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Past and Future of Microbiome Science James M. Tiedje Departments of Microbiology and Molecular Genetics and of Plant, Soil & Microbial Sciences Michigan State University Bioremediation - Expanding the Toolbox: Session I - The Microbiome

SLIDE 1

Past and Future of Microbiome Science

James M. Tiedje Departments of Microbiology and Molecular Genetics and of Plant, Soil & Microbial Sciences Michigan State University Bioremediation - Expanding the Toolbox: Session I - The Microbiome Webinar September 30, 2019

SLIDE 2

1012

Species*

Interactions with environment: biotic Molecular detail

f representative

To Understand the Microbial World

& abiotic

rganisms

*Locey and Lennon

PNAS 2016

SLIDE 3

Possible Shifts in Emphasis as the Field of Metagenomics Develops

Resources / Effort Sequencing Experimental

Single cell Single molecule Community metabolism Community signaling Community gene structure

Computational

Global patterns Evolution Ecological determinants Molecular structure + function

5 10

Now here NRC Report on “The

Years

New Science of Metagenomics” Shift in branding to “Microbiome” 2007

SLIDE 4

Era’s in Microbial Ecology: Technology Enabled

1960-70’s: Functions/processes; incubations, isolates; isotopes, gas/liq chromatography, auto analyzers 1980’s: Math models, kinetics; PCs 1990’s: Genes: DNA extraction, hybridization, 16S, machine sequencers, internet 2000’s: Genome sequences**: HTP sequencing, assembly 2010’s: MetaG, T, P, metabolomics: Low cost HTP sequence, HPCs 2020’s: ???

*Suflita, Horowitz, Shelton, Tiedje, 1982, Dehalogenation: Novel pathway for anaerobic biodegradation. Science, 218;1115 **Chain…Tiedje, et.al., 2006, Burkholderia xenovorans LB400 (PCB degrader) harbors a multi-replicon, 9.73-Mbp genome shaped for versatility. PNAS, 103:15280

SLIDE 5

My Themes

1. Untapped organismal and genetic diversity
2. Technology enabled, what is next?
3. Microbiomes; community-based activities

SLIDE 6

Genetic Traits Important to Biodegradation

1. Enzyme (the catalyst for bioconversion)
Its kinetics
2. Regulation (of gene expression)
3. Permease (transport into the cell)
4. Detergent production
5. Chemotaxis (movement to the pollutant)
6. Resistances
heavy metals
solvents
acidity, salt
heat
7. Attach or non-attaching microbes

SLIDE 7

Commonality of Midwest Soil Bacterial Populations

Samples from:

Rank of abundance

variable

Kansas, Iowa, Wisconsin

I1_13 1e+00 I2_13

Native prairie

IC0_01

Rank Abundance

>100 year of cultivation

Overlap by state in number

f sequences in common

phylotypes

Abundance

IC0_03 IC10_03

f 16S rRNA

IC1

phylotypes

IC10 IN0_01

log-log scale

IN1_03 IN10 1e−02

Abundance

IN1 IN2_03 KC0_01 KC10 KC1 KN0_01 KN10 1e−04

Community structure from 3 g of soil

KN1 WC0_01 WC10

ver 4 orders

WC1

f magnitude

WN0_01 WN10 WN1 1e+01 Rank 1e+03

Rank (log)

1e+05

SLIDE 8

How Diverse are Microbiomes at the Gene Level ?

GUT LAKES SOIL AMD

Iowa Soil

Practical outcomes are related to microbiome complexity
0.6 – 1 Tbp for U.S Midwest prairie soil*

Fig: Rodriquez-R and Konstantinidis, Bioinformatics, 2013 Nonpareil: a redundancy-based approach to assess the level of coverage in metagenomic datasets *Rodriguez_R, et.al, mSystems, 2018; an advanced algorithm

SLIDE 9

Comparison of Nonpareil sequence diversity (Nd) and 16S rRNA Shannon H’ taxonomic diversity indices on 86 metagenomes

16S rRNA-H’

Rodriguez-R et. al... Tiedje Konstantinidis mSystems, 2018

Metagenome Nd (α diversity)

SLIDE 10

The Microbial World Grows

~8,000 new genomes from metagenomes 17 new Bacterial Phyla Parks…Hugenholtz, Tyson 3 new Archaeal Phyla Nature Microbio, Sept 2017 Known as UBA’s Uncultured Bacteria & Archaea (from MAGs)

SLIDE 11

Nucl Acids Res, 2018

SLIDE 12 Camp Camp Camp Camp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Campylobacter jejuni subsp Camp Camp Campylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Campylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp ylobacter jejuni subsp Camp Camp Camp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Campylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp ylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Campylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Campylobacter jejuni subsp ylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Camp Campylobacter jejuni subsp Camp Camp Camp Camp Camp Camp Camp Camp Camp

Camp Camp

Camp Camp Camp Camp Camp Camp

Whole genome ANI relationships of 682 Campylobacter jejuni strains

At ANI cut-off of 95%, one species 32 ANI clades

ylobacter jejuni GCA 001223885 ylobacter jejuni CVM 41946 GCA 001419295 jejuni NZ CP010906 jejuni 2008 894 GCA 000254615 Campylobacter jejuni subsp jejuni GCA 000943485

Campylobacter jejuni subsp jejuni GCA 000943385

Campylobacter jejuni subsp jejuni GCA 000943475 jejuni GCA 000948105 ylobacter jejuni GCA 001234825 ylobacter jejuni subsp jejuni LMG 23211 GCA 000255095

Campylobacter jejuni subsp jejuni ATCC 33560 GCA 000254515

ylobacter jejuni GCA 001239325 Campylobacter jejuni GCA 001291825 ylobacter jejuni GCA 001234325 ylobacter jejuni GCA 001236325 ylobacter jejuni subsp jejuni 1213 GCA 000254935

Campylobacter jejuni GCA 001237205

Campylobacter jejuni GCA 001234425 ylobacter jejuni subsp jejuni BIGS0014 GCA 000314285 ylobacter jejuni NZ LN831025 Campylobacter jejuni subsp jejuni ATCC 33560 GCA 000251165 ylobacter jejuni GCA 001237885 ylobacter jejuni GCA 001227485

Campylobacter jejuni subsp jejuni 81 176 NC 008787 Campylobacter jejuni subsp doylei ATCC 49349 GCA 000701745

ylobacter jejuni GCA 001238945 Campylobacter jejuni GCA 001570705 doylei 269 97 NC 009707 Campylobacter jejuni subsp jejuni 81 176 UMCW7 GCA 000509895 jejuni 81 176 NZ AASL01000001 Campylobacter jejuni subsp jejuni 81 176 UMCW9 GCA 000511815 jejuni 129 258 GCA 000254255 jejuni 81 176 DRH212 GCA 000509875 Campylobacter jejuni GCA 001232425 ylobacter jejuni GCA 001230605 ylobacter jejuni GCA 001226425 Campylobacter jejuni GCA 001407995 Campylobacter jejuni GCA 001408075 ylobacter jejuni GCA 001226045 ylobacter jejuni GCA 001407575 ylobacter jejuni GCA 001238805 ylobacter jejuni GCA 001233645 ylobacter jejuni GCA 001229685 Campylobacter jejuni GCA 001406955 ylobacter jejuni GCA 001225345 ylobacter jejuni GCA 001226145

Campylobacter jejuni HB CJGB LXC GCA 001516425

ylobacter jejuni BJ CJD101 GCA 000440015 Campylobacter jejuni BJ CJGB96114 GCA 001516505 Campylobacter jejuni subsp jejuni 1997 11 GCA 000254795 ylobacter jejuni subsp jejuni HB93 13 GCA 000168175 ylobacter jejuni BJ CJGB96G25 GCA 001516485 ylobacter jejuni BJ CJGB95377 GCA 001516495 ylobacter jejuni CVM 41974 GCA 001419505 ylobacter jejuni GCA 001232165 Campylobacter jejuni GCA 001233625 ylobacter jejuni GCA 001228585 ylobacter jejuni GCA 001227845 Campylobacter jejuni GCA 001235365 ylobacter jejuni GCA 001233545 ylobacter jejuni GCA 001407895 Campylobacter jejuni GCA 001547595 ylobacter jejuni GCA 001239685 ylobacter jejuni GCA 001226025

Campylobacter jejuni subsp jejuni NZ CP012690

jejuni NZ CP012689 Campylobacter jejuni subsp jejuni 260 94 GCA 000168135 jejuni HN CJD07035 GCA 000438865 jejuni ICDCCJ07004 GCA 000355845 ylobacter jejuni K1 GCA 000686405 Campylobacter jejuni subsp jejuni ICDCCJ07002 GCA 000355825 ylobacter jejuni subsp jejuni ICDCCJ07001 CP002029 Campylobacter jejuni GCA 001292245 ylobacter jejuni subsp jejuni GCA 000943435 ylobacter jejuni GCA 001239485 ylobacter jejuni subsp jejuni LMG 23357 GCA 000254495 jejuni GCA 000943395 jejuni GCA 000948095 jejuni 1336 NZ CM000854 Campylobacter jejuni subsp jejuni GCA 000943445 ylobacter jejuni GCA 001226345 Campylobacter jejuni GCA 001236725 Campylobacter jejuni GCA 001488675 ylobacter jejuni subsp jejuni LMG 23216 GCA 000254295

Campylobacter jejuni GCA 001491675

ylobacter jejuni GCA 001225065 ylobacter jejuni subsp jejuni 55037 GCA 000254435 ylobacter jejuni GCA 001229945 Campylobacter jejuni GCA 001498975 ylobacter jejuni GCA 001233765 ylobacter jejuni GCA 001226545 ylobacter jejuni GCA 001237785 Campylobacter jejuni GCA 001291925 ylobacter jejuni GCA 001408215 ylobacter jejuni GCA 001407495 ylobacter jejuni NZ CP012149 ylobacter jejuni HB CJGB LC GCA 001516565 jejuni M1 NC 017280 ylobacter jejuni subsp jejuni NZ CP008787 ylobacter jejuni GCA 001233105 ylobacter jejuni GCA 001232725 ylobacter jejuni GCA 001228005 ylobacter jejuni GCA 001238825 Campylobacter jejuni GCA 001236805 ylobacter jejuni GCA 001235565 jejuni 81116 NC 009839 ylobacter jejuni GCA 001233905 Campylobacter jejuni subsp jejuni 327 GCA 000184845 ylobacter jejuni GCA 001233585 ylobacter jejuni GCA 001232265 Campylobacter jejuni GCA 001407915 ylobacter jejuni subsp jejuni BIGS0004 GCA 000314085 ylobacter jejuni GCA 001236445 ylobacter jejuni GCA 001232705

Campylobacter jejuni GCA 001230405

ylobacter jejuni GCA 001224665 ylobacter jejuni GCA 001236865 ylobacter jejuni GCA 001234085 ylobacter jejuni 4031 NC 022529 ylobacter jejuni GCA 001407055 ylobacter jejuni GCA 001236945 ylobacter jejuni GCA 001407675 Campylobacter jejuni GCA 001407975 ylobacter jejuni GCA 001238125 ylobacter jejuni GCA 001231105 Campylobacter jejuni CVM 41985 GCA 001419635 ylobacter jejuni CVM 41910 GCA 001418925 Campylobacter jejuni CVM 41934 GCA 001419185 ylobacter jejuni CVM 41914 GCA 001418985 ylobacter jejuni CVM 41905 GCA 001418905 ylobacter jejuni CVM 41964 GCA 001419625 ylobacter jejuni CVM 41936 GCA 001419195 ylobacter jejuni CVM 41908 GCA 001418915 Campylobacter jejuni CVM 41975 GCA 001419645 ylobacter jejuni NZ CP010465

Campylobacter jejuni CVM 41973 GCA 001419475

Campylobacter jejuni CVM 41933 GCA 001419165 ylobacter jejuni NZ CP010473 ylobacter jejuni NZ CP010492 ylobacter jejuni NZ CP010511 ylobacter jejuni NZ CP010503 ylobacter jejuni NZ CP010483 Campylobacter jejuni GCA 001490635 ylobacter jejuni NZ CP010491 ylobacter jejuni NZ CP010459 Campylobacter jejuni NZ CP010460 ylobacter jejuni NZ CP010481 ylobacter jejuni NZ CP010494 ylobacter jejuni NZ CP010507 ylobacter jejuni NZ CP010498 ylobacter jejuni NZ CP010488 ylobacter jejuni NZ CP010495 Campylobacter jejuni NZ CP010486 ylobacter jejuni NZ CP010500 ylobacter jejuni NZ CP010462 Campylobacter jejuni NZ CP010468 ylobacter jejuni NZ CP010458 ylobacter jejuni NZ CP010463 ylobacter jejuni NZ CP010501 Campylobacter jejuni NZ CP010471 ylobacter jejuni GCA 000821125 ylobacter jejuni CP010478 ylobacter jejuni NZ CP010510 ylobacter jejuni NZ CP010493 ylobacter jejuni NZ CP010490 Campylobacter jejuni NZ CP010469 ylobacter jejuni NZ CP010502

Campylobacter jejuni subsp jejuni LMG 9872 GCA 000255055

Campylobacter jejuni NZ CP010485 ylobacter jejuni NZ CP010467 ylobacter jejuni NZ CP010464 ylobacter jejuni NZ CP010474 Campylobacter jejuni GCA 001227305 ylobacter jejuni NZ CP010506 Campylobacter jejuni NZ CP010480 ylobacter jejuni NZ CP010461 ylobacter jejuni NZ CP010508 Campylobacter jejuni NZ CP010470 ylobacter jejuni NZ CP010489 ylobacter jejuni NZ CP010476 ylobacter jejuni NZ CP010477 ylobacter jejuni NZ CP010479 ylobacter jejuni NZ CP010472 ylobacter jejuni NZ CP010457 ylobacter jejuni NZ CP010504 ylobacter jejuni NZ CP010499 ylobacter jejuni NZ CP010475 Campylobacter jejuni NZ CP010484 ylobacter jejuni NZ CP010482 ylobacter jejuni NZ CP010466 ylobacter jejuni NZ CP010496 ylobacter jejuni NZ CP010509 Campylobacter jejuni NZ CP010487 ylobacter jejuni NZ CP010505 ylobacter jejuni NZ CP010497 Campylobacter jejuni 10186 GCA 000686225 ylobacter jejuni subsp jejuni CG8421 NZ CP005388 ylobacter jejuni 30318 GCA 000686305 Campylobacter jejuni GCA 001547615 ylobacter jejuni 255 GCA 000686265 ylobacter jejuni GCA 001547605 ylobacter jejuni 20176 GCA 000686445 ylobacter jejuni CVM 41902 GCA 001418855 ylobacter jejuni GCA 001407475 Campylobacter jejuni GCA 001236125 ylobacter jejuni GCA 001228865 Campylobacter jejuni GCA 001234745 ylobacter jejuni 10227 GCA 000686285 ylobacter jejuni NZ CP014344 ylobacter jejuni Cj1 GCA 000686345 ylobacter jejuni GCA 001407115 Campylobacter jejuni GCA 001291585 ylobacter jejuni subsp jejuni LMG 9081 GCA 000254535 ylobacter jejuni GCA 001408175 ylobacter jejuni GCA 001406935 Campylobacter jejuni GCA 001237585 ylobacter jejuni GCA 001236705 Campylobacter jejuni GCA 001230345 ylobacter jejuni GCA 001227125 ylobacter jejuni GCA 001230245 ylobacter jejuni GCA 001232365 ylobacter jejuni GCA 001406995

Campylobacter jejuni GCA 001224205

ylobacter jejuni GCA 001407815 ylobacter jejuni GCA 001407795 Campylobacter jejuni GCA 001408195 ylobacter jejuni GCA 001407075 Campylobacter jejuni GCA 001408115 ylobacter jejuni subsp jejuni R14 NZ CP005081 ylobacter jejuni GCA 001235145 ylobacter jejuni GCA 001225465 ylobacter jejuni GCA 001229085

Campylobacter jejuni subsp jejuni 2008 979 GCA 000254695

ylobacter jejuni GCA 001238105 ylobacter jejuni GCA 001234305 ylobacter jejuni GCA 001228305 Campylobacter jejuni GCA 001547675 ylobacter jejuni GCA 001229825

Campylobacter jejuni GCA 001491455

ylobacter jejuni GCA 001237545 ylobacter jejuni Cj3 GCA 000686385 ylobacter jejuni 30286 GCA 000685605 Campylobacter jejuni subsp jejuni CG8486 GCA 000168795 ylobacter jejuni GCA 001236605 ylobacter jejuni CVM 41900 GCA 001418835 ylobacter jejuni GCA 001225425 Campylobacter jejuni GCA 001226945 ylobacter jejuni GCA 001237005 ylobacter jejuni GCA 001227465 ylobacter jejuni GCA 001230045 Campylobacter jejuni CVM 41927 GCA 001419125 ylobacter jejuni HB CJGB XWM GCA 001516435 ylobacter jejuni subsp jejuni S3 NC 017281 ylobacter jejuni RM1221 NC 003912 Campylobacter jejuni GCA 001547625 ylobacter jejuni GCA 001236665 ylobacter jejuni GCA 001233325 Campylobacter jejuni GCA 001407635 ylobacter jejuni GCA 001224845 ylobacter jejuni GCA 001232125 ylobacter jejuni GCA 001230905

Campylobacter jejuni GCA 001236305

ylobacter jejuni GCA 001237745 Campylobacter jejuni GCA 001226085 ylobacter jejuni GCA 001232145 ylobacter jejuni subsp jejuni NW GCA 000234525 Campylobacter jejuni GCA 001442255 ylobacter jejuni subsp jejuni 87459 GCA 000254895 Campylobacter jejuni GCA 001442025 Campylobacter jejuni CVM 41921 GCA 001419065 ylobacter jejuni subsp jejuni LMG 23269 GCA 000254415 ylobacter jejuni GCA 001230365 ylobacter jejuni GCA 001227605 ylobacter jejuni GCA 001232545 Campylobacter jejuni GCA 001227425 ylobacter jejuni GCA 001442335 ylobacter jejuni subsp jejuni 1997 14 GCA 000254815 jejuni 53161 GCA 000254555 ylobacter jejuni subsp jejuni 51037 GCA 000254835 jejuni 51494 GCA 000254275 jejuni NZ CP010306 Campylobacter jejuni GCA 001442185 ylobacter jejuni GCA 001239645 ylobacter jejuni GCA 001420435 Campylobacter jejuni GCA 001237345 ylobacter jejuni GCA 001232465 ylobacter jejuni Cj5 GCA 000686325 Campylobacter jejuni GCA 001237125 ylobacter jejuni GCA 001234945 Campylobacter jejuni GCA 001231165 ylobacter jejuni GCA 001234065 ylobacter jejuni GCA 001236645 ylobacter jejuni GCA 001224805 ylobacter jejuni GCA 001224625 Campylobacter jejuni GCA 001232665

Campylobacter jejuni GCA 001239205

ylobacter jejuni GCA 001230165 ylobacter jejuni GCA 001228805 Campylobacter jejuni GCA 001407455 ylobacter jejuni GCA 001407095 ylobacter jejuni GCA 001224905 Campylobacter jejuni GCA 001235165 ylobacter jejuni GCA 001231765 ylobacter jejuni subsp jejuni LMG 23210 GCA 000255075 Campylobacter jejuni GCA 001226225 ylobacter jejuni GCA 001231625 ylobacter jejuni GCA 001226005

Campylobacter jejuni GCA 001224385

Campylobacter jejuni GCA 001239445 ylobacter jejuni GCA 001236085 ylobacter jejuni GCA 001236785 Campylobacter jejuni GCA 001407035 ylobacter jejuni GCA 001406895 ylobacter jejuni str NCCP No 15742 GCA 000397125 Campylobacter jejuni GCA 001233065 ylobacter jejuni GCA 001232485 ylobacter jejuni HB CJGB LL GCA 001516415 Campylobacter jejuni GCA 001234565 ylobacter jejuni GCA 001229985 ylobacter jejuni GCA 001226065 Campylobacter jejuni GCA 001230725 ylobacter jejuni GCA 001407345 ylobacter jejuni GCA 001407775 ylobacter jejuni subsp jejuni LMG 9217 GCA 000254575 Campylobacter jejuni GCA 001408155 ylobacter jejuni GCA 001234885 ylobacter jejuni GCA 001228825 Campylobacter jejuni GCA 001235925 ylobacter jejuni GCA 001234605 ylobacter jejuni GCA 001228225 ylobacter jejuni GCA 001235065 ylobacter jejuni GCA 001226845 Campylobacter jejuni GCA 001238565 ylobacter jejuni subsp jejuni BIGS0022 GCA 000314445 ylobacter jejuni GCA 001237605 ylobacter jejuni GCA 001233185 ylobacter jejuni GCA 001234165 ylobacter jejuni GCA 001233245 ylobacter jejuni GCA 001231685 ylobacter jejuni GCA 001232945 Campylobacter jejuni GCA 001235825 ylobacter jejuni GCA 001233665 ylobacter jejuni GCA 001231585 ylobacter jejuni GCA 001229485 ylobacter jejuni GCA 001225805 Campylobacter jejuni GCA 001232405

Campylobacter jejuni GCA 001494515

ylobacter jejuni GCA 001224985 ylobacter jejuni GCA 001420485 Campylobacter jejuni GCA 001496075 ylobacter jejuni GCA 001420455 ylobacter jejuni GCA 001235765 ylobacter jejuni GCA 001408055 Campylobacter jejuni GCA 001292185 ylobacter jejuni GCA 001226825

Campylobacter jejuni GCA 001292025

ylobacter jejuni GCA 001292285 ylobacter jejuni GCA 001291805 ylobacter jejuni GCA 001291505 Campylobacter jejuni GCA 001292385 ylobacter jejuni GCA 001292145 ylobacter jejuni GCA 001236525 Campylobacter jejuni GCA 001292045 ylobacter jejuni GCA 001231405 ylobacter jejuni subsp jejuni P854 GCA 000283215 ylobacter jejuni GCA 001238985 ylobacter jejuni GCA 001239265 Campylobacter jejuni CVM 41918 GCA 001419025 ylobacter jejuni GCA 001441735 ylobacter jejuni GCA 001235625

Campylobacter jejuni GCA 001238785

Campylobacter jejuni GCA 001234505 ylobacter jejuni GCA 001228445 ylobacter jejuni GCA 001233985 ylobacter jejuni GCA 001231185 ylobacter jejuni subsp jejuni LMG 23223 GCA 000254335

Campylobacter jejuni GCA 001225265

ylobacter jejuni GCA 001231205 ylobacter jejuni GCA 001229145 ylobacter jejuni GCA 001416835 Campylobacter jejuni GCA 001579555 ylobacter jejuni GCA 001417285 Campylobacter jejuni GCA 001239045 jejuni 60004 GCA 000254375 ylobacter jejuni subsp jejuni 86605 GCA 000254475 ylobacter jejuni GCA 001407695 ylobacter jejuni CVM 41943 GCA 001419245 Campylobacter jejuni GCA 001234685 ylobacter jejuni GCA 001408135 ylobacter jejuni subsp jejuni D2600 GCA 000234545 ylobacter jejuni NZ CP013116 ylobacter jejuni GCA 001233005 Campylobacter jejuni GCA 001233305 ylobacter jejuni subsp jejuni F38011 NZ CP006851 ylobacter jejuni GCA 001231045 ylobacter jejuni GCA 001225745 ylobacter jejuni GCA 001233225 Campylobacter jejuni GCA 001233805 ylobacter jejuni subsp jejuni LMG 23263 GCA 000254355 ylobacter jejuni GCA 001236205 Campylobacter jejuni GCA 001408095 ylobacter jejuni GCA 001407535 ylobacter jejuni GCA 001225025 ylobacter jejuni subsp jejuni 1854 GCA 000254995

Campylobacter jejuni GCA 001229065

Campylobacter jejuni GCA 001228125 ylobacter jejuni subsp jejuni BIGS0030 GCA 000314525 ylobacter jejuni GCA 001225205 ylobacter jejuni GCA 001228545 Campylobacter jejuni GCA 001237645 ylobacter jejuni subsp jejuni 1997 1 GCA 000254675 ylobacter jejuni GCA 001230525 ylobacter jejuni GCA 001228625 ylobacter jejuni GCA 001223845 Campylobacter jejuni GCA 001237965 ylobacter jejuni subsp jejuni 2008 988 GCA 000254655 ylobacter jejuni GCA 001231605 ylobacter jejuni GCA 001227765 Campylobacter jejuni GCA 001237385 ylobacter jejuni GCA 001224345 ylobacter jejuni subsp jejuni P110B GCA 000242375 jejuni H22082 GCA 000242395 Campylobacter jejuni GCA 001239105 ylobacter jejuni GCA 001235245 ylobacter jejuni GCA 001227805 ylobacter jejuni GCA 001232325 ylobacter jejuni GCA 001407415 Campylobacter jejuni GCA 001406915 ylobacter jejuni GCA 001407755 ylobacter jejuni GCA 001239365 ylobacter jejuni GCA 001231085 ylobacter jejuni GCA 001227345 ylobacter jejuni subsp jejuni 1997 4 GCA 000254735 ylobacter jejuni Cj2 GCA 000686365 Campylobacter jejuni GCA 001442295 Campylobacter jejuni GCA 001488115 ylobacter jejuni GCA 001441755 ylobacter jejuni CVM 41922 GCA 001419085 ylobacter jejuni GCA 001225325 ylobacter jejuni GCA 001225045 ylobacter jejuni GCA 001225565 ylobacter jejuni GCA 001228745 ylobacter jejuni GCA 001232925 ylobacter jejuni GCA 001224585 ylobacter jejuni GCA 001225185 Campylobacter jejuni GCA 001237145

Campylobacter jejuni GCA 001232205

Campylobacter jejuni subsp jejuni LMG 23218 GCA 000254315 ylobacter jejuni GCA 001233285 ylobacter jejuni GCA 001235585 ylobacter jejuni GCA 001231385 Campylobacter jejuni GCA 001236845 ylobacter jejuni GCA 001236765 ylobacter jejuni GCA 001230945 ylobacter jejuni GCA 001231785 ylobacter jejuni GCA 001227525 ylobacter jejuni GCA 001225785 Campylobacter jejuni GCA 001407935 ylobacter jejuni GCA 001407135 ylobacter jejuni GCA 001407655 Campylobacter jejuni GCA 001417165 ylobacter jejuni subsp jejuni LMG 23264 GCA 000254395 ylobacter jejuni GCA 001228425 ylobacter jejuni GCA 001224025 ylobacter jejuni GCA 001407615 ylobacter jejuni GCA 001229545 ylobacter jejuni GCA 001228245 ylobacter jejuni GCA 001231745 ylobacter jejuni GCA 001226385 ylobacter jejuni GCA 001225105 Campylobacter jejuni GCA 001239345

Campylobacter jejuni GCA 001230285

ylobacter jejuni GCA 001407555 Campylobacter jejuni GCA 001407595 ylobacter jejuni GCA 001407195 ylobacter jejuni GCA 001229665 ylobacter jejuni subsp jejuni 1577 GCA 000254955 Campylobacter jejuni GCA 001235045 ylobacter jejuni 32488 NC 021834 ylobacter jejuni GCA 001225685 jejuni 1798 GCA 000254975 jejuni 2008 872 GCA 000254635 ylobacter jejuni subsp jejuni BIGS0013 GCA 000314265 ylobacter jejuni GCA 001231705 ylobacter jejuni subsp jejuni 1997 7 GCA 000254755 Campylobacter jejuni GCA 001234965 ylobacter jejuni GCA 001407715 Campylobacter jejuni GCA 001231505

Campylobacter jejuni GCA 001232585

ylobacter jejuni GCA 001223725 Campylobacter jejuni GCA 001234665 Campylobacter jejuni GCA 900000215 ylobacter jejuni GCA 001225845 ylobacter jejuni GCA 001233405 ylobacter jejuni GCA 001229365 ylobacter jejuni GCA 001234865 jejuni 140 16 GCA 000254915 ylobacter jejuni subsp jejuni 1893 GCA 000255015 ylobacter jejuni GCA 001223585 ylobacter jejuni subsp jejuni 1928 GCA 000255035 jejuni NZ CP010307 jejuni NZ CP010058 Campylobacter jejuni GCA 001224105 ylobacter jejuni GCA 001407735 jejuni 00 2538 NC 022351 ylobacter jejuni GCA 001407015 ylobacter jejuni GCA 001407515 jejuni LMG 9879 GCA 000254455 jejuni 00 2425 NC 022362 jejuni 00 2426 NC 022352 jejuni 84 25 GCA 000168195 jejuni 00 2544 NC 022353 ylobacter jejuni subsp jejuni 110 21 GCA 000254855 ylobacter jejuni GCA 001227405 ylobacter jejuni GCA 001229465 Campylobacter jejuni subsp jejuni 305 GCA 000184825 ylobacter jejuni GCA 001236385 ylobacter jejuni GCA 001229505 ylobacter jejuni GCA 001225405 ylobacter jejuni GCA 001229225 jejuni IA3902 NC 017279 ylobacter jejuni subsp jejuni NZ CP010072 jejuni NZ CP010301 ylobacter jejuni GCA 001407855 Campylobacter jejuni GCA 001494015 ylobacter jejuni subsp jejuni 04197 GCA 000285735 ylobacter jejuni GCA 001237085 ylobacter jejuni GCA 001489095 ylobacter jejuni GCA 001230705 Campylobacter jejuni GCA 001231905 ylobacter jejuni GCA 001230125 jejuni NCTC 11168 mfK12E5 NZ CP006688 jejuni NCTC 11168 mcK12E5 NZ CP006687 jejuni NCTC 11168 Kf1 NZ CP006686 jejuni NCTC 11168 K12E5 NZ CP006685 ylobacter jejuni subsp jejuni NCTC 11168 GSv NZ CP006689 jejuni NCTC 11168 BN148 NC 018521 jejuni NCTC 11168 ATCC 700819 NC 002163 Campylobacter jejuni g113 GCA 000633065 ylobacter jejuni GCA 001224325

Campylobacter jejuni GCA 001225885

Campylobacter jejuni GCA 001224405 ylobacter jejuni subsp jejuni 6399 GCA 000285715 ylobacter jejuni GCA 001225165 jejuni xy259 GCA 000285675 ylobacter jejuni GCA 001232965 ylobacter jejuni GCA 001227285 Campylobacter jejuni GCA 001235005 ylobacter jejuni GCA 001232825 ylobacter jejuni GCA 001227065 Campylobacter jejuni GCA 001230085 ylobacter jejuni GCA 001235965 jejuni 04199 GCA 000285755 ylobacter jejuni GCA 001407365 Campylobacter jejuni subsp jejuni DFVF1099 GCA 000184805 jejuni RB922 GCA 000285695 ylobacter jejuni GCA 001235125 Campylobacter jejuni GCA 001407955 ylobacter jejuni HB CJGB QYT GCA 001516405 Campylobacter jejuni GCA 001227665 ylobacter jejuni GCA 001224005 ylobacter jejuni subsp jejuni CF93 6 GCA 000168115 ylobacter jejuni BJ CJGB96299 GCA 001516515 ylobacter jejuni GCA 001238165 ylobacter jejuni GCA 001237565 ylobacter jejuni GCA 001223745 Campylobacter jejuni GCA 001233745 ylobacter jejuni GCA 001236545 ylobacter jejuni GCA 001238245 ylobacter jejuni GCA 001233965 ylobacter jejuni GCA 001234225 ylobacter jejuni GCA 001229025 ylobacter jejuni GCA 001224865 ylobacter jejuni GCA 001229325 ylobacter jejuni GCA 001231425 Campylobacter jejuni GCA 001238885 Campylobacter jejuni GCA 001228605 ylobacter jejuni GCA 001228665 ylobacter jejuni GCA 001236105 ylobacter jejuni GCA 001226745 ylobacter jejuni GCA 001224125 ylobacter jejuni GCA 001230865 ylobacter jejuni GCA 001238185 Campylobacter jejuni GCA 001230685 Campylobacter jejuni GCA 001237865 ylobacter jejuni GCA 001229645 ylobacter jejuni GCA 001236165 ylobacter jejuni subsp jejuni 2008 831 GCA 000254715 ylobacter jejuni GCA 001228525 ylobacter jejuni subsp jejuni JCM 2013 GCA 000614815 ylobacter jejuni GCA 001408235 Campylobacter jejuni GCA 001232565 ylobacter jejuni GCA 001224265 ylobacter jejuni GCA 001407315 ylobacter jejuni GCA 001238765 Campylobacter jejuni GCA 001238065 ylobacter jejuni GCA 001234145

Campylobacter jejuni GCA 001233465

ylobacter jejuni GCA 001229245 ylobacter jejuni subsp jejuni 87330 GCA 000254875 ylobacter jejuni NZ CP012696 Campylobacter jejuni X GCA 000466925 Campylobacter jejuni GCA 000753095 ylobacter jejuni NZ CP014744 ylobacter jejuni GCA 001231985 ylobacter jejuni GCA 001230585 ylobacter jejuni subsp jejuni 2008 1025 GCA 000254595 ylobacter jejuni NZ CP014742 ylobacter jejuni CVM 41923 GCA 001419105 ylobacter jejuni CVM 41912 GCA 001418935 ylobacter jejuni GCA 001407435 ylobacter jejuni GCA 001406975 ylobacter jejuni GCA 001233685 Campylobacter jejuni GCA 001236225 ylobacter jejuni subsp jejuni PT14 NC 018709 ylobacter jejuni GCA 001239025 Campylobacter jejuni GCA 001232745 ylobacter jejuni GCA 001228565 ylobacter jejuni GCA 001228165 ylobacter jejuni GCA 001223925 ylobacter jejuni GCA 001223765 ylobacter jejuni GCA 001236345 Campylobacter jejuni GCA 001236905 ylobacter jejuni GCA 001224965 ylobacter jejuni GCA 001234285 ylobacter jejuni GCA 001239305 Campylobacter jejuni GCA 001232905 ylobacter jejuni GCA 001226245 ylobacter jejuni GCA 001233085 Campylobacter jejuni GCA 001237265 ylobacter jejuni GCA 001226665 ylobacter jejuni GCA 001224465 ylobacter jejuni GCA 000759575 ylobacter jejuni GCA 001230545 Campylobacter jejuni GCA 001232885 ylobacter jejuni GCA 000752375 ylobacter jejuni GCA 000753015 ylobacter jejuni GCA 000753175 ylobacter jejuni GCA 000753295 ylobacter jejuni GCA 000752795 Campylobacter jejuni GCA 000752275 Campylobacter jejuni GCA 001228485 ylobacter jejuni GCA 001226485 ylobacter jejuni GCA 001230565 ylobacter jejuni GCA 001408015 ylobacter jejuni GCA 001231345 ylobacter jejuni GCA 001228945 ylobacter jejuni GCA 001227785 Campylobacter jejuni GCA 001235985 ylobacter jejuni GCA 001230205 ylobacter jejuni GCA 001233025 Campylobacter jejuni subsp jejuni BH 01 0142 GCA 000172355 jejuni 1997 10 GCA 000254775 ylobacter jejuni GCA 001237465 ylobacter jejuni GCA 001231005 Campylobacter jejuni GCA 001227265 ylobacter jejuni GCA 001225925 ylobacter jejuni GCA 001231245 Campylobacter jejuni GCA 001291965 ylobacter jejuni GCA 001225005 ylobacter jejuni GCA 001224485

Campylobacter jejuni GCA 001229445

Campylobacter jejuni GCA 001224185 ylobacter jejuni GCA 001225385 ylobacter jejuni GCA 001231845 ylobacter jejuni GCA 001238965 ylobacter jejuni GCA 001225645 Campylobacter jejuni GCA 001407395 ylobacter jejuni GCA 001407155

ylobacter jejuni subsp jejuni 414 CM000855 Campylobacter jejuni GCA 001407875

Campylobacter jejuni GCA 001407175 Campylobacter jejuni K5 GCA 000686425

ylobacter jejuni GCA 001407215

ylobacter jejuni GCA 001408035 ylobacter jejuni GCA 001291705 ylobacter jejuni GCA 001291565 ylobacter jejuni GCA 001292465

Campylobacter jejuni GCA 001291685

ylobacter jejuni GCA 001291625 Campylobacter jejuni GCA 001291945 ylobacter jejuni GCA 001291645

SLIDE 13

Genetic image of a species

All MiGA tools in the Enveomics Collection at: www.enve-omics.gatech.edu

SLIDE 14

Ecological Framework for Community Outcome

What determines microbial community structure?

Deterministic Stochastic

Major drivers - Subtle* - Physiological - Pure chance Substrate use & its kinetics, adaptions to stressful conditions

spores/dormancy, attachment/biofilms, phage attack, interactions

1. What features are deterministic?
2. How much is deterministic?
3. How much of the unexplained is the unmeasured?
4. How much is due to non-uniform inoculation (dispersal)?
5. Is system dynamics a primary determinant of this distribution?

* Laurent Philippot “Time for noise to become a signal”

How to ‘herd’ the community structure for desired outcomes

SLIDE 15

Protein tree

f dioxygenases

OT-I contains small clusters that attack dioxin (gray) and dibenzofuran (black) compounds (clusters 71, 107, 96, 108, 83). OT-II is mainly phthalate dioxygenases (30)

Iwai, Johnson…Tiedje 2011, AEM, Appl. 77: 3551

SLIDE 16

B

h

t a i n e d s e q u e n c e s Only obtained sequences a n d r e f e r e n c e s e q u e n c e s O n l y r e f e r e n c e s e q u e n c e s

Known and novel sequence clusters

Clusters with 0.6 distance

Obtained sequences were distributed in

many clusters

33 and 13 novel clusters were obtained

Iwai, S…..Tiedje, 200, ISMEJ 4, 279

for BPHD-f3 and BPHD-r1, respectively

SLIDE 17

Dioxygenase cluster comparison of two sites

BPHD-f3 sequence results

Unique clusters

Novel clusters Clusters with reference genes

8% of total Unique clusters 4% of total

Czech Republic soil Passaic River, New Jersey

More than 90% of the total sequences were in common clusters
Novel clusters found in Czech sample were also found in Passaic River
Passaic River sequences were clustered more with reference genes

SLIDE 18

Stable Isotope Probing : Selecting Sub-Populations

Separates and concentrates nucleic acids from populations that

actively assimilate the labeled substrate

Use 13C-biphenyl, an analogue of PCB, as substrate.

Describe & Identify

Organisms (16S rRNA genes)
Functional genes (ARHDs)

12C-DNA & RNA 13C-labeled substrates 13C-DNA & RNA

13C-Metagenome

Microarray Cosmid Library: Recovering genes

Sul, WJ......Tiedje, et.al. 2009, AEM, 75:5501

SLIDE 19

Structure and PCB Dioxyenase Activity of the Metagenomic Clone

Regions responsible for substrate specificity

I II III IV

f BphA

13C enriched clone L11E10

Dioxygenase activity
n PCB congeners
L11E10-bphAE attack PCB

rings that are not substituted on one ring or substituted at the para position only.

Sul,...Tiedje, et.al. 2009, AEM, 75:5501

SLIDE 20

Rank Order of Degradation Kinetics of 3-Chlorobenzoate Degraders

Rhodes....Tiedje et. al. AEM, 2013

SLIDE 21

Many Strains Produce Intermediate Products (red and green colors)

Rhodes…Tiedje et. al. AEM, 2013

SLIDE 22

Advantages of long read sequencing: gene context

ARG gene coverage: distribution of Blastn hits

PacBio Nanopore Reads: 232,649 1,920,385 # Sequences >10 ARGs & MGEs 212 49

13

SLIDE 23

Percentage of ARGs associated with MGEs

Trimethoprim Sulfonamide Others Chloramphenicol Beta-lactam Quinolone Tetracycline Peptide MLS Aminoglycoside Multidrug/Metal Biocide Multidrug Metal Sequence contain ARGs Sequenec contain ARGs and MGEs

Type of antibiotic resistance

1.1% 25.1% 77.0% 70.6% 57.4% 22.8% 2.3%

Pacbio

Metal resistance, antimicrobial biocide resistance and Multidrug resistance genes are dominant

4000 8000 12000 16000 20000 24000

Number of sequences

15 A small proportion of multidrug and metal resistance genes appear transferable

SLIDE 24

What About the Future?

SLIDE 25

What is Next for Our Science: 5 Years

Return to the questions of the 1970s, 80s
Process measures, modeling, culture-based resolution
But with more resolving methods
Hypothesis-driven microbiome science:
Ecology, Evolution, Function, Mechanism
Long-read sequencing technology becomes standard
Use of advanced microscopies, at the microbes’ scale
image analysis
More use of isotope methods, link to function
Better habitat characterization & its reporting
SAGs and MAGs become part of organized taxonomy
Use of whole genome information, not only 16S rRNA
Meta analyses of the decade of ‘diversity’ data

SLIDE 26

Longer–term Future Prospects

In field, rapid, e-connected diagnostics, AI guidance
Image analysis
enhanced metabolite/chemical analyses
multiple omics markers
AI, Machine Learning, Deep learning neural networks
HTP culturing: ecol, physio & biochem characterization
DNA markers for kinetic properties; competition
HTP direct protein sequencing
Real-time, e-linkage of omics with function
CRISPR-cas9 directed microbe design
Particular microbiome & management ownership
Synthetic ecobiology; designer communities
Quantum computing: complex system-complex

solutions

SLIDE 27