Biofilm development and cell death in the marine bacterium Pseudoalteromonas tunicata

Abstract

The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of P. tunicata and its interaction with marine surfaces, we examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of P. tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J. S. Webb et al., J. Bacteriol. 185:4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by P. tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment. A Delta alpP mutant derivative of P. tunicata was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P. tunicata and subsequent dispersal of surviving cells within the marine environment.

FIG. 1.

Biofilm development and cell death of the P. tunicata wild-type strain. Biofilms were stained with the BacLight LIVE/DEAD bacterial viability kit. Red propidium iodide-stained cells have a compromised cell membrane and are dead. Time points after inoculation are shown as follows: (A) 1 h; (B) 24 h; (C) 48 h; (D) 72 h; (E) 144 h; (F) 168 h. Bars, 50 μm.

FIG. 2.

The P. tunicata AlpP mutant does not show cell death during biofilm development. Biofilms were stained with the LIVE/DEAD BacLight bacterial viability kit. Time points after inoculation are shown as follows: (A) 1 h; (B) 24 h; (C) 48 h; (D) 72 h; (E) 144 h; (F) 168 h. Bars, 50 μm.

FIG. 3.

Addition of purified of AlpP to P. tunicata biofilms. Biofilms were stained with the LIVE/DEAD BacLight bacterial viability kit. (A) Add-back of AlpP to P. tunicata ΔalpP 48-h biofilms; (B) P. tunicata ΔalpP mutant biofilm plus buffer control (20 mM Tris, 0.3 M NaCl); (C) AlpP added back to young (30 h) P. tunicata wild-type biofilms before the normal onset of killing within the biofilm; (D) P. tunicata wild-type biofilm plus buffer control (20 mM Tris, 0.3 M NaCl). Bars, 50 μm. Similar results were obtained in four replicate experiments.