Positive Selection during Niche Adaptation Results in Large-Scale and Irreversible Rearrangement of Chromosomal Gene Order in Bacteria

Abstract

Analysis of bacterial genomes shows that, whereas diverse species share many genes in common, their linear order on the chromosome is often not conserved. Whereas rearrangements in gene order could occur by genetic drift, an alternative hypothesis is rearrangement driven by positive selection during niche adaptation (SNAP). Here, we provide the first experimental support for the SNAP hypothesis. We evolved Salmonella to adapt to growth on malate as the sole carbon source and followed the evolutionary trajectories. The initial adaptation to growth in the new environment involved the duplication of 1.66 Mb, corresponding to one-third of the Salmonella chromosome. This duplication is selected to increase the copy number of a single gene, dctA, involved in the uptake of malate. Continuing selection led to the rapid loss or mutation of duplicate genes from either copy of the duplicated region. After 2000 generations, only 31% of the originally duplicated genes remained intact and the gene order within the Salmonella chromosome has been significantly and irreversibly altered. These results experientially validate predictions made by the SNAP hypothesis and show that SNAP can be a strong driving force for rearrangements in chromosomal gene order.

Keywords: Salmonella Typhimurium; SNAP hypothesis; chromosome rearrangements; experimental evolution.

Fig. 1.

The SNAP hypothesis. (a) Schematic overview of the SNAP model. The gene under selection for duplication is shown in dark blue and the regions of homology flanking the ends of the duplicated region are indicated by blue rectangles. Red crosses designate copies of genes that are deleted. (b) Change in duplication size during the stages of the SNAP model. The light blue area indicates the progress of the Mte^{+, 2000G} isolate along the SNAP trajectory (31% of duplication remains). (c) Whole-genome alignment (with a 5 kb block size) of Salmonella Typhimurium (NC_003197) with Escherichia coli (NC_000913) and Proteus mirabilis (NC_010554). Homologous regions displayed below each black line are inverted relative S. typhimurium.

Fig. 2.

Selection and analysis of the Mte⁺ duplication. (a) Comparison of wild-type (WT) and Mte⁺ colonies on minimal malate plates. (b) Normalized read depth coverage of the Mte⁺ isolate. The locations of the six genes involved in malate utilization within the duplicated region are indicated. (c) Schematic overview of the protein functions of the six malate utilization genes included in the duplicated region. (d) Individual genes involved in malate utilization were deleted and Mte⁺ colonies were selected. The read depth analysis of the Mte⁺ colonies carrying deletions of yhiT, meaB, and STM3081 is shown below. (e) Overview of the construction process of single-gene duplications using lambda-red recombineering. (f) Growth curves of strains in minimal malate medium. The curves show the average density ± 95% confidence interval of five biological replicates.

Fig. 3.

Long-term evolution of the Mte⁺ isolate. (a) Schematic overview of the evolution experiment. (b) Read depth and mutation analysis of four strains along an evolutionary trajectory. Nonsynonymous mutations in the coding sequences of genes are indicated by black (nonessential genes) and red (essential genes) lines. (c) Example of mutation analysis in the duplicated region (26% of reads are shown). The consensus nucleotide and amino acid sequences are shown above (glyS wild-type reads) and below (glyS K138* reads). (d) The mutation rate of wild-type and ΔmutL Salmonella in minimal malate medium. (e) Overview of the number of duplicate genes within the Mte⁺ duplication region in the strains along an evolutionary trajectory. (f) Duplication stability assay in four strains along an evolutionary trajectory. Lines indicate the average of three biological replicates. The Mte^{+, 1500G} and Mte^{+, 2000G} isolates are both fully stabilized. (g) Comparison of gene order of wild-type genes between Salmonella (top) and the Mte^{+, 2000G} isolate (bottom). Only genes with a mutated/deleted allele within the duplication region are shown. Changes in the linear order of genes are indicated by gray triangles.

Fig. 4.

Identification of the locations of the large deletion and the mutations in the evolved isolates. (a) The chromosome of the Mte^{+, 500G} isolate was classified into five sections (a–e) and two deletions (ΔL and ΔR) were designed in order to identify the location of section C. The chromosomal order and theoretical result of the two deletions are shown for the case that section C is located in the left copy (left) or the right copy (right) of the duplication. The gray crosses indicate chromosomal structures that are nonviable due to the absence of essential genes. The dashed box shows the number of colonies acquired from the two transformation experiments. (b–g) Schematic overview of deletions designed to identify the location of mutations (left) and read depth coverage analysis of the resulting Mte^{+, 1500G} (b–e) or Mte^{+, 2000G} (f–g) isolates after transformation (right). The dotted black lines indicate the location of the originally duplicated section and the green stars indicate sequences that were used for the localization of the selected mutations.