Mobile genetic elements (MGEs) are key factors responsible for dissemination of virulence determinants and antimicrobial-resistance genes amongst pathogenic bacteria. Conjugative MGEs are notable for their high gene loads donated per transfer event, broad host ranges and phylogenetic ubiquity amongst prokaryotes, with the subclass of chromosomally inserted integrative and conjugative elements (ICEs) being particularly abundant. The focus on a small number of model systems has biased the study of ICEs towards those conferring readily selectable phenotypes to host cells, whereas the identification and characterization of integrated cryptic elements remains challenging. Even though antimicrobial resistance and horizontally acquired virulence genes are major factors aggravating neisserial infection, conjugative MGEs of Neisseria gonorrhoeae and Neisseria meningitidis remain poorly characterized. Using a phenotype-independent approach based on atypical distributions of DNA uptake sequences (DUSs) in MGEs relative to the chromosomal background, we have identified two groups of chromosomally integrated conjugative elements in Neisseria : one found almost exclusively in pathogenic species possibly deriving from the genus Kingella , the other belonging to a group of Neisseria mucosa -like commensals. The former element appears to enable transfer of traditionally gonococcal-specific loci such as the virulence-associated toxin–antitoxin system fitAB to N. meningitidis chromosomes, whilst the circular form of the latter possesses a unique attachment site ( attP ) sequence seemingly adapted to exploit DUS motifs as chromosomal integration sites. In addition to validating the use of DUS distributions in Neisseriaceae MGE identification, the >170 identified ICE sequences provide a valuable resource for future studies of ICE evolution and host adaptation.

Neisseria gonorrhoeae , the causative agent of gonorrhoea, is a major burden on global healthcare systems, with an estimated ~80–90 million new global cases annually. This burden is exacerbated by increasing levels of antimicrobial resistance, which has greatly limited viable antimicrobial therapies. Decreasing gonococcal drug susceptibility has been driven largely by accumulation of chromosomal resistance determinants, which can be acquired through natural transformation, whereby DNA in the extracellular milieu is imported into cells and incorporated into the genome by homologous recombination. N. gonorrhoeae possesses a specialized system for DNA uptake, which strongly biases transformation in favour of DNA from closely related bacteria by recognizing a 10–12 bp DNA uptake sequence (DUS) motif, which is highly overrepresented in their chromosomal DNA. This process relies on numerous proteins, including the DUS-specific receptor ComP, which assemble retractile protein filaments termed type IV pili (T4P) extending from the cell surface, and one model for neisserial DNA uptake proposes that these filaments bind DNA in a DUS-dependent manner before retracting to transport DNA into the periplasm. However, conflicting evidence indicates that elongated pilus filaments may not have such a direct role in DNA binding uptake as this model suggests. Here, we quantitatively measured DNA binding to gonococcal T4P fibres by directly visualizing binding complexes with confocal fluorescence microscopy in order to confirm the sequence-specific, comP-dependent DNA binding capacity of elongated T4P fibres. This supports the idea that pilus filaments could be responsible for initially capturing DNA in the first step of sequence-specific DNA uptake.