Metabolic modelling of a bacterial/animal symbiosis
Download
1 / 21

Metabolic modelling of a bacterial/animal symbiosis - PowerPoint PPT Presentation


  • 156 Views
  • Updated On :

Metabolic modelling of a bacterial/animal symbiosis. Sandy Macdonald and Gavin Thomas Department of Biology University of York Angela Douglas Department of Entomology Cornell University USA. Outline. The Buchnera / aphid symbiosis The Buchnera genome

Related searches for Metabolic modelling of a bacterial/animal symbiosis

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Metabolic modelling of a bacterial/animal symbiosis' - sheba


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Slide1 l.jpg

Metabolic modelling of a bacterial/animal symbiosis

Sandy Macdonald and Gavin Thomas

Department of Biology

University of York

Angela Douglas

Department of Entomology

Cornell University

USA


Outline l.jpg
Outline

  • The Buchnera / aphid symbiosis

  • The Buchnera genome

  • Construction and analysis of the Buchnera metabolic model


Slide3 l.jpg

Many insects have obligate bacterial symbionts

  • Aphid symbionts

  • Buchnera sp. APS – Acyrthosiphon pisum

  • Buchnera sp. SG - Schizaphis graminum

  • Buchnera sp. BP - Baizongia pistacea

  • Ant symbionts

  • Blochmannia floridanus

  • Blochmannia pennsylvanicus

  • Tsetse fly symbionts

  • Wigglesworthia glossinidia brevipalpis


Slide4 l.jpg

Aphids – phloem sap feeding pests

The pea aphid, Acyrthosiphon pisum.

  • About 5000 different species

  • Major crop pests.

  • Restricted diet of phloem sap.

  • All contain a primary symbiont and often a secondary symbiont.


Slide5 l.jpg

Buchnera aphidicola sp. APS is the primary symbiont of the pea aphid

  • The bacteria are located in specialised insect cells called bacteriocytes in their body cavity.

  • They are surrounded by an aphid-derived bacteriocycte membrane.

  • TEM of bacteriocyte cytoplasm, showing coccoid Buchnera.

  • The Buchnera are unculturable

  • Vertically transmitted to aphid offspring via the ovary.

  • The function of the symbiosis is nutritional.

  • Phloem sap poor in essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine).

  • These are known to be provided by the symbiont.


Slide6 l.jpg

  • The Buchnera APS genome

  • Small - 0.64 Mb

  • 607 genes (569 protein coding genes)

  • AT rich - 73 % AT

  • Almost 90% of the genes have known functions in E. coli K-12.

  • Virtually no transcription regulation.

  • No IS or phage elements

  • Genome reannotated in BuchneraBASE.


Slide7 l.jpg

Free-living

Gene loss and gene gain

Intracellular

Gene loss only

E. coli K-12

4.2 Mb

Buchnera sp. APS

0.6 Mb

The genome sequences of primary insect symbionts reveal clear descent from an ancestral -proteobacterium

Ancestor ~3.5 Mb

200 My ago…


Slide8 l.jpg

The Buchnera genome has undergone reductive evolution by a series of deletion and inactivations of existing genes

  • Initial large deletions removed large regions of the genome.

  • Followed by smaller deletions and gene inactivation.

  • Genomes are still relatively co-linear (syntenic).



Understanding the symbiosis l.jpg

Understanding the symbiosis

Exploiting E. coli to rapidly produce a high quality metabolic reconstruction of Buchnera APS


Slide11 l.jpg

The iJR904 metabolic model of E. coli K-12

Metabolic model which contains the reactions catalysed by 904 gene products from E. coli.

  • Built from biochemical/genetics literature for E. coli

  • High quality reconstruction

  • Contains 931 unique biochemical reactions

  • Mass and charge balanced

  • e.g.

  • [c]h2o + sl26da --> 26dap-LL + succ

  • [c]26dap-LL <==> 26dap-M

  • [c]26dap-M + h --> co2 + lys-L

Central

metabolism


Slide12 l.jpg

Construction of the metabolic network

  • Method

  • Map all orthologues between APS and K-12.

  • Manually assign reaction codes for APS gene products.

  • Store all relationships in BuchneraBASE.

www.buchnera.org


Slide13 l.jpg

Construction of the metabolic network (2)

Removing isolated reactions

During the reductive evolution, some pathways are in the process of being lost and still have remnants left.

APS has a number of isolated enzymes, e.g. SerC, which were included in the original mapping.


Slide14 l.jpg

Construction of the metabolic network (3)

Missing reactions

Some pathways for EAAs are not complete from the in silico reconstructions, but are known to function in vivo. Infer promiscuous enzymes to fill these gaps.

Full gene complement for synthesis of 4 EAAs (histidine, tryptophan, threonine and lysine), as well as the non-essentials arginine, cysteine and glycine.

Also very short of transporters. Many have been inferred.


Slide15 l.jpg

Carbon-skeleton based manual visualisation of iGT196

196 gene products

240 compounds (39% of iJR904)

263 reactions (27% of iJR904)

35% of reactions for EAA biosynthesis.

Key

Red hexagon – high flux precursor Blue square - EAA

Red circle – low flux precursor Blue circle – biomass component

Grey triangle – inferred reaction

Thomas et al., (2009) BMC Systems Biology 3:24.


Understanding the symbiosis16 l.jpg

Understanding the symbiosis

Analysis of the reconstruction using constraint-based modelling (flux balance analysis)


Slide17 l.jpg

Exchange

Intracellular

Biomass

Exchange

Flux balance analysis

The steady-state assumption states that for each metabolite the sum of the fluxes producing that metabolite is equal to the sum of the fluxes consuming that metabolite.


Slide18 l.jpg

Running FBA with the Buchnera model

FBA is essentially an optimisation problem solved using linear programming, i.e. it finds the optimal route of fluxes through the network to get the desired output (the objective function).

Used reduced version of the ‘biomass’ reaction of E. coli as the objective function.

(0000050) ACP + (0000050) fmnh2 + (0000050) pnto-R + (0000050) gthrd + (0000050) thmpp + (0000050) sheme + (0000050) btn + (0000050) hemeO + (0203000) gtp + (0126000) ctp + (0136000) utp + (0027600) murein5p5p[p] + (0025400) dctp + (0176000) phe-L + (0241000) thr-L + (0054000) trp-L + (0326000) lys-L + (0146000) met-L + (0428000) leu-L + (0090000) his-L + (0276000) ile-L + (0281000) arg-L + (0087000) cys-L + (0582000) gly + (0402000) val-L + (0007000) spmd + (0000010) fad + (0025400) dgtp + (0024700) dttp + (0024700) datp + (45731800) atp + (0001000) amp + (0002150) nad + (0000050) nadh + (0000130) nadp + (0000400) nadph + (0000006) coa + (0000050) accoa + (0000003) succoa + (0730200) pi + (0730200) ppi

To make this a ‘symbiotic’ model the biomass reaction modified to include the exported EAA component.

An estimate of EAA export was obtained empirically using the pea aphid-Buchnera symbiosis reared on chemically defined diets. It varied among the amino acids, from 22% (histidine and tryptophan) to 50% (threonine) of the amount synthesised.

Model required significant tweaking to get it to function – had to build iteratively.


Slide19 l.jpg

The Buchnera metabolic network is highly constrained

The primary constraint that is used to assess the output of the network are the uptake fluxes - 5 main precursors used by the network.

Optimal growth flux (5.21) is unusually only reached by essentially a single ‘solution’, i.e. a single distribution of internal fluxes, as judged by flux variability analysis.

  • A number of ‘dead end’ metabolites are produced in significant quantities in the 5.21 model by Buchnera

    • Adenine by product of spermidine biosynthesis

    • 5-methylribose by product of spermidine biosynthesis

    • S-ribosylhomocysteine by product of cysteine biosynthesis

    • Succinate by product of lysine biosynthesis


Slide20 l.jpg

The Buchnera metabolic network is fragile

  • Dogma is that biological networks have evolved to be robust, i.e. tolerant of some disruption.

  • Deleted individual genes in silico and measured resulting growth flux.

  • E. coli iJR904 serves as a control.

  • Deletion of 84% of the genes in iGT196 led to a >99% decrease in growth flux (19% for iJR904).

  • Similar results using linear minimisation of metabolic adjustment (linearMOMA).


Slide21 l.jpg

Can the aphid control the metabolic function of its symbiont?

Designed a qualitative experiment to demonstrate the principle that the aphid could manipulate the metabolic output of the bacterium just by changing the inputs.

If this network is ‘always on’ then this is a plausible way to control the symbiosis.

Possibly at the level of transporter activity in the bacteriocyte membrane.