The Role of the DNA Uptake Sequence in Natural Transformation of the Obligate Human Pathogen Neisseria gonorrhoeae

Natural transformation is the main means of horizontal genetic exchange in the obligate human pathogen Neisseria gonorrhoeae. Neisseria spp. have been shown to preferentially take up and transform their own DNA by recognizing the nonpalindromic 10- or 12-nucleotide sequence 5'-ATGCCGTCTGAA-3', termed the DNA uptake sequence (DUS). This thesis investigates the effects of the DUS on transformation and DNA uptake for two laboratory strains of N. gonorrhoeae. I found that both strains showed efficient transformation of DUS containing DNA but that the level of transformation with DNA lacking a DUS (DUS0) was variable in the different strains. The DUS-enhanced transformation was 20-fold in strain FA1090, but was approximately 150-fold in strain MS11. Both strains tested provide some level of DUS0 transformation, and DUS0 transformation was type IV pilus dependent. Competition with plasmid DNA revealed that transformation of MS11 was enhanced by the addition of excess plasmid DNA containing a DUS while FA1090 transformation was competitively inhibited. Although FA1090 was able to mediate much more efficient transformation of DNA lacking a DUS than was MS11, DNA uptake experiments showed similar levels of uptake of DNA containing and lacking a DUS in FA1090 and MS11. DNA uptake was competitively inhibited in both FA1090 and MS11; supporting the notion that DNA uptake is similar in both FA1090 and MS11 and is enhanced by the DUS. Transported DNA is processed in the periplasm and the resultant nucleotides are incorporated into the chromosome during replication. DUS recognition leading to DNA uptake is dependent on the inner membrane protein ComA in FA1090, but not in MS11, suggesting other strain-dependent differences during transformation. Investigations into single-stranded DNA (ssDNA) transformation revealed that ssDNA transformation is less efficient than matched dsDNA transformation, and there is a recombination bias for the coding strand of the transforming DNA gyrB1. Furthermore, one strand of the DUS is much more active to enhance ssDNA transformation compared to the other strand of the DUS. Taken together, these data indicate that the role of the DUS during DNA transformation is variable between strains of N. gonorrhoeae and that the DUS has multiple roles during transformation.