Biological adaptation to DHABs

ADAPTATION STRATEGIES OF BACTERIA TO EXTREME CONDITIONS

Genome mutation Gene acquisition

Genetic variability in microbial populationMutants adapted to the new environment

Horizontal Gene Exchange

• 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 2hBannock 69 10 daysUrania 131 13 daysL’Atalante 54 144 days

gamma-proteobacteri

aBacillus

1,00E+00

1,00E+02

1,00E+04

1,00E+06

1,00E+08

1,00E+10

1,00E+12

tempo (settimane)0 5 201510 20days 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

0 days 38

0

50

100

150

200

250

C.C.C

O.C.

L’ATALANTE BANNOCK DISCOVERY URANIA

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

0

25

50

75

100%

0 7 17 28 38 0 7 17 28 38 0 7 17 28 38 0 7 17 28 38

RELA

TIV

E Q

UA

NTIT

Y

days in brine

DNA SURVIVAL IN THE BRINES

Does the DNA maintain the biological properties (transforming ability)?

L’ATALANTE

BANNOCK

DISCOVERY

URANIA

0 days 38

1.E+04

1.E+05

1.E+06

0 10 20 30 40

days of incubation of DNA in brinestr

an

sfo

rma

tio

n y

ield

(u

fc/n

g D

NA

)

l'atalante

bannock

discovery

urania

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

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 CRUISEalong the depth profile in the 4 basins:

estimation of live/dead cells

estimation of quantity/diversity of extracellular DNA

THERE ARE DEAD CELLS ALONG THE DEPTH PROFILE ?

0

5

10

15

20

25

30

1.00

E+

03

1.00

E+

04

1.00

E+

05

1.00

E+

06

1.00

E+

07

DAPI total cells/ml Ipropidio dead cells/ml

0

5

10

15

20

25

30

0 20 40 60 80

% dead cells/ total DAPI cells

%dead/total

Staining of

Total cells with DAPI

Dead cells with damaged membrane with PROPIDIUM IODIDE

TOTAL

DEAD

BANNOCK

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

THERE ARE DEAD CELLS ALONG THE DEPTH PROFILE ? Staining of

Total cells with DAPI

Dead cells with damaged membrane with PROPIDIUM IODIDE

URANIA

3

6

9

12

15

18

1.E+04 1.E+05 1.E+06 1.E+07

salin

ity %

DEAD cells DAPI cells

3

6

9

12

15

18

0 20 40 60 80 100

%dead/total

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

TOTAL

DEAD

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 11.614.4

16.919 22.4

24.6SW

5m

SW 3

00m

28 Brin

e

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

THERE IS NAKED EXTRACELLULAR DNA ALONG THE DEPTH PROFILE ? DGGE-fingerprinting of Bannock basin cellular and extracellular DNA

3.9 6 9 11.614.4

16.919 22.4

24.63.9 ext

race

llular

28 Brin

e

28 Brin

e extra

cellu

lar

Cellular DNA

GRADIENT

dissolved DNA contains few populations poorly represented by the live populations

3.9 6 9 11.614.4

16.919 22.4

24.6SW

5m

SW 300m

28 Brin

e

Extracellular DNA

GRADIENT

dead populations decrease along with the salinity increase: are species not adapted to hypersaline environment?

bands sequencing in progress

CONCLUSIONS

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

IN VITRO EXPERIMENTS IN SAMPLE OBSERVATIONS

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