Team:Newcastle/Results/Chemotaxis
Exploring Bacterial Chemotaxis
As proof of concept, the project aimed to introduce a plasmid containing an operon for naringenin biosynthesis. Naringenin belongs to a group of chemicals named flavonoids which play an important role in plant-microbe interactions.
Through the introduction of a naringenin biosynthesis operon into our root-colonising Pseudomonas sp. chassis, it was hypothesised that the a microbial community could be engineered. This would be done by attracting selected free-living nitrogen fixing bacteria (FLNFB) in order to localise nitrogen fixation around the root. As a result of this, one would be able to alleviate the over usage of synthetic fertilisers which have numerous detrimental impacts on the environment, ranging from eutrophication [1] to degradation of soil health [2].
We selected Azorhizobium caulinodans (ORS571), Azospirillum brasilense (SP245), and Herbaspirillum seropedicae (Z67) as our FLNFB as they each form different interactions with plant roots. For example, A. caulinodans has been shown to fix nitrogen when free living and when in symbiosis with Sesbania rostrata, a semi-aquatic tree [3]. H. seropedicae on the other hand is a root endophyte, much like our Pseudomonas spp. and commonly colonises popular crops such as wheat and maize [4].
These bacteria demonstrate potential to provide nitrogen nourishment in different ways. However, for the proof of concept, it was important to demonstrate that bacterial behaviour could be influenced by naringenin exposure. As such, a series of assays regarding naringenin’s ability to influence a microbiome were a core aspect of our project.
Characterising Bacterial Behaviour in a Laboratory Environment
Prior to conducting in depth assays, we first needed to identify how our FLNFB would behave in our laboratory. The team approached this by identifying what areas were integral to understanding changes in bacterial behaviour. We identified that we needed to characterise growth in liquid culture and on solid agar. Growth on agar was further divided into colony morphology and average diameter after 24 or 48 hours (species dependent). We also characterised the change in growth rate in the presence of our selected chemoattractant –naringenin – relative to standard growth in LB.
As a measure of our methods, we refer to our Escherichia coli (DH5α) control. This is as E. coli has been very thoroughly studied and so we can refer how our E. coli behaves in relation to the literature.
Assessing the Impact of Naringenin on Bacterial Behaviour
To assess chemotactic behaviour in response to naringenin, our team adopted 3 approaches that were identified from established literature. These approaches included agar-based, capillary-based and microscopy-based techniques. The aim of all variants was to demonstrate a change in behaviour in the presence of different chemicals; said behaviour would then give a representation of chemotaxis in our species.
As previously mentioned, the Alternative Root proof of concept involves naringenin’s ability to influence the microbiome. Because of this, bacterial behaviour in the presence of naringenin was assessed. Observed behaviour would then be compared to a blank control (distilled water or 1.5% ethanol) and, when appropriate, a positive control (malate).
REFERENCES & Attributions
1. Yang X-e, Wu X, Hao H-l, & He Z-l (2008) Mechanisms and Assessment of Water Eutrophication. Journal of Zhejiang University. Science. B 9(3):197-209.
2. Usman MN, MG; Musa, I (2015) Effect of Three Levels of NPK Fertilizer on Growth Parameters and Yield of Maize-Soybean Intercrop. International Journal of Scientific and Research Publications 5(9).
3. Liu W, et al. (2017) Azorhizobium caulinodans Transmembrane Chemoreceptor TlpA1 Involved in Host Colonization and Nodulation on Roots and Stems. Frontiers in Microbiology 8:1327..
4. Pedrosa FO, et al. (2011) Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses. PLoS Genetics 7(5):e1002064.
Attributions: Connor Trotter