Hidden microbial diversity at high temperatures

Anna-Louise Reysenbach [email protected] www.alrlab.pdx.edu

The microbial world still has a lot of surprises • – 30-50% coding capacity unknown • Estimates of <1% grown in the lab • High biochemical diversity – Promise for improving processes – Production of high value chemicals/biofuels • Unknown provides ideal targets for discovery using a host of different approaches including creative culturing, metabolic modeling etc. • Consortia/more complex systems are now tractable (& help maintain optimal conditions for production) • Historical- where there is , microbes will ‘engineer themselves… Silicon chips, PCBs etc..

Overview:

• Generally, what do we know about the diversity in high temp systems? (who are some of the key players? 50- 80C thermophily happens)

• Examples of targeting discovery

• Any interesting targets out there? – Next wave of pyrosequencing insights

• Any conclusions?

1 Kamchatka

No close relative in culture, Iceland new genus

New species

2 Costa Rica, new species from Rincon volcano

intra-cytoplasmic structures

And so on…. 0.5 µm

•only explored with undersea vehicles less than 1 % of the mid-oceanic ridge zone which accounts for more than 90 % of the earth's volcanic activity

•Yet relatively little is known about microbial diversity (surprising as many of our thermophilic archaeal isolates are from these systems)

3 Emerging patterns of

Dominated by epsilon Uncultivated groups

Others, e.g. Aquificales,Thermotogales, Thermales

Archaea Methanogens Thermococcales Archaeoglobales Korarchaeota, Nanoarchaeum Uncultured groups--- Deep- Groups, especially, DHVE2

All cultures are neutrophiles, at best acido tolerant or mildly acidophilic (pH 6.0)

DHVE2 Uncultivated

For many years we and others asked…

• 1. Who are the DHVE2?

• 2. Why no cultured from deep- sea vents?

4 Major vent sites, and sites where the DHVE2 have been detected

•1. Who are the DHVE2?

Eastern Lau Spreading Center

Lau Vent Fluids Vai Lili Subsurface deposition? [Mg] = 14 – 46 mM Substrate control?

Vai Lili [Fouquet et al., 1991] For fluids [Mg] ≤ 3 mM Basalt Basalt/Andesite Andesite

Data from TUIM05MV; C.G. Wheat, J. Seewald, M. Mottl, Preliminary Data.

5 Then serendipity enters the search for DHVE2

• Mariner- one of the lowest pH measurements for deep-sea vents pH <2.5 • Clone libraries revealed an unusual sequence from an --rich microbial mat, 600C • never before detected at vents, related to Picrophilus, ,

Never before detected at vents

Could we grow the Thermoplasma-like phylotype?

6 Not yet.. But we have somebody else even more interesting… • The devilheterotrophventblob –DHVE-2 horns

- “ boonei”

Genome has been sequenced. Draft and now being closed by JGI. Second isolate being sequenced too.. Reysenbach et al. 2006. Nature 442:444-447 Reysenbach and Flores, 2008. Geobiology. 6:331-336

Acidic and anaerobic zones

2+ + Fe + 2H2S FeS2 + H2 + 2H

+ 2+ + Cu + Fe + 2H2S Cu FeS2 + 0.5H2 + 3H

2- + H2S + 2O2 SO4 + 2H S0 Epsilon Proteobacteria

Lots of [CHO]n Fe3+, S0

7 MAR08-237A (Rainbow) MAR08-276 (Lucky Strike) MAR08-339 (Lucky Strike) MAR08-361 (Lucky Strike) MAR08-641 (TAG) DQ451875, T469 EPR07-159 (9°N East Pacific Rise) M38637, AF339746, X84901, U20163, Pyrococcus furiosus Z70244, Thermococcus hydrothermalis

0.10

Fig. 2 Neighbor joining tree of cultured representatives of the DHVE2 (colors) based on 1261 unambiguously aligned nucleotides of 16S rRNA gene sequences. Halobacterium salinarum was used as the outgroup to root the tree (not shown). Scale bar represents 10 substitutions per 100 nucleotides.

More …

'MAR08-307R_Full_16S' 'MAR08-368R_Full_16S' EP5 Y18292, Hippea maritima KM1 AM712337, uncultured delta proteobacterium Y16943, multipotens Y16942, Desulfurella propionica X72768, Y16941, Desulfurella kamchatkensis X99234, Desulfonatronovibrio hydrogenovorans AF524933, Desulfonauticus submarinus AF418177, Desulfobacterium autotrophicum DQ826724, Desulfatibacillus olefinivorans AJ237607, Desulfobacterium indolicum DQ146482, Desulfatirhabdium butyrativorans

0.10

MAR08-307R ≈70 bp

MAR08-307R MAR08-368R Hippea maritima “Uncultured delta” “Uncultured delta” ≈100 bp ≈80 bp

8 Overview:

• Generally, what do we know about the diversity in high temp systems? (who are some of the key players?)

• Examples of targeting discovery

• Any interesting targets out there? – Next wave of pyrosequencing insights

• Any conclusions?

Bacterial

9 Dominated by Sulfurovum or Dominated by Sulfurimonas Caminibacter, Nautiales

Epsilons

Pacific 100

80 60 Korarchaeota 40 DHVE6 20

40 Archaeoglobus0 35 Atlantic 30 25 Methanocaldococcus 20 15 KM 10 MA R130 Thermococcus TC1275 0 MAR08_13 TC126c MAR08_2 um yr p d ro fie MAR08_5 Desulfurococcales TC126be si A s ed la ifi 2 nc s E MAR08_6 u as V e- l H s TC126a a nc D bu ce -u lo MAR08_7 a ae og is ob e e d gl c G se d o ca _ fie e c ae si s ha co rt s u MAR08_7 rc ro e la cc A fu nc nc o d l _i u c ie su ra e- o if MAR08_5 e e a rm ss us D n ce he la b ge a t c lo _ c o n ro us ta oc an -u y c MAR08_13 o c th e P c e o e ea co m ha an M c o ilu rc th tia rm of a Only 1 sample Archaeoglobus dominates ra e ic e m t o M d h r ae K ro T e is y Th n P ca ul #5/6 methanogens dominate with Thermococcus** V #2 Vulcanisaeta and other Desulfucoccales** #7 Desulfurococcales and Thermoproteales #2 and 7 Korearchaeota 2,5,7,13 Nanoarchaeum

Rare vent Archaea? 14000 12000 10000 8000 6000

4000 TC126a 2000 TC127 0 TC126c Mar130 ; 1 globi rotei KM o p VE II TC126a e o KM a p ta a rm ;DH GMEG i ia rch a A ou o er 1 TC126b t ae er C The o a;S ArchaeaGr h V ta;A ; ae t bact ta h ne rc o eo o n lobact e rc rya ta;DH TC126c a Mari u tha Ha h rya ; e ; rc u archaeo ta a Archaea Archaea;Korarchaeota o ea;E uryarcha a;E ren ae a a;M aot E rena C h archaeo C ae ; eot che a; ; rc Arch ren e aea ar ea h ryarchaeota;Methanomicrobiarya ry ;C Arch u archa ha rc y ea Archa rc A r Eu A ea;E a; ha ;Eu ae rc Sulfolobales?! ha ea A Archaea;Eu Arc Arch rcha Picrophilus?! A BacteriaHalophiles too!!

10 Conclusions • Lots of potential targets still in the environment • Understanding the ecology… when and where of interest occur, coexist etc. • Multiple *informed*, coordinated approaches will allow for selective enrichment of of interest… • Consortia for novel metabolisms, synthrophy • Low biodiversity systems- meta- transcriptome, proteome

Thanks..

Yitai Liu, Gilbert Flores, Mircea Podar

NSF, JGI, ICoMM

The crew, pilots and scientific parties

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