Identification of a virulent phage infecting species of Nitrosomonas

Abstract

In the first and limiting step of nitrification, ammonia (NH₃) is oxidised to nitrite (NO₂^-) by the action of some prokaryotes, including bacteria of the Nitrosomonas genus. A potential approach to nitrification inhibition would be through the application of phages, but until now this method has been unexplored and no virulent phages that infect nitrifying bacteria have been described. In this study, we report the isolation of the first phage infecting some Nitrosomonas species. This polyvalent virulent phage (named ΦNF-1) infected Nitrosomonas europaea, Nitrosomonas communis, and Nitrosomonas nitrosa. Phage ΦNF-1 has the morphology of the Podoviridae family, a dsDNA genome of 41,596 bp and a 45.1 % GC content, with 50 predicted open reading frames. Phage ΦNF-1 was found to inhibit bacterial growth and reduce NH₄⁺ consumption in the phage-treated cultures. The application of phages as biocontrol agents could be a useful strategy for nitrification inhibition without the restrictions associated with chemical inhibitors.

Fig. 1. Growth of Nitrosomonas cultures in the presence of phage ΦNF-1 and the effect of the phage on the NH₄⁺ uptake in the cultures.

The growth of cultures in the presence of Φ NF-1 was monitored by the variation in the number of amoB gene copies in N. europaea (A), N. nitrosa (B), and N. communis (C) (orange circle). ΦNF-1 propagation was monitored by the increase in the number of copies of a non-coding fragment of the ΦNF-1 genome (orange cross). The control culture contained only bacteria (green). The NH₄⁺ in cultures of the three strains of N. europaea (D), N. nitrosa (E), and N. communis (F) was measured in the presence (orange) or absence (green) of ΦNF-1. Controls included ΦNF-1 alone with AOB medium (black) and sterile AOB medium (blue). Results are the average of three to five independent experiments.

Fig. 2. Morphological and genetic characterization of phage ΦNF-1.

Electron micrographs of phage particles in phage suspensions: ΦNF-1 (A) and suspension #2 (B) infecting N. europaea. In (B) N. europaea cell can be seen with phage ΦNF-1 attached to the surface. In (b) a 2.5X amplification shows phage capsids in detail. C Suspension #4 infecting N. communis, and (D) suspension #5 infecting N. nitrosa. Bar = 100 nm. (E) Genetic map of phage ΦNF-1. Each arrow corresponds to an open reading frame (ORF) drawn in scale considering the total phage genome size of 41,596 bp. White arrows correspond to unidentified ORFs. Annotated ORFs have been assigned to a function within the phage genome (structural, packaging, lysis or phage replication).