Evolution of Genome Architecture in Archaea: Spontaneous Generation of a New Chromosome in Haloferax volcanii

Abstract

The common ancestry of archaea and eukaryotes is evident in their genome architecture. All eukaryotic and several archaeal genomes consist of multiple chromosomes, each replicated from multiple origins. Three scenarios have been proposed for the evolution of this genome architecture: 1) mutational diversification of a multi-copy chromosome; 2) capture of a new chromosome by horizontal transfer; 3) acquisition of new origins and splitting into two replication-competent chromosomes. We report an example of the third scenario: the multi-origin chromosome of the archaeon Haloferax volcanii has split into two elements via homologous recombination. The newly generated elements are bona fide chromosomes, because each bears "chromosomal" replication origins, rRNA loci, and essential genes. The new chromosomes were stable during routine growth but additional genetic manipulation, which involves selective bottlenecks, provoked further rearrangements. To the best of our knowledge, rearrangement of a naturally evolved prokaryotic genome to generate two new chromosomes has not been described previously.

Fig. 1.

Genome rearrangement of Δorc5 strain. (A) Location of replication origins and adjacent orc genes on Haloferax volcanii main chromosome (+pHV4). Positions of the two rRNA loci are indicated with black arrows. The integrated pHV4 mini-chromosome is indicated by a thick line. The eight replichores representing the direction of replication forks are shown by colored arrows, corresponding to their respective origins. SfaAI sites are indicated by tick marks. (B) Replication profiles of the Δorc5 mutant H1689 and a reference wild-type (WT) laboratory strain H26. The number of reads is plotted against the chromosomal location. The linearized H. volcanii chromosome showing positions of oriC and orc genes is shown below (colored as in A). Two discontinuities in the Δorc5 replication profile are indicated by vertical arrows. (C) Restriction fragment length polymorphisms in WT and Δorc5 strain as shown by digestion with SfaAI and PFGE. The 390 kb SfaAI fragment (shown on the map in panel A) is absent from the digest of Δorc5 DNA, and a novel 579 kb SfaAI fragment is present; these bands are indicated by arrows.

Fig. 2.

Novel genome architecture of Δorc5 strain. (A) Scheme for outcome of recombination between sod1 and sod2 genes to split the main chromosome (+pHV4) and generate two new chromosomes (new chr 1 and new chr 2). (B) PFGE and Southern blot confirming two new chromosomes in Δorc5 strain. Intact genomic DNA of wild isolate DS2, WT H26 and Δorc5 H1689 strains was probed with sod1 and sod2 sequences. (C) Recombination of sod1 and sod2 genes in Δorc5 strain H1689 was confirmed by end-point PCR using primers to unique sequences flanking sod1 and sod. The identity of the PCR products was validated by DNA sequencing.

Fig. 3.

Genome architecture of the Δorc5 strain is polymorphic. (A) Southern blot conforming location of breakpoints of genome rearrangement in Δorc5 strain. Genomic DNA of WT H26 and Δorc5 H1689 was digested with StyI or EcoRV and probed with sequences adjacent to sod1 or sod2, respectively. A WT-sized band is present in the Δorc5 lanes. (B) Southern blot of PFGE confirming relocation of oriC1 to new chr 2 in Δorc5 strain. SfaAI-digested DNA of WT H26 and Δorc5 H1689 strains was probed with sequences adjacent to oriC1. Relevant SfaAI sites are indicated on the maps, the new chr 1 does not hybridize with oriC1 (map not shown). A faint 390 kb WT-sized band is present in the Δorc5 lane. (C) PFGE confirming new genome architecture of Δorc5 strain. Genomic DNA of WT H26 and Δorc5 H1689 was digested with AvrII or SwaI. Relevant AvrII and SwaI sites are indicated on the outside and inside of chromosome maps, respectively. The 417 bp SwaI fragment is found on pHV3 (not shown), which is not affected by the genome rearrangement.

Fig. 4.

Deletion of orc5 does not increase the rate of genome rearrangement. (A) Scheme showing new replichores in the absence of orc5 (replichores and rRNA loci indicated as in fig. 1A). (B) SfaAI restriction fragment length polymorphisms were not seen in unrelated strains with different combinations of orc and oriC deletion. Strain genotypes are indicated below. (C) SfaAI-digested genomic DNA of 25 independently derived Δorc4 mutants and 25 independently derived Δorc5 mutants. Representative images, the Δorc4 clone and Δorc5 clone with a genome rearrangement are indicated by an asterisk.

Fig. 5.

New genome architectures of Δorc5 derivatives. (A) AvrII and SfaAI digests of genomic DNA from derivatives of Δorc5 strain H1689 identifying four different genome states. Strain genotypes and genome architecture state is indicated below, polymorphic and monomorphic refer to strains with H1689-type genome rearrangements. The monomorphic Δorc5 Δorc3 strain H2202 is indicated. (B) Southern blots showing that additional genome rearrangements in derivatives of Δorc5 strain H1689 did not involve recombination of the sod gene region. Genomic DNA was digested with StyI or EcoRV and probed with sequences adjacent to sod1 or sod2, respectively (for key to restriction fragments, see fig. 3A). (C) Replication profile of Δorc5 Δorc3 strain H2202 (lane 9 in panels A and B) where the genome is in a monomorphic state. Labeled as in figure 1B, the two discontinuities in the replication profile are indicated by vertical arrows. (D) Replication profile of Δorc5 Δorc3 strain H2202 remapped to sequences corresponding to new chr 1 and new chr 2.