Extracelular DNA in pure cultures

Studenteropgave: Speciale (inkl. HD afgangsprojekt)

  • Rasmus Clemmensen
DNA has previously been assumed as being an exclusively intracellular information storage material, but the discovery of extracellular DNA has changed this. Extracellular DNA is found to accomplish many functions like stabilizing the structure of a biofilm, provide protection against foreign DNA and function as nourishment for bacteria during periods of starvation etc. The production of eDNA has so far mostly been investigated in biofilms, however in this project the main focus was on eDNA in pure cultures of Thermotoga neapolitana, Pseudomonas aeruginosa, Staphylococcus aureus, Tsukamurella spumae and Bacillus mycoides and in complex environmental samples obtained from rocks inside two different streams. The experiments performed during the making of this Master’s thesis were organized into three parts where the first one consisted of experiments on the thermophile bacteria T. neapolitana. The second part included experiments on pure cultures and biofilms of P. aeruginosa, S. aureus, T. spumae and B. mycoides. The third part concerned experiments on the complex environmental samples from streams mentioned above. The presence and development of eDNA in the investigated samples was confirmed by imaging and genetic approaches and it was found that the production of eDNA in pure cultures were varying and depended on the growth phase of the culture and probably also was influenced by different stress factors. The found eDNA was compared to genomic DNA extracted from the same cultures, using fragmentation with the restriction enzymes HaeIII and MspI. The results from these experiments were inconclusive since unfortunately no genomic DNA was detectable from the bacteria, making a comparison impossible. Another method of comparing eDNA and genomic DNA was tested using DNase I and ethidium momoazide bromide (EMA) to degrade and block the eDNA respectively so the genomic DNA that was released after cell lysis would be the only available genetic material. This method showed promising results, although it was not possible to amplify genomic DNA after cell lysis in all cases. The eDNA and genomic DNA was also compared for the presence of the three different housekeeping genes encoding 16S rRNA, gyrB and rpoB and not all primers gave the expected bands for four investigated bacteria types. However the general observation was that the eDNA bands were clearer than the bands originating from genomic DNA. Since the polymerase chain reaction (PCR) products from eDNA contained the same genetic information as that obtained from genomic DNA, this indicates that eDNA is most likely was a copy of the genomic DNA.
Antal sider68
Udgivende institutionAAU
ID: 18832318