Biological adaptation to DHABs

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

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

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

DNA SURVIVAL IN THE BRINES What happens to DNA when released into the brines by the decaying cells? L’ATALANTE BANNOCK DISCOVERY URANIA days 38 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 25 50 75 100% 7 17 28 RELATIVE QUANTITY days in brine

DNA SURVIVAL IN THE BRINES Does the DNA maintain the biological properties (transforming ability)? L’ATALANTE BANNOCK DISCOVERY URANIA days 38 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

No natural competent strains demonstrated until now 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

“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

Dead cells with damaged membrane with PROPIDIUM IODIDE THERE ARE DEAD CELLS ALONG THE DEPTH PROFILE ? Staining of Total cells with DAPI Dead cells with damaged membrane with PROPIDIUM IODIDE 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

Dead cells with damaged membrane with PROPIDIUM IODIDE THERE ARE DEAD CELLS ALONG THE DEPTH PROFILE ? Staining of Total cells with DAPI Dead cells with damaged membrane with PROPIDIUM IODIDE 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

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 3.9 6 9 11.6 14.4 16.9 19 22.4 24.6 SW 5m SW 300m 28 Brine Dissolved DNA was in very low amount, impossible to be directly quantified, but in sufficient amount to obtain PCR product Basing on PCR efficiency, in the upper part of the salinity gradient there is the higher amount of extracellular DNA GRADIENT

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

IN SAMPLE OBSERVATIONS 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 Seawater/brine interface could constitute an “hot spot” for genetic exchange as a strategy for bacteria to adapt to the extreme environment