Evolutionary Trajectory of the Replication Mode of Bacterial Replicons

Abstract

As typical bacterial replicons, circular chromosomes replicate bidirectionally and circular plasmids replicate either bidirectionally or unidirectionally. Whereas the finding of chromids (plasmid-derived chromosomes) in multiple bacterial lineages provides circumstantial evidence that chromosomes likely evolved from plasmids, all experimentally assayed chromids were shown to use bidirectional replication. Here, we employed a model system, the marine bacterial genus Pseudoalteromonas, members of which consistently carry a chromosome and a chromid. We provide experimental and bioinformatic evidence that while chromids in a few strains replicate bidirectionally, most replicate unidirectionally. This is the first experimental demonstration of the unidirectional replication mode in bacterial chromids. Phylogenomic and comparative genomic analyses showed that the bidirectional replication evolved only once from a unidirectional ancestor and that this transition was associated with insertions of exogenous DNA and relocation of the replication terminus region (ter2) from near the origin site (ori2) to a position roughly opposite it. This process enables a plasmid-derived chromosome to increase its size and expand the bacterium's metabolic versatility while keeping its replication synchronized with that of the main chromosome. A major implication of our study is that the uni- and bidirectionally replicating chromids may represent two stages on the evolutionary trajectory from unidirectionally replicating plasmids to bidirectionally replicating chromosomes in bacteria. Further bioinformatic analyses predicted unidirectionally replicating chromids in several unrelated bacterial phyla, suggesting that evolution from unidirectionally to bidirectionally replicating replicons occurred multiple times in bacteria.IMPORTANCE Chromosome replication is an essential process for cell division. The mode of chromosome replication has important impacts on the structure of the chromosome and replication speed. Bidirectional replication is the rule for bacterial chromosomes, and unidirectional replication has been found only in plasmids. To date, no bacterial chromosomes have been experimentally demonstrated to replicate unidirectionally. Here, we showed that the chromids (plasmid-derived chromosomes) in Pseudoalteromonas replicate either uni- or bidirectionally and that a single evolutionary transition from uni- to bidirectionality explains this diversity. These uni- and bidirectionally replicating chromids likely represent two stages during the evolution from a small and unidirectionally replicating plasmid to a large and bidirectionally replicating chromosome. This study provides insights into both the physiology of chromosome replication and the early evolutionary history of bacterial chromosomes.

Keywords: Pseudoalteromonas; chromid; chromosome evolution; chromosome replication; unidirectional replication.

FIG 1

Survey of replication directions for publicly available complete bacterial genomes. (a) Prediction of replication direction of large replicons (>200 kb) based on GC skew analyses. (b) Predicted unidirectionally replicating replicons. (c) Comparison of the ratio of Chr2 size to Chr1 size for bidirectionally and unidirectionally replicating Chr2 from bacteria with multiple chromosomes and Chr2 from Pseudoalteromonas.

FIG 2

Comparison of the main chromosome and chromid structure for species P. tunicata (a) and P. spongiae (b). The ori site (blue circle), dif site (red rectangle), and genes related to main chromosome/chromid replication and maintenance are indicated on the outer circle. GC skew is shown as the inner circle. Predicted replication directions are shown with blue (clockwise) and green (counterclockwise) arrows.

FIG 3

Analyses of replication direction and synchronization mode by deep genome sequencing. (a and b) Gradient changes of sequencing coverage from the replication origin (ori site) to the replication terminus (represented by dif) along a replicating replicon for P. tunicata DSM 14096^T (a) and P. spongiae JCM 12884^T (b) at the exponential phase. Data were presented in bins of 1,000 bp and were corrected using data from the stationary phase (see Materials and Methods). Note that the y axis was set to a base 2 logarithmic scale. Arrows indicate the replication direction (blue for clockwise and green for counterclockwise). (c) Sequencing coverage for the replication origin regions (ori1 and ori2) and the terminus regions (ter1 and ter2). Sequencing coverage of ter1 and ter2 was represented by the coverage near the dif1 and dif2 sites, respectively.

FIG 4

Phylogenomics analysis of Pseudoalteromonas spp. and comparison of the replicating directions of the chromids of different clades. (a) A maximum-likelihood tree reconstructed based on the concatenated amino acid sequences of shared single-copy genes on the main chromosome. The genus diverged into four major clades: I, II, III, and IV. The replication direction of the chromid was marked for all clades and their ancestors. All branches are supported by the IQ-Tree ultrafast bootstrap percentages of 100%. The bar represents 0.05 substitution per site. See details in Fig. S2a for the complete tree. (b) Colinear analysis of uni- and bidirectionally replicating chromids of Pseudoalteromonas spp. A total of 82 common single-gene families located on the chromids were found among the 43 genomes studied and are indicated using lines between chromids. Genes parA2 and parB2 and the locus ori2 are shown in blue. Genes repA and tus are shown in green. The ter2 region is represented by dif2. The locus dif2 is shown in red. The remaining genes are shown in gray. It is clear that the neighboring region of dif2 is highly conserved. Arrows indicate the replication directions. Asterisks indicate the predicted insertion sequences and integrons, and boxes indicate the predicted prophage-like regions.