CoNISMa, Consorzio Nazionale

Interuniversitario Scienze del

Mare

Partner 1c

DISTAM

Università degli Studi di Milano

Daniele Daffonchio

Tullio Brusa

Sara Borin

Adriana Bona

Enrica Canzi

Claudia Sorlini

WP6: genetic characterization of isolated microorganisms and of enzymes interesting for biotechnological application

Catabolic genes

characterization

19% of the isolates are able to grow on oil and catechol as unique

carbon sources

crude oil degradation

0

20

40

Urania

Banno

ck

Discov

ery

L'Ata

lante

W/B

inte

rface

B/S in

terfa

ce

sedim

ent

% a

ctiv

e st

rain

s

Research for xyl E-type genes

(catechol 2,3 dioxigenase) gene via PCR

Isolated strains

Community DNA

All negatives

DHABs cultivable/uncultivable bacteria do not clevage aromatic ring via common extradiol cleavage

Plasmid profile characterization

Plasmid profile has been characterised for several 2001 aerobic isolates

isolates harbouring plasmids

0

25

50

75

100

% s

tra

ins

0

25

50

75

100

gamma-proteobacteria

bacillaceae

Plasmids (mobile genetic elements?) are abundant among Discovery isolates, and absent in L’Atalante strainsPlasmids are equally distributed between the most represented phylogenetic groups

“Fishing” of functional genes in the metagenomic DNA

Plasmids exogenous isolation

Plasmids donor:cells recovered filtrating Discovery water/brines interface concentratedPlasmids hosts:Pseudomonas putida

Search for transconiugants

donor

host

MATING

Screening for:Hg resistanceNaCl toleranceOil degradationNaphtalene degradation

NEGATIVE

Screening for natural competent strains

Horizontal gene transfer (HGT) occurs in many natural environments

Natural competent cells are naturally able to acquire naked DNA via natural transformation

Extreme conditions imposed by the brines could stimulate competent cells to exchange DNA in order to adapt to the environment

In DHABs, is there free DNA?

In DHABs, are there natural competent bacteria?Could brines stimulate the competence in these strains?

Could brines enhance natural transformation and HGT?

250n

g50

ng10

ng 2ng

1min

6hou

rs1d

ay2d

ays

8day

s

DNA stability in brines

DNA is detectable in Discovery brine for at least 1 day:

after exposure to brines DNA maintains the trasforming ability?

Trasformation experiments

donor plasmid pZR80:broad host range plasmidconfers resistance to ampicillin and kanamycine

host strains:isolated strains of the -proteobacteria group

Washed cellsApS, KmS

Incubation with pZR80

Plating on Ap and

Km

Screening for ApR KmR colonies

Natural competent

strain

5D 18BAlteromonas macleodii Halomonas meridiana

NaCl 5% 0.E+00 3.E-08PCB 0.E+00 8.E-10OTB 0.E+00 1.E-08filter 1.E-09 1.E-09brine 0.E+00 8.E-05

Trasformation experiments

Natural competent strain?

No plasmid could be detected in Ap/Km resistant strains