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Milano May 2004

Milano May 2004. Latest from Essex. 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 MgCl 2

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Milano May 2004

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  1. Milano May 2004 Latest from Essex 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 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

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

  4. ASM Library

  5. US13 AS22 AS2 and AS3 AN-BS10B Virgibacillus panthothenicus AN-BS5A BS18 Bacillus halodenitrificans 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) Bacillus jeotgali DS5 Bacillus sp. MB-5 Bacillus benzoevorans Bacillus firmus AN-BS8 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 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 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. US13, US14 AS22 AS2, AS3, AS4 AN-BS10B Anaerobic growth by fermentation AN-BS5A Virgibacillus pantothenticus BS18 Bacillus halodenitrificans AN-AS13 AN-BS1B Gracilibacillus halotolerans BS16 BS17 BS14 Halobacillus litoralis Halobacillus trueperi US16 LC16 Halobacillus halophilus LC39 Anaerobic growth by respiration Bacillus halophilus Strain NO3- NO2- SO3- S2O32- MnIV fumarate DMSO TMAO AS2, AS3, AS4 – + + – +/ – – + – US13, US14, AS22– + – +/ – +/ – + – +

  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

  11. 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 5384-98 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 Maximum likelihood 1298 bp. 10 nucleotide substitutions per 100 bases

  12. 10 mm 10 mm An Important Team in the Interface • Isolated from the same enrichment from Bannock interface • Anaerobic • Moderate halophiles 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

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

  14. 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)

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

  16. 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.

  17. 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?

  18. Can organisms grow in 5 Molar MgCl2?

  19. Selection of Isolates to Test for Growth at Different MgCl2 Concentrations

  20. Growth of Isolates on Different Concentrations of NaCl and MgCl2 • All strains prefer NaCl • No growth beyond 1.05 M MgCl2 Virgibacillus pantothenticus

  21. But, these isolates had been pre-grown on NaCl-rich media • Also, we had onlya bulk sample of Discovery interface • And so, obtained a dissection of Discovery interface and inoculated into MgCl2-rich media (November 2003 CruiseR/V Urania)

  22. Interface fraction RI Approx. [NaCl] (mM) Mg2+ concentration in medium (mM) 48 420 840 1260 1680 2101 5.10 1.340 700 + + ? ? - - 5.7 1.342 825 + + ? - - - 5.6 1.345 1300 + + ? - - - Atalante Interface 5.5 1.355 2450 + + - - - - 5.4 1.367 3875 ? ? - - - - 5.3 1.370 4225 - - - - - - 5.2 1.373 4575 - - - - - - 5.1 1.376 4925 - - - - - - 10.3 1.378 5050 - - - - - - 10.1 1.382 5200 - - - - - - Interface fraction RI Approx. [MgCl2] (mM) Mg2+ concentration in medium (mM) Samples Inoculated into CPS medium (unbuffered) RI = refractive index - = no growth + = growth ? = possible growth 1000 mM MgCl2 = 95.2 gl-1 48 420 840 1260 1680 2101 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 - - - - - - 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

  23. 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

  24. 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

  25. PhD Studentship at Essex

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