of known DSB repair pathways. The endjoining repair pathway represents a simple ligation with another available DNA end while the second repair pathway involves recombination with an intact homologous copy. The endjoining repair pathway can lead to deletion and even to gross chromosomal rearrangements [23]. The recombination repair pathway requires RecA and RecBCD in E. coli and the Rad52 pathway in yeast [24]. In Streptomyces, analysis of several deletion endpoints has revealed that illegitimate, i.e., endjoining, or homologous recombination events are involved in genomic instability. Such observations, along with the location of genome rearrangements at the extremities of the chromosomal DNA, suggest that the instability could result from the generation of DSBs at the chromosomal extremities. 3. Modulation of recombination according to the developmental state of the S. ambofaciens colony In S. ambofaciens, genomic instability is related to the instability of the pigmentation of colonies leading to four main phenotypes in the progeny which arise from pigmented colonies (WT): pigmented colonies, pigmentdefective colonies, pigmented colonies with pigmentdefective sector and/or papillae (figure 2). Deletions were analyzed in Pig–col, Pig–sec and Pig–pap mutants (pigment-defective mutants derived from pigment-defective colony, sector and papilla, respectively) arising from five WT subclones. Pig–col may result from a mutational event occurring during the sporulation process, Pig–sec from a mutation during vegetative mycelium development and Pig–pap from a mutational event incurred during aerial mycelium formation [25] (figure 2). Pig–sec and Pig–col mutants are not phenotypically distinguishable (figure 2): the degree of colony pigment production varies among these mutants and some of them are also unable to produce antibiotics and/or spores. However, the molecular characterization of these mutants has revealed that the deletion frequencies at the chromosome extremities are about 10-fold lower in Pig–sec than in Pig–col mutants [15]. These results suggest a relationship between the stage of development and the occurrence of rearrangements and hence, of recombination at the chromosomal extremities. The Pig–pap mutants are quite different from the Pig–sec and Pig–col mutants. They harbor a white mycelium, are unable to sporulate and no genomic rearrangements are detected [26, 27]. As explained above, the Pig–sec mutants are likely derived from mutation during mycelium vegetative

DNA rearrangements of the S. ambofaciens linear chromosome

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Figure 1. Different types of mutant chromosomes generated by genomic instability in Streptomyces. A. Mutants arising from a recombination event leading to either a linear chromosomal DNA with reduced TIR (lower part of the figure) or to TIR loss and the subsequent circularization of the chromosome (top of the figure). B. Mutant chromosomal DNA retaining a linear conformation but exhibiting increased TIR.

growth while the Pig–col mutants arise from a mutation during the sporulation process. Development of vegetative mycelium occurs on medium where nutrients are not limited, hence this phase might be associated with rapid growth. Conversely, the sporulation process is observed in a situation where nutrients have become limited. This last phase of differentiation ensures the dissemination of the species and hence, its survival. During this phase, genome

rearrangements might be favored to insure species survival through diversification. Such a developmental control of diversity is observed in prokaryotic and eukaryotic organisms. Thus, the stationary phase of E. coli corresponds to a period of differentiation during which mutation frequencies increase [28]. In eukaryotic organisms, the overall rate of recombination in germinal cells can be 100- to 1000-fold higher than in vegetative cells, contri-

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Figure 2. Formation of S. ambofaciens pigment-defective mutants at different stages of development. Pig-col, Pig-sec and Pig-pap mutants arise from a pigment-defective colony, sector and papilla, respectively.

buting to the genetic diversity of the gametes [29]. In the preceding section we have evoked the possibility that genome plasticity results from DSB formation. It must be noted that DSBs can be specifically generated during certain growth phases [29]. Finally, two alternate hypotheses could account for the characteristics of Pig–pap mutants. The first involves an epigenetic modification leading to the switching off of specific genes responsible for the sporulation process as in the eukaryotes silencing phenomena. It must be emphasized that a variation of the methylation state of DNA, which is known to influence gene expression and recombination, occurs during the life cycle of Streptomyces [30]. The second hypothesis is that the Pig–pap mutants result from classic mutational events. 4. Modulation of recombination in S. ambofaciens according to mutator state(s) We have shown that the number of papillae per colony does not randomly fluctuate but is genetically determined. Thus, a colony exhibiting a high number of papillae (> 20;

hyperpap colony) (figure 3) yields more hyperpap colonies than a colony having few papillae. In 12/12 cases, a mutator strain has been isolated from a hyperpap colony. The mutator clones produce far more Pig–col (more than 10%) and hyperpap colonies (almost all colonies are hyperpap) than their WT ancestor (about 1% Pig–col and hyperpap). The molecular characterization of Pig–col mutants derived from five independent WT clones (isolated from the same S. ambofaciens strain) and three mutators (derived from three of the five WT clones) revealed a variation in the deletion frequencies at the chromosome ends [15, 27]. In each WT progeny, the deleted Pig–col have deletions in either the right chromosomal extremity or in both extremities. Although multiple recombination sites existed, mutants have been regrouped in two categories according to the location of these mutations. The first category corresponds to mutants deleted in the right chromosomal arm and the second to mutants deleted in both extremities. Hence, we have observed that two WT clones predominantly produce mutants deleted on the right chromosomal arm, suggesting that, in these clones, the recombination sites located on the right chromosomal arm are used more frequently than in

DNA rearrangements of the S. ambofaciens linear chromosome

Figure 3. Hyperpap colony of S. ambofaciens. This colony (of about 14 mm) was observed after 14 days of growth at 30 °C on solid medium.

the three other WT clones. These results suggest that within the same strain, the zone where genome rearrangements preferentially occur might differ from one clone to another [15]. Taken together, these results show the existence of an endogeneous control of genome rearrangements. We proposed that a mutator state of variable intensity arises during colony development. Such a mutator state would vary from a low level (as is seen in WT subclones) to a high level (as is seen in mutator strains). Although the molecular mechanism of such a mutator state remains unknown, the quantitative nature of this phenomenon points towards a number of hypotheses. One explanation is that an epigenetic modification such as DNA methylation is responsible for the control of rearrangement frequency. An alternative explanation may lie in a quantitative modification of the nucleotide sequence such as repetition of a nucleotide motif in gene(s) controlling genome integrity. Because some human diseases are associated with both expansion of nucleotide triplets and modification of the methylation pattern [31], we propose that these two hypotheses are not conflicting. References [1] Louarn J.M., Louarn J., Francois V., Patte J., Analysis and possible role of hyperrecombination in the termination region of the Escherichia coli chromosome, J. Bacteriol. 173 (1991) 5097–5104. [2] Pryde F.E., Gorham H.C., Louis E.J., Chromosome ends: all the same under their caps, Curr. Opin. Genet. Dev. 7 (1997) 822–828. [3] Louis E.J., Naumova E.S., Lee A., Naumov G., Haber J.E., The chromosome end in yeast: its mosaic nature and influence on recombinational dynamics, Genetics 136 (1994) 789–802. [4] Leblond P., Decaris B., Unstable linear chromosomes: the case of Streptomyces, in: Charlebois R.L. (Ed.), Organization of the prokaryotic genome, American Society for Microbiology, Washington D.C., 1999, in press. [5] Musialowski M.S., Flett F., Scott G.B., Hobbs G., Smith C., Oliver S.G., Functional evidence that the principal DNA replication origin of the Streptomyces coelicolor chromosome is close to dnaA-gyrB region, J. Bacteriol. 176 (1994) 5123–5125.

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