Generating Knock-Out and Complementation Strains of Neisseria meningitidis
The human-restricted pathogens Neisseria meningitidis and Neisseria gonorrhoeae are naturally competent for DNA uptake. This trait has been exploited extensively for genetic manipulation of these bacteria in the laboratory. Most transformation protocols w
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1. Introduction Neisseria meningitidis, a causative agent of sepsis and meningitis, is a human-restricted Gram-negative bacterium which is naturally competent for transformation throughout its entire life cycle (1). This trait is illustrated by the extensive horizontal genetic exchange occurring in vivo and has also greatly facilitated the generation of mutants in the laboratory. Meningococci discriminate between self and nonself DNA, in that DNA containing a 10-bp sequence, called DUS for DNA Uptake Sequence, is taken up with much higher efficiency than DNA lacking this sequence. This DUS (5¢ GCCGTCTGAA 3¢) occurs around 2,000 times in the average 2.3 Mbp genomes of N. meningitidis, occupying as much as 1% of the chromosome. Addition of two semiconserved residues 5¢ of the 10-mer DUS, yielding the 12-mer DUS 5¢ATGCCGTCTGAA 3¢, increases transformation efficiency in some strains (2). Myron Christodoulides (ed.), Neisseria meningitidis: Advanced Methods and Protocols, Methods in Molecular Biology, vol. 799, DOI 10.1007/978-1-61779-346-2_4, © Springer Science+Business Media, LLC 2012
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V. van Dam and M.P. Bos
Transformation involves multiple steps, including DNA uptake, processing, and chromosomal integration (3). The basis of transformation in the closely related species N. gonorrhoeae has been studied extensively (4, 5). Uptake of genus-specific DNA (6) is mediated by type IV pili at the bacterial surface. In N. gonorrhoeae, double-stranded plasmid DNA is linearized during DNA uptake and cleaved in a way which is probably not site specific (7). Next, incoming DNA is converted, in part, to a single-stranded intermediate (8), while the subsequent events are not well defined. Not as much is known about transformation in N. meningitidis (9), but it is generally thought to resemble the process observed in N. gonorrhoeae. The most straightforward way to create mutants is to replace the relevant gene with an antibiotic resistance marker, allowing for easy selection. However, for obvious safety reasons, it is recommended to limit the number of different antibiotic resistance markers in a meningococcal strain. One way to avoid creating multiresistant strains is to construct markerless mutants. For N. gonorrhoeae, it was shown that incubation of a small number of bacteria with an excess amount of DNA results in such high transformation efficiencies that no selection is necessary as at least 20% of the resulting colonies is correctly transformed (10). Unfortunately, we did not obtain similar success with N. meningitidis using this approach. A different way to construct markerless mutants in N. gonorrhoeae was developed by Johnston and Cannon (11) by exploiting the counter-selective properties of the rpsL gene encoding ribosomal protein 12. This strategy involves the use of a two-gene cassette that contains both a selectable marker (ermC, conferring erythromycin resistance) and a counter-selectable marker (rpsL). Streptomycin resistance can be mediated by a particular allele of the rpsL gene, rpsLR. The sensitive rpsL
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