Chromosome evolution in the Thermotogales: large-scale inversions and strain diversification of CRISPR sequences

Abstract

In the present study, the chromosomes of two members of the Thermotogales were compared. A whole-genome alignment of Thermotoga maritima MSB8 and Thermotoga neapolitana NS-E has revealed numerous large-scale DNA rearrangements, most of which are associated with CRISPR DNA repeats and/or tRNA genes. These DNA rearrangements do not include the putative origin of DNA replication but move within the same replichore, i.e., the same replicating half of the chromosome (delimited by the replication origin and terminus). Based on cumulative GC skew analysis, both the T. maritima and T. neapolitana lineages contain one or two major inverted DNA segments. Also, based on PCR amplification and sequence analysis of the DNA joints that are associated with the major rearrangements, the overall chromosome architecture was found to be conserved at most DNA joints for other strains of T. neapolitana. Taken together, the results from this analysis suggest that the observed chromosomal rearrangements in the Thermotogales likely occurred by successive inversions after their divergence from a common ancestor and before strain diversification. Finally, sequence analysis shows that size polymorphisms in the DNA joints associated with CRISPRs can be explained by expansion and possibly contraction of the DNA repeat and spacer unit, providing a tool for discerning the relatedness of strains from different geographic locations.

FIG. 1.

Whole-genome amino acid alignments between T. maritima strain MSB8 and T. neapolitana strain NS-E. The Promer algorithm was used to calculate and plot the amino acid percentage identity of maximally unique matching subsequences of at least 5 amino acids between the two genomes. A point (x,y) indicates a sequence that occurs once within each genome, at location x in one genome and at location y in the other genome. The matching sequences may occur on either the forward or the reverse strand; in either case, the locations indicate the 5′ end of the sequences. The point 0,0 corresponds to the putative origin of replication for each genome. Panel A shows the alignment at the whole-genome level. Two regions of interest, boxes B and C, are shown in more detail in panels B and C, respectively.

FIG. 2.

Cumulative GC skew and ORF orientation in the T. maritima strain MSB8 and T. neapolitana strain NS-E genomes. (A and B) Plots of cumulative GC skew calculated with 1-kb windows. (C and D) Plots of the running sum of ORF orientation. (E and F) Expanded plots of GC skew for the pink regions displayed in panels A and B and calculated with a 100-bp window. The four regions in T. maritima (E) and T. neapolitana (F) are putative inverted segments revealed by the whole-genome alignment (Fig. 1). The asterisks in panels A and B correspond to the putative origin of DNA replication, as described by Lopez and coworkers (21). The roman numerals correspond to the nomenclature of the different chromosomal regions displayed in Fig. 1.

FIG. 3.

CRISPR motifs in the genomes of T. maritima strain MSB8 and T. neapolitana strain NS-E. The CRISPR sequences were generated from three different multiple sequence alignments that were compiled by DNA HMM searches. Note the overlapping identity between the two 30-mers in the different Thermotoga species. Also, all occurrences of the 29-mer are located in a single region in T. neapolitana which is absent from T. maritima.

FIG. 4.

An ethidium bromide-stained agarose gel of PCR products for each of the T. neapolitana species at the 15 DNA joints described in Materials and Methods.

FIG. 5.

Two-step model of successive inversions to produce an inversion of one DNA segment (B to B′) and a concomitant translocation of an adjoining DNA segment (C). Two alternative pathways, which differ in the order of the larger and smaller inversions, are proposed: the green path begins with a large inversion, and the red path ends with a large inversion. In both pathways, segment C is inverted in the first step, and it is proposed that there was positive selective pressure favoring a subsequent cell population in which segment C is restored to its original orientation after the second step.