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					 Functional Encyclopedia of Bacteria and Archaea




                         Matthew Blow

Deutschbauer lab, LBNL        JGI
          Adam Deutschbauer         Cindi Hoover
          Morgan Price              Feng Chen
          Kelly Wetmore             Jim Bristow
          Adam Arkin
                                                   mjblow@lbl.gov
1. Gene function annotation using transposon
   mutagenesis and sequencing (TnSeq)

2. A ‘Functional Encyclopedia of Bacteria and
   Archaea’ (FEBA)
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
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.
  Our solution: Experimental evidence based
            annotation of genomes
Develop a rapid experimental pipeline to:
  1) Assess phenotypic capability
      via growth assays (~300                  3) Predict gene function with TnSeq in
  metabolic and stress conditions)                 multiple conditions per microbe

                                                                        Nucleus


                                                                     Synthetic light
   2) Correct gene structure and                                     collecting structure
 identify promoters with RNAseq




In D. vulgaris, 507 gene revisions and 1,124
promoters at single nucleotide resolution.
         Gene function annotation by TnSeq

                                                                       Identify mutant fitness
                                                                       effects by PCR and
  Microbe of interest                         Condition A
                                                                       sequencing


       Is there evidence that this approach
i) Transposon
Mutagenesis

         works to annotate gene function?
ii) Recovery
                                              Condition B
iii) Antibiotic
selection




                                                   …              essential in           essential in
                                                    …             condition B            condition C
                                                      …
                                            Selection under                   essential in
    Mutant population                                                        all conditions
 Millions cells, 1 random mutant per cell   100’s of conditions
      Proof of principle: Gene function annotation using
   Transposon mutagenesis and microarray based analysis

                                                Condition 1



                                                Condition 2



                                                Condition 3
                                         …etc
S. Oneidensis MR-1          Mutant      Growth under            Assay selected
Metal reducing bacteria   population   ~300 conditions          populations on
Bio-remediation
                                                                  microarray




                                                (Deutschbauer et al PLoS Genetics 2011)
   Proof of principle: Gene function annotation using
Transposon mutagenesis and microarray based analysis

                                              290 diverse conditions
                                                                                 3,355
                                                                             Genes with Tn
                                                                               mutants
               (average 7 mutants per gene)




                                                                                 1,230
 3,355 genes




                                                                        Genes with significant
                                                                            phenotypes




                                                                                    40
                                                                         Genes with proposed
                                                   –ve fitness effect   annotations of specific
                                                   No fitness effect      molecular function
                                                   +ve fitness effect
                                                                        (Deutschbauer et al PLoS Genetics 2011)
1. Gene function annotation using transposon
   mutagenesis and sequencing (TnSeq)

2. A ‘Functional Encyclopedia of Bacteria and
   Archaea’ (FEBA)
                A Functional Encyclopedia of
                Bacteria and Archaea (FEBA)
   ~50 Phylogeneticaly                                    TnSeq under 50 growth
diverse organisms (GEBA)                                       conditions
                              *       *                        Phosphorou
                    *                     *                             Environmen
                                                               s sources, 8
            *                                 *                         tal stresses
                                                                Sulfur (temp, pH,
                                                             sources, 12 salinity), 9
                                                                                          Carbon
                                                  *                                     sources, 96
    *                     Bacterial
                        phylogenetic                  *
                            tree
        *
                                                  *          Small
                *                                          molecule
                    *                                      stresses
                          *               *                (metals,                         Nitrogen
                                  *                       antibiotics),                   sources, 48
*= GEBA / candidate F-GEBA                                    165
Phylogeny approach to maximize functional diversity                 300 possible growth conditions

        Outcome: 1000’s of novel gene function annotations
           Plans for a FEBA pilot project
Aim 2
Culturing and transposon mutagenesis of ~40 diverse bacteria




                                                                   ..etc

                                                 Aim 1
                                                 a) Work through the entire
                                                    functional annotation
   Growth assays   RNASeq             TnSeq         pipeline for one bacteria
                                                    (P. Stutzeri)
                                                 b) Expand to ~10 bugs
             Analysis / integration

        Functional genome annotation
           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
 Strategy for identifying transposon insertions
                                                     PCR primer contains
                                                     adapter arm and
                                                  5’ index sequence                 3’
                                                        3’                         5’


                                                  5’                                3’             4. PCR
     1. Isolate genomic DNA                                                                        using Tn
     from mutant population                                                                        specific
                                             3’                                                    primer
                                                                          Tn specific
                                                                                                     Tn specific primer
                                                                                        5’
                       2. Sonicate DNA                                    primer
                                                  5’
                                                                                             3’                    3’                    5’

                                             3’                                         5’
                                                                    etc                            Transposon               Illumina universal
                                                                                                   complementary            adapter
                                                                                                   sequence
                                                                           5. Sequencing
                       3. Ligate custom                                    (HiSeq or MiSeq)
                       truncated illumina                                                                     Random 5mer
                       adapter                         Read 2 primer
       5’                               3’        5’                                          3’
3’                                5’         3’                                         5’
             DNA / Tn junction                         Index Read              Read 1 primer




                   +                   3’
                                                  6. Mapping to reference genome
        5’                                        and counting

3’                               5’

     Genomic DNA only inserts are
     not amplifiable by downstream
     PCR
      Does this sequencing strategy work?

Can we use it to identify function of known genes?
          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
TnSeq specifically identifies Tn insertions and
           is highly reproducilbe

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

                                         150
           Tn inserts per gene (Rep 1)




                                         100



                                          50


                                                        Pearson correlation 0.99
                                          0
                                               0          50         100        150
                                                    Tn inserts per gene (Rep 2)
“Essential” genes appear as transposon free regions


                   Transposon           Insertion            Transposon
                    insertions           free site            insertions
           230
Illumina
    read
   depth    0
 Genes


                 Non-essential genes                   Non-essential genes



                                 Essential gene:
                           dihydroxy-acid dehydratase
                         (required for biosynthesis of amino acids)
Top 20 genes advantageous for survival in minimal media
                                                           Tn insertion ratio
Gene
                                                            (LB / minimal)
Phosphoribosylanthranilate_isomerase                              7.0
phosphoserine_phosphatase_SerB                                    6.2
3-isopropylmalate_dehydrogenase                                   5.0
Predicted_membrane_protein                                        4.7
Putative_threonine_efflux_protein                                 3.8
O-succinylhomoserine_sulfhydrylase                                3.5
Chemotaxis_protein_histidine_kinase_and_related_kinases           3.4
tryptophan_synthase,_beta_subunit                                 3.2
Indole-3-glycerol_phosphate_synthase                              3.2
hypothetical_protein                                              3.1
anthranilate_phosphoribosyltransferase                            3.1
ATP_phosphoribosyltransferase,_regulatory_subunit                 3.0
methionine_biosynthesis_protein_MetW                              3.0
5,10-methenyltetrahydrofolate_synthetase                          2.9
Membrane_protease_subunits,_stomatin/prohibitin_homologs          2.8
3-isopropylmalate_dehydratase,_large_subunit                      2.8
anthranilate_synthase_component_I                                 2.7
Predicted_integral_membrane_protein                               2.7
Imidazoleglycerol-phosphate_dehydratase                           2.7
5,10-methylenetetrahydrofolate_reductase                          2.6
Top 20 genes advantageous for survival in minimal media
                                                           Tn insertion ratio
Gene
                                                            (LB / minimal)
Phosphoribosylanthranilate_isomerase                              7.0
phosphoserine_phosphatase_SerB                                    6.2
3-isopropylmalate_dehydrogenase                                   5.0
Predicted_membrane_protein                                        4.7
Putative_threonine_efflux_protein                                 3.8
O-succinylhomoserine_sulfhydrylase                                3.5
Chemotaxis_protein_histidine_kinase_and_related_kinases           3.4
tryptophan_synthase,_beta_subunit                                 3.2
Indole-3-glycerol_phosphate_synthase                              3.2
hypothetical_protein                                              3.1
anthranilate_phosphoribosyltransferase                            3.1
ATP_phosphoribosyltransferase,_regulatory_subunit                 3.0
methionine_biosynthesis_protein_MetW                              3.0
5,10-methenyltetrahydrofolate_synthetase                          2.9
Membrane_protease_subunits,_stomatin/prohibitin_homologs          2.8
3-isopropylmalate_dehydratase,_large_subunit                      2.8
anthranilate_synthase_component_I                                 2.7
Predicted_integral_membrane_protein                               2.7
Imidazoleglycerol-phosphate_dehydratase                           2.7
5,10-methylenetetrahydrofolate_reductase                          2.6

    Red = known role in amino acid biosynthesis
    Blue = known role in purine biosynthesis
  Conclusion:
  - TnSeq strategy works
  - Identifies genes required for growth in minimal media


 The next experiment:



 P. Stutzeri
Mutant library

                                          Synthesis of      Sequencing of
                  Selection under      libraries in plate      pooled
                 multiple conditions     based format        experiments
           Plans for a FEBA pilot project
Aim 2
Culturing and transposon mutagenesis of ~40 diverse bacteria




                                                                   ..etc

                                                 Aim 2
                                                 a) Work through the entire
                                                    functional annotation
   Growth assays   RNASeq             TnSeq         pipeline for one bacteria
                                                    (P. Stutzeri)
                                                 b) Expand to ~10 bugs
             Analysis / integration

        Functional genome annotation
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?
MiSeq analysis of transposon mutant libraries
            from four new bugs

   Tn mutants of four marine bacteria with similar culturing conditions



   Alcanivorax       Dinoroseobacter       Kangiella             Phaeobacter
    jadensis             shibae           aquimarina             gallaeciensis



                                                         Isolate and pool DNA


                                                         PCR Tn inserts and
                                                         sequence on MiSeq


                                                         Map to four genomes

    Alcanivorax      Dinoroseobacter        Kangiella            Phaeobacter
      jadensis            shibae           aquimarina            gallaeciensis
     insertions         insertions          insertions            insertions
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
                                                                                                                                                                  TA = insertion site
                                                                                       20
                                                                                            Kangiella aquimarina (639 potential insertions)
                  (Insertion dinucleotide frequency / genome dinucleotide frequency)

                                                                                                                                                                  preference of
                                                                                                                                                                  pHIMAR transposon
                                                                                       10


                                                                                        0



Conclusion:                                                                            20
                                                                                             Phaeobacter gallaeciensis (158 potential insertions)
Fold enrichment




                                                                                       10


- We are able to culture and mutagenize diverse bacteria
       0

- Need to demonstrate that we can generate high
      40
         Dinoroseobacter shibae (170 potential insertions)
diversity mutant libraries
      30
                                                                                       20
                                                                                       10
                                                                                        0

                                                                                       20
                                                                                            Alcanivorax jadensis (161 potential insertions)
                                                                                       10


                                                                                        0
                                                                                             AA   AC   AG   AT   CA   CC   CG    CT   GA      GC   GG   GT   TA   TC   TG   TT

                                                                                                        Dinucleotide sequence of Tn insertion site
                                Summary
                                We are developing high throughput
                                experimental approaches to
                                annotate gene function

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

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

                                                             mjblow@lbl.gov
                         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
Arg biosynthesis genes




                           Does SO_3749 catalayze
                           missing step in Arg
                           biosynthesis?




                          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
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)
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           Further growth
WORK PROPERLY              (Vector is lost)

				
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posted:6/27/2014
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