Identification of DNA motifs implicated in maintenance of bacterial core genomes by predictive modeling

Abstract

Bacterial biodiversity at the species level, in terms of gene acquisition or loss, is so immense that it raises the question of how essential chromosomal regions are spared from uncontrolled rearrangements. Protection of the genome likely depends on specific DNA motifs that impose limits on the regions that undergo recombination. Although most such motifs remain unidentified, they are theoretically predictable based on their genomic distribution properties. We examined the distribution of the "crossover hotspot instigator," or Chi, in Escherichia coli, and found that its exceptional distribution is restricted to the core genome common to three strains. We then formulated a set of criteria that were incorporated in a statistical model to search core genomes for motifs potentially involved in genome stability in other species. Our strategy led us to identify and biologically validate two distinct heptamers that possess Chi properties, one in Staphylococcus aureus, and the other in several streptococci. This strategy paves the way for wide-scale discovery of other important functional noncoding motifs that distinguish core genomes from the strain-variable regions.

Figure 1. The E. coli Chi site, 5′-GCTGGTGG-3′, Is Significantly Overrepresented and Skewed on E. coli Backbone DNA

Statistical scores of overrepresentation and skew are plotted for all octamers whose overrepresentation scores are higher than 5 (corresponding to a p-value < 10⁻⁷). Chi and one of the motifs of the KOPS family [6] are indicated.

Figure 2. Motif C, 5′-GAAGCGG-3′, Is the S. aureus Chi Site

(A) Overrepresentation and skew of heptamers on the S. aureus backbone. Plots are represented as in Figure 1. Only heptamers with an overrepresentation score higher than 5 (corresponding to a p-value < 10⁻⁷) are shown. The four best candidates (motifs A to D) are indicated. (B) Experimental validation that motif C has Chi activity in S. aureus. Motif C was cloned in plasmid pRC and evaluated for HMW accumulation, an indicator of Chi activity [27]. Total genomic DNA was hybridized with a probe specific to the RC plasmid. Plasmid monomer (mn), dimer (dm), and open circle (oc) forms, and HMW are indicated by arrows. Motif C (lane 2, 5′-GAAGCGG-3′) is HMW-positive, but it gives a negative signal when cloned in the reverse orientation (lane 3) or when it carries a mismatch (lanes 4–6).

Figure 3. Chi Sequence Conservation through Evolution

The tree shows phylogenetic relationships of the eight bacterial species in which Chi is known, based on 16S RNA, and was constructed using the tree-builder command of the Ribosomal Database Project (https://rdp.cme.msu.edu/). E. coli, H. influenzae, L. lactis, and B. subtilis Chi sites were previously identified [5,25,26]. Chi sites of S. aureus and the three streptococci were identified by predictive modeling and confirmed experimentally (this work). Partial conservation of Chi correlates with phylogenetic conservation among bacteria.