Metabolome profiling reveals metabolic cooperation between Bacillus megaterium and Ketogulonicigenium vulgare during induced swarm motility

Abstract

The metabolic cooperation in the ecosystem of Bacillus megaterium and Ketogulonicigenium vulgare was investigated by cultivating them spatially on a soft agar plate. We found that B. megaterium swarmed in a direction along the trace of K. vulgare on the agar plate. Metabolomics based on gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS) was employed to analyze the interaction mechanism between the two microorganisms. We found that the microorganisms interact by exchanging a number of metabolites. Both intracellular metabolism and cell-cell communication via metabolic cooperation were essential in determining the population dynamics of the ecosystem. The contents of amino acids and other nutritional compounds in K. vulgare were rather low in comparison to those in B. megaterium, but the levels of these compounds in the medium surrounding K. vulgare were fairly high, even higher than in fresh medium. Erythrose, erythritol, guanine, and inositol accumulated around B. megaterium were consumed by K. vulgare upon its migration. The oxidization products of K. vulgare, including 2-keto-gulonic acids (2KGA), were sharply increased. Upon coculturing of B. megaterium and K. vulgare, 2,6-dipicolinic acid (the biomarker of sporulation of B. megaterium), was remarkably increased compared with those in the monocultures. Therefore, the interactions between B. megaterium and K. vulgare were a synergistic combination of mutualism and antagonism. This paper is the first to systematically identify a symbiotic interaction mechanism via metabolites in the ecosystem established by two isolated colonies of B. megaterium and K. vulgare.

Fig. 1.

Principal-component analysis results for different samples of intracellular metabolites, including monoculture of B. megaterium (B.m) monoculture of K. vulgare (K.v), and coculture, at different times. (a) Score plot. The samples of a monoculture of B. megaterium are in red, the samples of a monoculture of K. vulgare are in blue, and cocultured samples are in green. Samples in different time spots also have different symbols; 24 h, cross; 48 h, circle; and 72 h, asterisk. (b) Loading plot. The horizontal axis in both figures was defined as the 1st principal component, and the vertical axis was defined as the 2nd principal component.

Fig. 2.

Comparison of important primary metabolites in B. megaterium (solid bars) to those in K. vulgare (hatched bars) at 24 h. Relative abundance was calculated by normalization of the peak area of each metabolite to the internal standard and to the dry weight. **, P < 0.01; ***, P < 0.001. The error bars represent standard deviations.

Fig. 3.

Swarming pattern of the ecosystem via chemotaxis of species and exchange of metabolites. The photographs show monocultures of B. megaterium and K. vulgare and coculture after 48 h of growth at 30°C on soft agar.

Fig. 4.

Comparison of amino acids and nutrition compounds for B. megaterium (gray bars), K. vulgare (hatched bars), and medium (open bars). (a) Comparison of amino acids and nutrition compounds released by K. vulgare compared to those released by B. megaterium. **, P < 0.01; ***, P < 0.001. (b) Change in amino acids after migration. Relative abundance was calculated by normalization of the peak area of each metabolite to the internal standard and to the dry weight. The maximal concentration of each compound was set as 100%. The error bars represent standard deviations.

Fig. 5.

Comparison of erythrose, erythritol, guanine,and inositol in different agar extracts. Relative abundance was calculated by normalization of the peak area of each metabolite to the internal standard and to the dry weight. The maximal concentration of each compound was set as 100%. The error bars represent standard deviations.

Fig. 6.

Comparison of the levels of sugar acids in the agar extracts of K. vulgare compared to those of the coculture. Relative abundance was calculated by normalization of the peak area of each metabolite to the internal standard and to the dry weight. The maximal concentration of each compound was set as 100%. The error bars represent standard deviations.

Fig. 7.

Comparison of DPA in B. megaterium (“brick” bars) to that in the coculture (stippled bars). Relative abundance was calculated by normalization of the peak area of each metabolite to the internal standard and to the dry weight. The maximal concentration of DPA in the mixed sample at 72 h was set as 100%. The error bars represent standard deviations.

Fig. 8.

Comparison of amino acids in the coculture (stippled bars) with those in B. megaterium (“brick” bars) at 48 h (a) and 72 h (b). Relative abundance was calculated by normalization of the peak area of each metabolite to the internal standard and to the dry weight. The maximal concentration of each amino acid in B. megaterium was set as 100%. The error bars represent standard deviations.