Honours Projects 2013
Dr Jason Terpolilli
Centre for Rhizobium Studies
J.Terpolilli@murdoch.edu.au
Background
My research focuses on plant-microbe interactions and in particular on the root-nodule bacteria.
These organisms are unique in their ability to convert atmospheric nitrogen (N2) to plant-available
nitrogen by forming symbiotic associations with legumes. My work seeks to understand how these
organisms, referred to as rhizobia, completely change their way of life and cellular machinery to
ultimately be converted into N2-fixing ‘factories’ (called bacteroids) for the plant’s benefit. The drive
to understand how these microbes achieve N2 fixation, how it is regulated and what determines how
efficient the process is derives from two things:
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Its crucial role in the biosphere, being the principle means by which atmospheric nitrogen is
made available to higher organisms for growth and development and
The critical role N2 fixation plays in raising soil fertility without the need for using energyexpensive and CO2-producing industrially synthesised fertilisers.
Below are outlined four possible honours projects. Please have a look at them and if you they
interest you then I encourage you to come and discuss them with me.
Also, if you have other projects that you would be interested in pursuing, then I would be more than
happy to chat with you about them.
Cheers
Jason
1. Why are some rhizobia better at fixing N2 than others?
While there are rhizobia that are highly efficient fixers, there are many more that fix little or no N.
This has crucial implications in agriculture where suboptimal fixers are prevalent, ultimately leading
to less N-fixed and an overall reduction in the soil fertility and sustainability.
In this project, you will investigate the phenomenon of suboptimal fixation using the recently
sequenced model legume Medicago truncatula. You will make genomic libraries of effective
microsymbionts (such as WSM419 and WSM1022) by cloning them into a suitable broad-host range
vector. You will then develop an electroporation-based method to mobilise these constructs into a
partially effective strain (such as Sm1021) and then inoculate the Sm1021 library-carrying strain onto
the original host plant and look for isolates with increased N2 fixation abilities, relative to the wildtype strain.
In this project will receive training in: PCR, cloning, gel electrophoresis, library construction,
glasshouse experiments, plant propagation and microbiology.
For enquiries, please contact Dr Jason Terpolilli (J.Terpolilli@murdoch.edu.au)
2. How effective are bacteroids from the Medicago-Sinorhizobium symbiosis?
The Medicago truncatula- Sinorhizobium meliloti (Sm1021) symbiosis serves as a model system for
N2 fixation and plant-microbe interaction research. Although we know that Sm1021 cannot fix N2 on
M. truncatula to the same extent as other strains, such as WSM419 or WSM1022 at the whole plant
level, we do not know whether this is due to (a) individual Sm1021 bacteroids being less efficient at
N2 fixation than their effective counterparts (i.e. a Sm1021 bacteroid does not fix as much N2 as a
WSM419/WSM1022 bacteroid); (b) there being less Sm1021 bacteroids per infected plant cell,
relative to the effective strains. The bacteroids from all three strains may fix at the same rate, but
there may be fewer active Sm1021 bacteroids, resulting in a net reduction in the total amount of
N2fixed; (c) the number of infected plant cells may be reduced in the M. truncatula-Sm1021
symbiosis, so that although Sm1021 bacteroids fix at the same rate and there are as many bacteroids
per plant cell, the total available N2 fixing volume of the plant cells could be reduced in a Sm1021
infected nodule.
In this project will receive training in: gas chromatography, protein assays, gel electrophoresis,
microscopy, glasshouse experiments, plant propagation and microbiology.
For enquiries, please contact Dr Jason Terpolilli (J.Terpolilli@murdoch.edu.au)
3. Is bacterial gluconeogenesis important to N2 fixation?
Rhizobia transform into N2 fixing bacteroids in the root nodules of legumes and acquire carbon from
the plant principally in the in the form of malate. This malate is then used by the bacteroids to
generate energy to power the N2-fixing enzyme nitrogenase. Accumulated evidence suggests that in
some N2-fixing relationships, the plant also supplies sugars to the bacteroids, whereas in other
symbioses, the bacteria are fed exclusively malate and have to make sugars themselves by
gluconeogenesis. Whether or not bacteroids receive sugars from plants could significantly affect
bacteroid metabolism and therefore alter the efficiency of N2 fixing symbioses.
The aim of this project is to therefore construct a series of mutations in enzymes of central carbon
metabolism in key rhizobial strains. You will then evaluate the growth phenotype of these mutants
in the free-living state and characterise their symbiotic phenotype on their legume host plants. In
particular, you will measure rates of N2 fixation and compare rates of root infection, looking for key
affects on and symbiotic performance.
In this project will receive training in: cloning, gene inactivation and verification, gel
electrophoresis, gas chromatography, protein assays, gel electrophoresis, microscopy, glasshouse
experiments, plant propagation and microbiology.
For enquiries, please contact Dr Jason Terpolilli (J.Terpolilli@murdoch.edu.au)
4. Can we engineer symbiotic bacteria to fix more N2?
Nitrogenase is the enzyme responsible for fixing N and is found only in prokaryotes (such as the root
nodule bacteria). Nitrogenase requires homocitrate as a cofactor and in free-living nitrogen-fixers,
this biologically unusual molecule is produced by the enzyme homocitrate synthase. But, in all the
rhizobia that associate with the major crop and pasture legumes (e.g. soybean, bean, peas, clover),
the ability to synthesise homocitrate is absent from the bacteria. Instead, they rely on the host plant
synthesising it for them.
In preliminary experiments, pea plants inoculated with a strain carrying a construct expressing the
homocitrate synthase gene, were able to fix more nitrogen than plants inoculated with the wild-type
strain. This is an interesting result and needs to be explored further.
In this project you would modify the construct and transfer this modified version to other strains of
root nodule bacteria. You would then test in the glasshouse whether the engineered strains had
increased levels of fixation. There would also be an opportunity to use metabolomic techniques to
confirm the production of homocitrate in the transformed strain.
In this project will receive training in: PCR, cloning, gas chromatography, glasshouse experiments,
plant propagation and microbiology.
For enquiries, please contact Dr Jason Terpolilli (J.Terpolilli@murdoch.edu.au)