Two Lineages of Pseudomonas aeruginosa Filamentous Phages: Structural Uniformity over Integration Preferences

Abstract Pseudomonas aeruginosa filamentous (Pf) bacteriophages are important factors contributing to the pathogenicity of this opportunistic bacterium, including biofilm formation and suppression of bacterial phagocytosis by macrophages. In addition, the capacity of Pf phages to form liquid crystal structures and their high negative charge density makes them potent sequesters of cationic antibacterial agents, such as aminoglycoside antibiotics or host antimicrobial peptides. Therefore, Pf phages have been proposed as a potential biomarker for risk of antibiotic resistance development. The majority of studies describing biological functions of Pf viruses have been performed with only three of them: Pf1, Pf4, and Pf5. However, our analysis revealed that Pf phages exist as two evolutionary lineages (I and II), characterized by substantially different structural/morphogenesis properties, despite sharing the same integration sites in the host chromosomes. All aforementioned model Pf phages are members of the lineage I. Hence, it is reasonable to speculate that their interactions with P. aeruginosa and impact on its pathogenicity may be not completely extrapolated to the lineage II members. Furthermore, in order to organize the present numerical nomenclature of Pf phages, we propose a more informative approach based on the insertion sites, that is, Pf-tRNA-Gly, -Met, -Sec, -tmRNA, and -DR (direct repeats), which are fully compatible with one of five types of tyrosine integrases/recombinases XerC/D carried by these viruses. Finally, we discuss possible evolutionary mechanisms behind this division and consequences from the perspective of virus–virus, virus–bacterium, and virus–human interactions.


Table S3
Characteristics of accessory proteins (n=773) from the Pf prophages.

Figure S2
Pf prophages with duplicated and/or atypical location of attP sites.

Figure S3
Alignment of PA0718 variants present in the Pf prophages.

Figure S4
Comparison of core gene similarity of the Pf (pro)phages (n=108) from the lineage I (outer rings) and the lineage II (inner ring). Genes from the Pf4 prophage (the outermost black circle) were used as references. The red, blue, orange, purple and green circles represent Pf-tRNA-Gly, Pf-tRNA-Met, Pf-tRNA-Sec, Pf-tmRNA and Pf-DR prophages, respectively. The comparison was performed using BRIG software with blastn algorithm (evalue 0.1, word size 7 options). Identical Pf prophages, occupying the same branch of the tree presented in Fig. 4, were excluded for the clarity. The position of reference Pf prophages: Pf1, Pf6, Pf5, Pf7 and Pf-LESB58, are indicated by coloured triangles (yellow, black, green, red and dark blue) at the PA0721 gene position.

Figure S5a
Accessory proteins of the Pf prophages classified into Cellular Component (CC) category (n=210).

Figure S5b
Accessory proteins of the Pf prophages classified into Biological Function (BF) category (n=95).

Figure S5c
Accessory proteins of the Pf prophages classified into Molecular Function (MF) category (n=168).

Figure S6
ParDE toxin-antitoxin operon and its possible origin.

Figure S7
Operon composed of a putative Cro/Cl family transcriptional regulator, GNAT family Nacetyltransferase and adenylate kinase present in certain Pf-tRNA-Met prophages, and its possible origin.

Figure S8
Distribution of CRISPR-Cas spacer sequences in genes of the Pf (pro)phages (n=108) from the lineage I (outer rings) and the lineage II (inner rings). List of the spacer sequences, their affiliation to CRISPR-Cas type and P. aeruginosa strains carrying them are included in the table below. The red, blue, orange, purple and green circles represent Pf-tRNA-Gly, Pf-tRNA-Met, Pf-tRNA-Sec, Pf-tmRNA and Pf-DR prophages, respectively. The comparison was performed using BRIG software with blastn algorithm (evalue 0.1, word size 7 options). Identical Pf prophages, i.e. occupying the same branch of the tree presented in Fig. 4, were excluded for the clarity. The list of Pf prophages was included in supplementary figure S4, Supplementary Material online.

Figure S10
Comparison of three prophages from the lineage I (Pf-tRNA-Met-I and Pf-tRNA-Gly-I) and lineage II (Pf-tRNA-Sec-II) present in P. aeruginosa strain X78812 (CP008872).
Genes from the Pf4 prophage (the outermost black circle) were used as references. The red, blue, orange, purple and green circles represent Pf-tRNA-Gly, Pf-tRNA-Met, Pf-tRNA-Sec, Pf-tmRNA and Pf-DR prophages, respectively. The comparison was performed using BRIG software with blastn algorithm (evalue 0.1, word size 7 options). Identical Pf prophages, occupying the same branch of the tree presented in Fig. 4, were excluded for the clarity. The position of reference Pf prophages: Pf1, Pf6, Pf5, Pf7 and Pf-LESB58, are indicated by coloured triangles (yellow, black, green, red and dark blue) at the PA0721 gene position.       S11. Visualization of TM-score (a metric for measuring the similarity of two protein structures) superposition of P. aeruginosa Phd (white thick wirefames) and Phd/YefM family antitoxin from Pf prophages (white thin wirefames), red colour denotes the residue pairs of distance < 5.0 Angstrom (https://zhanglab.ccmb.med.umich.edu/TM-score/). 3D models of the proteins were predicted with SWISS-MODEL tool (https://swissmodel.expasy.org/).