The impact of the neisserial DNA uptake sequences on genome evolution and stability

Abstract

Background: Efficient natural transformation in Neisseria requires the presence of short DNA uptake sequences (DUSs). Doubts remain whether DUSs propagate by pure selfish molecular drive or are selected for 'safe sex' among conspecifics.

Results: Six neisserial genomes were aligned to identify gene conversion fragments, DUS distribution, spacing, and conservation. We found a strong link between recombination and DUS: DUS spacing matches the size of conversion fragments; genomes with shorter conversion fragments have more DUSs and more conserved DUSs; and conversion fragments are enriched in DUSs. Many recent and singly occurring DUSs exhibit too high divergence with homologous sequences in other genomes to have arisen by point mutation, suggesting their appearance by recombination. DUSs are over-represented in the core genome, under-represented in regions under diversification, and absent in both recently acquired genes and recently lost core genes. This suggests that DUSs are implicated in genome stability rather than in generating adaptive variation. DUS elements are most frequent in the permissive locations of the core genome but are themselves highly conserved, undergoing mutation selection balance and/or molecular drive. Similar preliminary results were found for the functionally analogous uptake signal sequence in Pasteurellaceae.

Conclusion: As do many other pathogens, Neisseria and Pasteurellaceae have hyperdynamic genomes that generate deleterious mutations by intrachromosomal recombination and by transient hypermutation. The results presented here suggest that transformation in Neisseria and Pasteurellaceae allows them to counteract the deleterious effects of genome instability in the core genome. Thus, rather than promoting hypervariation, bacterial sex could be regenerative.

Figure 1

Visual representation of M-GCAT's multiple alignment of six Neisseria genomes. The horizontal lines correspond to a linear representation of each genome sequence. The vertical polygons represent the 79 M-GCAT clusters of average length 28,153 nucleotides. Evident from the visual representation of the alignment is that there are many rearrangements throughout the genome comparison. Inverted vertical polygons depict an inversion between two of the genome sequences. The small rectangles overlapping the horizontal lines correspond to the standardized MUSCLE alignment score for each respective M-GCAT cluster; darker intensities indicate better scores. In total, 82.5% of the original genome sequences are covered by the multiple alignment, and 17.5% was left unaligned. kb, kilobases.

Figure 2

Consistent neisserial phylogenetic trees. (a) From the concatenated ubiquitous gene alignment (numbers indicate nonparametric bootstrap results in percentage out of 1,000 experiments); (b) from the concatenated 1,000 nucleotides regions (± 500 nucleotides) surrounding ubiquitous DNA uptake sequence (DUS) sites in the M-GCAT multiple alignment; and (c) entire concatenated M-GCAT multiple alignment. Distance matrices were computed from the alignments using Tree-Puzzle [85] by maximum likelihood with the HKY+Γ model and trees computed with BIONJ [86].

Figure 3

Distribution of lengths of gene conversion events. We used GENECONV to predict gene conversion events in each pair of sequences of the multiple alignment. (a) Gene conversion events were predicted only for Neisseria meningitidis sequence pairs (six total), in which we identified 1,469 gene conversion fragments with an average length of 1,728 nucleotides. (b) We identified 2,717 putative gene conversion fragments in all pairs of sequences (15 total) with an average length of 1,127 nucleotides (nt; indicated by the vertical dashed line). In both figures, the height of each bar in the figure indicates the number of gene conversion fragments with the specified length.

Figure 4

DUS degeneracy. (a) Histogram of the degeneracy of DNA uptake sequence (DUS) elements in Neisseria gonorrhoeae that are exact DUSs (nondegenerate) in all of the other five genomes. These DUSs have most likely degenerated in the N. gonorrhoeae lineage. The number of substitutions (blue striped bars) and gaps (red striped bars) present in each degenerate DUS site in N. gonorrhoeae were calculated. The x-axis labels are the number of each type of mutation for each case, for example 1 to 10 substitutions or gaps. (b) The same analysis as shown in panel a but for DUSs that are present in at least one strain of Neisseria meningitidis but are absent in both N. gonorrhoeae and Neisseria lactamica. Most of these DUSs are not expected to be ancestral. The degeneracy in this case was measured in the N. lactamica sequences facing the N. meningitidis DUS, and is similar when N. gonorrhoeae is used instead. (c) Weblogo [87] of the degeneracy of DUS sites in one N. meningitidis strain. A weblogo is a graphical representation of a multiple sequence alignment in which the height of the bases in each position indicates their relative frequency, whereas the overall weight of the stack indicates the conservation of that position in the motif. Similar weblogos are found for the other genomes.

Figure 5

DUS alignment sites. Two examples of aligned DNA uptake sequence (DUS) sites in the multiple alignment of the genomes. The red rectangle surrounds the columns that delimit the DUS sequence, which is presented in bold. The positions are positive if the sequence is in the published strand and in negative if they are in the complementary strand. (a) A region of the multiple alignment containing the 3'-TTCAGACGGC-5' reverse complement of the DUS exclusively in Neisseria gonorrhoeae FA1090. (b) A second region from the multiple alignment containing the 3'-GCCGTCTGAA-5' DUS sequence in N. gonorrhoeae FA1090, and showing an altered DUS with one substitution in the remaining sequences.

Figure 6

Within the core genome the DUS proximal regions accumulate more substitutions. The black line represents the percent identity for regions surrounding all exactly conserved DNA uptake sequence (DUS) in the multiple alignments. The red line corresponds to the percentage identity for regions surrounding randomly selected DUS-less sites in the multiple alignment. (a) All DUSs analyzed. (b) Only DUSs contained inside protein coding sequences.

Figure 7

Distribution of the values of DUSp among all positions in the N. meningitidis Z2491 genome. All DNA uptake sequence (DUS) positions in N. meningitidis Z2491 were excluded from the histogram. DUSp, DUS proximity.