Isolation and characterization of a phage collection against Pseudomonas putida

Abstract

The environmental bacterium, Pseudomonas putida, possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production as a cell factory, as well as in bioremediation strategies to degrade various aromatic pollutants. For P. putida to flourish in its environment, it must withstand the continuous threats posed by bacteriophages. Interestingly, until now, only a handful of phages have been isolated for the commonly used laboratory strain, P. putida KT2440, and no phage defence mechanisms have been characterized. In this study, we present a new Collection of Environmental P. putida Phages from Estonia, or CEPEST. This collection comprises 67 double-stranded DNA phages, which belong to 22 phage species and 9 phage genera. Our findings reveal that most phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require an intact lipopolysaccharide for P. putida infection. Furthermore, we show that cryptic prophages present in the P. putida chromosome provide strong protection against the infection of many phages. However, the chromosomal toxin-antitoxin systems do not play a role in the phage defence of P. putida. This research provides valuable insights into the interactions between P. putida and bacteriophages, which could have significant implications for biotechnological and environmental applications.

Figure 1. Phage isolation process by using Pseudomonas putida devoid of 13 TA systems and four prophages.

(A) Schematic representation of P. putida PaW85 genome. The location of 13 TA systems and 4 prophages that are deleted from the phage isolation strain P. putida Δ13TAΔ4φ are indicated. The figure was made with SnapGene software (www.snapgene.com). (B) A scheme of phage isolation process: environmental samples were incubated with the host P. putida Δ13TAΔ4φ overnight at 20°C. The culture was then cleared and overlay agar plates poured to visualize phage plaques. Individual plaques were purified three times before making a final phage monoculture stock.

Figure 2. The phylogenetic and morphological analysis of phages in CEPEST collection.

(A) Midpoint-rooted phylogenetic tree of 22 representative phage species from 9 clusters (G1–G9). The tree is based on multiple alignment of four concatenated protein sequences (major head protein, DNA primase, spanin, and terminase large subunit), calculated with IQ-TREE using ModelFinder and visualized using iTOL online tool. Bootstrap values >70 are shown. Dark colours mark different VIRIDIC genus clusters, and lighter tones of each colour show species belonging to the corresponding clusters. (B) Morphology of phages in CEPEST collection. A negative staining TEM image of a representative phage from each genus cluster in the CEPEST collection (G1—NoPa, G2—Vasula, G3—Amme-1, G4—Emajogi, G5—Laguja-2, G6—Luke-3, G7—Kompost-2, G8—Kurepalu-1 and G9—Kurepalu-2).

Figure 3. Temperature sensitivity of CEPEST phages.

The infection efficiency of each phage species representative was tested at temperatures from 15 to 37°C. A: Qualitative infectivity of phages from genus clusters G6, G7, G8, and G9. (B) Heatmap of infection of EOP of each phage at a certain temperature. Zero level shows the most efficient infection, and numbers (and decreasing intensity of grey colour) represent a 10-time decrease in EOP. DL (white) is the detection limit of the experiment, no infection could be detected.

Figure 4. Pseudomonas putida PaW85 prophages provide protection against several CEPEST collection phages.

Quantitative EOP measurement of phage infection on bacterial lawns of (A) P. putida PaW85 (wt), (B) P. putida PaW85 lacking 13 TA systems (Δ13TA), (C) P. putida PaW85 lacking four prophages (Δ4φ), (D) P. putida PaW85 lacking 13 TA systems and four prophages (Δ13TAΔ4φ). Then, 1.5 μl drops of 10-fold serial dilutions of phages were spotted on the strain to be tested, and plates were incubated overnight at 20°C. EOP was counted from the plaques in dilution spots.

Figure 5. Host specificity of CEPEST phages.

(A) Qualitative assay of infection of all 22 phage species representatives (indicated in the colour matrix) on different Pseudomonas hosts. (B) Quantitative EOP measurement of phage infection on their Pseudomonas hosts. Then, 1.5 μL drops of 10-fold serial dilutions of phages were spotted on the strain to be tested, and EOP was counted from the plaques. (C) EOP of G5 phages on P. putida PaW85, its ancestral strain mt-2 containing the pWW0 plasmid, and P. putida PaW85 with the pWW0 plasmid. (D) A heatmap of host specificity of phages. Zero level shows the infection of PaW85 reference strain, and numbers (and decreasing or increasing intensity of grey colour) represent 10-fold changes in EOP. DL (white) is the detection limit of the experiment, no infection could be detected. Plates were incubated for 24 h at 20°C.

Figure 6. Most CEPEST phages require intact LPS for infection.

(A) A qualitative assay of phage infection of all 22 phage species representatives on P. putida PaW85 wild type (wt) and its wbpL or wbpM deficient strains and wbpL deletion derivative in the Δ13TAΔ4φ background. (B) EOP of phages that can infect mutants with deficient LPS. Plates were incubated for 24 h at 20°C before recording the result.