Metabolic characterisation of Magnetospirillum gryphiswaldense MSR-1 using LC-MS-based metabolite profiling

Abstract

Magnetosomes are nano-sized magnetic nanoparticles with exquisite properties that can be used in a wide range of healthcare and biotechnological applications. They are biosynthesised by magnetotactic bacteria (MTB), such as Magnetospirillum gryphiswaldense MSR-1 (Mgryph). However, magnetosome bioprocessing yields low quantities compared to chemical synthesis of magnetic nanoparticles. Therefore, an understanding of the intracellular metabolites and metabolic networks related to Mgryph growth and magnetosome formation are vital to unlock the potential of this organism to develop improved bioprocesses. In this work, we investigated the metabolism of Mgryph using untargeted metabolomics. Liquid chromatography-mass spectrometry (LC-MS) was performed to profile spent medium samples of Mgryph cells grown under O₂-limited (n = 6) and O₂-rich conditions (n = 6) corresponding to magnetosome- and non-magnetosome producing cells, respectively. Multivariate, univariate and pathway enrichment analyses were conducted to identify significantly altered metabolites and pathways. Rigorous metabolite identification was carried out using authentic standards, the Mgryph-specific metabolite database and MS/MS mzCloud database. PCA and OPLS-DA showed clear separation and clustering of sample groups with cross-validation values of R²X = 0.76, R²Y = 0.99 and Q² = 0.98 in OPLS-DA. As a result, 50 metabolites linked to 45 metabolic pathways were found to be significantly altered in the tested conditions, including: glycine, serine and threonine; butanoate; alanine, aspartate and glutamate metabolism; aminoacyl-tRNA biosynthesis and; pyruvate and citric acid cycle (TCA) metabolisms. Our findings demonstrate the potential of LC-MS to characterise key metabolites in Mgryph and will contribute to further understanding the metabolic mechanisms that affect Mgryph growth and magnetosome formation.

Fig. 1. Effect of O₂ limitation on the physiology of Mgryph. Cultures were grown under O₂-limited and O₂-rich conditions and characterised at 48 h. (A) OD₅₆₅ (black bars) and cellular magnetism (C_mag; pale grey bars). Error bars are standard deviation (n = 6). Fluorescence of cells stained with Pyr-546 and transmission electron micrographs of magnetic (B) and non-magnetic (C) cells.

Fig. 2. PCA scores plot of FSM (flask standard media, control) and spent media of non-magnetosome (O₂-rich) and magnetosome-producing (O₂-limited) cells analysed with LC-MS (cross validation: R²X = 0.81 and Q² = 0.61).

Fig. 3. OPLS-DA scores plots of the spent media of magnetosome (O₂-limited), non-magnetosome producing (O₂-rich) cells and FSM controls analysed with LC-MS (cross-validation: (A) R²X = 0.83, R²Y = 0.99 and Q² = 0.96; (B) R²X = 0.76, R²Y = 0.99 and Q² = 0.98).

Fig. 4. Summary of the detected features, metabolite identification and pathway analysis of the significantly altered metabolites in the spent media of magnetosome compared to non-magnetosome producing cells. (A) Venn diagram presenting the common and the significantly changed features detected in the spent media samples, (B) pie-chart showing the significantly changed and the common number of identified metabolites (Level 1 and 2), putatively identified metabolites (Level 3 and 4) and the unidentified metabolite features between the two sets of samples; and (C) pathway analysis: the top pathways were ranked by the gamma-adjusted p-values for permutation per pathway (y-axis) and the total number of hits per pathway (x-axis). Colour graduated from white to yellow, orange and red, circle size (large > small) as well as the values of both x and y increase represents the degree of significance.

Fig. 5. Relative levels expressed as peak area (arbitrary units) of significantly altered metabolites in the spent media of Mgryph grown on O₂-limited cultures (n = 6, magnetosome producing cells) compared to O₂-rich cultures (n = 6, non-magnetosome producing cells).

Fig. 6. Pathway mapping of metabolites detected in the spent medium samples of non-magnetosome (NM), magnetosome (M) producing Mgryph cells and control FSM. Green circle indicates that the metabolite was detected in the samples while the bar graph shows the relative abundance of metabolites expressed as peak areas (arbitrary units).