Transcript Nessun titolo diapositiva - ULR CoNISMa di Milano

Studies on DHABs biological evolution
Adaptation strategies of bacteria to extreme conditions
Microbial populations under
environmental stress
Induction of error-prone DNA polymesares
inefficiency of DNA repair systems
Genome mutation
MUTATOR
phenotype
Genetic variability in
microbial population
Activation of
transposition
Mutants adapted to e new environment
Gene acquisition
Horizontal Gene
Exchange
Transformation
Natural competent cells
Acquisition of free DNA
Conjugation
Cell to cell contact
Transduction
Bacterial association
Syntrophy
Bacteriophage
mediated
Adaptation strategies of bacteria to extreme conditions
Microbial populations under
environmental stress
Induction of error-prone DNA polymesares
inefficiency of DNA repair systems
Genome mutation
MUTATOR
phenotype
Genetic variability in
microbial population
Activation of
transposition
Mutants adapted to e new environment
Gene acquisition
Horizontal Gene
Exchange
Transformation
Natural competent cells
Acquisition of free DNA
Conjugation
Cell to cell contact
Transduction
Bacterial association
Bacteriophage
mediated
Strategies of the work
CELLS SURVIVAL IN THE BRINES
What happens to water/brine isolates
when they sink in the brines?
 Survival rates
 Viable But Not Culturable State
 Mutations in the survived populations
 Transfer of mobile genetic elements (IS, transposons)
Strategies of the work
HORIZONTAL GENE TRANSFER BY TRANSFORMATION
This mechanism involves the acquisition of naked DNA free in the environment
by naturally competent bacteria, in active state or in starvation
 Is the DNA released into the brines by decaying cells stable?
 Released DNA can still transform bacterial cells?
 In DHABs, is there naked DNA?
 In DHABs, are there natural competent bacteria?
 Could brines stimulate the competence in these strains?
Strategies of the work
HORIZONTAL GENE TRANSFER BY CONJUGATION
This mechanisms involves the transfer of conjugative plasmids between
metabolically active cells
In DHABs, are there conjugative plasmids?
Can the isolates exchange conjugative plasmids?
Isolates
Bulk DNA
Cells recovered from
DHABs without cultivation
CELLS SURVIVAL IN THE BRINES
Results
1.E+10
11D
2
Bacillus firmus
1.E+06
1
1.E+04
OD60
cfu/ml
1.E+08
cfu/ml
OD600
in Discovery brine
1.E+02
1.E+00
0
0
1
10
20
30
60
11A
120
Halobacillus trueperi
minutes
in L’Atalante brine
1.E+12
1.E+12
1.E+09
1.E+06
1.E+03
1.E+00
1.E+10
Discovery
Bannock
Urania
L’Atalante
B
1 day 2h
69 10 days
131 13 days
54 144 days
1
0
1
10
minutes
20
30
2
OD600

3
2
0
1.E+08
cfu/ml
Sterile brines:
3
1.E+06
1.E+04
1
1.E+02
1.E+00
20d
93d
0
144days
CELLS SURVIVAL IN THE BRINES
Results
sopravvivenza Bacilli
1,00E+12
Bacillaceae
ufc/ml
1,00E+10
Discovery
1,00E+08
L'Atalante
1,00E+06
Bannock
1,00E+04
Urania
1,00E+02
1,00E+00
sopravvivenza
gamma-proteobatteri
10
15
20
5
0
tempo (settimane)
1,00E+12
ufc/ml
weeks
1,00E+10
Discovery
1,00E+08
L'Atalante
Bannock
-proteobacteria
1,00E+06
Urania
1,00E+04
1,00E+02
1,00E+00
0
5
10
tempo (settimane)
15
20
20
Gamma-proteobacteria survive for a longer period than Bacillaceae
Results
CELLS SURVIVAL IN THE BRINES
10 minutes in Bannock brines
60 minutes in Bannock brines
SYBR Green:
total cells
Propidium iodide:
dead cells
A large fraction of the cells are died
the brines have bacteriolytic effect
DNA SURVIVAL IN THE BRINES
Results
250
L’ATALANTE
BANNOCK
DISCOVERY
URANIA
200
L’ATALANTE
150
C.C.C
O.C.
100
50
0
BANNOCK
DISCOVERY
DNA is degraded in L’Atalante, where the cells survive
longer time (DNAse activity? Brines are 1 year old)
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
Results
L'ATALANTE
60
1.E+04
40
20
0
1.E+03
T7
T 17
T 28
100
O.C.
80
1.E+05
60
C.C.C.
1.E+04
40
transformants
20
0
T 38
days of incubation
T7
T 17
1.E+05
100
80
1.E+04
60
40
20
0
1.E+03
T 28
T 28
T 38
140
% plasmid forms
120
T 17
transformants
1.E+05
1.E+04
0
T 38
increases with the convertion
1.E+06
60
C.C.C.
40
transformants
20
brine maintains its biological
The transformation activity
120
100
O.C.
80
DNA after exposure to the
activity
URANIA
transformants/ng DNA
140
T7
C.C.C.
days of incubation
DISCOVERY
T0
O.C.
1.E+03
T0
transformants/ng DNA
T0
1.E+06
120
transformants/ng DNA
1.E+05
80
% plasmid forms
100
140
transformants/ng DNA
1.E+06
120
% plasmid forms
% plasmid forms
140
BANNOCK
O.C.
into the OC form
C.C.C.
transformants
1.E+03
T0
days of incubation
T7
T 17
T 28
T 38
days of incubation
1.E+06
1.E+05
urania
discovery
bannock
l'atalante
1.E+04
1.E+03
T0
T7
T 17
T 28
T 38
Transforming activity is
more in brines with less
degrading activity
TRANSFORMATION
Results
Screening of aerobic isolates for natural competence
donor plasmids:
broad host range plasmids
confer resistance to diverse antibiotics
Green fluorescent protein
Transformation protocol:
Optimized protocols for the transformation of naturally competent strains
(Acinetobacter BD413, B. subtilis 1A1): on solid surfaces, in liquid media
In brines
Washed cells
ApS, KmS
Incubation with
pZR80gfp(Ap Km gfp)
Plating
on Ap and Km
Screening for colonies
ApR KmR gfp
Natural competent
strains
TRANSFORMATION
Results
NaCl 5%
PCB
OTB
filter
brine
5D
Alteromonas macleodii
0.E+00
0.E+00
0.E+00
1.E-09
0.E+00
18B
Halomonas meridiana
3.E-08
8.E-10
1.E-08
1.E-09
8.E-05
Natural competent strain?
No plasmid could be detected in Ap/Km
resistant strains
CONJUGATION
Results
Several strains showed to host plasmids
isolates harbouring plasmids
% strains
100
75
50
25
0
nno
Ba
ck
co v
Dis
ery
L
ala
'At
nt e
Ura
nia
EXPERIMENTS IN PROGRESS
Mating experiments to
transfer by conjugation:
Plasmids from Biodeep strains to selected
receiving strains (Pseudomonas KT2442gfp)
Known broad host range plasmids (p???gfp) from
Pseudomonas KT2442 to the Biodeep strains
Triparental mating experiments will be done to transfer non self-trasmissible plasmids, using
helper strains which harbour genes codyfing for the mobilization of such plasmids.
PROPOSAL FOR THE NOVEMBER CRUISE
If the HPSS will be available
Preliminar study to indagate cells survival and natural competence at in situ conditions:
Inoculation in the HPSS:
•Broad host range plasmid harbouring antibiotic resistance genes
•Selected strains sensible to the antibiotics
•Deploying the HPSS into the brines (L’Atalante or Urania)
•Recover the HPSS, incubate some hours
•During incubation, decompress a subsample (if possible) and plate in solid
media with and without the antibiotic, to recover the cells which survived and
the ones that received the plasmid