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.

Major Intercontinentally Distributed Sequence Types of Kingella kingae and Development of a Rapid Molecular Typing Tool

“…DNA was extracted from specimens with the BioRobot EZ1 workstation using the EZ1 DNA tissue kit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations and stored at Ϫ80°C. The DUS, previously described for K. kingae (19), was used to design the primer king3DUS (5=-AAGCAGCCT GCA-3=). DNA PCR amplification was performed in a 50-l reaction mixture that contained 25 l of multiplex PCR master mix and 10 l of Q-Solution (Qiagen), 1 l of primer stock solution (50 M), and 2 l of DNA (5 ng/l).…”

“…Therefore, the development of a rapid and cost-effective method is still pending. The DNA uptake sequence (DUS) is a short sequence required by certain bacteria, notably Neisseriaceae, for the acquisition of extracellular DNA (19,20). The DUS, recently identified in K. kingae, consists of 12 nucleotides that are present on either the positive or negative DNA strand and are repeated in 2,787 copies in the ATCC 23330 K. kingae type strain genome (19).…”

“…The DNA uptake sequence (DUS) is a short sequence required by certain bacteria, notably Neisseriaceae, for the acquisition of extracellular DNA (19,20). The DUS, recently identified in K. kingae, consists of 12 nucleotides that are present on either the positive or negative DNA strand and are repeated in 2,787 copies in the ATCC 23330 K. kingae type strain genome (19). Given that the genome size of K. kingae is approximately 2 Mb (21,22), the DUS should be randomly repeated, on average, every 500 to 1,000 bp; therefore, we hypothesized that these 12 nucleotides may serve as a potential PCR target for studying the genomic polymorphism of the strains using a one-primer amplification method.…”

Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism

“…Single-stranded DNA is transported through a pore formed by ComA through the inner membrane (28). The probability of DNA uptake is strongly enhanced by the presence of the DNA uptake sequence (DUS) (29). This 12-bp-long sequence occurs at high frequency on the gonococcal genome, conveying species-specific recognition of DNA.…”

Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations

“…Interestingly, some species (including Neisseriaceae and Haemophilus influenzae) strongly select for self-DNA by preferentially taking up DNA with a recognition sequence (DNA Uptake Sequence, DUS). [13,14] For Neisseriaceae, specificity is achieved by binding to a minor pilin, ComP, that is most likely part of the competence pilus. [15,16] For Vibrio cholerae, there is no evidence for sequence-preference during DNA uptake.…”