1. 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

2. Database of Strains from BIODEEP
Some information missing from database
% 16S rRNA similarity to closest cultivated relative
Next relative with standing in nomenclature +
Isolation medium
Link to maintenance medium

3. Paper 1 Isolates B - hypersaline brine: 15 S - sediment: 218
I - seawater-brine interface: 162
Paper 1
Isolates

4. ASM Library

5. Phylogenetic tree of Bacillales based on 16S rRNA sequences
US13
AN-BS10B
AS2 and AS3
AN-BS5A
BS18
Virgibacillus panthothenicus
AN-BS1B
AN-AS13
Bacillus halodenitrificans
Gracilibacillus halotolerans
BS16
BS17
BS14
Halobacillus trueperi
Halobacillus litoralis
LC16
US16
Halobacillus halophilus
Bacillus halophilus
AS28
LC39
Bacillus niacini
Bacillus jeotgali
BS29 (similar: US7)
DS5
Bacillus sp. MB-5
Bacillus benzoevorans
Bacillus firmus
US1
AN-BS8
Bacillus cohnii
Bacillus horikoshii
„Bacillus aquaemaris“
Bacillus licheniformis
Bacillus subtilis
Bacillus pumilus
DS1
US2 (=US4)
BS3
Bacillus marinus
Jeotgalibacillus alimentarius
„Bacillus aminovorans“
AS13
DS10
DS9
BS19
Bacillus sp. MK03
Bacillus simplex
AS12
BS12
AS7
Bacillus pseudofirmus
Bacillus sp. OS-5
Bacillus baekryungensis
AS5
DS16
Bacillus barbaricus
Bacillus alcalophilus
Bacillus sp. NRRLB-14851
BS25
BS6 (similar: AS6)
Bacillus decolorationis
Bacillus halodurans
AN-BS10A
Phylogenetic tree of Bacillales based on 16S rRNA sequences
Strains from the DHABs are distributed throughout the order Bacillales

6. 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

7. Strain US13 doesn’t grow below 5% w/v NaCl
10%
15%
20%
22%
25%
27%
30%
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

8. 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
0.88 1
Jaccard's Coefficient

9. Anaerobic growth by fermentation
AS22
US13, US14
AN-BS10B
AS2, AS3, AS4
AN-BS5A
BS18
Virgibacillus pantothenticus
AN-BS1B
AN-AS13
Bacillus halodenitrificans
Gracilibacillus halotolerans
BS16
BS17
BS14
Halobacillus trueperi
Halobacillus litoralis
LC16
US16
Halobacillus halophilus
Bacillus halophilus
LC39
Anaerobic growth by fermentation
Strain NO3- NO2- SO3- S2O32- MnIV fumarate DMSO TMAO
AS2, AS3, AS4 – – +/ – – + –
US13, US14, AS22 – + – +/ – +/ – + – +
Anaerobic growth by respiration

10. 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 3
Taxonomy of halophilic, anaerobic Bacillales
Paper 2
Bacillales diversity

12. Bacteroidetes AN-BI4 e-Proteobacteria AN-BI3A
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
Bacteroidetes
Empedobacter brevis ATCC 14234
Rikenella microfusus ATCC 29728T
Anaerophaga thermohalophila DSM 12881T
Marinilabilia salmonicolor DSM 6480T
Cytophaga fermentans ATCC 19072T
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
e-Proteobacteria
AN-BI3A
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.

13. An Important Team in the Interface
Isolated from the same enrichment from Bannock interface
Anaerobic
Moderate halophiles
10 mm
10 mm
AN-BI3A (e-Proteobacteria)
Organic acids as carbon and energy source
Uses a variety of S-compounds as terminal electron acceptors
AN-BI4 (Bacteroidetes)
Equivalent tRFs in brine and interface samples
Fermentative growth
Paper 4 Taxonomy / ecology of Bacteroidetes and e-Proteobacteria

14. Solar Salterns on the shore of the Great Salt Lake

15. 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)

17. Water activities of the brines
Gillion, J., McGenity, T.J and Hallsworth, J.E.

18. 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.

19. 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?

20. Can organisms grow in 5 Molar MgCl2?

21. Selection of Isolates to Test for Growth at Different MgCl2 Concentrations
Strain
Origin
Rationale
Phylogenetic group
Next cultured relative
AN-AI3 AI
I, inc DI; common; fac an
Archaea - Haloarchaea
Halorhabdus utahensis
DS 16 DS D
Firmicutes - Bacillales
Bacillus alcalophilus
DS 9
Bacillus simplex
DI 1 DI
I, inc DI
Bacillus sp
DS 5
I, inc DI; common
Bacillus sp. MB-5
DS 14
Bacillus sp. MB-9
DS 1
Bacillus sp. MN-003
DS 10
Glacial ice bacterium G200-N5
AS 2 AS
fac an
Halobacillus salinus
US 13 US
Virgibacillus marismortui
AN-BS5A BS
I, inc DI; common; an enrich
Virgibacillus pantothenticus
AN-AS6E
fac an; halotol
Firmicutes - Clostridiales
Thermohalobacter berrensis
AN-BI2D BI
I; an enrich
Alpha -
Alpha proteobacterium GMDJE10F1
LC 25
Gamma - Alteromonadaceae
Alteromonas macleodii
LC 7 DB I
Pseudoalteromonas sp. A28
DS 8
common
Gamma - Halomonadaceae
Halomonas aquamarina
LC 26
Halomonas sp. MBIC2031
AN-BI1D
Gamma - Idiomarina
Slope strain DIII1c
AN-BI1B
I; common; an enrich
Gamma - Marino-Alcani
Marinobacter hydrocarbonoclasticus
AN-BI7C
Gamma proteobacterium GMD16F03
AN-BI1C
Gamma - Vibrionales
Vibrio natriegens

22. Growth of Isolates on Different Concentrations of NaCl and MgCl2
Conc NaCl in non-buffered media (M)
Conc MgCl2 in non-buffered media (M)
Strain
0.43
0.86
1.72
2.59
3.45
4.31
5.17
0.26
0.52
0.79
1.05
1.47
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
All strains prefer NaCl
No growth beyond 1.05 M MgCl2
Virgibacillus pantothenticus

23. 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)

24. Mg2+ concentration in medium (mM)
Interface
fraction RI
Approx. [MgCl2] (mM)
Mg2+ concentration in medium (mM) 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
2.10
1.344
~450 + + + - - -
2.9
1.344
~450 + + + - - -
2.8
1.345
~500 + + + ? - -
Discovery Interface
2.7
1.348
600 + + + - - -
2.6
1.355
900 + + + - - -
2.5
1.360
1150 ? + ? - - -
2.4
1.365
1450 - ? - - - -
2.3
1.370
1700 - - - - - -
2.2
1.375
1900 - - - - - - -
5200
1.382
10.1
5050
1.378
10.3
4925
1.376
5.1
4575
1.373
5.2
4225
1.370
5.3 ?
3875
1.367
5.4 +
2450
1.355
5.5
1300
1.345
5.6
825
1.342
5.7
700
1.340
5.10
2101
1680
1260
840
420 48
Mg2+ concentration in medium (mM)
Approx. [NaCl] (mM) RI
Interface
fraction
Atalante Interface
2.1
1.377
2025 - - - - - -
9.3
1.384
2400 - - - - - -
9.2
1.393
2950 - - - - - -
9.1
1.398
3250 - - - - - -
Growth only from the top of Discovery interface
Discovery microbes grow in media with higher MgCl2 concentrations

25. 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

26. 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

27. PhD Studentship at Essex