Shadowing the actions of a predator: backlit fluorescent microscopy reveals synchronous nonbinary septation of predatory Bdellovibrio inside prey and exit through discrete bdelloplast pores

Abstract

The Bdellovibrio are miniature "living antibiotic" predatory bacteria which invade, reseal, and digest other larger Gram-negative bacteria, including pathogens. Nutrients for the replication of Bdellovibrio bacteria come entirely from the digestion of the single invaded bacterium, now called a bdelloplast, which is bound by the original prey outer membrane. Bdellovibrio bacteria are efficient digesters of prey cells, yielding on average 4 to 6 progeny from digestion of a single prey cell of a genome size similar to that of the Bdellovibrio cell itself. The developmental intrabacterial cycle of Bdellovibrio is largely unknown and has never been visualized "live." Using the latest motorized xy stage with a very defined z-axis control and engineered periplasmically fluorescent prey allows, for the first time, accurate return and visualization without prey bleaching of developing Bdellovibrio cells using solely the inner resources of a prey cell over several hours. We show that Bdellovibrio bacteria do not follow the familiar pattern of bacterial cell division by binary fission. Instead, they septate synchronously to produce both odd and even numbers of progeny, even when two separate Bdellovibrio cells have invaded and develop within a single prey bacterium, producing two different amounts of progeny. Evolution of this novel septation pattern, allowing odd progeny yields, allows optimal use of the finite prey cell resources to produce maximal replicated, predatory bacteria. When replication is complete, Bdellovibrio cells exit the exhausted prey and are seen leaving via discrete pores rather than by breakdown of the entire outer membrane of the prey.

FIG. 1.

Bdellovibrio filamentous growth-phase cells septate synchronously within the bdelloplast, forming both odd and even numbers of progeny. (Aa) Frequency measurement of the number of mature Bdellovibrio cells within 146 E. coli S17-1::pMAL-p2_mCherry fluorescent bdelloplasts. Average prey cell length was 4.67 μm ± 1.37 and width was 0.97 μm ± 0.09 (n = 167). (Ab) Examples of septated and mature growth-phase Bdellovibrio cells within fluorescently labeled bdelloplasts, with images used to illustrate frequency plot shown in panel Aa and described above. (Ba) Sketch showing key points in Bdellovibrio cell development within a bdelloplast. (Bb and c) Selected frames from time-lapse movies showing synchronously dividing growth-phase cells in fluorescent bdelloplasts. (Bd) Selected frames from time-lapse movies showing two synchronously dividing growth-phase cells forming different amounts of progeny (3 and 4) within a fluorescent bdelloplast. Fluorescence mCherry activity is false-colored green for clarity. Bars = 1 μm. Selected time-lapse movies are provided in Movies S1 and S2 in the supplemental material.

FIG. 2.

Bdellovibrio cells produce both odd and even numbers of progeny within the bdelloplast, as visualized by electron microscopy. (a) Distribution of number of progeny within E. coli DFB225 bdelloplasts at 3 h postinfection (n = 77). (b) Examples of septated Bdellovibrio progeny within bdelloplasts at 3 h postinfection, showing (from left to right) 3, 4, and 5 progeny cells. Cells were stained with 2% PTA (pH 7.0). Bars = 500 nm. The average E. coli DFB225 prey cell length in this experiment was 2 μm.

FIG. 3.

Rate of septation of a growth-phase Bdellovibrio cell is independent of the number of progeny (n = 47) (A), whereas Bdellovibrio maturation and bdelloplast lysis time decrease as the number of progeny increases (n = 79) (B). Time points were calculated from time-lapse movies using image time stamp data from SimplePCI software. Error bars indicate standard deviations.

FIG. 4.

Cell length and width measurements of mature attack-phase cells and immature Bdellovibrio progeny from bdelloplasts. The average length of the cells before infection (white) was 1.66 ± 0.24 μm (n = 100), while that of progeny cells in bdelloplasts at 3 h after infection (black) was 0.87 ± 0.12 μm (n = 89). The average cell width is similar between recently divided progeny cells (0.350 ± 0.05 μm) and mature attack-phase cells (0.316 ± 0.04 μm). We also show two representative cells of the two types, side by side, under the same negative staining conditions and using a just-released cell as the immature progeny example to avoid bdelloplast debris. Bar = 1 μm.