Functional Encyclopedia of Bacteria and Archaea

Functional Encyclopedia of Bacteria and Archaea

Matthew Blow

[email protected]

Adam DeutschbauerMorgan PriceKelly WetmoreAdam Arkin

Cindi HooverFeng ChenJim Bristow

Deutschbauer lab, LBNL JGI

1. Gene function annotation using transposon mutagenesis and sequencing (TnSeq)

2. A ‘Functional Encyclopedia of Bacteria and Archaea’ (FEBA)



Problem: Computational annotation of microbial genomes is imperfect

Nucleus

Isolate Sequence Predict gene structure and function Incomplete modelCurrent computational genome annotation pipeline:

Limitations of homology:

• New experimental approaches are necessary to rapidly annotate and characterize microbial genomes.

• Median bacterial genome: 3261 protein coding genes 971 “hypothetical” protein coding genes

Nucleus

Synthetic light collecting structure

Develop a rapid experimental pipeline to:1) Assess phenotypic capability

via growth assays (~300 metabolic and stress conditions)

2) Correct gene structure and identify promoters with RNAseq

3) Predict gene function with TnSeq in multiple conditions per microbe

In D. vulgaris, 507 gene revisions and 1,124promoters at single nucleotide resolution.

Our solution: Experimental evidence based annotation of genomes

Gene function annotation by TnSeq

i) TransposonMutagenesis

ii) Recovery

iii) Antibiotic selection

Mutant populationMillions cells, 1 random mutant per cell

Condition A

Condition B

Selection under100’s of conditions

……

… essential in condition B

essential in all conditions

essential in condition C

Microbe of interest

Identify mutant fitness effects by PCR and sequencing

Is there evidence that this approach works to annotate gene function?

S. Oneidensis MR-1Metal reducing bacteria Bio-remediation

Mutantpopulation

(Deutschbauer et al PLoS Genetics 2011)

Proof of principle: Gene function annotation using Transposon mutagenesis and microarray based analysis

…etc

Growth under ~300 conditions

Condition 1

Condition 2

Condition 3

Assay selected populations on

microarray

3,35

5 ge

nes

(ave

rage

7 m

utan

ts p

er g

ene)

290 diverse conditions3,355

Genes with Tn mutants

40Genes with proposed

annotations of specific molecular function

1,230Genes with significant

phenotypes

(Deutschbauer et al PLoS Genetics 2011)

Proof of principle: Gene function annotation using Transposon mutagenesis and microarray based analysis

–ve fitness effectNo fitness effect

+ve fitness effect



~50 Phylogeneticaly diverse organisms (GEBA)

** *

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*

*

**

*

Bacterial phylogenetic

tree

*

**

*

*

**

*= GEBA / candidate F-GEBAPhylogeny approach to maximize functional diversity

Carbon sources; 96

Nitrogen sources; 48

Small molecule stresses (metals, an-

tibiotics); 165

Sulfur sources; 12

Phosphorous sources, 8 Environmental

stresses (temp, pH, salinity); 9

TnSeq under 50 growth conditions

300 possible growth conditions

Outcome: 1000’s of novel gene function annotations

A Functional Encyclopedia of Bacteria and Archaea (FEBA)

Plans for a FEBA pilot project

Aim 1a) Work through the entire

functional annotation pipeline for one bacteria (P. Stutzeri)

b) Expand to ~10 bugs

Aim 2Culturing and transposon mutagenesis of ~40 diverse bacteria

..etc

Growth assays RNASeq TnSeq

Analysis / integration

Functional genome annotation





..etc


?

Strategy for identifying transposon insertions

1. Isolate genomic DNA from mutant population

2. Sonicate DNA

Read 1 primer

Read 2 primer

Index Read

5’ 3’

3’ 5’

5. Sequencing (HiSeq or MiSeq)

6. Mapping to reference genome and counting

DNA / Tn junction

Genomic DNA only inserts are not amplifiable by downstream PCR

3. Ligate custom truncated illumina adapter

+

5’

3’ 5’

3’

5’

3’ 5’

3’

Tn specificprimer

4. PCR using Tn specific primer

Random 5mer

Transposon complementary sequence

Illumina universal adapter

Tn specific primer

5’3’

PCR primer contains adapter arm and index sequence

5’ 3’3’ 5’

5’

5’ 3’

3’

3’ 5’

5’ 3’

etc

Does this sequencing strategy work?

Can we use it to identify function of known genes?

Select in LB

Select in minimal media

Proof of principle: Identification of genes required for survival in minimal media in Pseudomonas Stutzeri

P.StutzeriSoil bacteria with a potential applications in bioremediation

>106 mutant cells

TransposonMutagenesis

Compare

Map to the genome Tn insertion is at TAReplicate 1 99.91% 97.81%Replicate 2 99.92% 97.80%

TnSeq specifically identifies Tn insertions and is highly reproducilbe

0

50

100

150

150100500

Tn in

sert

s pe

r gen

e (R

ep 1

)

Tn inserts per gene (Rep 2)

Pearson correlation 0.99

“Essential” genes appear as transposon free regions

0

230Illumina read

depthGenes

Non-essential genes Non-essential genes

Essential gene:dihydroxy-acid dehydratase

(required for biosynthesis of amino acids)

Transposon insertions

Transposon insertions

Insertion free site

Top 20 genes advantageous for survival in minimal media

Gene Tn insertion ratio(LB / minimal)

Phosphoribosylanthranilate_isomerase 7.0phosphoserine_phosphatase_SerB 6.23-isopropylmalate_dehydrogenase 5.0Predicted_membrane_protein 4.7Putative_threonine_efflux_protein 3.8O-succinylhomoserine_sulfhydrylase 3.5Chemotaxis_protein_histidine_kinase_and_related_kinases 3.4tryptophan_synthase,_beta_subunit 3.2Indole-3-glycerol_phosphate_synthase 3.2hypothetical_protein 3.1anthranilate_phosphoribosyltransferase 3.1ATP_phosphoribosyltransferase,_regulatory_subunit 3.0methionine_biosynthesis_protein_MetW 3.05,10-methenyltetrahydrofolate_synthetase 2.9Membrane_protease_subunits,_stomatin/prohibitin_homologs 2.83-isopropylmalate_dehydratase,_large_subunit 2.8anthranilate_synthase_component_I 2.7Predicted_integral_membrane_protein 2.7Imidazoleglycerol-phosphate_dehydratase 2.75,10-methylenetetrahydrofolate_reductase 2.6

Top 20 genes advantageous for survival in minimal media

Gene Tn insertion ratio(LB / minimal)

Phosphoribosylanthranilate_isomerase 7.0phosphoserine_phosphatase_SerB 6.23-isopropylmalate_dehydrogenase 5.0Predicted_membrane_protein 4.7Putative_threonine_efflux_protein 3.8O-succinylhomoserine_sulfhydrylase 3.5Chemotaxis_protein_histidine_kinase_and_related_kinases 3.4tryptophan_synthase,_beta_subunit 3.2Indole-3-glycerol_phosphate_synthase 3.2hypothetical_protein 3.1anthranilate_phosphoribosyltransferase 3.1ATP_phosphoribosyltransferase,_regulatory_subunit 3.0methionine_biosynthesis_protein_MetW 3.05,10-methenyltetrahydrofolate_synthetase 2.9Membrane_protease_subunits,_stomatin/prohibitin_homologs 2.83-isopropylmalate_dehydratase,_large_subunit 2.8anthranilate_synthase_component_I 2.7Predicted_integral_membrane_protein 2.7Imidazoleglycerol-phosphate_dehydratase 2.75,10-methylenetetrahydrofolate_reductase 2.6

Red = known role in amino acid biosynthesisBlue = known role in purine biosynthesis

Conclusion: - TnSeq strategy works- Identifies genes required for growth in minimal media

P. StutzeriMutant library

The next experiment:

Selection under multiple conditions

Synthesis of libraries in plate

based format

Sequencing of pooled

experiments


Aim 2a) Work through the entire

functional annotation pipeline for one bacteria (P. Stutzeri)

b) Expand to ~10 bugs




..etc


Progress toward culturing and mutagenesis of ~40 bacteria

44 bugs (9 phyla)In hand at LBNL

15 bugs (5 phyla) Cultured

9 bugs (2 phyla)Tn mutagenesis

attempted

Was mutagenesis successful?

Tn mutants of four marine bacteria with similar culturing conditions

MiSeq analysis of transposon mutant libraries from four new bugs

Alcanivorax jadensis

Dinoroseobacter shibae

Kangiella aquimarina

Phaeobacter gallaeciensis

Isolate and pool DNA

PCR Tn inserts and sequence on MiSeq

Alcanivorax jadensis

insertions

Dinoroseobacter shibae

insertions

Kangiella aquimarina insertions

Phaeobacter gallaeciensis

insertions

Map to four genomes

MiSeq analysis of transposon mutant libraries from four new bugs

96% reads map to unincorporated transposon!

But…….

Candidate transposon insertions from all 4 bugs

Candidate transposon are at expected TA dinucleotides

0

10

20

0

10

20Chart Title

010203040 Chart Title

AA AC AG AT CA CC CG CT GA GC GG GT TA TC TG TT0

10

20Chart Title

Fold

enr

ichm

ent

(Inse

rtio

n di

nucl

eotid

e fr

eque

ncy

/ gen

ome

dinu

cleo

tide

freq

uenc

y) Kangiella aquimarina (639 potential insertions)

Phaeobacter gallaeciensis (158 potential insertions)

Dinoroseobacter shibae (170 potential insertions)

Alcanivorax jadensis (161 potential insertions)

Dinucleotide sequence of Tn insertion site

TA = insertion site preference of pHIMAR transposon

Conclusion: - We are able to culture and mutagenize diverse bacteria- Need to demonstrate that we can generate high diversity mutant libraries

Summary

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Bacterial phylogenetic

tree

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**The ‘FEBA’ project will provide functional annotation for 50 diverse organisms / 1000s novel genes

[email protected]

We are developing high throughput experimental approaches to annotate gene function

Future ‘product’ of JGI? Keen to target bugs of interest to DOE and to JGI user community

Example of specific novel gene function annotation from transposon mutagenesis

Gene S0_3749 = Hypothetical gene with no homology based annotation

Arg

bio

synt

hesi

s ge

nes

Conditions

Strong –ve fitness effectNo fitness effect

Functional evidence from mutant assays

Does SO_3749 catalayze missing step in Arg biosynthesis?

2. Function confirmed in complementation assay

Conclusion: SO374 encodes a functional acetyl-ornithine deacetylaseNo homology to the functional ortholog (argE) in E.Coli

Transposon mutagenesis through bacterial conjugation

Target cell E. Coli ‘donor’ cell

Vector carrying transposon

Conjugation

Growth in absence of DAP(E. Coli dies)

Further growth(Vector is lost)

Transposon mutagenesis through bacterial conjugation

Target cell E. Coli ‘donor’ cell

Vector carrying transposon

Conjugation

Growth in absence of DAP(E. Coli dies)

Further growth(Vector is lost)

THIS STEP DIDN’T WORK PROPERLY

Documents

Functional Encyclopedia of Bacteria and Archaea