2024 RTP round - Dissecting the functions of a quorum-sensing controlled network of non-coding RNAs in the genus Mesorhizobium

Status: Closed

Applications open: 7/07/2023
Applications close: 25/08/2023

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About this scholarship
Description/Applicant information

Mesorhizobium spp. are soil bacteria capable of forming a range of nitrogen-fixing symbioses with agriculturally important legumes. In Australia and New Zealand, high performing Mesorhizobium spp. strains are selected for use as agricultural inoculants for chickpea and clover, providing an economically and environmentally-friendly alternative to chemical fertilisation. However, the benefits of introduced inoculants can be short-lived in Australian soils as introduced strains are outcompeted by native Mesorhizobium spp., which lack the genetic capacity for symbiosis. Therefore, development of Mesorhizobium spp. strains that are competitive with indigenous species would be agriculturally beneficial. We have discovered a distinct quorum-sensing system (MQS) conserved in the genus Mesorhizobium, which allows Mesorhizobium spp. to communicate specifically with each other through the secretion and response to a novel signalling molecule (5Z)-N-[(3S)-2-Oxotetrahydro-3-furanyl]-5-dodecenamide (5-cis-C12-HSL). Despite the ubiquitous presence of the MQS locus in the genus, the cellular functions controlled by MQS are unclear. Intriguingly, strains deleted for the MQS system initially outcompete wild-type strains in co-culture experiments but the wild-type strain ultimately prevails in nutrient-limited stationary-phase conditions. RNA sequencing of MQS mutant strains led to the discovery that the primary output of the MQS system is to activate transcription five novel non-coding RNA (ncRNA) genes, which are also highly conserved throughout the genus. The proximity of ncRNA genes and the MQS locus with genes involved in phosphate uptake, storage and metabolism suggests they may control phosphate utilisation. Australian soils are deficient in phosphorus, so understanding how introduced legume symbionts manage and utilise phosphate may provide additional avenues for inoculant improvement. This project will explore the molecular functions of the ncRNA genes and test the hypothesis that these ncRNA are involved in phosphate metabolism. 

Aim 1: Accurately define the ncRNA genes using 5'-end RNAseq.
While our previous RNA sequencing identified the position of the ncRNA genes in the genome, generic RNAseq protocols do not generate accurate predictions for the transcriptional start sites. Moreover, given that the targets of ncRNA are most often the 5' untranslated leader sequences of protein-coding mRNAs, accurate genome-wide identification of transcriptional start sites is critical for identification of ncRNA targets. We will carry out RNA sequencing using Tobacco Acid Pyrophosphatase which depletes processed mRNA carrying a 5' monophosphate, but not nascently transcribed mRNA carrying a 5' triphosphate. This aim will identify the precise genome-wide transcriptional start sites for ncRNA and mRNA.
Aim 2: Identification of ncRNA targets using iTRAQ proteomics
In bacteria ncRNAs often regulate the translation of targets through binding to the 5'UTRs of mRNAs and inhibiting access to the ribosome. Our previous RNAseq experiments revealed that while MQS mutants were abolished for expression of the five ncRNAs, there were very few differences in the abundances of other RNAs. This suggests the ncRNAs likely regulate phenotypes at the level of translation. iTRAQ will be carried out on MQS mutant and ncRNA-mutant strains to identify potential targets of ncRNA regulation. The post-transcriptional regulation of each putative target will be confirmed by constructing translational fusions with the lacZ gene and measuring β-galactosidase expression.
Aim 3: Characterising phenotypes controlled by the MQS-regulated ncRNAs
We suspect the ncRNAs are involved in the uptake, utilization and/or storage of phosphate and will begin testing this hypothesis immediately using colourmetric bioassays. Mutant strains have been constructed carrying individual deletions in each of the five ncRNAs and these will be assayed. It is likely we will identify additional potential phenotypes regulated by the ncRNAs during the course of Aim 2. Additional experiments will be designed to test each of the mutant strains. 

This project will contribute fundamental data to increase our molecular and genetic understanding of Mesorhizobium spp. in Australian soils that are critical for nitrogen fixation. This will ultimately aid in the selection and generation of highly effective agricultural legume inoculants. This research is an extension of findings made in ARC grant FT170100235 and data gleaned from this work will be used as a basis for future ARC applications. 

An internship opportunity will be explored with local biotechnology companies. 

Student type

• Future Students

Faculty

• Faculty of Health Sciences

Course type

• Higher Degree by Research

Citizenship

• Australian Citizen
• Australian Permanent Resident
• New Zealand Citizen
• Permanent Humanitarian Visa
• International Student

Scholarship base

• Merit Based

Value

The annual scholarship package (stipend and tuition fees) is approx. $60,000 - $70,000 p.a.

Successful HDR applicants for admission will receive a 100% fee offset for up to 4 years, stipend scholarships at the 2023 RTP rate valued at $32,250 p.a. for up to a maximum of 3 years, with a possible 6 month completion scholarship. Applicants are determined via a competitive selection process and will be notified of the scholarship outcome in November 2023. 

For detailed information, visit: Research Training Program (RTP) Scholarships | Curtin University, Perth, Australia.

Scholarship Details
Maximum number awarded

1

Eligible courses

All applicable HDR courses

Eligibility criteria

Experience in Molecular biology and microbiology and an understanding of microbial genetics. 

Application process

This project has identified a preferred candidate and is no longer available.  Please review remaining scholarships projects .

Enrolment Requirements

Eligible to enrol in a Higher Degree by Research Course at Curtin University by March 2024.

Recipients must complete their milestone 1 within 6 month of enrolment and remain enrolled on a full-time basis for the duration of the scholarship.

Enquiries

The Project lead has identified a preferred candidate and is no longer accepting applications. Please click here to review remaining scholarships projects.

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