Scent of danger: floc formation by a freshwater bacterium is induced by supernatants from a predator-prey coculture

Abstract

We investigated predator-prey interactions in a model system consisting of the bacterivorous flagellate Poterioochromonas sp. strain DS and the freshwater bacterium Sphingobium sp. strain Z007. This bacterial strain tends to form a subpopulation of grazing-resistant microscopic flocs, presumably by aggregation. Enhanced formation of such flocs could be demonstrated in static batch culture experiments in the presence of the predator. The ratio of aggregates to single cells reached >0.1 after 120 h of incubation in an oligotrophic growth medium. The inoculation of bacteria into supernatants from cocultures of bacteria and flagellates (grown in oligotrophic or in rich media) also resulted in a substantially higher level of floc formation than that in supernatants from bacterial monocultures only. After separation of supernatants on a C(18) cartridge, the aggregate-inducing activity could be assigned to the 50% aqueous methanolic fraction, and further separation of this bioactive fraction could be achieved by high-pressure liquid chromatography. These results strongly suggest the involvement of one or several chemical factors in the induction of floc formation by Sphingobium sp. strain Z007 that are possibly released into the surrounding medium by flagellate grazing.

FIG. 1.

Experimental setup: Sphingobium sp. strain Z007 as the control and Sphingobium sp. strain Z007 and Poterioochromonas sp. strain DS in direct-contact experiments (first panel), after centrifugation and freezing in supernatant experiments (second panel), after the first separation step on a C₁₈ cartridge (third panel), and after further separation step on an HPLC C₁₈ ODS-A reverse-phase column (fourth panel). Fr, fraction.

FIG. 2.

Cytogram (90° light scatter versus UV fluorescence) of Sphingobium sp. strain 007 after staining of the cells with DAPI. The gate in the upper right corner of the graph comprises events that were counted as bacterial cell aggregates. Gray dots depict cells of Poterioochromonas sp. strain DS in the same sample. (Inset) Cytogram used for the counting of Poterioochromonas sp. strain DS (90° light scatter versus green fluorescence). Agg, aggregates; SC, single cells; P, Poterioochromonas cells.

FIG. 3.

(A) Time course of changes in abundance of Poterioochromonas sp. strain DS and of single cells of Sphingobium sp. strain Z007 in controls (bacteria only) and in coculture with the flagellate during batch culture incubation in artificial lake water medium. (B) Ratios of aggregates to single cells of Sphingobium sp. strain Z007 in controls and in coculture with the flagellate and abundance of aggregates of Sphingobium sp. strain Z007 in controls and in coculture with the flagellate. Error bars indicate the standard errors of three replicates. Asterisks, significantly higher than control treatments at P < 0.01.

FIG. 4.

Abundance of aggregates at 24 h (t_{24 h}; upper panels) and differences in single-cell numbers between time zero (t_{0 h}) and 24 h (lower panels) of Sphingobium sp. strain Z007 in the supernatants of pure bacterial cultures (black bars; SP) and in supernatants of cocultures with Poterioochromonas sp. strain DS (white bars; SP+P). Supernatants were derived from experiments that were performed using ALW and rich medium (DSMZ 7) and from direct-contact experiments at different time points (72 h, 96 h, 120 h). Error bars indicate the standard errors of three replicates. Asterisk, significantly higher than other fractions at P < 0.01.

FIG. 5.

Abundance of single cells and ratios of aggregates to single cells of Sphingobium sp. strain Z007 after 48 h of incubation in DSMZ 7 medium amended with fractions obtained by C₁₈ cartridge separation of supernatants from pure bacterial cultures (A1 and A2) and in supernatants from cocultures (B1 and B2). Error bars indicate the standard errors of three replicates. Asterisk, significantly higher than the other fractions at P < 0.05.

FIG. 6.

(Upper panel) Ratios of aggregates to single cells of Sphingobium sp. strain Z007 after 48 h of incubation in DSMZ 7 medium amended with four subfractions obtained by HPLC separation of the 50% aq. MeOH fraction of the supernatant of the bacteria and flagellate coculture. Error bars indicate the standard errors of three replicates. Asterisk, significantly higher than the other fractions at P < 0.05. (Lower panel) HPLC chromatograms (at 210 nm and 278 nm) of the 50% aq. MeOH fraction with the marked collected fractions (Fr1 to Fr4).