1. Biological adaptation to DHABs

2. 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)

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. 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 days
Urania days
L’Atalante 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?

5. 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

6. 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

7. 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

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. 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

10. 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

11. 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

12. 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

13. 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