Azospirillum brasilense sp7 for plant-growth promoting applications in future climates

Abstract

As the planet experiences more extreme environmental conditions such as rising global temperature leading to drought, the productivity of agricultural land is threatened by changes to soil temperature, moisture, pH and the increasing emergence of plant pathogens. With future climates becoming more unpredictable it is important welook towards nature-based solutions for more sustainable agricultural practices such as using microbial-based products for the abiotic and biotic stress management of agricultural land. Members of the genus Azospirillum are some of the best characterised plant-growth-promoting bacteria, primarily known for nitrogen fixation and phytohormone production in the rhizosphere of a remarkable range or leguminous and non-leguminous plants. A. brasilense is the most well-studied species of the genera with a multitude of biological mechanisms responsible for the tolerance of associated plants to abiotic and biotic stress. Using synthetic biology we can exploit the versatile potential of A. brasilense for engineering applications in biofertilisation, biocontrol and biostimulation in environmental conditions related to climate change. In this work I characterise a suite of genetic tools for the high-resolution control of transcription and translation in Azospirillum brasilense sp7. In Chapter 2, I use a novel bioinformatic approach to design and test a diverse library of constitutive promoters, characterised a tightly regulated inducible promoter system and tested a leak-free set of terminators, all within a broad host range plasmid. The overarching aim of Chapter 3 was to artificially mimic the conditions of natural evolution in A. brasilense sp7 using synthetic biology. To do so, I used synthetic sRNAs for the post-transcriptional regulation of the methyl-directed mismatch repair (MMR) system proteins, MutS, MutL and MutH. I demonstrated the ability of synthetic sRNAs to repress target mRNA translation into protein in A. brasilense sp7 and E. coli. Finally, in Chapter 4 i designed and established a platform for the evolution of A. brasilense sp7 with pipelines encompassing adaptive laboratory evolution, UV-mediated mutagenesis and engineering with synthetic biology. The aim of this was to generate evolved A. brasilense sp7 strains with distinct phenotypes highly relevant to commercial agriculture in the 21st Century. Evolved strains were screened for growth, temperature tolerance, phytohormone production and biocontrol activity. The work described in these chapters reveals a cohesive story of how synthetic biology can be applied within the context of evolution for the engineering of an agronomically-important bacteria for climate-mitigating applications.

Date of Award	22 May 2025
Original language	English
Awarding Institution	Northumbria University
Supervisor	Ciaran Kelly (Supervisor)