PhD Thesis proposal form
Discipline
(Biology)
Doctoral School
ED 145: Plant Sciences / Sciences du Végétal
http://www.ed-sciences-du-vegetal.u-psud.fr/en/ecoledoctorale.htm
Thesis subject title:
Integration of the urea cycle in diatom metabolism
 Laboratory name and web site
Laboratory name: Genome Responses to Environmental Signals in Photosynthetic Organisms
Address: Environmental and Evolutionary Genomics Section, Institut de Biologie de l’Ecole
Normale Supérieure (IBENS), Paris
Web site: http://www.ibens.ens.fr/spip.php?article86&lang=en
 PhD supervisor (contact person):
 Name: Chris Bowler
 Position: Director of Research (first class) at CNRS and Head of Environmental and
Evolutionary Genomics Section at the Institut be Biologie de l’Ecole Normale Supérieure
(IBENS), Paris
 email: Chris Bowler <[email protected]>
 Phone number: +33 1 44 32 35 25/24
 Thesis proposal (max 1500 words):
a) Background
Carbon export by phytoplankton in marine ecosystems is governed and balanced by assimilation of
nitrogen. Among marine phytoplankton, diatoms are often the most responsive to mixing events
where nutrient laden water is upwelled into the sunlit euphotic zone, yet the cellular basis for this
competitive advantage is poorly understood. Our current understanding of nitrogen metabolism in
photosynthetic eukaryotes is largely based on physiological, biochemical and genome sequence data
from the green alga Chlamydomonas reinhardtii and higher plants. However, diatoms appear to
utilize a variety of biochemical components previously only observed in metazoans or bacteria. Most
notably, a complete metazoan-like ornithine -urea cycle (OUC) appears to have been acquired from
the diatom exosymbiont ancestor (Allen et al 2006). In metazoans, the OUC is involved in the
catabolism of amino acids and the generation of urea for export. In diatoms, the presence of the urea
degrading enzyme urease, acquired from an endosymbiont ancestor, strongly suggests an alternative
function. Furthermore, nitrate is far too precious in an oceanic context to be eliminated by a
phytoplankton cell. We therefore hypothesize that the OUC functions in an anabolic capacity to
repackage and recycle inorganic C and N from both endogenous and exogenous sources. Transgenic
diatom lines in which we knocked down the activity of carbamoyl phosphate synthase (unCPS),
whose gene product provides the inputs into the urea cycle, are indeed impaired in their response to
nutrient additions (Allen et al 2011). Metabolomic analyses indicate that OUC intermediates are
particularly depleted and that both the tricarboxylic acid cycle (TCA) and the glutamine
synthetase/glutamate synthase (GS-GOGAT) cycles are directly linked with the OUC. Several other
metabolites found to be depleted appear to be generated from OUC intermediates by the products of
genes laterally acquired from bacteria. This metabolic coupling of bacterial and exosymbiont-derived
gene products is likely to be fundamental to diatom physiology because impacted compounds include
the diatom cell wall components polyamines and proline (Kröger et al., 2008), the latter being also
the major osmolyte in diatoms (Krell et al., 2007). The diatom OUC therefore represents a key
pathway for anaplerotic carbon fixation into nitrogenous compounds that are essential for diatom
metabolism.
b) Objectives and Methodology
Utilizing traditional physiological techniques in concert with genome-enabled resources, the PhD
student will evaluate a suite of basic hypotheses concerning the role of mitochondria in nitrogen
assimilation and metabolism in marine diatoms. The objectives of the PhD project are: 1) To trace the
flux of NH4+ through the diatom urea cycle and determine the primary cellular assimilation
pathways for urea, NH4+ and NO3-, 2) To evaluate the occurrence and role of a putative
mitochondrial diatom GSGOGAT cycle, 3) To examine the roles of CK, hybrid cluster protein,
agmatinase, and OCD. The first objective will be addressed using stable-isotope pulse-chase studies
of 15N incorporation into a broad suite of 20 metabolites. For objective 2, cellular localization and
associated physiological studies of GS proteins will be conducted using transgenic diatoms
expressing RNAi constructs and fluorescent reporter protein fusions. Objective 3 will be addressed
using traditional biochemical methods following heterologous protein expression and purification,
and the role of individual biochemical components will be examined using parallel full genome
transcriptome and metabolite studies in transgenic diatoms overexpressing or underexpressing
specific genes.
 Publications of the laboratory in the field (max 5):
 Allen, A. E., Dupont, C. L., Oborník, M., Horák, A., Nunes-Nesi, A., McCrow, J. P., Zheng,
H., Hu, H., Fernie, A. R. and Bowler, C. Evolution and metabolic significance of the urea
cycle in photosynthetic diatoms. Nature 473: 203-207 (2011).
 Moustafa, A., Beszteri, B., Maier, U. G., Bowler, C., Valentin, K., and Bhattacharya, D.
Genomic footprints of a cryptic plastid endosymbiosis in diatoms. Science 324: 1724-1726
(2009). 40 citations.
 Bowler, C., et al. (77 authors) The Phaeodactylum genome reveals the evolutionary history of
diatom genomes. Nature, 456: 239-244 (2008). 119 citations.
 Allen, A. E., La Roche, J., Maheswari, U., Lommer, M., Schauer, N., Lopez, P. J., Finazzi,
G., Fernie, A. R., and Bowler, C. Whole-cell response of the pennate diatom Phaeodactylum
tricornutum to iron starvation. Proc. Natl. Acad. Sci. USA 105: 10438-10443 (2008). 30
citations.
 Armbrust, E.V., Berges, J.B., Bowler, C., et al. The genome of the diatom Thalassiosira
pseudonana: Ecology, evolution, and metabolism. Science 304: 79-86 (2004). 461 citations.
 Specific requirements to apply, if any: