Download a multi-omics approach to alleviating

Project proposal form – 2017 entry
Project title: Bacterial biofertilisation of phosphorus in the rhizosphere of agricultural crops: a multi-omics
approach to alleviating food security
Host institution: University of Warwick
Theme: Organisms, omics and biogeochemistry
Key words: metagenomics, metaproteomics, Pi-scavenging, exoenzymes, rhizosphere
Supervisory team:
Professor Elizabeth Wellington, School of Life Sciences, University of Warwick,
[email protected], Dr Alex Jones, School of Life Sciences, University of Warwick,
[email protected] ; Dr Rob Finn, European Institute of Bioinformatics, [email protected]
Project Highlights:
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Cutting-edge multi-omics approach
Microbial physiology linked to crop
production
Scaling from the laboratory to the field
Overview:
This project focuses on bacterial extracellular (exo)
enzymes involved in the remineralisation and
solubilsation of complex organic phosphates and
insoluble inorganic phosphates. These processes are
thought to be involved in soil fertility and thus provide
agricultral crops with inorganic phosphate (Pi)
required for growth. Since the solubility of Pi salts is
poor, and phopshorus (P) present in organic forms
(Po) is not directly available for uptake by the roots,
the supply of Pi in many soils is insufficient to
maintain plant growth. Whilst bacteria have long been
exploited for their enzymes there is a critical need to
consider all the pathways for Pi mobilisation in soil in
order to manipulate the plant microbiome to reduce
our reliance on rock phopshate (Pi), which is a finite
commodity. The alkaline and acid phosphatases,
phytases, nucleases and phosphonatases are regarded
as key microbial enzymes involved in microbial
phosphate mineralisation. Recent work in our lab
using Warwicks cutting-edge proteomics facility
revealed a number of exoenzymes secreted from the
cell in response to Pi-depletion in various
rhizobacterial strains (RBS) from the genus
Pseudomonas and Flavobacteria (Lidbury et al. 2016,
Fig. 1). We now aim to take a novel approach to study
in situ the enzymes involved in Pi mobilization by
using metaproteomics and applying our newly
developed method of metaexoproteomics (Johnson-
Rollings et al., 2014). Our aim is to focus on the
extracellular proteins in soil, as enzymes involved in
breakdown of insoluble polymers and organic
complexes must be in the extracellular milieu in order
to act on these substrates. Wilmes and Bond [3]
pioneered protein extraction from environmental
samples. The soil metaexoproteome (SMEP) alongside
the soil metaproteome (SMP) will indicate enzymes
involved in Pi mobilisation and combined with
metagenomics will provide insights into the microbial
phosphatome in soil.
50%
45%
40%
35%
30%
25%
20%
15%
10%
Low Pi
5%
High Pi
0%
DSM4166 - replete DSM4166 - deplete
Figure 1:
Analysis of the exoproteome of
Pseudomonas stutzeri DSM4166. Proteins in the
exoprtoeome were semi-quantified Left panel from
mass-spectroscopy data generated from the protein
gel right panel.
Methodology:
We will use various RBS as inoculants in soil
experiments to determine the validity of our protein
extraction process. and also to asceretain whether or
not the same exoproteins are secreted in response to
low Pi in situ as in vitro. We will first use laboratory
control pot experiments before moving into fields
plots. Warwick is currently running several field trials
where wheat and oil seed rape (OSR) have been
grown in differing rotations. The metagenome (MG)
and metatranscriptome (MT) of the soil +/- Pi will also
be obtained from deep sequence analysis of total
community DNA/RNA and this data will be used to
inform analysis of the soil SMP and SMEP.
Objectives
1. Determine the phosphatome of various
rhizobacterial strains in situ
2. Use soil microcosms to determine the key proteins
responsible for Pi mobilisation in the rhizopshere of
OSR and wheat in different soils.
3. Determine the key proteins responsible Pi
mobilisation in field plots.
Training and skills:
CENTA students will attend 45 days training
throughout their PhD including a 10-day placement. In
the first year, students will be trained as a single
cohort on environmental science, research methods
and core skills. Throughout the PhD, training will
progress from core skills sets to master classes specific
to the student's projects and themes.
Extensive training in experimental techniques related
to molecular analysis of environmental samples will
be provided in our lab by a senior research technician
and post-doctoral research assistant working on a
project similar to this. This includes developing skills in
the field of bioinformatics, which has become a staple
tool in microbial ecology. This project will provide a
unique opportunity to work with skilled
bioninformaticians at the European Bionformatics
Institute (EBI), where a training course is available.
The student will join a metagenomics network
(ComMet) and gain access to training workshops and
meetings in the UK.
Partners and collaboration (including CASE):
The experimental expertise in Wellington lab will be
complimented by detailed expert knowledge provided
by Jones, the director of Warwick Proteomics Centre
where protein extracts will be analysed. Current work
with Jones aims to improve resolution of peptides
using a gel-independent Thermo Scientific™ Orbitrap
Fusion Tribrid mass spectrometer. Bioinformatics
expertise offered by Rob Finn will assist in resolving
the protein identities from peptide hits using
metagenome data bases. Data bases of enzymes
predicted to be in the phosphatome are currently
being developed by Finn and Wellington using in vitro
data derived from published studies and genomic data
bases.
Possible timeline:
Year 1: Use various RBS to generate data on the in situ
expression of Pi responsive exoenzymes.
Year 2: Work with plant-soil systems to analyse
rhizosphere MG in response to Pi using HiSeq and use
EBI portal to resolve gene diversity. Use this database
to assist in resolving SMP and SEMP developed from
protein extracts of rhizosphere.
Year 3: Field trial experiments on wheat and OSR
using the same methods developed in years 1 and 2.
Further reading:
1) Lidbury IDEA, Murphy ARJ, Scanlan DJ, Bending GD,
Jones AME, Moore JD et al (2016). Comparative
genomic, proteomic and exoproteomic analyses of
three Pseudomonas strains reveals novel insights into
the phosphorus scavenging capabilities of soil
bacteria. Environ Microbiol doi:10.1111/14622920.13390.
2) Roca A, Pizarro-Tobías P, Udaondo Z, Fernández M,
Matilla MA, Molina-Henares MA, Molina L, Segura A,
Duque E, Ramos JL. (2013). Analysis of the plant
growth-promoting properties encoded by the genome
of the rhizobacterium Pseudomonas putida BIRD-1.
Environ Microbiol. 15, 780-94.
3) Johnson-Rollings, A. S., Wright, H., Masciandaro, G.,
Macci, C., Doni, S., Calvo-Bado, L. A., Slade, S. E., Vallin
Plou, C., Wellington, E. M. H. (2014). Exploring the
functional soil-microbe interface and exoenzymes
through soil metaexoproteomics. ISME J. 8, 2148-50.
4) Wilmes, P.; Bond, P. L., (2006). Metaproteomics:
studying functional gene expression in microbial
ecosystems. Trends Microbiol, 14, 92-97.
Further details:
Professor E M H Wellington
School of Life Sciences
The University of Warwick
Coventry CV4 7AL
United Kingdom
Tel: 00442476 523184
Fax: 00442476 523701
Email: [email protected]
http://www2.warwick.ac.uk/fac/sci/lifesci/people/ew
ellington/