Transcript Nessun titolo diapositiva - ULR CoNISMa di Milano

Partner 1c
CoNISMa, Consorzio Nazionale
Interuniversitario Scienze del Mare
DISTAM
Università degli Studi di Milano
Daniele Daffonchio
Tullio Brusa
Adriana Bona
Sara Borin
Enrica Canzi
Claudia Sorlini
WP4: Determination of the distribution, taxonomy and diversity of microorganisms
from DHABs, and isolation of strains with biotechnological potential
Urania 2001
samples
2001 isolates collection
Strict
anaerobes
aerobes
URANIA
25
URANIA
BANNOCK
23
BANNOCK
DISCOVERY
21
DISCOVERY
1
L’ATALANTE
23
L’ATALANTE
2
95
Transferred to Proteus
Characterized for diversity
Species identification
Screened for activity
3
Species identification
Molecular typing of isolated strains
Isolated
strains













ITS-PCR
species/subspecies
specific
16S rDNA sequencing
identification
Group 1
Halobacillus
Group 2
Halomonas
Group 3
Alteromonas
Group 4
nnn
Group 5
nnn
Group n
nnn
Rep-PCR
interspecies
diversity
characterization
Molecular typing of isolates based on
16S-23S rDNA Intergenic Transcribed Spacer PCR
46 different ITS groups
Only 3 groups found in different basins
31 Groups with only 1 isolate
Very high subspecies diversity
Identification of ITS groups
Most abundant groups
GR ITS
STRAINS
SEQ. STRAIN
% HOM. CLOSEST RELATIVES
RELATIVES ORIGIN
1
8B-9A-15B-13U-10B-18B-9B
9A
99
Halomonas sp.
PAH degrading bacteria, sediments
4
11A-15U-14D-15D-13D-23D
13D
100
Halobacillus trueperi
type strain DSM 10404T
17
16U-16Ubis-10U-11U-11Ubis
10U
97
Bacillus acidogenesis
acidophilic bacillus, acidic sphagnum peat bog
Isolates, all aerobic/facultative anerobic, have a wide subspecies diversity
but belong to few phylogenetic groups:
100
-proteobacteria
75
-proteobacteria
Enterococcaceae
%
Bacillus
50
25
0
gammaproteobac
Bacillaceae
alfaproteobac
Enterococcaceae
Phylogenetic groups
L'ATALANTE
Alteromonas macleodii (2)
Alteromonas marina (4)
Bacillus licheniformis
Bacillus sp.
Exiguobacterium auriantiacum
Halobacillus trueperi
Halomonas meridiana
Idiomarina abyssalis
Pseudoalteromonas sp.
Virgibacillus pantotenicus
BANNOCK
DISCOVERY
Aererehalobacter ostenderi
Alteromonas macleodii (3)
alfa proteobacterium
Bacillus baekryungensis (2)
Bacillus licheniformis
Bacillus firmus (3)
Halomonas alimentaria (2)
Bacillus panthotenticus (2)
Halomonas meridiana (5)
Bacillus sp.
Idiomarina baltica
Halobacillus trueperi (3)
Marinobacter hydrocarbonoclasticus
Halomonas
(6)
cupida (5)
Rhodospirilaceae bact
Virgibacillus pantotenicus (2)
Virgibacillus pantotenticus
URANIA
Bacillus acidogenesis (5)
Bacillus aquaemaris (3)
Bacillus sp.
Citrobacter sp.
Enterococcus gallinarum
Exiguobacterium auriantiacum
Halobacillus karajiensis
Halobacillus trueperi (4)
Halomonas meridiana
Idiomarina ioihiensis (2)
Pseudoalteromonas porphyrae
Pseudoalteromonas sp.
Salinisphaera shabanense
Virgibacillus carmonensis
Bacillaceae and proteobacteria in all the basins
Phylogenetic groups
WB INTERACE
Exiguobacterium auriantiacum
alfa proteobacterium
Alteromonas macleodii (5)
Alteromonas marina (4)
Bacillus baekryungensis
Bacillus pantothenticus
Bacillus sp.
Citrobacter sp.
Enterococcus gallinarum
Halobacillus trueperi
Halomonas cupida (5)
Halomonas meridiana (2)
Idiomarina abyssalis
Idiomarina baltica
Idiomarina ioihiensis (2)
Marinobacter hydrocarbonoclasticus (6)
Pseudoalteromonas porphyrae
Pseudoalteromonas sp.(2)
Salinisphaera shabanense
proteobacteria in
water/brine interface
BS INTERFACE
Aererehalobacter ostenderi
Bacillus acidogenesis (3)
Bacillus aquaemaris
Bacillus baekryungensis
Bacillus firmus (3)
Bacillus licheniformis
Bacillus panthotenticus
Bacillus sp.
Exiguobacterium auriantiacum
Halobacillus karajiensis
Halobacillus trueperi (4)
Halomonas alimentaria
Halomonas meridiana (3)
Virgibacillus pantotenicus
SEDIMENT
Bacillus licheniformis
Bacillus acidogenesis (2)
Bacillus jeotgali (2)
Bacillus sp.
Halobacillus trueperi (3)
Halomonas alimentaria
Halomonas meridiana (2)
Rhodospirilaceae bact
Virgibacillus carmonensis
Virgibacillus pantotenticus
Bacillaceae in sediments
New species:
99% similarity with unclassified
sediment -proteobacterium
95% similarity with Ruegeria
algicola
97% similarity with unclassified
PAH-degrading -proteobacterium
96% similarity with
Rhodospirillum
Aerobic isolates
intra-species diversity
based on BOX-PCR
fingerprinting
5Bt WB
7B WB
8B WB
15B S
10B BS
23D S
14D S
15D S
9B WB
18B S
8D BS
30D WB
19D WB
19Db WB
2D WB
4D WB
5D WB
11Ub BS
10U BS
5A WB
2B WB
11A BS
14B BS
6D WB
22U BS
1D WB
24U WB
4A WB
6A WB
13D BS
13U BS
9A BS
In each ITS group the strains display a
different degree of genomic variability.
Strains belonging to the same ITS
group, are not grouped according to the
basin of origin, or according to the same
sample type.
17B S
16B S
11U BS
12D BS
11D BS
5Bbis WB
16Ubis S
15U S
16U S
In some cases strains of the same origin
show relatively high similarity
6
29
53
% similarity
76
100
Qualitative data on the environmental significance of
cultivable microflora:
DGGE fingerprint
Isolated strains do not seem to represent the total eubacterial community
inhabiting the water/brine interface
Quantitative data on eubacteria/arhcaea prevalence in
Urania and Bannock basins:
Real Time PCR
Real time quantification
02
03
04
05
06
07
08
E+
E+
E+
E+
E+
E+
E+
.
.
.
.
.
.
.
1
1
1
1
1
1
1
0
Real time quantification
1,E+04
1,E+05
1,E+06
0
Water-brine interface is
an enrichment phase for
bacteria
5
5
10
10
Eubacteria are 1-3
magnitude orders higher
than archaea in the 2
basins.
15
20
15
25
20
30
35
25
eubacteria cells
Urania
archaea cells
1,E+07
arc cells
Bannock
eub cells
Aerobic strains isolated from water/brine interface: are adapted to
survive in the brines?
Kinetics of survival in the brines
3,5
1 ,E+ 1 0
3
2,5
1 ,E+ 0 8
2
OD
ufc/m l
1 ,E+ 0 6
1,5
1 ,E+ 0 4
1
1 ,E+ 0 2
0,5
1 ,E+ 0 0
0
Brines have bactericidal activity
0
1m
i
1 n
ho
2 ur
ho
ur
s
1
di
e
2
di
e
1
w
ee
k
9d
2
w ie
ee
ks
19
di
e
30
di
e
-proteobacteria
6 A A l te r o m o n a s m a r i n a
1 ,E+ 1 2
u fc / m l
O D600
e x p o su r e ti m e
Cells in 10 minutes decrease of 9
magnitude orders
3
1 ,E+ 1 0
2,5
1 ,E+ 0 8
2
1 ,E+ 0 6
1,5
1 ,E+ 0 4
1
1 ,E+ 0 2
0,5
1 ,E+ 0 0
0
L’Atalante brines
OD
1 ,E+ 1 2
0
1m
in
1
ho
ur
2
ho
ur
s
1
di
e
2
di
e
1
w
ee
k
9d
ie
2
w
ee
ks
19
di
e
30
di
e
ufc/m l
Bacillus
1 1 A H a l o b a c i l l u s tr u e p e r i
u fc / m l
O D600
A fraction of the cells remains
vital for variable range of time (2
weeks or more than 30 days)
Aerobic strains isolated from water/brine interface: are adapted to
survive in the brines?
L’Atalante brines
3
di
e
30
ks
di
e
ee
19
s
di
e
1
w
2
2
ur
ur
ho
m
in
ho
m
in
exposure time
di
e
1 ,E+ 1 0
2,5
O D600
1 ,E+ 0 8
2
1,2
1 ,E+ 0 8
1
0,8
OD
0
di
e
u fc / m l
30
ks
di
e
ee
19
s
di
e
1
w
2
ur
ur
ho
2
m
in
ho
1
30
m
in
m
in
u
O D600
10
30
19
ie
9d
ee
Bannock brines
0,2
1 ,E+ 0 0
u fc / m l
0
0
0,4
1 ,E+ 0 2
m
in
1 ,E+ 0 0
0,6
1 ,E+ 0 4
1
0,5
OD
1 ,E+ 0 2
di
e
1
di
e
1 ,E+ 0 4
ks
1,5
ufc/m l
1 ,E+ 0 6
1 ,E+ 0 6
Discovery brines
OD
0
OD600
di
e
1,E+00
0
u fc / m l
1 ,E+ 1 0
20
O D600
0,5
ufc/ml
1 1 D B a c i l l u s fi r m u s
1 ,E+ 1 2
w
u fc / m l
1
30
1
m
in
u
20
di
e
30
19
ie
ks
9d
ee
di
e
e x p o su r e ti m e
1,E+02
30
0
1
1,E+04
19
1 ,E+ 0 0
O D600
2
30
ee
19
ie
ee
9d
w
2
2
w
1
1
ur
ho
w
1
0
ufc/m l
1 ,E+ 0 0
OD
0,5
di
e
1 ,E+ 0 2
ks
1
di
e
1 ,E+ 0 4
k
1,5
di
e
1 ,E+ 0 6
di
e
2
2
0,5
u fc / m l
e x p o su r e ti m e
0
1m
in
1
ho
ur
2
ho
ur
s
1
di
e
2
di
e
1
w
ee
k
2
1 ,E+ 0 8
s
2,5
ur
1 ,E+ 1 0
0
1m
in
3
ho
1 ,E+ 0 2
1 2 B B a c i l l u s l i c h e n i fo r m i s
1 ,E+ 1 2
1
ufc/m l
1
2
di
e
ee
k
9d
2
w ie
ee
ks
19
di
e
30
di
e
2
w
s
di
e
ur
ur
1
ho
0
1m
in
ho
1
1
O D600
e x p o su r e ti m e
1 1 A H a l o b a c i l l u s tr u e p e r i
Bacillus
1 ,E+ 0 4
0
1 ,E+ 0 0
u fc / m l
1,5
1,5
0
0
1 ,E+ 0 0
0,5
2
1,E+06
1 ,E+ 0 6
9d
ie
we
ek
s
1 ,E+ 0 2
0,5
0
1m
in
ho
ur
2
ho
ur
s
1
di
e
2
di
e
1
w
ee
k
1 ,E+ 0 2
1,E+08
2
1
1
2,5
2
di
e
we
ek
1 ,E+ 0 4
1 ,E+ 0 4
1,E+10
1
1,5
3
2,5
di
e
2
1 ,E+ 0 6
m
in
1,5
10
ufc/m l
OD
2
1 ,E+ 0 6
1 ,E+ 0 8
ufc/m l
2,5
1 ,E+ 0 8
OD
2,5
1 ,E+ 1 0
2
3
1
1 ,E+ 1 0
1,E+12
ho
ur
ho
ur
s
3
3
2
1 ,E+ 1 0
1 ,E+ 1 2
1m
in
3,5
1
1 ,E+ 1 2
ufc/ml
3,5
OD
1 ,E+ 1 2
1 ,E+ 0 8
13U Halomonas meridiana
5 D A l te r o m o n a s m a c l e o d i i
18B H a lo m o n a s m e rid ia n a
6 A A l te r o m o n a s m a r i n a
ufc/m l
-proteobacteria
Kinetics of cells survival in the brines
O D600
Urania brines
Discovery has the most aggressive brines: death of all the inoculated cells in 1 day
Urania has the less aggressive brines: surviving of a fraction of the cells over 1 month
Urania 2002
samples
Mixed cultures obtained
SAMPLES:
Sulfur red bac
suphate red bac
Sulfur red bac
Methane bac
Brine+substrate
Geobacter
Methane ox bac
Sulphur ox bac
Brines
Brine/sediment interface
Sediments
Mud pit
1
2,1
2,2
2,3
2,4
2,5
2,6
3,1
3,2
3,3
3,4
3,5
3,6
4
5
6,1
6,2
6,3
7
8
9
10
Bannock
brine
BS
1
High salinity
Anaerobiosis
L'Atalante
brine
BS
Discovery
brine
BS
Urania
brine
BS
1
2.2
2.3
2.4
2.4
2.5
3.3
3.4
3.5
3.6
4
5
6.1
6.2
6.3
7
8
3.1
3.2
3.3
3.4
5
6.1
6.2
4
5
6.1
6.2
6.3
7
8
5
4
5
6.1
47 mixed
cultures
obtained
3.6
4
5
6.3
7
7
8
8
9
10
Isolated strains
Medium
mixed
culture
isolated
strains
23 isolated strains
Identified strains
basin
sample
1
Sulfur-reducing
bacteria
2
17
Methane bacteria
Anaerobic
methanotrophic
bacteria
18
19
20
21
22
23
24
25
26
27
38
39
40
Haloanaerobiaceae
41
Geobacter
42
43
44
45
1UB02
2UB02
3UB02
1BBS02
6BBS02
7BBS02
7UB02
8UB02
6UB02
1ABS02
4DBS02
2ABS02
9DBS02
5DBS02
6DBS02
1DB02
10UB02
9UB02
10BBS02
11BBS02
12BBS02
2BBS02
Haloanaerobium prevalens
Haloanaerobium prevalens
Uncultured bacterium
Thermobacillus barrensis
Haloanaerobium prevalens
Bacillus pantotenticus
Bacillus licheniformis
Bacillus licheniformis
Bacillus licheniformis
Haloanaerobium prevalens
Bacillus pantotenticus
Bacillus pantotenticus
Bacillus pantotenticus
Bacillus pantotenticus
C sorce
Elemental sulfur
CO2, Sodium Sodium acetate,
acetate, Sodium
Sodium
formiate
formiate
CO2
U
U
U
B
B
B
U
U
U
A
D
B
B
B
BS
BS
BS
B
B
B
BS
BS
A
D
D
D
D
U
BS
BS
BS
BS
B
B
CH4
Peptone, Meat
extract
U
B
B
B
B
B
BS
BS
BS
BS
Glucose, Yeast Glucose, Yeast
extract,
extract,
Trypticase
Trypticase
B
BS
e- acceptors
Sodium acetate;
Sodium acetate;
Yeast extract;
Ferrous
Ferrous
ammonium
ammonium
citrate
citrate
Citrate
4BBS02
e- donors
CH4
Citrate
Na2SO4
Organic or
inorganic
compounds
Fe3+
other
(traces)
Yeast
extract,
Triptone