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