José R. De La Torre San Francisco State University [email protected] We Live on a Microbial Planet

José R. De La Torre San Francisco State University Jdelator@Sfsu.Edu We Live on a Microbial Planet

Living under extreme conditions: Understanding how microorganisms in high temperature environments use and recycle Nitrogen

José R. de la Torre San Francisco State University [email protected] We Live On A Microbial Planet

• Liquid water! Life is based on aqueous chemistry

• Something to “eat” (electron donor) must be a reduced chemical compound

• Something to “breathe” (electron acceptor) must of an oxidized chemical compound

• Building blocks (nutrients)

• Capacity to perform chemistry

Phosphorus

Hydrogen

Carbon

Microbes catalyze key geochemical processes

N2O Anammox Assimilation Denitrification

NH3 biomass NO

— — NO2 NO2

— NO3

• pH • Temperature • Salinity • Water • Pressure • Oxygen • Nutrients • Radiation • Toxic chemicals (e.g., heavy metals)

TERM An organism that grows best at… Acidophile low pH Alkaliphile high pH Thermophile high temperature (>40˚C) Hyperthermophile VERY high temperature (>80˚C) Psychrophile low temperatures (<15˚C) Halophile high salt concentrations Xerophile very low water activity Piezophile (barophile) high pressure

Anaerobe in the absence of oxygen (O2) Oligotroph under limiting nutrient concentrations

To st udy microbes, the MUST be in pure culture!

Unfortunately, we now know this cannot be done for the vast majority of microbes: they just won’t grow in the lab!

Sediments 0.25% Soil 0.3% Freshwater 0.25% Activated sludge 1-15%

< 0.1%

Seawater 0.001-0.1%

Carl Woese (Univ. of Illinois) used ribosomal RNA (rRNA) genes to determine “natural” or evolutionary relationships among organisms.

Carl Woese (1928-2012)

“Molecules as documents of evolutionary history” Zuckerkandl and Pauling (1965) J. Theoret. Biol., 8:357-366

post-anal • molecular homologies tail • universal genetic code • eukaryote chromosomes same structure • chlorophyll a • cytochrome C • nucleic acid and amino acid comparisons • much more about this in a few weeks Hemoglobin Small subunit ribosomal RNA

• 16S rRNA in archaea & bacteria

• 18S rRNA in eucaryotes

• Contains regions with 100% conservation in all organisms

• Comparing the sequence of the gene allows us to establish evolutionary relationships

BACTERIUM ARCHAEON

Escherishia coli Nitrosopumilus maritimus EUCARYOTE

Homo sapiens subsp. Bieber

Natural taxonomy: based on evolutionary relatedness i.e, allows phylogeny to be predictive

--> Related organisms should have similar properties (not that we always know “which” properties those are) Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. Norman Pace: Cultivation-independent identification of microorganisms

BACTERIA

ARCHAEA

• Use molecular biology (PCR) to obtain and sequence rRNA genes directly from environment

• Enables cataloging of microbial species in any environment

• Revolutionized our view of microbiology

• All cellular life is related (one origin of Life!) chloroplasts • Three domains of life, not two: Bacteria, Archaea, Eukarya

• Most life is microbial You are here (still)

• Eukaryotic nuclear line of descent as old as Archaea

• Bacterial origins of both mitochondrion and chloroplast

• The organisms closest to the “Root” of the Big Tree are all thermophiles

• This suggests that Life may have originated in a high temperature environment

Marine Thaumarchaeota at the Hawaii Ocean Time Series

Karner, DeLong & Karl (2001), Nature 409:507-10

• Discovered in 1992 by Jed Fuhrman (USC) and Ed DeLong (WHOI) • Could not be cultivated, so idea of what their metabolism could be. • 20% of all microorganisms in the ocean (1028 cells)! • Impact on global geochemical/nutrient cycles?

Anne Bernhard Shedd Aquarium (Chicago, IL) (Conn. College) • Highly active, nitrifying marine filtration systems

• No molecular evidence of known ammonia-oxidizing bacteria Plum Island Sound Estuary (MA)

• Similar finding: lots of ammonia oxidation but no known bacteria detected

What organism(s) are oxidizing the ammonia?

Anne Martin Bernhard Könneke gravel substratum

Temp.: 24˚C + [NH4 ]: ~0.5 µM

– + NH3 + 1½ O2 à NO2 + H + H2O Martens-Habbena et al., (2009) Nature Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. CryoEM Tomography of N. maritimus

100 nm

(Grant Jensen & Zhiheng Yu, Caltech)

Glycerol

Biphytanyl Fatty Acids

Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. How widespread is ammonia- oxidation within the Archaea? Are there any thermophilic ammonia-oxidizing archaea?

YNP

• Found ample evidence of thermophilic AOA throughout the Park.

• Predominantly in neutral to alkaline springs Imperial Geyser Octopus Spring (pH 7.5 to 9.0) x x • Ranged in temperature x from 65˚C to 85˚C Heart Lake x Shoshone • Only present in the Geyser Basin sediment layers (not water column) 10 km

Virginia Russell

de la Torre et al., 2008

Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. N. yellowstonii has no intracellular compartments Nitrosocaldus yellowstonensis A B Nitrosomonas europaea Emily Tung

Photo by Yuichi Suwa 150 nm C D Nitrosococcus oceanus

100 nm Watson et al. (1989) Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. How does the activity of AOA shape the function and composition of hot spring microbial communities? Nitrogen Limitation in Alkaline Geothermal Systems

Despite N limitation: Long Microbial Filaments!

Willm Martens- Habbena

Do AOA exacerbate the problem of Nitrogen k = 0.139 µM m limitation in microbial communities?

Martens-Habbena et al. (2009), Nature 461:976-9

Eric Trinity Boyd Hamilton

N • The primary mode of 2

N O interaction between AOA and 2 Anammox

diazotrophs in circumneutral Assimilation Denitrification

NH3 biomass geothermal systems is NO + competition for NH4

— — NO2 NO2

— + NO3 • AOA high affinity for NH4 + functions to keep bioavailable NH4 pool low

• This in turn forces diazotrophs to divert reductant/energy away from CO2 fixation and towards N2 fixation

Eric Trinity Boyd Hamilton Acetylene Reduction Activity

(Proxy for N2 Fixation) Ethylene

Hamilton et al., 2014 Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. If we inhibit AOA metabolism with ATU, other cells in the community incorporate more carbon Eric Trinity Boyd Hamilton 14 C-CO2 incorporation

• Dominant autotrophs are likely aerobic

+ • Depletion of NH4 pool due to AOA metabolism results in diversion of energy from CO2 fixation to N2 fixation

• Major producers of N2O! • Biochemistry of archaeal ammonia- oxidation remains unknown • No specialized compartment to carry out toxic chemical reactions

NH + O NH OH NO – + H+ 3 2 2 ? 2

– AMO e cyt aa3

Genome Size 1.43 Mbp Hope Matt incl. ECE/virus 50 kb Gray Ashby

G+C Content 37%

Number of Genes 1630 (53) Protein coding 1586 (53) amo cluster 1 16S rRNA 1 23S rRNA 1 tRNAs 38

Percentage coding 91%

Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. Can we use comparative genomics to identify the components of ammonia oxidation in archaea? Thermophilic AOA enrichments

Yellowstone (5)

Great Basin, Nevada (4) Tengchong, China (14)

Madagascar (1)

cultures (us) cultures (others) sequences Copyright © 2018 José R. de la Torre – May not be reproduced inHeather any manner withoutAmy express Jo writtenTalia permission. Casey Diego Madeline Virginia Average nucleotide identity (ANI) suggests that N. American & Chinese strains might be different genera

Nitrosphaera evergladensis SR1 Nitrosphaera viennensis EN76 Nitrosphaera gargensis Ga9.2 61.2% - SOIL 64.5% Nitrosphaera sp. GerE (NEVADA) 99.9% Nitrosphaera sp. GerD (NEVADA) Nitrosotalea devanaterra Nd1 (ACID SOIL) ACID SOIL Nitrosotenuis uzonensis N4 Nitrosopumilus maritimus SCM1 MARINE Nitrosopelagicus brevis CN25 Nitrosocaldus yellowstonensis HL72 Nitrosocaldus yellowstonensis D5 93.5% 98.2% - - 99.9% Nitrosocaldus yellowstonensis DSB YELLOWSTONE 72.8% - 93.8% 73.4% Nitrosocaldus yellowstonensis HL4 Nitrosocaldus yellowstonensis ISA2 93.5% - Nitrosocaldus gerlachensis GBS-F 99.9% NEVADA 93.8% 61.2% - Nitrosocaldus gerlachensis GBS-B 64.5% Nitrosothermus tenchongensis CNB Nitrosothermus tenchongensis CNU1 Nitrosothermus tenchongensis JZ Nitrosothermus tenchongensis GsxB Nitrosothermus tenchongensis JZ25 98.3% - 72.8% - Nitrosothermus tenchongensis JZ1 CHINA 99.8% 73.4% Nitrosothermus tenchongensis CNM1 Nitrosothermus tenchongensis DRC1 Nitrosothermus tenchongensis QQP Nitrosothermus tenchongensis DRC2 Nitrosothermus tenchongensis CNU2 0.1 Nitrosothermus tenchongensis JM JZ JM JZ1 CNB QQP JZ25 GsxB CNU1 CNU2 DRC2 DRC1 CNM1 GBS-F GBS-B Color Key & Histogram vis CN25 400 300 ergladensis SR1 GerE (NEVADA) GerD (NEVADA) v ellowstonensis D5 ellowstonensis HL4 ellowstonensis DSB ellowstonensis ISA2 ellowstonensis HL72 200 Count 100 Nitrosotenuis uzonensis N4 vanaterra Nd1 (ACID SOIL) Nitrosphaera gargensis Ga9.2 Nitrosopelagicus br e Nitrosphaera vienn ensis EN76 0 Nitrosopumilus maritimus SCM1 Nitrosphaera e Nitrosothermus tenchongensis Nitrosothermus tenchongensis Nitrosocaldus y Nitrosphaera sp. Nitrosphaera sp.

Nitrosocaldus gerlachensis 60% 70% 80% 90% 100% Nitrosocaldus gerlachensis Nitrosothermus tenchongensis Nitrosocaldus y Nitrosothermus tenchongensis Nitrosocaldus y Nitrosothermus tenchongensis Nitrosothermus tenchongensis Nitrosocaldus y Nitrosothermus tenchongensis Nitrosocaldus y % Average Nucleotide Identity (ANI) Nitrosothermus tenchongensis Nitrosothermus tenchongensis Nitrosothermus tenchongensis Nitrosothermus tenchongensis Nitrosothermus tenchongensis Nitrosotalea d e

0.1 Nitrosopumilus maritimus SCM1 NdevNd1 Nitrosopumilus salaria BD31 1.000 NituzN4 16S rRNA Nitrosoarchaeum koreensis MY1 marine 0.630 NbrevCN25 Nitrosoarchaeum limnia SFB1 0.740 Csym Cenarchaeum symbiosum NkorMY1 Nitrosotalea devanaterra Nd1 acidic soil 0.580 1.000NlimBG20 Nitrososphaera sp. JG1 Nitrososphaera viennensis sp. EN76 1.000 1.000 NlimSFB1 Nitrososphaera gargensis Ga9.2 NsalBD31 soil 1.000 NkorAR1 GerD (Nevada) 1.000 GerE (Nevada) NmarSCM1 Soil fosmid clone 54D9 NexqG61 NeverSR1 HL4 (YNP) 1.000 1.000 ISA2 (YNP) NvieEN76 Nitrosocaldus yellowstonii HL72 (YNP) 1.000 NgarGa9-2 Yellowstone Lake clone YLA060 (YNP) 1.000GerD GerE GBS-B (Nevada) GBSB GBS-F (Nevada) GBSF GBS clone SSE_L4_B03 (Nevada) ISA2 CNM1 (China) NyelHL72 DRC1 (China) NyelD5 DRC2 (China) 1.000 NyelDSB JM1 (China) hot HL4 CNU2 (China) QQRM GxsB (China) springs QQ-P (China) QQP CNU1 (China) CNU2 QQ-L (China) CNM1 QQ-RM (China) DRC2 JZ (China) JM GxsB metagenome (China) QQL CNB (China) CNB CNB_new (China) CNU1 DRC1 SRBZ-4 (China) GsxB JZ Fosmid JFF045_C05 (Japan) JZ1 JZ25 0.10 Caldiarchaeum subterraneum (Japan) Csub Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. Two geographically separated species(?) of thermophilic AOA

Nitrosocaldus

Pro1 ECE H2ase glycosylation NyelDSBDSB

NyelD5D5

NyelHL72HL72

HL4HL4 YELLOWSTONE YELLOWSTONE ISA2ISA2 GBS-FGBS-F

NEV. NEV. GBS-BGBS-B

DRC1

DRC2

CNM1

CNU2

CNU1 QQL GsxB

CNB

JM JZ

JZ1

JZ25 Comparative Genomics

hot springs marine

soil

euryarchaeota crenarchaeota Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. Comparative genomics to discover conserved genes found only in AOA

MARINE AOA (15)

Genes also present in SOIL non-AOA archaea AOA THERMO (5) AOA Genes variably present across AOA (26) lineages Genes conserved and unique to AOA

NON-AOA ACIDIC ARCHAEA SOIL AOA (4)

100000 Yellowstone & China ThAOA “Soil” Acidic “Marine” AOA non- Nevada ThAOA AOA Soil AOA AOA

10000

1000 # of genes

100

pan genome core genome 10 175 genes (122 AOA-specific)

JZ JM JZ1 HL4 ISA2 CNB GsxB JZ25 QQL QQP GerE Csub GBSF GBSB DRC1 CNM1 CNU1 CNU2 DRC2 QQRM GerD Csym NyelD5 ieEN76 NyelDSB NituzN4 NexqG61NoleMY3 NdevNd1 NkorAR1NkorMY1 NsalBD31 NyelHL72 NgarGa9-2NeverSR1 Nv NlimBG20 NmarSCM1ThaumDS1 ThaumFn1 Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. NbrevCN25 Summary

• After nearly 15 years of work, we still do not know how AOA oxidize ammonia! • Thermophilic AOA are present and can be cultivated from a variety of springs in the U.S. and China • Chinese strains are significantly different in nucleotide sequence, and may be another genus of thermophilic AOA

• Genomic comparisons have allowed us to identify ~100 core genes that unique to all AOA. • N. yellowstonensis in hot springs causes Nitrogen limitation, affecting rates of carbon and nitrogen fixation.

• At high temperatures, non-AOA autotrophs may be N limited • N. yellowstonensis does not seem to take up free amino acids

Copyright © 2018 José R. de la Torre – May not be reproduced in any manner without express written permission. SF State University UC Santa Cruz Talia Jewell Todd Lowe Hope Gray David Bernick Amy Jo Johnson Robert Theis UNLV Emily Tung Brian Hedlund Donne Estipona Jeremy Dodsworth Casey Bowers Madeline Cassani Montana State Univ. Diego Gelsinger Eric Boyd Virginia Russell Trinity Hamilton Chris Condry Roxanne Bantay Univ. Miguel Hernández Brittany Baker Paco Rodriguez-Valera Elizabeth Winters Mario López Pérez Louis Contreras Riccardo Rosselli Jose Manuel Haro Genome Sequencing Moreno Matt Ashby (Taxon) Joe DeRisi (UCSF) Steven Quake (Stanford) Iwijn De Vlaminck Jad Kanbar

Northern Arizona Univ. Bruce Hungate Paul Dijkstra Jamie Brown