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Simulated microgravity influences on Sinorhizobium medicae WSM419 growth kinetics and exometabolome

Hillman, Colin Johnson (2011) Simulated microgravity influences on Sinorhizobium medicae WSM419 growth kinetics and exometabolome. Honours thesis, Murdoch University.

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Symbiotic nitrogen fixation (SNF) is a key component of the nitrogen cycle. The majority of this fixed nitrogen is provided by leguminous plants in a tightly controlled symbiosis with a specialised array of root nodule bacteria (RNB) collectively known as rhizobia. Central to the successful establishment of the symbiotic pairing is the recognition of root-exuded isoflavones by the RNB. In response, the RNB synthesise nodulation factors to initiate symbiotic partnership. Due to the importance of the legume-rhizobia system within the biological, industrial and agricultural sectors, research has been focussed on studying the symbiotic partnership, the communication dynamics involved during the symbiosis and the partners in terrestrial contexts. However, research on the symbionts or into the symbiosis has not been extended to extra-planetary environments. As a prelude to such studies, information is required regarding the ability of the symbiotic partners to cope in microgravity conditions.

Understanding how the legume-rhizobia symbiosis copes under reduced gravity environments would present numerous benefits, primarily in permitting the development of efficient food production initiatives for future space colonisation programmes. The impetus behind this is simple; plants recycle animal wastes to provide animal nutrients and vice versa. Nitrogen recycling will be one of the key aspects involved in these processes. Unfortunately, space-based gravity studies are almost prohibitively expensive and prolonged investigations can seldom be conducted (Hoson et al., 1997). Clinostats can be used to mimic some microgravity-associated aspects and help to provide valuable initial data sets and predictor models for future gravity-oriented studies; although there have been noted criticisms regarding their usefulness (Brown, 1979; Brown et al., 1976).

Before future gravitropic studies can be conducted, basic parameters need to be addressed. Considerations include the effects of microgravity on rhizobial growth kinetics, extracellular metabolite flux rates and how this might affect the ability of the host-microsymbiont pairing to become firmly established. Central to these parameters will be the requirement to reliably measure these variables. The overall objective of this research thesis was to measure the growth kinetics and repertoire of chemical signals emitted by Ensifer medicae WSM419 (hereafter referred to as Sinorhizobium medicae WSM419), during episodes of simulated microgravity onboard 2-dimensional, vertical clinostats.

First, two metabolite procedures were optimised; an intracellular one and an extracellular protocol. Before intracellular metabolite analysis could be initiated, it was crucial to assess the effects of quenching procedures on the viability of S. medicae WSM419. This was especially important for the intracellular method, owing to the rapid turnover rates of intracellular metabolites and their lower concentrations compared to exometabolites. One suitable quenching agent to test was cold-glycerol saline (Villas-Boas and Bruheim, 2007).

S. medicae WSM419 cells were quenched using cold-glycerol saline, which has been described as the most reliable quenching agent developed to date. However, compared to the saline control treatments, the quenched WSM419 cells consistently displayed markedly reduced viability (between 5% and 20% survival rates) and delayed growth, even after 1 week. These viability results question the applicability of cold glycerol-saline as a suitable quenching agent. The intracellular metabolites were therefore not extracted with this protocol, in light of these findings. The extracellular protocol involved culturing the WSM419 cells, removing them via filtration and then processing the extracellular supernatant. In contrast to the intracellular method, the extracellular protocol generated high-quality, dependable data sets for both the technical and biological replicates. Visualisation of the data sets using Principal Component Analysis (PCA) confirmed the ability of the extracellular protocol to produce dependable and consistent data sets.

With the extracellular protocol verified as a dependable method, the growth kinetics experiments were next undertaken. Free-living WSM419 cells were cultured both on a gyratory shaker and a vertical clinostat. To simulate the presence of the host legume, Medicago truncatula, the isoflavone luteolin was aliquoted into one sub-group of eachtreatment to determine how the growth rate of WSM419's would be influenced. The cell treatment replicates on the gyratory shaker had identical mean generation times (9.5 h) and consistently displayed low mean growth variability (<0.04 and <0.05 OD600nm variation at most). In contrast, cell replicates incubated on the vertical clinostats exhibited far greater growth rate variability (~0.47 and ~0.01 OD 600nm at most) during their charted growth. While the non-luteolin imbibed treatment had a similar MGT (11 h) to the gyratory treatments, the luteolin-imbibed treatment on the clinostat possessed the longest MGT (25 h). Although the optical density readings accurately measured the growth kinetics of all treatments, this approach could not determine how the exometabolite profiles of the WSM419 treatments were being influenced by either microgravity or the luteolin. The possibility of methanol toxicity was also considered.

Therefore, the protocol that was adopted to analyse extracellular metabolites was employed to determine what types of compounds had been secreted into the external medium. Visual results from the PCA plots displayed four relatively distinct groups, indicating different metabolite compositions between the four treatments (gyratory shaker and clinostats, with a sub-group each experiment with added luteolin). Metabolite variation between individual replicates appeared consistent with the growth kinetic studies. For example, the low growth variability displayed by shaker-cultured cells not imbibed with luteolin clustered tightly on the PCA chart, suggesting that there was a correlation between growth rate and metabolite signature. Direct comparisons between individual replicates, elimination of the PCA outliers and the new consignment to the PCA plots was not carried out due to time constraints. A sub-group of 25 metabolites were identified as displaying the widest fluctuations across all samples. However the absence of their corresponding mass spectral tags in the internal-library prevented positive identification. Four of the metabolites were tentatively identified as sugars. Manual editing and data analysis presented major bottlenecks for the metabolite study. Even relatively small data sets necessitate thorough scrutiny, particularly when metabolite peaks fail to be properly registered by the analytical software. Again, time constraintsprevented more detailed analyses from being conducted, although the effectiveness of the extracellular metabolite method was verified.

Overall, this study suggests that the clinostats used to mimic microgravity and the addition of luteolin may exert considerable influence on WSM419 growth kinetics and external metabolite fluctuations in some situations. The devised GC-MS protocol will offer the capability to generate reliable and reproducible data sets, which could subsequently be used to identify metabolites of interest, particularly when pure standards of additional compounds are run to ensure positive identification. Furthermore, these methods can be readily applied to different bacterial species, and would have applications outside of legume-rhizobia biology. Deposition of any newly documented compounds might assist in developing more accurate theories on bacterial behaviour in exotic conditions such as microgravity.

Item Type: Thesis (Honours)
Murdoch Affiliation: School of Biological Sciences and Biotechnology
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): UNSPECIFIED
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