Abstract
Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination.
Highlights
Competence for transformation in the pathogenic Neisseria meningitidis and Neisseria gonorrhoeae has been studied for more than half a century and is a highly evolved and complex process where multiple proteins at the bacterial surface, in the membranes and in the cytoplasm are in contact with the transforming DNA [1,2,3], for reviews see [4,5,6]
A BLAST search with the amino acid sequence of NMB1032, the NlaIV protein in N. meningitidis MC58, revealed that this gene is common in N. gonorrhoeae strains and present in N. lactamica, N. flavescens and N. polysacchareae strains, but absent in all other sequenced species of the genus Neisseria
DNA Uptake Sequence (DUS) is generally required for efficient transformation, DUS-independent transformation has been documented in certain gonococcal strains [29,32]
Summary
Competence for transformation in the pathogenic Neisseria meningitidis and Neisseria gonorrhoeae has been studied for more than half a century and is a highly evolved and complex process where multiple proteins at the bacterial surface, in the membranes and in the cytoplasm are in contact with the transforming DNA [1,2,3], for reviews see [4,5,6]. Since the stretch of DNA involved in gene conversion events was found to be longer in strains within PCs than between different PCs, an intriguing model was proposed in which RM is an important player in the genesis and persistence of PCs. The impact of RMSs in the evolution of the highly recombinogenic meningococci makes a strong case for RMS as an efficient barrier towards DNA exchange and a major driver of sexual isolation and speciation [12], at least in meningococci. The impact of RMSs in the evolution of the highly recombinogenic meningococci makes a strong case for RMS as an efficient barrier towards DNA exchange and a major driver of sexual isolation and speciation [12], at least in meningococci Another factor to influence the fluidity of DNA by way of transformation in meningococci and gonococci is exerted by the DNA Uptake Sequence (DUS) [16,17]. Substantial variation between strains of N. gonorrhoeae with regard to DUS specificity and transformation efficacy has been documented, and it has been proposed that DUS may influence multiple steps during transformation [29] which may obscure the study of DUS activity and specificity
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