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Role and visualization of the single-stranded and double-stranded DNA in the biofilm of Neisseria gonorrhoeae
Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhoea, is a Gram-negative human-adapted diplococcus. 80% of the clinical N. gonorrhoeae isolates encode an unusual Type IV secretion system (T4SS) within the horizontally acquired region - the Gonococcal Genetic Isla...
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| Format: | Dissertation |
| Sprache: | Englisch |
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Philipps-Universität Marburg
2013
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| Zusammenfassung: | Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhoea, is a Gram-negative human-adapted diplococcus. 80% of the clinical N. gonorrhoeae isolates encode an unusual Type IV secretion system (T4SS) within the horizontally acquired region - the Gonococcal Genetic Island (GGI). Next to the Single Stranded DNA binding protein (SSB) encoded on the chromosome, a second SSB, SsbB, is encoded within the GGI. SSBs are highly conserved, essential proteins found in all kingdoms of life. They bind single stranded DNA (ssDNA) with high affinity, have low sequence specificity and are involved in DNA recombination, DNA replication and DNA repair. A second copy of SSBs can be encoded on the chromosome or on a plasmid. These SSBs can be involved in diverse mechanisms like natural competence, plasmid segregation and DNA transport. We analyzed the physiological role of SsbB and characterized its function biochemically. We found that close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins. Remarkably, in contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB formed a stable tetramer. Electrophoretic mobility shift assays, fluorescence titration assays, as well as atomic force microscopy demonstrated that SsbB binds ssDNA specifically with high affinity. SsbB binds single stranded DNA with minimal binding frames of 15 and 70 nucleotides for one or two SsbB tetramersrespectively. The binding mode was independent of increasing Mg2+ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity. We propose that these novel SsbBs play an unknown role in the maintenance of genetic islands.
Remarkably the T4SS of N.gonorrhoeae was shown to secrete ssDNA directly into the medium. Currently nothing is known about the exact function of the secreted DNA. Studies have shown that not only exopolysaccharides but also extracellular DNA (eDNA) can play an important role in the initial establishment of biofilms. The composition and the origin of the eDNA are not completely understood. N. gonorrhoeae biofilms contain large amounts of extracellular DNA which play an important role in biofilm formation. To study the role of ssDNA in biofilm formation, the development of biofilms of N. gonorrhoeae strain MS11 was compared with a MS11 ΔtraB strain, which is impaired in ssDNA secretion and the MS11 ΔtraB::traB complementation strain in which ssDNA secretion is restored. Furthermore, the role of ssDNA in biofilm formation was studied by treating biofilms with Exonuclease I which specifically degrades ssDNA. These experiments demonstrated that the secreted ssDNA strongly stimulated biofilm formation especially during initial attachment.
Furthermore, we developed a unique technique to separately detect ssDNA and dsDNA. To visualize ssDNA, SSB proteins, which specifically bind ssDNA with high affinity in a sequence-independent manner, were employed. The highly stable SSB protein from Thermoanaerobacter tengcongensis, and different cysteine mutants within this protein were purified to homogeneity. The different cysteines containing proteins were labelled with environmentally sensitive fluorescent probes. Specific combinations of cysteine mutants and fluorescent probes were selected to obtain proteins that showed a strongly increased fluorescence upon binding of ssDNA. To visualize dsDNA the thermostable double stranded DNA binding protein Sac7d of Sulfolobus acidocadarius was used. Both proteins were applied to visualize single- and double-stranded DNA in biofilms and planktonic cultures. Remarkably, only dsDNA could be detected in N. gonorrhoeae biofilms using this approach. We conclude that ssDNA plays an important role in biofilm formation, but that the amount of ssDNA necessary is much lower than the amount of dsDNA found in mature biofilms. |
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| DOI: | 10.17192/z2013.0373 |