Project title: Metaomic analysis of the bacterial community phosphatome
Project code:
Host institution: University of Warwick
Theme: Organisms, omics and biogeochemistry
Key words: metagenomics, metaproteomics, phytases, phosphate uptake, rhizosphere,
Supervisory team: Professor Elizabeth Wellington, School of Life Sciences, University of Warwick,
e.m.h.wellington@warwick.ac.uk, Dr Alex Jones, School of Life Sciences, University of Warwick,
alex.jones@warwick.ac.uk; Dr Ian Lidbury, School of Life Sciences I.Lidbury@warwick.ac.uk, University of
Warwick; Dr Rob Finn, European Institute of Bioinformatics, rdf@ebi.ac.uk
Overview:
The focus on bacterial enzymes involved in
metabolism of insoluble organic phosphates relates to
the importance of these degradative reactions in soil
fertility. Whilst bacteria have long been exploited for
their enzymes there is a critical need to consider all
the pathways for phopshate (P) mobilisation in soil in
order to manipulate the plant microbiome to reduce
reliance on rock phopshate (Pi). The acid
phosphatases, phytases and phosphonases are
regarded as key microbial enzymes involved in
microbial phosphate mineralisation and were
identified in rhizobacteria (PGRB) (Roca et al., 2013 ).
We aim to take a novel approach to study in situ the
enzymes involved in P mobilization by using
metaproteomics and applying our newly developed
method of metaexoproteomics (Johnson-Rollings et
al., 2014; Fig 1). 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. Since the solubility of Pi
salts is poor, and 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. Wilmes and Bond [3] pioneered protein
extraction from environmental samples. The soil
metaexoproteome (SMEP) will indicate enzymes
involved in Pi mobilisation and combined with
metagenomics allow insights into the microbial
phosphatome in soil. The soil metaproteome (SMP) is
all proteins both intra and extracellular and with
SMEP provides bioinformatic challenges for protein
identification.
Figure 1: Comparison of soil bacterial community
structure and function using 16S rRNA,
metaproteomics and chitinase gene diversity (ref 2).
Methodology:
We will use Pseudomonas putida as inoculant and
standard to determine depth and coverage for
sequencing. Initial experiments will determine
expression of phosphatases, phytases and
phosphonases in vitro experiments under differing
conditions of growth and P availability. Subsequent
experiments will be done in soil to develop
metagenomic profile of the rhizoplane and
rhizosphere community developing on the roots of
Brassica rapa inoculated with P.putida with and
without Pi fertiliser. The metagenome and
metatranscriptome of the soil +/- Pi will be obtained
from deep sequence analysis of total community DNA
used to inform analysis of the soil metaproteome
(SMP) and SMEP.
Objectives
1. Determine phosphatome of P. putida in vitro
2. Use soil microcosms to determine colonisation of B.
rapa with P. putida as inoculant.
3. Exploit bioinformatic data derived from secretome
data in vitro and metaomic analysis of the soil to
resolve SMP amd SMEP in response to Pi regimes.
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 and the chance to develop new
approaches to identification of proteins derived from
metaproteomics using metagenomic data bases. The
will provide a unique opportunity to develop new
bioinformatic tools for application in metaomics and
work with skilled scientists at EBI. The student will join
a metagenomics network (ComMet) and gain access
to training workshops and meetings in the UK. A
training course in metagenomics at EBI will be
available. Training in environmental microbiology,
statistical procedures and plant sciences will
complement the bioinformatics work.
Partners and collaboration (including CASE):
The experimental expertise in Wellington lab will be
complimented by detailed expert knowledge provided
by Jones 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: Work with P. putida to study secretome and
resolve proteome under differing P availability then
develop protein extraction methods using study site
soil inoculated with P. putida. Further develop
phosphatome data base.
Year 2: Work with plant-soil systems to analyse
rhizosphere metagenome using HiSeq and use EBI
portal to resolve gene diversity. Use this data base to
assist in resolving metaproteome developed from
protein extracts of rhizosphere.
Year 3:Further develop rhizosphere phosphatome
using gene expression data in plant rhizosphere from
metatranscriptomics to resolve enzymes involved in P
mobilisation by comparison of activity in gradients of
P availability.
Further reading:
1) 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.
2) 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.
3) 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: e.m.h.wellington@warwick.ac.uk
http://www2.warwick.ac.uk/fac/sci/lifesci/people/ew
ellington/
Nikki Glover
Deputy Student and Academic Services Manager
School of Life Sciences
Ext. 23502