Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil

Abstract

Bacillus is commonly isolated from soils, with organisms of Bacillus cereus sensu lato being prevalent. Knowledge of the ecology of B. cereus and other Bacillus species in soil is far from complete. While the older literature favors a model of growth on soil-associated organic matter, the current paradigm is that B. cereus sensu lato germinates and grows in association with animals or plants, resulting in either symbiotic or pathogenic interactions. An in terra approach to study soil-associated bacteria is described, using filter-sterilized soil-extracted soluble organic matter (SESOM) and artificial soil microcosms (ASM) saturated with SESOM. B. cereus ATCC 14579 displayed a life cycle, with the ability to germinate, grow, and subsequently sporulate in both the liquid SESOM extract and in ASM inserted into wells in agar medium. Cells grew in liquid SESOM without separating, forming multicellular structures that coalesced to form clumps and encasing the ensuing spores in an extracellular matrix. Bacillus was able to translocate from the point of inoculation through soil microcosms as shown by the emergence of outgrowths on the surrounding agar surface. Microscopic inspection revealed bundles of parallel chains inside the soil. The motility inhibitor L-ethionine failed to suppress outgrowth, ruling out translocation by a flagellar-mediated mechanism such as swimming or swarming. Bacillus subtilis subsp. subtilis Marburg and four Bacillus isolates taken at random from soils also displayed a life cycle in SESOM and ASM and were all able to translocate through ASM, even in presence of L-ethionine. These data indicate that B. cereus is a saprophytic bacterium that is able to grow in soil and furthermore that it is adapted to translocate by employing a multicellular mode of growth.

FIG. 1.

Morphology of B. cereus growing in LB broth (A to E) or SESOM+ (F to J) at 30°C with shaking at 150 rpm for 4 h (A and F), 6 h (B and G), 10 h (C and H), 24 h (D and I), and 48 h (E and J). Culture samples were stained with Live/Dead BacLight stain and visualized by epifluorescence microscopy.

FIG. 2.

Morphology of soil Bacillus isolates 9 (A), 20 (B), 12 (C), and 17 (D) and B. subtilis subsp. subtilis Marburg (E) cultured in SESOM+ for 24 h at 30°C with shaking at 150 rpm. Culture samples were stained with Live/Dead BacLight stain and visualized by epifluorescence microscopy.

FIG. 3.

Culturable counts (A) and percentage of spores (B) of B. cereus in SESOM+-saturated artificial soil microcosms located in wells in SESOM+ or LB agar and incubated for 96 h at 30°C. Data are averages of three separate experiments, and error bars indicate standard errors of the means.

FIG. 4.

Translocation of B. cereus and other soil Bacillus species through artificial soil microcosms by elongation and division of cells in chains. B. cereus point inoculated at the center of a soil microcosm (A) was able to translocate through the soil matrix containing a 1 mM concentration of the motility inhibitor l-ethionine, as demonstrated by outgrowths onto the surrounding agar surface (B). The bundles of chains observed in ASM at 72 h of incubation (C) were reminiscent of the multicellularity as induced in liquid SESOM after 10 h of incubation with shaking at 30°C (D). Samples were stained with Live/Dead BacLight stain and viewed by CLSM.

FIG. 5.

Swimming (A and B) and swarming (C and D) motility of B. cereus ATCC 14579 in SESOM swim and swarm agar without (A and C) and with (B and D) a 1 mM concentration of the motility inhibitor l-ethionine, viewed after incubation at 30°C for 72 h.