Functional Encyclopedia of Bacteria and Archaea

1. Functional Encyclopedia of Bacteria and Archaea Matthew Blow Deutschbauer lab, LBNL JGI Adam Deutschbauer Morgan Price Kelly Wetmore Adam Arkin Cindi Hoover Feng Chen Jim Bristow mjblow@lbl.gov

2. Gene function annotation using transposon mutagenesis and sequencing (TnSeq) A ‘Functional Encyclopedia of Bacteria and Archaea’ (FEBA)

3. Gene function annotation using transposon mutagenesis and sequencing (TnSeq) A‘Functional Encyclopedia of Bacteria and Archaea’ (FEBA)

4. Problem: Computational annotation of microbial genomes is imperfect Current computational genome annotation pipeline: Isolate Sequence Predict gene structure and function Incomplete model Nucleus Limitations of homology: • Median bacterial genome: • 3261 protein coding genes • 971 “hypothetical” protein coding genes • New experimental approaches are • necessary to rapidly annotate • and characterize microbial genomes.

5. Oursolution: Experimental evidence based annotation of genomes Develop a rapid experimental pipeline to: 1) Assess phenotypic capability via growth assays (~300 metabolic and stress conditions) 3) Predict gene function with TnSeq in multiple conditions per microbe Nucleus Synthetic light collecting structure 2) Correct gene structure and identify promoters with RNAseq In D. vulgaris, 507 gene revisions and 1,124 promoters at single nucleotide resolution.

6. Gene function annotation by TnSeq Identify mutant fitness effects by PCR and sequencing Condition A Microbe of interest Is there evidence that this approach works to annotate gene function? i) Transposon Mutagenesis ii) Recovery iii) Antibiotic selection Condition B essential in condition B essential in condition C … … … Selection under 100’s of conditions essential in all conditions Mutant population Millions cells, 1 random mutant per cell

7. Proof of principle: Gene function annotation using Transposon mutagenesis and microarraybased analysis S. Oneidensis MR-1 Metal reducing bacteria Bio-remediation Condition 1 Condition 2 Condition 3 …etc Mutant population Growth under ~300 conditions Assay selected populations on microarray (Deutschbauer et al PLoS Genetics 2011)

8. Proof of principle: Gene function annotation using Transposon mutagenesis and microarraybased analysis 290 diverse conditions 3,355 Genes with Tn mutants 1,230 Genes with significant phenotypes 3,355 genes (average 7 mutants per gene) 40 Genes with proposed annotations of specific molecular function –ve fitness effect No fitness effect +ve fitness effect (Deutschbauer et al PLoS Genetics 2011)

9. Gene function annotation using transposon mutagenesis and sequencing (TnSeq) A‘Functional Encyclopedia of Bacteria and Archaea’ (FEBA)

10. A Functional Encyclopedia of Bacteria and Archaea (FEBA) ~50 Phylogeneticaly diverse organisms (GEBA) TnSeq under 50 growth conditions * * * * * * * * Bacterial phylogenetic tree * * * * * * * * *= GEBA / candidate F-GEBA Phylogeny approach to maximize functional diversity 300 possible growth conditions Outcome: 1000’s of novel gene function annotations

11. Plans for a FEBA pilot project Aim 2 Culturing and transposon mutagenesis of ~40 diverse bacteria ..etc Aim 1 Work through the entire functional annotation pipeline for one bacteria (P. Stutzeri) Expand to ~10 bugs Growth assays RNASeq TnSeq Analysis / integration Functional genome annotation

12. Plans for a FEBA pilot project Aim 1 Culturing and transposon mutagenesis of ~40 diverse bacteria ..etc ? Growth assays RNASeq TnSeq Analysis / integration Functional genome annotation

13. Strategy for identifying transposon insertions PCR primer contains adapter arm and index sequence 5’ 3’ 3’ 5’ 3’ 5’ 5’ 5’ 4. PCR using Tn specific primer 3’ 3’ 1. Isolate genomic DNA from mutant population 3’ Tn specific primer 2. Sonicate DNA 5’ 5’ 3’ etc 5’ 3’ Tn specific primer 5. Sequencing (HiSeq or MiSeq) 3’ 3. Ligate custom truncated illumina adapter 5’ Transposon complementary sequence Illumina universal adapter DNA / Tnjunction Random 5mer + 3’ 5’ 6. Mapping to reference genome and counting Read 2 primer 3’ 5’ 5’ 3’ Genomic DNA only inserts are not amplifiable by downstream PCR 5’ 3’ Read 1 primer Index Read

14. Does this sequencing strategy work? Can we use it to identify function of known genes?

15. Proof of principle: Identification of genes required for survival in minimal media in Pseudomonas Stutzeri P.Stutzeri Soil bacteria with a potential applications in bioremediation Transposon Mutagenesis Select in LB Compare >106 mutant cells Select in minimal media

16. TnSeq specifically identifies Tn insertions and is highly reproducilbe 150 100 Tn inserts per gene (Rep 1) 50 Pearson correlation 0.99 0 0 50 100 150 Tn inserts per gene (Rep 2)

17. “Essential” genes appear as transposon free regions Transposon insertions Insertion free site Transposon insertions 230 Illumina read depth 0 Genes Non-essential genes Non-essential genes Essential gene: dihydroxy-acid dehydratase (required for biosynthesis of amino acids)

18. Top 20 genes advantageous for survival in minimal media

19. Top 20 genes advantageous for survival in minimal media Red = known role in amino acid biosynthesis Blue = known role in purine biosynthesis

20. Conclusion: - TnSeq strategy works - Identifies genes required for growth in minimal media • The next experiment: P. Stutzeri Mutant library Synthesis of libraries in plate based format Sequencing of pooled experiments Selection under multiple conditions

21. Plans for a FEBA pilot project Aim 2 Culturing and transposon mutagenesis of ~40 diverse bacteria ..etc Aim 2 Work through the entire functional annotation pipeline for one bacteria (P. Stutzeri) Expand to ~10 bugs Growth assays RNASeq TnSeq Analysis / integration Functional genome annotation

22. 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

23. Was mutagenesis successful?

24. MiSeq analysis of transposon mutant libraries from four new bugs Tn mutants of four marine bacteria with similar culturing conditions Alcanivoraxjadensis Dinoroseobactershibae Kangiellaaquimarina Phaeobactergallaeciensis Isolate and pool DNA PCR Tn inserts and sequence on MiSeq Map to four genomes Alcanivoraxjadensis insertions Dinoroseobactershibae insertions Kangiellaaquimarina insertions Phaeobactergallaeciensis insertions

25. MiSeq analysis of transposon mutant libraries from four new bugs 96% reads map to unincorporated transposon! But……. Candidate transposon insertions from all 4 bugs

26. Candidate transposon are at expected TA dinucleotides TA = insertion site preference of pHIMAR transposon Kangiellaaquimarina (639 potential insertions) Conclusion: - We are able to culture and mutagenize diverse bacteria - Need to demonstrate that we can generate high diversity mutant libraries Phaeobactergallaeciensis(158 potential insertions) Fold enrichment (Insertion dinucleotide frequency / genome dinucleotide frequency) Dinoroseobactershibae (170 potential insertions) Alcanivoraxjadensis(161 potential insertions) Dinucleotide sequence of Tn insertion site

27. Summary We are developing high throughput experimental approaches to annotate gene function * * * * * * • The ‘FEBA’ project will provide functional annotation for 50 diverse organisms / 1000s novel genes * Bacterial phylogenetic tree * * * * • Future ‘product’ of JGI? Keen to target bugs of interest to DOE and to JGI user community * * * * * mjblow@lbl.gov

29. Example of specific novel gene function annotation from transposon mutagenesis Gene S0_3749 = Hypothetical gene with no homology based annotation Functional evidence from mutant assays 2. Function confirmed in complementation assay Conditions Does SO_3749 catalayze missing step in Arg biosynthesis? Arg biosynthesis genes Strong –ve fitness effect No fitness effect Conclusion: SO374 encodes a functional acetyl-ornithine deacetylase • No homology to the functional ortholog (argE) in E.Coli

30. Transposon mutagenesis through bacterial conjugation Vector carrying transposon Target cell E. Coli ‘donor’ cell Conjugation Growth in absence of DAP (E. Coli dies) Further growth (Vector is lost)

31. Transposon mutagenesis through bacterial conjugation Vector carrying transposon Target cell E. Coli ‘donor’ cell Conjugation Growth in absence of DAP (E. Coli dies) THIS STEP DIDN’T WORK PROPERLY Further growth (Vector is lost)