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Biological adaptation to DHABs

Biological adaptation to DHABs. ADAPTATION STRATEGIES OF BACTERIA TO EXTREME CONDITIONS. Gene acquisition. Genome mutation. Horizontal Gene Exchange. Genetic variability in microbial population Mutants adapted to the new environment. Cell-cell transfer (conjugation)

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Biological adaptation to DHABs

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  1. Biological adaptation to DHABs

  2. ADAPTATION STRATEGIES OF BACTERIA TO EXTREME CONDITIONS Gene acquisition Genome mutation Horizontal Gene Exchange Genetic variability in microbial population Mutants adapted to the new environment • Cell-cell transfer (conjugation) • Acquisition of naked DNA (transformation)

  3. IN VITRO -EX SAMPLE EXPERIMENTS • survival of cells into the brines • “survival” of DNA into the brines • screening for isolates able to acquire naked DNA via natural transformation

  4. WB isolates survive into the brines at different rates gamma-proteobacteria Bacillus Discovery 1 day 2h Bannock 69 10 days Urania 131 13 days L’Atalante 54 144 days When exposed to the brines most of the cells die, while a fraction of the population survive for a period of time 20 days gene acquisition? mutations? CELLS SURVIVAL IN THE BRINES What happens to water/brine isolates when they sink into the brines?

  5. L’ATALANTE BANNOCK DISCOVERY URANIA L’ATALANTE 100% 0 days 38 75 50 RELATIVE QUANTITY BANNOCK 25 0 0 0 days in brine 0 0 7 17 28 38 7 17 28 38 7 17 28 38 7 17 28 38 DISCOVERY • DNA is degraded in L’Atalante, where the cells survive longer time • DNA is preserved in Discovery, where the cells survive only 1 day • Urania is the less aggressive toward DNA • The CCC form is converted in OC URANIA DNA SURVIVAL IN THE BRINES What happens to DNA when released into the brines by the decaying cells?

  6. L’ATALANTE 0 days 38 BANNOCK DISCOVERY URANIA DNA SURVIVAL IN THE BRINES Does the DNA maintain the biological properties (transforming ability)? • DNA after exposure to the brine maintains its biological activity • The transformation activity increases with the conversion into the OC form • Transforming activity is higher in brines with high degrading activity: in Urania brine the total amount of DNA is preserved but it is degraded at molecular level

  7. THERE ARE ISOLATES ABLE TO ACQUIRE NAKED DNA? • Screening of aerobic isolates for natural competence • Donor plasmids: • broad host range plasmids • confer resistance to diverse antibiotics • Green fluorescent protein Transformation protocol: • Optimised protocols for the transformation of naturally competent strains • In brines No natural competent strains demonstrated until now • widespread antibiotic resistance • lack of suitable Ori/promoters • possible integration of plasmids

  8. “IN SAMPLE” EXPERIMENTS • 2003 CRUISE • along the depth profile in the 4 basins: • estimation of live/dead cells • estimation of quantity/diversity of extracellular DNA

  9. Total cells with DAPI Staining of Dead cells with damaged membrane with PROPIDIUM IODIDE THERE ARE DEAD CELLS ALONG THE DEPTH PROFILE ? TOTAL DEAD • 30-70% of the cells present along the depth profile are dead • in the water/brine interface there are 4 105/ml dead cells: high quantity of released DNA that could be acquired via natural transformation BANNOCK

  10. Total cells with DAPI Staining of Dead cells with damaged membrane with PROPIDIUM IODIDE THERE ARE DEAD CELLS ALONG THE DEPTH PROFILE ? TOTAL DEAD • 30-100% of the cells present along the depth profile are dead • in the interface the ratio of dead cell in lower than in seawater and brine URANIA

  11. Dissolved DNA was in very low amount, impossible to be directly quantified, but in sufficient amount to obtain PCR product GRADIENT SW 300m 28 Brine SW 5m 11.6 14.4 16.9 22.4 24.6 3.9 19 6 9 Basing on PCR efficiency, in the upper part of the salinity gradient there is the higher amount of extracellular DNA THERE IS NAKED EXTRACELLULAR DNA ALONG THE DEPTH PROFILE ? Filtered samples from brine and interface have been desalted by dialysis on board and concentrated 100X

  12. GRADIENT 28 Brine extracellular 3.9 extracellular 28 Brine SW 300m 28 Brine SW 5m 11.6 14.4 16.9 22.4 24.6 3.9 19 11.6 14.4 16.9 22.4 24.6 6 9 3.9 19 6 9 Extracellular DNA Cellular DNA • dead populations decrease along with the salinity increase: are species not adapted to hypersaline environment? • bands sequencing in progress THERE IS NAKED EXTRACELLULAR DNA ALONG THE DEPTH PROFILE ? DGGE-fingerprinting of Bannock basin cellular and extracellular DNA GRADIENT • dissolved DNA contains few populations poorly represented by the live populations

  13. Seawater/brine interface could constitute an “hot spot” for genetic exchange as a strategy for bacteria to adapt to the extreme environment CONCLUSIONS IN VITRO EXPERIMENTS IN SAMPLE OBSERVATIONS • most of the cells sinking into the brines die releasing their DNA, but a fraction of the population survives for 5 weeks • DNA is preserved in the brines and maintains transformation ability • 30-70% of the cells present in the basins are dead • small quantities of dissolved extracellular DNA are detectable in the WB interface, composed by populations poorly represented by the live populations

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