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Terry McGenity, Andrea Sass, Gayle Harris, Jenny Gillion, Boyd McKew, Flavia Alves, John Hallsworth, Ken Timmis Milano May 2004 Latest from Essex Strain.

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Presentation on theme: "Terry McGenity, Andrea Sass, Gayle Harris, Jenny Gillion, Boyd McKew, Flavia Alves, John Hallsworth, Ken Timmis Milano May 2004 Latest from Essex Strain."— Presentation transcript:

1 Terry McGenity, Andrea Sass, Gayle Harris, Jenny Gillion, Boyd McKew, Flavia Alves, John Hallsworth, Ken Timmis Milano May 2004 Latest from Essex Strain database Quick recap and new information about isolates - All isolates - Bacillales - Interface consortium Life in MgCl 2 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 B - hypersaline brine:15 S - sediment: 218 I - seawater-brine interface: 162 Paper 1 Isolates

4 ASM Library

5 AS22 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 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 25 strains studied in detail 23/25 strains grow at 12 o C 7/25 strains grow at 20% NaCl 2/25 strains grow anaerobically (by fermentation) Salt tolerance of selected LAtalante Isolates Can they grow in situ?

7 5% w/v NaCl 10% 15% 20% 22% 25% 27% 30% Strain US13 doesnt grow below 5% w/v NaCl 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 AS2 AS3 US13 AS6 BS6 AS7 BS3 US2 US4 AS28 BS29 US7 DS10 DS9 US1 BS17 BS18 AS12 BS12 DS16 AS5 US16 BS25 DS1 DS5 UPGMA analysis showing phenotypic diversity of 25 Bacillales strains (based on 108 tests) Jaccard's Coefficient

9 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 StrainNO 3 - NO 2 - SO 3 - S 2 O 3 2- Mn IV fumarateDMSOTMAO AS2, AS3, AS4 – ++ –+/ – –+ – US13, US14, AS22 –+ –+/ – +/ –+ –+ Anaerobic growth by respiration

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

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12 AN-BI4 AN-BI3A Desulfovibrio vulgaris DSM 644 T Wolinella succinogenes ATCC T Helicobacter pylori 181 Thiomicrospira denitrificans DSM 1251 T Arcobacter nitrofrigilis DSM 7299 T Campylobacter jejuni SSI Sulfurospirillum arcachonense DSM 9755 T Chlorobium limicola DSM 245 T Sulfurospirillum barnesii DSM T Sulfurospirillum deleyianum DSM 6946 T Sulfurospirillum sp. strain 18.1 Uncultured hydrothermal vent bacterium C1_B008 Uncultured hydrothermal vent bacterium P. palm A/C 22 Cytophaga fermentans ATCC T Marinilabilia salmonicolor DSM 6480 T Anaerophaga thermohalophila DSM T Rikenella microfusus ATCC T Empedobacter brevis ATCC Chryseobacterium gleum ATCC T Tenacibaculum maritimum IFO T Flavobacterium aquatile ATCC T Cytophaga marinoflava ATCC19326 T Flexibacter aggregans ssp. catalaticus IFO Bacteroides fragilis ATCC T Porphyromoas asaccharolytica ATCC T Sporocytophaga myxococcoides DSM T 10 nucleotide substitutions per 100 bases Maximum likelihood 1298 bp. Bacteroidetes -Proteobacteria

13 10 m AN-BI4 (Bacteroidetes) AN-BI3A ( -Proteobacteria) Isolated from the same enrichment from Bannock interface Anaerobic Moderate halophiles 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 An Important Team in the Interface Paper 4 Taxonomy / ecology of Bacteroidetes and -Proteobacteria

14 Solar Salterns on the shore of the Great Salt Lake

15 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) Haloarchaea enhance halite precipitation

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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 MgCl 2 ( ), NaCl ( ) (± se, n= 2) Gillion, J., McGenity, T.J and Hallsworth, J.E.

19 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? Life in Discovery hypersaline brine

20 Can organisms grow in 5 Molar MgCl 2 ?

21 StrainOriginRationalePhylogenetic groupNext cultured relative AN-AI3AII, inc DI; common; fac anArchaea - HaloarchaeaHalorhabdus utahensis DS 16DS D Firmicutes - BacillalesBacillus alcalophilus DS 9DS D Firmicutes - BacillalesBacillus simplex DI 1DII, inc DIFirmicutes - BacillalesBacillus sp DS 5DSI, inc DI; commonFirmicutes - BacillalesBacillus sp. MB-5 DS 14DS D Firmicutes - BacillalesBacillus sp. MB-9 DS 1DS D Firmicutes - BacillalesBacillus sp. MN-003 DS 10DS D Firmicutes - BacillalesGlacial ice bacterium G200-N5 AS 2ASfac anFirmicutes - BacillalesHalobacillus salinus US 13USfac anFirmicutes - BacillalesVirgibacillus marismortui AN-BS5ABSI, inc DI; common; an enrichFirmicutes - BacillalesVirgibacillus pantothenticus AN-AS6EASfac an; halotolFirmicutes - ClostridialesThermohalobacter berrensis AN-BI2DBII; an enrichAlpha -Alpha proteobacterium GMDJE10F1 LC 25DII, inc DI; commonGamma - AlteromonadaceaeAlteromonas macleodii LC 7DBIGamma - AlteromonadaceaePseudoalteromonas sp. A28 DS 8DScommonGamma - HalomonadaceaeHalomonas aquamarina LC 26BIIGamma - HalomonadaceaeHalomonas sp. MBIC2031 AN-BI1DBII; an enrichGamma - IdiomarinaSlope strain DIII1c AN-BI1BBII; common; an enrichGamma - Marino-AlcaniMarinobacter hydrocarbonoclasticus AN-BI7CBII; an enrichGamma - Marino-AlcaniGamma proteobacterium GMD16F03 AN-BI1CBII; an enrichGamma - VibrionalesVibrio natriegens Selection of Isolates to Test for Growth at Different MgCl 2 Concentrations

22 Conc NaCl in non-buffered media (M) Conc MgCl 2 in non-buffered media (M) Strain AN-BI1D LC DS DI DS AN-BI1C DS AN-BI2D LC LC AS DS AN-BS5A US AN-BI1B AN-BI7C DS DS D D Virgibacillus pantothenticus All strains prefer NaCl No growth beyond 1.05 M MgCl2 Growth of Isolates on Different Concentrations of NaCl and MgCl 2

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 MgCl 2 -rich media (November 2003 Cruise R/V Urania)

24 ? ?+? ?+++ ~ ~ ~ Mg 2+ concentration in medium (mM) Approx. [MgCl 2 ] (mM) RI Interface fraction Discovery Interface Samples Inoculated into CPS medium (unbuffered) RI = refractive index - = no growth + = growth ? = possible growth 1000 mM MgCl 2 = 95.2 gl -1 Growth only from the top of Discovery interface Discovery microbes grow in media with higher MgCl 2 concentrations

25 Preliminary data indicate that there is a point beyond which microbes cannot grow (~1.5 M MgCl 2 ), but: Only a restricted number of media tested This is MgCl 2 not Discovery basin Microbes continue to surprise us However, beyond this point life is extremely stressful

26 Examine isolates tRFLP across interface Quantitative real-time PCR to look at distribution of key isolates Other? Future Work Paper 5 Why there is microbial life in Discovery brine, but 1 to 1.5 Molar MgCl 2 seems to inhibit life

27 PhD Studentship at Essex


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