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. 2023 Oct 14;3(1):110.
doi: 10.1038/s43705-023-00318-5.

Establishment of a transparent soil system to study Bacillus subtilis chemical ecology

Carlos N Lozano-Andrade  1 , Carla G Nogueira  1 , Nathalie N S E Henriksen  1 , Mario Wibowo  1 , Scott A Jarmusch  1 , Ákos T Kovács  2   3
Affiliations

Establishment of a transparent soil system to study Bacillus subtilis chemical ecology

Carlos N Lozano-Andrade et al. ISME Commun. .

Abstract

Bacterial secondary metabolites are structurally diverse molecules that drive microbial interaction by altering growth, cell differentiation, and signaling. Bacillus subtilis, a Gram-positive soil-dwelling bacterium, produces a wealth of secondary metabolites, among them, lipopeptides have been vastly studied by their antimicrobial, antitumor, and surfactant activities. However, the natural functions of secondary metabolites in the lifestyles of the producing organism remain less explored under natural conditions, i.e. in soil. Here, we describe a hydrogel-based transparent soil system to investigate B. subtilis chemical ecology under controllable soil-like conditions. The transparent soil matrix allows the growth of B. subtilis and other isolates gnotobiotically and under nutrient-controlled conditions. Additionally, we show that transparent soil allows the detection of lipopeptides production and dynamics by HPLC-MS, and MALDI-MS imaging, along with fluorescence imaging of 3-dimensional bacterial assemblages. We anticipate that this affordable and highly controllable system will promote bacterial chemical ecology research and help to elucidate microbial interactions driven by secondary metabolites.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overview of the hydrogel bead system for studying bacterial chemical ecology.
A Hydrogel beads preparation protocol. A polymeric solution containing a mixture of Phytagel and sodium alginate is dropped into a solution of CaCl2. Then, the formed beads are soaked in 0.1 × TSB for 2 h. The excess of liquid is drained, and the beads are transferred into falcon tubes for subsequent experiments. B Hydrogel beads and size distribution. C Overview of the experimental approaches followed with the transparent soil microcosm and its possible applications. Beads within a given sample were mixed before downstream applications to reduce sampling heterogeneity. D Beads inoculated with P5_B1gfp inspected under fluorescence microscope.
Fig. 2
Fig. 2. The transparent soil microcosm supports the growth of B. subtilis and other bacterial isolates.
A Changes in B. subtilis P5_B1 populations (spore and total number of cells) on transparent soil microcosm were monitored as CFU/g over time (n = 3). The solid lines represent adjusted curve from a generalized model using the function stat_smooth in R. The gray area represents the dispersion given as confidence interval at 95%, and the points the actual count of each replicate. B Fluorescence intensity plot of gfp-labeled B. subtilis cells harvested from the beads at day 5 of inoculation. The gates were constructed from the non-fluorescent control samples. The GFP signal was detected using a 488 nm laser. C Endpoint population changes of four bacterial species on the soil microcosms. Population growth after day 1 and day 11 post inoculation were estimated by CFU/g (n = 3). D Culture-independent population dynamic estimation. A taxonomic summary showing the relative abundance of the five bacterial species inoculated into the transparent soil over 15 days (n = 3).
Fig. 3
Fig. 3. Surfactin and plipastatin are produced at detectable levels in the transparent soil microcosms.
A Base peak chromatograms (BPC) of B. subtilis lipopeptides detected on the hydrogel matrix at day 5: (1) lipopeptides detection (m/z 1000–1600), (2) Plipastatin B C17 (m/z 753.4287 ± 10 ppm), (3) Plipastatin A C17 (m/z 739.4131 ± 10 ppm), (4) Surfactin-C15 (m/z 1036.6904 ± 10 ppm), (5) Surfactin-C14 (m/z 1022.6748 ± 10 ppm). Beads within a given sample were mixed before extraction to reduce sampling heterogeneity. B MSI spectrometry reveals the presence/absence of isoforms of the surfactin and plipastatin families in each B. subtilis variant. Scale bar indicates 5 mm. C Surfactin and plipastatin dynamics over the time measure as the peak area of each compound. D Refined molecular network of the metabolic profile of P5_B1 propagated on the beads. Molecular network after filtering the complete network with parent masses between 700 and 1600, RTmean from 6 to 11 min.
Fig. 4
Fig. 4. Tomato roots colonized by B. subtilis P5_B1gfp.
The panel shows the P5_B1 colonization dynamics over 10 days (A to D: 1, 4, 7, and 10 days, respectively). The tomato seedlings were grown on the hydrogel beads and imaged by CLSM. Images are representative of three independent tomato seedlings and showed actively colonized root tips. The scale bars are indicated in each image.
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