Latest from Essex
Milano May 2004
Terry McGenity, Andrea Sass, Gayle Harris, Jenny Gillion,
Boyd McKew, Flavia Alves, John Hallsworth, Ken Timmis
•Strain database
•Quick recap and new information about isolates
- All isolates
- Bacillales
- Interface consortium
•Life in MgCl2
•Publication plans
Database of Strains from BIODEEP
http://www2.geo.unimib.it/BioDeep/Project.html
Some information missing from database
•% 16S rRNA similarity to closest cultivated relative
•Next relative with standing in nomenclature +
•Isolation medium
•Link to maintenance medium
Location of different phylogenetic/physiological groups in the DHABs
Phylogenetic group
AI AB AS BI BB BS UI UB US DI DB DS SW
Archaea - Halobacteriales
1 0 1 0
0 0 0
0
0 0
0
0
0
Archaea - Methanosarcinales
0 0 4 0
0 0 0
0
0 0
0
0
0
Firmicutes - Bacillales
2 0 42 1
0 42 0
2 29 7
1 31
1
Firmicutes - Clostridiales
0 0 17 0
0 6 0
0
1 0
0
0
0
Firmicutes - Lactobacillales
0 0 0 0
0 1 1
0
0 0
0
0
0
1 0 1 2
0 1 0
0
0 0
0
0
0
 Proteobacteria
0 0 0 0
0 1 1
0
0 0
0
0
0
 Proteo - unknown
9 0 0 21
0 0 3
0
5 4
1
1
0
 Proteo - Alteromonadaceae
0 0 0 3
0 0 14
0
0 0
0
0
0
 Proteo - Chromatiaceae
0 0 0 2
0 0 1
0
0 0
0
0
0
 Proteo - Enterobacteriales
2 0 2 4
0 7 1
4
3 7
0
1
0
 Proteo - Halomonadaceae
1 0 0 3
0 0 4
0
0 0
0
0
0
 Proteo - Idiomarina
1 1 2
1
1 0
0
0
4
 Proteo - Marinobacter-Alcanivorax 4 1 3 40
1 0 0 1
0 6 1
0
0 0
0
0
0
 Proteo - Pseudomonadaceae
3 0 0 2
0 0 0
0
0 0
0
0
0
 Proteo - Vibrionales
0 0 0 1
0 0 0
0
0 0
0
0
0
 Proteobacteria
Actinobacteria
0 0 2 0
0 2 0
0
0 0
0
0
0
Bacteroidetes
0 0 0 2
0 0 0
0
0 0
0
0
0
Halanaerobiales
5 0 6 2
0 0 3
3
0 0
1
1
0
Total
29 1 78 84
1 67 31 10 39 18
3 34
5
B - hypersaline brine:
S - sediment:
I - seawater-brine interface:
15
218
162
? Total
0
2
0
4
0 158
0
24
0
2
0
5
0
2
1
45
0
17
0
3
0
31
1
9
5
63
0
9
0
5
0
1
0
4
0
2
0
21
7 407
Paper 1
Isolates
Location of different phylogenetic/physiological groups in the DHABs
Phylogenetic group
AI AB AS BI BB BS UI UB US DI DB DS SW
Archaea - Halobacteriales
1 0 1 0
0 0 0
0
0 0
0
0
0
Archaea - Methanosarcinales
0 0 4 0
0 0 0
0
0 0
0
0
0
Firmicutes - Bacillales
2 0 42 1
0 42 0
2 29 7
1 31
1
Firmicutes - Clostridiales
0 0 17 0
0 6 0
0
1 0
0
0
0
Firmicutes - Lactobacillales
0 0 0 0
0 1 1
0
0 0
0
0
0
1 0 1 2
0 1 0
0
0 0
0
0
0
 Proteobacteria
0 0 0 0
0 1 1
0
0 0
0
0
0
 Proteo - unknown
9 0 0 21
0 0 3
0
5 4
1
1
0
 Proteo - Alteromonadaceae
0 0 0 3
0 0 14
0
0 0
0
0
0
 Proteo - Chromatiaceae
0 0 0 2
0 0 1
0
0 0
0
0
0
 Proteo - Enterobacteriales
2 0 2 4
0 7 1
4
3 7
0
1
0
 Proteo - Halomonadaceae
1 0 0 3
0 0 4
0
0 0
0
0
0
 Proteo - Idiomarina
1 1 2
1
1 0
0
0
4
 Proteo - Marinobacter-Alcanivorax 4 1 3 40
1 0 0 1
0 6 1
0
0 0
0
0
0
 Proteo - Pseudomonadaceae
3 0 0 2
0 0 0
0
0 0
0
0
0
 Proteo - Vibrionales
0 0 0 1
0 0 0
0
0 0
0
0
0
 Proteobacteria
Actinobacteria
0 0 2 0
0 2 0
0
0 0
0
0
0
Bacteroidetes
0 0 0 2
0 0 0
0
0 0
0
0
0
Halanaerobiales
5 0 6 2
0 0 3
3
0 0
1
1
0
Total
29 1 78 84
1 67 31 10 39 18
3 34
5
ASM Library
? Total
0
2
0
4
0 158
0
24
0
2
0
5
0
2
1
45
0
17
0
3
0
31
1
9
5
63
0
9
0
5
0
1
0
4
0
2
0
21
7 407
US13
AS22
AS2 and AS3
AN-BS10B
AN-BS5A
Virgibacillus panthothenicus
BS18
Bacillus halodenitrificans
Phylogenetic tree of Bacillales based
on 16S rRNA sequences
AN-AS13
AN-BS1B
Gracilibacillus halotolerans
BS16
BS17
BS14
Halobacillus litoralis
Halobacillus trueperi
US16
LC16
Halobacillus halophilus
LC39
Bacillus halophilus
AS28
Bacillus niacini
BS29 (similar: US7)
DS5
Bacillus jeotgali
Bacillus sp. MB-5
Bacillus benzoevorans
Bacillus firmus
AN-BS8
Strains from the DHABs are distributed
throughout the order Bacillales
Bacillus cohnii
Bacillus horikoshii
US1
„Bacillus aquaemaris“
Bacillus pumilus
BS3
Bacillus subtilis
Bacillus licheniformis
US2 (=US4)
DS1
„Bacillus aminovorans“
DS10
Jeotgalibacillus alimentarius
Bacillus marinus
AS13
BS19
Bacillus simplex
DS9
Bacillus sp. MK03
AS12
BS12
AS7
Bacillus baekryungensis
Bacillus sp. OS-5
AS5
Bacillus pseudofirmus
DS16
Bacillus alcalophilus
BS25
Bacillus barbaricus
Bacillus sp. NRRLB-14851
BS6 (similar: AS6)
Bacillus halodurans
Bacillus decolorationis
AN-BS10A
Can they grow in situ?
•25 strains studied in detail
•23/25 strains grow at 12 oC
•7/25 strains grow at 20% NaCl
•2/25 strains grow anaerobically (by fermentation)
Salt tolerance of selected L’Atalante Isolates
AS2
AS3
0:00
AS5
0:00
0:00
2:00
td(h)
1:00
td(h)
td(h)
1:00
1:00
3:00
3:00
2:00
4:00
0
5
10
15
4:00
0
20
5
10
15
20
0
AS6
1:00
2:00
5
10
% NaCl
15
20
20
0:00
td(h)
td(h)
2:00
15
AS12
0:00
1:00
10
% NaCl
AS7
0:00
0
5
% NaCl
% NaCl
td(h)
2:00
1:00
2:00
3:00
0
5
10
% NaCl
15
20
0
5
10
%NaCl
15
20
Strain US13 doesn’t grow below 5% w/v NaCl
0.5
Series1
5% w/v NaCl
Series2
10%
0.4
O.D.
Series3
15%
0.3
Series4
20%
Series5
22%
0.2
Series6
25%
0.1
27%
Series7
30%
Series8
0
0
100
200
300
400
Time (h)
US13 (and related strains) is phenotypically similar to AS2 (and related strains), e.g.:
•use as sole source of carbon and energy of:
n-acetyl-glucosamine
glycerol
trehalose
UPGMA analysis showing phenotypic diversity of 25
Bacillales strains (based on 108 tests)
DS5
DS1
BS25
US16
AS5
DS16
BS12
AS12
BS18
BS17
US1
DS9
DS10
US7
BS29
AS28
US4
US2
BS3
AS7
BS6
AS6
US13
AS3
AS2
0.28
0.4
0.52
0.64
0.76
Jaccard's Coefficient
0.88
1
US13, US14
AS22
AS2, AS3, AS4
AN-BS10B
AN-BS5A
Virgibacillus pantothenticus
Anaerobic growth by fermentation
BS18
Bacillus halodenitrificans
AN-AS13
AN-BS1B
Gracilibacillus halotolerans
BS16
BS17
BS14
Halobacillus litoralis
Halobacillus trueperi
US16
LC16
Halobacillus halophilus
LC39
Bacillus halophilus
Anaerobic growth by respiration
Strain
NO3-
NO2-
SO3-
S2O32-
MnIV
fumarate DMSO
TMAO
AS2, AS3, AS4
–
+
+
–
+/ –
–
+
–
US13, US14, AS22
–
+
–
+/ –
+/ –
+
–
+
Bacillales Summary
•Bacillus-like strains predominatly from sediments
•Most unable to grow under in-situ conditions
•New halophilic facultatively anaerobic strains have the
potential to be active in situ
Paper 2
Bacillales diversity
Paper 3
Taxonomy of halophilic,
anaerobic Bacillales
Location of different phylogenetic/physiological groups in the DHABs
Phylogenetic group
AI AB AS BI BB BS UI UB US DI DB DS SW
Archaea - Halobacteriales
1 0 1 0
0 0 0
0
0 0
0
0
0
Archaea - Methanosarcinales
0 0 4 0
0 0 0
0
0 0
0
0
0
Firmicutes - Bacillales
2 0 42 1
0 42 0
2 29 7
1 31
1
Firmicutes - Clostridiales
0 0 17 0
0 6 0
0
1 0
0
0
0
Firmicutes - Lactobacillales
0 0 0 0
0 1 1
0
0 0
0
0
0
1 0 1 2
0 1 0
0
0 0
0
0
0
 Proteobacteria
0 0 0 0
0 1 1
0
0 0
0
0
0
 Proteo - unknown
9 0 0 21
0 0 3
0
5 4
1
1
0
 Proteo - Alteromonadaceae
0 0 0 3
0 0 14
0
0 0
0
0
0
 Proteo - Chromatiaceae
0 0 0 2
0 0 1
0
0 0
0
0
0
 Proteo - Enterobacteriales
2 0 2 4
0 7 1
4
3 7
0
1
0
 Proteo - Halomonadaceae
1 0 0 3
0 0 4
0
0 0
0
0
0
 Proteo - Idiomarina
1 1 2
1
1 0
0
0
4
 Proteo - Marinobacter-Alcanivorax 4 1 3 40
1 0 0 1
0 6 1
0
0 0
0
0
0
 Proteo - Pseudomonadaceae
3 0 0 2
0 0 0
0
0 0
0
0
0
 Proteo - Vibrionales
0 0 0 1
0 0 0
0
0 0
0
0
0
 Proteobacteria
Actinobacteria
0 0 2 0
0 2 0
0
0 0
0
0
0
Bacteroidetes
0 0 0 2
0 0 0
0
0 0
0
0
0
Halanaerobiales
5 0 6 2
0 0 3
3
0 0
1
1
0
Total
29 1 78 84
1 67 31 10 39 18
3 34
5
? Total
0
2
0
4
0 158
0
24
0
2
0
5
0
2
1
45
0
17
0
3
0
31
1
9
5
63
0
9
0
5
0
1
0
4
0
2
0
21
7 407
Sporocytophaga myxococcoides DSM 11118T
Porphyromoas asaccharolytica ATCC 25260T
Bacteroides fragilis ATCC 25285T
Flexibacter aggregans ssp. catalaticus IFO 15977
Cytophaga marinoflava ATCC19326T
Flavobacterium aquatile ATCC 11947T
Tenacibaculum maritimum IFO 15946T
Chryseobacterium gleum ATCC 35910T
Empedobacter brevis ATCC 14234
Rikenella microfusus ATCC 29728T
Anaerophaga thermohalophila DSM 12881T
Marinilabilia salmonicolor DSM 6480T
Cytophaga fermentans ATCC 19072T
Bacteroidetes
AN-BI4
Uncultured hydrothermal vent bacterium P. palm A/C 22
Uncultured hydrothermal vent bacterium C1_B008
Desulfovibrio vulgaris DSM 644T
Wolinella succinogenes ATCC 29543T
Helicobacter pylori 181
Thiomicrospira denitrificans DSM 1251T
Arcobacter nitrofrigilis DSM 7299T
Campylobacter jejuni SSI 5384-98
AN-BI3A
-Proteobacteria
Sulfurospirillum arcachonense DSM 9755T
Sulfurospirillum sp. strain 18.1
Sulfurospirillum deleyianum DSM 6946T
Sulfurospirillum barnesii DSM 10660T
Chlorobium limicola DSM 245T
10 nucleotide substitutions per 100 bases
Maximum likelihood 1298 bp.
An Important Team in the Interface
•Isolated from the same enrichment from Bannock interface
•Anaerobic
•Moderate halophiles
10 mm
AN-BI3A (-Proteobacteria)
10 mm
AN-BI4 (Bacteroidetes)
•Equivalent tRFs in brine and
interface samples
•Fermentative growth
•Organic acids as carbon and
energy source
•Uses a variety of S-compounds
as terminal electron acceptors
Paper 4 Taxonomy /
ecology of Bacteroidetes
and -Proteobacteria
Solar Salterns on the shore of the Great Salt Lake
Haloarchaea enhance halite precipitation
Halite crystals forming around lab-grown
colonies of halococci
(Sabine Castanier, University of Nantes)
Halite crystal forming around microbial
cells in the Berre salt works
(Sabine Castanier, University of Nantes)
Water activities of the brines
Mean water activity of brines at 25 C (+/- 2 s.e, n= 3)
1
0.9
0.8
0.7
aw
0.6
0.5
0.4
0.3
0.2
0.1
0
Sea water
Discovery
Gillion, J., McGenity, T.J and Hallsworth, J.E.
l'Atalante
Sample
Urania west
Bannock
Percentage of highest initial rate of reaction (%)
100
90
80
70
60
50
40
30
20
10
0
0
0.5
1
1.5
2
2.5
3
3.5
4
Total concentration of salts (M)
Activity of glucose-6-phosphate dehydrogenase from
Leuconostoc mesenteroides in the presence of different
concentrations of MgCl2 (♦), NaCl (■) (± se, n= 2)
Gillion, J., McGenity, T.J and Hallsworth, J.E.
4.5
Life in Discovery hypersaline brine
Indications of metabolic activity and growth:
•Ectoenzyme activity in Discovery Basin (Marseille)
•Methanogenesis in Discovery Basin (Marseille)
•Sulphate reduction in Discovery Basin (Marseille)
Indications of the presence of cells/DNA (not necessarily signs of life):
•DNA in Discovery Basin (Several)
•Cells in Discovery Basin (Milano)
Indications of microbial survival in Discovery brine:
•Isolates from Discovery Basin (Several)
Has anyone isolated RNA from Discovery Basin?
Can organisms grow in 5 Molar MgCl2?
Selection of Isolates to Test for Growth at Different MgCl2 Concentrations
Strain
AN-AI3
DS 16
DS 9
DI 1
DS 5
DS 14
DS 1
DS 10
AS 2
US 13
AN-BS5A
AN-AS6E
AN-BI2D
LC 25
LC 7
DS 8
LC 26
AN-BI1D
AN-BI1B
AN-BI7C
AN-BI1C
Origin
AI
DS
DS
DI
DS
DS
DS
DS
AS
US
BS
AS
BI
DI
DB
DS
BI
BI
BI
BI
BI
Rationale
I, inc DI; common; fac an
D
D
I, inc DI
I, inc DI; common
D
D
D
fac an
fac an
I, inc DI; common; an enrich
fac an; halotol
I; an enrich
I, inc DI; common
I
common
I
I; an enrich
I; common; an enrich
I; an enrich
I; an enrich
Phylogenetic group
Archaea - Haloarchaea
Firmicutes - Bacillales
Firmicutes - Bacillales
Firmicutes - Bacillales
Firmicutes - Bacillales
Firmicutes - Bacillales
Firmicutes - Bacillales
Firmicutes - Bacillales
Firmicutes - Bacillales
Firmicutes - Bacillales
Firmicutes - Bacillales
Firmicutes - Clostridiales
Alpha Gamma - Alteromonadaceae
Gamma - Alteromonadaceae
Gamma - Halomonadaceae
Gamma - Halomonadaceae
Gamma - Idiomarina
Gamma - Marino-Alcani
Gamma - Marino-Alcani
Gamma - Vibrionales
Next cultured relative
Halorhabdus utahensis
Bacillus alcalophilus
Bacillus simplex
Bacillus sp. 19500
Bacillus sp. MB-5
Bacillus sp. MB-9
Bacillus sp. MN-003
Glacial ice bacterium G200-N5
Halobacillus salinus
Virgibacillus marismortui
Virgibacillus pantothenticus
Thermohalobacter berrensis
Alpha proteobacterium GMDJE10F1
Alteromonas macleodii
Pseudoalteromonas sp. A28
Halomonas aquamarina
Halomonas sp. MBIC2031
Slope strain DIII1c
Marinobacter hydrocarbonoclasticus
Gamma proteobacterium GMD16F03
Vibrio natriegens
Growth of Isolates on Different Concentrations of NaCl and MgCl2
Strain
AN-BI1D
LC26
DS8
DI1
DS9
AN-BI1C
DS5
AN-BI2D
LC25
LC7
AS2
DS1
AN-BS5A
US13
AN-BI1B
AN-BI7C
DS16
DS10
10D
21D
0
+
+
+
+
+
+
+
+
+
+
+
Conc NaCl in non-buffered media (M)
0.43 0.86 1.72 2.59 3.45 4.31
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Virgibacillus pantothenticus
5.17
-
Conc MgCl2 in non-buffered media
(M)
0
0.26 0.52 0.79 1.05 1.47
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
•All strains prefer NaCl
•No growth beyond 1.05 M MgCl2
•But, these isolates had been pre-grown on NaCl-rich
media
•Also, we had only a bulk sample of Discovery interface
•And so, obtained a dissection of Discovery interface and
inoculated into MgCl2-rich media (November 2003 Cruise
R/V Urania)
Atalante Interface
Discovery Interface
Interface
fraction
RI
Approx.
[MgCl2]
(mM)
Mg2+ concentration in medium (mM)
48
2.10
2.9
2.8
2.7
2.6
2.5
2.4
2.3
2.2
2.1
9.3
Interface
9.2
fraction
9.1
5.10
5.7
5.6
5.5
5.4
5.3
5.2
5.1
10.3
10.1
~450
1.344
~450
1.344
~500
1.345
600
1.348
900
1.355
1150
1.360
1450
1.365
1700
1.370
1900
1.375
2025
1.377
1.384 Approx.
2400
2950
1.393
RI
[NaCl]
(mM)
3250
1.398
1.340
1.342
1.345
1.355
1.367
1.370
1.373
1.376
1.378
1.382
700
825
1300
2450
3875
4225
4575
4925
5050
5200
+
+
+
+
+
?
-
420
840
1260
1680
2101
+
+
+
+
+
+
?
+
+
+
+
+
?
?
- medium
- (mM)
Mg2+- concentration
in
-
-
48
420
840
1260
1680
2101
+
+
+
+
?
-
+
+
+
+
?
-
?
?
?
-
?
-
-
-
Samples Inoculated
into CPS medium
(unbuffered)
RI = refractive index
- = no growth
+ = growth
? = possible growth
1000 mM MgCl2 =
95.2 gl-1
•Growth only from
the top of
Discovery interface
•Discovery
microbes grow in
media with higher
MgCl2
concentrations
Preliminary data indicate that there is a point beyond
which microbes cannot grow (~1.5 M MgCl2), but:
•Only a restricted number of media tested
•This is MgCl2 not Discovery basin
•Microbes continue to surprise us
However, beyond this point life is extremely stressful
Future Work
•Examine isolates
•tRFLP across interface
•Quantitative real-time PCR to
look at distribution of key isolates
•Other?
Paper 5 Why there is
microbial life in Discovery
brine, but 1 to 1.5 Molar MgCl2
seems to inhibit life
PhD Studentship at Essex