Metagenomics and Metaproteomics of Polysaccharide-Degrading Bacteria

Mike Cox, Jennifer Edwards, Clive Edwards and Alan McCarthy

Cellulose

Chitin

using an integrated ‘omic’ approach to examine the presence and diversity of polysaccharide-degrading microorganisms that colonise baits in situ.

Metagenomics

Metaproteomics

SIP

Isolation

Diversity

MetaprotogenomoSIPalomics

Screening

Expression Probing

Probe design problems

Protein Databases

Modular Enzymes

Large Numbers

Diverse DomainsSubstrate Specificity

GH5 Family Architecture

And Solutions…

Perl script Downloads accession RPS Blast (Protein PSSMs from PFAM) Locates domain and trims Outputs DNA.gb

ARB GH 5, 6, 8, 9, 12, 18, 19, 48 CBM 2, 3, 4, 6, 10, 17, 33 Library almost ready for screening

Metaproteomics

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300M/z0

100

%

J EN 1J EN1 A c c Ma s s 60 (Ce n,1, 80.00, Ht,5000.0,0.00,1.00) 2: TOF MS MS 776.85ES +

R A S F F G E E N D N 684.35

y 6

480.25y 4

333.19y 3

240.14a175.12

y 1

87.06N

268.13b

382.17b1

463.22

497.20b2

627.33y 5

594.23549.21

813.39y 7

740.29b4

775.42

942.43y 8

924.42814.39

909.34

1056.46y 9

1039.38

943.51

1171.51y 10

1056.52

1154.46

1153.521136.41

1172.56 1285.56y 111268.57

1297.761385.59

13C Cellulose did not degrade

13C – O bond 16-18 x stronger

Jennifer EdwardsClive EdwardsAlan McCarthyRob LockhartPaul LoughnaneJames McDonaldAlex de Menezes

Lab H, University of LiverpoolAquatic Metagenomics Consortium

Bela TiwariNEBCNERCSGM