Strains, plasmids, and growth conditions

Comprehensive details of the strains and plasmids used in this study are shown in Table S1 in the supplementary materials. All primers used for polymerase chain reaction (PCR) in this study are listed in Table S2. P. aeruginosa was cultured in blood plate or Luria-Bertani (LB) broth medium at 37℃. Carbenicillin (Car, 300 µg/mL) and gentamicin (Gen, 200 µg/mL) (Wenzhou Kangtai Biological Technology Co., Zhejiang, China) were added as needed.

Isolation of the phage

Referring to the methodology outlined in a previous study [19], the phage vB_Pae_PLY was isolated from the sewage of the First Affiliated Hospital of Wenzhou Medical University using PAO1 as the host strain and enriched to reach a titer of 1 × 1010 plaque-forming units (PFU)/mL. The purified phage lysate was stored in SM buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 8 mM MgSO4) at 4℃ for later use. Then, the double-layer agar plate method was used to determine phage titer. Briefly, 100 µL of 109 colony-forming units (CFU)/mL bacterial host culture was mixed with 100 µL of ten-fold serially diluted phage lysate and incubated for 10 min at room temperature. Subsequently, 8 mL of melted 0.4% soft agar maintained at 45 °C was added to the phage–bacteria mixture. After mixing, the contents of the tube were immediately poured over a pre-prepared confluent monolayer of 1.5% agar. After incubation at 37℃ for 8–12 h, the number of PFUs was calculated. Serially diluted phage suspensions were prepared in triplicate for each dilution factor. The titer of the phage stock (PFU/mL) was calculated as follows:

Titer (PFU/mL) = Number of PFUs × 10 (Volume conversion factor) × Dilution factor.

Analysis of biological characteristics of the phageHost range determination

Spot assays were performed to determine the host range of the vB_Pae_PLY phage against an additional 40 strains of P. aeruginosa clinical isolates. The presence of a clear zone and lysis plaque was recorded as the tested strain being susceptible to the phage [20].

One-step growth curve

The one-step phage growth was performed as previously described but with few modifications [21]. The phage vB_Pae_PLY was mixed with the host P. aeruginosa PAO1 with a multiplicity of infection (MOI) of 0.01. After adsorption at 37℃ for 10 min, the mixture was centrifuged at 12,000 × g for 2 min to remove the unadsorbed phages in the supernatant. The pellet was washed twice with LB broth medium, resuspended in 25 mL of fresh LB broth medium, and incubated with shaking at 37°C, 180 rpm. The samples were then collected at 0, 10, 20, 30, 40, 60, 90, 120 and 240 min, respectively, and the phage titer was determined via the double-layer agar method. The burst size was calculated as the ratio of the final phage titer to the number of initial bacterial cells infected.

Transmission Electron Microscopy (TEM)

TEM imaging was performed following a previously established method to observe the morphology of phages [19].

Whole genome analysis of phageGenomic structural analysis and functional annotation

The genomic DNA of vB_Pae_PLY was extracted using λ phage genomic DNA extraction kit (column type) (Beijing Abigen Biotechnology Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Genome sequencing and analyses were performed at Shanghai Personal Biotechnology Co., Ltd. The genome was annotated using Prokka and eventually corrected manually by using BLASTP to search for similar proteins in the NCBI non-redundant database. The genomic architecture of vB_Pae_PLY was analyzed using Proksee (https://proksee.ca/) and the genome map was visualized [22]. Virulence factors and antibiotic resistance-encoding genes were searched using the Virulence Factor Database (VFDB, http://www.mgc.ac.cn/VFs/main.htm) and the Comprehensive Antibiotic Resistance Database (CARD, https://card.mcmaster.ca/analyze/rgi) webservers, respectively. In addition, phage genome-based life cycle identification was performed using the PhageAI online platform (https://phage.ai/) [19]. The complete genome sequence of vB_Pae_PLY was deposited in the GenBank database under accession number OR689712.

Phylogenetic tree analysis

ViPTree (https://www.genome.jp/viptree/) was used to generate viral proteomic tree and classify viruses based on genome-wide similarity. A total of 349 genome sequences of all Pseudomonadota phages of the family Autophagoviridae were selected for the phylogenetic tree analysis by iTOL (https://itol.embl.de/).

Identification of the phage adsorption receptorPeriodate and proteinase K treatments

Bacterial cell surface LPS and outer membrane proteins were destroyed using periodate and proteinase K, respectively [23, 24]. The PAO1 strain was cultured overnight in LB broth medium at 37 °C to an OD600 of 1 (approximately 1 ~ 3 × 109 CFU/mL). The bacterial culture was then mixed with 100 mM sodium periodate (NaIO4) and incubated at 25 °C in the dark for 2 h. Similarly, a 0.2 mg/mL proteinase K solution (QIAGEN, Germany) was added to the bacterial culture, and the mixture was incubated at 37 °C for 3 h. Following both treatments, the mixture was centrifuged at 12,000 × g for 1 min. The cells were washed and re-suspended in 1 mL LB broth medium. A control without NaIO4 or proteinase K was included.

The phage adsorption rate was then measured: 10 µL of phage suspension (1 × 108 PFU/mL) was added to 1 mL each of the above bacterial suspensions respectively and incubated at 37 °C with shaking at 180 rpm for 10 min to allow sufficient adsorption of phages. After centrifugation at 12,000 × g for 1 min, the phage titer of in the supernatant was measured using the double-layer agar plate method as described above. As a control, the phage titer in the LB broth medium alone was set to 100%. The phage adsorption rate was calculated as follows: Phage adsorption rate (%) = [(Control phage titer - Residual phage titer in supernatant)/ Control phage titer] ×100%.

LPS adsorption assay

LPS was isolated from P. aeruginosa PAO1 using the LPS Extraction Kit. The LPS adsorption assay was performed with minor modifications, as previously described [18]. Briefly, 50 µL of the extracted LPS was added to 850 µL of LB broth medium, and mixed with 100 µL of phage solution (105 PFU/mL) at 37 °C for 10 min to allow adsorption. For the control sample, 50 µL of phosphate-buffered saline (PBS) was added to the LB broth medium and mixed with the phage. The phage titer in the supernatant and the phage adsorption rate were determined as described above.

Analysis of the effect of lasR on phage infectionConstruction of the lasR knockout mutant (PaΔlasR)

The gene knockout of lasR was performed using a previously described method from PAO1 [25]. The plasmids and primers used for the gene knockout are listed in Table S1 and S2, respectively. The lasR mutant strain was screened by colony PCR and the target fragment was sequenced (Beijing Luhe Huada Gene Technology Co.,Ltd, China). Finally, transcriptome sequencing technology was used to verify the success of the lasR gene knockout.

Spot assay

To evaluate phage sensitivity, 3 µL aliquots of 10-fold serial dilutions (105–107 PFU) of each phage were spotted on a bacterial lawn on double-layer agar plates [26]. After incubation at 37 °C for 12 h, plaque formation was observed.

Adsorption rate assay

The phage adsorption assay was conducted following a previous methodology with minor modifications [27]. 100 µL of 107 PFU/mL phage solutions were mixed with 900 µL of PAO1 and PaΔlasR bacterial cultures (1 × 108 CFU/mL) separately at an MOI of 0.01, and incubated at 37 °C for 10 min to allow sufficient phage adsorption. The titer of free phage in the supernatant was determined and phage adsorption rates were calculated as described above.

Phage-killing experiment

To further compare the effect of the lasR gene on phage infection efficiency, time-killing experiments were performed to detect the number of viable bacteria during the phage-killing process with minor modifications based on previous method [19]. Firstly, the PAO1 and PaΔlasR strains were cultured overnight in fresh LB broth medium, and the bacterial suspensions were adjusted to 0.5 McFarland. Then, 30 µL of bacterial suspensions were added to 3 mL of LB broth medium, followed by the addition of 30 µL of phage (1 × 109 PFU/mL). Further, the mixture was incubated at 37 °C with shaking at 180 rpm for 12 h. Following this, 20 µL of the culture was collected at 4, 8, and 12 h intervals, respectively, for viable colony counts.

Real-Time quantitative PCR (RT-qPCR)

RT-qPCR was performed referring to a previous study [28]. Individual colonies of freshly cultured PAO1 and PaΔlasR strains were grown to the logarithmic phase in the LB broth medium. Then, bacterial RNA was extracted using RNAiso Plus. The total cDNA was synthesized using the PrimeScript™ RT Reagent Kit, and Real-time PCR was performed following the manufacturer’s instructions (Takara Biomedical Technology Co., Ltd.). The levels of mRNA of the target genes were normalized against rpsL. Using PAO1 as the control strain, the relative expression was calculated by the 2−ΔΔCt method. All primers used in this study are listed in Table S2.

Statistical analyses

All the data were expressed as mean ± standard deviation of at least three independent experiments. Statistical significance was determined using an independent two-tailed t-test. The P-values of < 0.05, < 0.01, and < 0.001 were denoted by *, **, and ***, respectively, whereas insignificance was denoted by ns. The GraphPad Prism 8.0 software was used for the statistical analysis.