Bacterial Consumption of T4 Phages

Abstract

The bacterial consumption of viruses not been reported on as of yet even though bacteria feed on almost anything. Viruses are widely distributed but have no acknowledged active biocontrol. Viral biomass undoubtedly reintegrates trophic cycles; however, the mechanisms of this phase still remain unknown. ¹³C-labelled T4 phages monitor the increase of the density of the bacterial DNA concomitant with the decrease of plaque forming units. We used ¹²C T4 phages as a control. T4 phage disappearance in wastewater sludge was found to occur mainly through predation by Aeromonadacea. Phage consumption also favours significant in situ bacterial growth. Furthermore, an isolated strain of Aeromonas was observed to grow on T4 phages as sole the source of carbon, nitrogen, and phosphorus. Bacterial species are capable of consuming bacteriophages in situ, which is likely a widespread and underestimated type of biocontrol. This assay is anticipated as a starting point for harnessing the bacterial potential in limiting the diffusion of harmful viruses within environments such as in the gut or in water.

Keywords: Aeromonas; bacteriophage; stable isotope probing.

Figure A1

Phylogenetic tree of Aeromonas_Isolate_007_151020.

Figure A2

Schematic representation of the capture, digestion, and absorption of the T4 phages by Aeromonas sp. Since T4 phages are large particles (not to scale in the figure), we assume that Aeromonas captures the T4 phages due to its similarity with the cell wall of E. coli. Once captured, the proteins of the capsid may be digested by the extracellular proteases present in Aeromonas sp. and the DNA could be degraded as well. The products of these degradations could be ingested by Aeromonas via common transporters.

Figure A3

Purity of the bacteriophage preparation. We checked the purity of the bacteriophage preparation by means of electronic microscopy. Materials were directly adsorbed onto a carbon film membrane on a 300-mesh copper grid, stained with 1% uranyl acetate, dissolved in distilled water, and dried at room temperature. Grids were examined with a Hitachi HT7700 electron microscope operated at 80kV (Elexience—France), and the images were acquired with a charge-coupled device camera (AMT).

Figure 1

Bacterial growth on T4 phages. (A); Identification of ¹³C-labeled bacteria: the ¹³C-labeled T4 (red) were incubated with a microbial community of a wastewater treatment plant in the same conditions as the ¹²C control (blue). (B); Bacteria present in each sample: the bar plots show the growth of each ASVs based on the 16S rDNA copies, detailing the nine bacteria assimilating T4 phages.

Figure 2

The amount of DNA for the top nine ASVs (in ng) is plotted against the density of the Cesium gradient (in g/mL). The blue color is in the control bottle. The red color indicates the amount of DNA in the bottle supplemented with 3.2 µg of ¹³C in the ¹³C-labeled T4 bacteriophages. The dots indicate the actual measures performed after 24 h, i.e., the amount of DNA of each ASV (Qbit was combined with the 16S rDNA sequencing) in each fraction, the density of which was measured by means of refractometry. The lines indicate the Gaussian distributions to accurately estimate the mean buoyant density. The mean buoyant density of ASV 2 (Tolumonas sp.) was estimated with the theoretical value in the ¹²C bottle, as ASV2 did not grow sufficiently in that bottle to fit a reliable Gaussian fit. The pie charts indicate the ratios of the nine ¹³C-labeled ASV in the ¹³C and ¹²C bottles after 24 h, and their proportion in the initial sample is reported in the X axis. For example, the pie of Aeromonas is roughly balanced because ASV 1 (Aeromonas sp.) represents 19% and 8% in the 12C and 13C bottles, respectively. In contrast, ASV 2 (Tolumonas sp.) only grew substantially in the ¹³C bottle (red).

Figure 3

Aeromonas sp. growth on T4 phages: Aeromonas_isolate_007 isolated on the Aeromonas Isolation Agar medium grew on T4 phages as the sole carbon and nitrogen source. When a few Aeromonas cells were incubated with 10¹¹ T4 phages, the colony forming units (red) increased while the plaque-forming units (blue) decreased. The Aeromonas cell control in SM buffer without phages (grey) confirms that Aeromonas cannot use the Tris from the SM buffer as a carbon source.