Dialects of the DNA uptake sequence in Neisseriaceae

Abstract

In all sexual organisms, adaptations exist that secure the safe reassortment of homologous alleles and prevent the intrusion of potentially hazardous alien DNA. Some bacteria engage in a simple form of sex known as transformation. In the human pathogen Neisseria meningitidis and in related bacterial species, transformation by exogenous DNA is regulated by the presence of a specific DNA Uptake Sequence (DUS), which is present in thousands of copies in the respective genomes. DUS affects transformation by limiting DNA uptake and recombination in favour of homologous DNA. The specific mechanisms of DUS-dependent genetic transformation have remained elusive. Bioinformatic analyses of family Neisseriaceae genomes reveal eight distinct variants of DUS. These variants are here termed DUS dialects, and their effect on interspecies commutation is demonstrated. Each of the DUS dialects is remarkably conserved within each species and is distributed consistent with a robust Neisseriaceae phylogeny based on core genome sequences. The impact of individual single nucleotide transversions in DUS on meningococcal transformation and on DNA binding and uptake is analysed. The results show that a DUS core 5'-CTG-3' is required for transformation and that transversions in this core reduce DNA uptake more than two orders of magnitude although the level of DNA binding remains less affected. Distinct DUS dialects are efficient barriers to interspecies recombination in N. meningitidis, N. elongata, Kingella denitrificans, and Eikenella corrodens, despite the presence of the core sequence. The degree of similarity between the DUS dialect of the recipient species and the donor DNA directly correlates with the level of transformation and DNA binding and uptake. Finally, DUS-dependent transformation is documented in the genera Eikenella and Kingella for the first time. The results presented here advance our understanding of the function and evolution of DUS and genetic transformation in bacteria, and define the phylogenetic relationships within the Neisseriaceae family.

Figure 1. Alignment of eight distinct DUS dialects.

Alignment of the eight different DUS dialects identified in the genomes of Neisseriaceae family members. Two nucleotides at the 5′-end of DUS are included and the numbering is given on the top. Relative shading indicate the level of sequence conservation.

Figure 2. Phylogenetic tree based on the core genome and DUS dialect distribution.

The phylogenetic tree obtained by the EDGAR service based on the core genome sequences of representative bacteria used in this study. The occurrences of the DUS dialects evolved are indicated. The scale bar represents 0.01 substitutions per nucleotide site. Chromobacterium violaceum was used as an outgroup. The core genome for this tree consisted of 474 coding sequences. On the right is the 16SrDNA based cladogram with the connectors of the dots showing the relation to the phylogram on the left.

Figure 3. Effects of point mutations in the DUS on quantitative transformation and DNA binding and uptake of N. meningitidis MC58.

(A) Quantitative transformation of Neisseria meningitidis strain MC58 with plasmid DNA containing a panel of modified DUS sequences. Transformation rates are shown as percentage relative to the AT-DUS. Standard deviations from 7 independent experiments are indicated by bars. (B) DNA binding (gray bars) and uptake (white bars) assay with radiolabelled plasmid DNA. Average values from 4 independent experiments are shown as percentage of total DNA added. T-test results for (A) transformation rates compared to AT-DUS and (B) for DNA binding versus uptake are indicated by stars (p≤0.2 = *, p≤0.05 = **, p≤0.001 = ***). DUS sequence transversions are given as abscissa labels. The sequence logo is based on the 2742 occurrences of DUS, with a single nucleotide divergence allowed, found in the genome of N. meningitidis MC58.

Figure 4. Quantitative transformation of N. meningitidis and binding and uptake of DNA with DUS from other Neisseriaceae.

(A) Quantitative transformation of Neisseria meningitidis strain MC58 with plasmid DNA containing different DUS dialects and with the Haemophilus influenzae USS. Transformation rates are shown as percent relative to the AT-DUS. The standard deviations from 6 independent experiments are indicated by bars. (B) DNA binding (gray bars) and uptake (white bars) assay with radiolabelled plasmid DNA. Average values from 3 independent experiments are shown as percentage of total DNA added. Abscissa labels give the DUS variant. Statistic analysis as in Figure 3.

Figure 5. Quantification of DNA binding and uptake of Neisseriaceae with different DUS variants.

Total DNA binding shown as gray bars and benzonase resistant DNA (uptake) shown as white bars plotted as percentage of DNA added. The species tested are Neisseria mucosa (A), Neisseria elongata subsp. glycolytica (B), Kingella oralis (C) and Eikenella corrodens (C). Abscissa labels give the DUS variant. Represented are the results from 3 (A, D) and 5 (B, C) independent experiments. Student's t-test values are indicated (p≤0.1 = *, p≤0.05 = **, p≤0.001 = ***).