Molecular Characterization and Genome Mechanical Features of Two Newly Isolated Polyvalent Bacteriophages Infecting Pseudomonas syringae pv. garcae

Abstract

Coffee plants have been targeted by a devastating bacterial disease, a condition known as bacterial blight, caused by the phytopathogen Pseudomonas syringae pv. garcae (Psg). Conventional treatments of coffee plantations affected by the disease involve frequent spraying with copper- and kasugamycin-derived compounds, but they are both highly toxic to the environment and stimulate the appearance of bacterial resistance. Herein, we report the molecular characterization and mechanical features of the genome of two newly isolated (putative polyvalent) lytic phages for Psg. The isolated phages belong to class Caudoviricetes and present a myovirus-like morphotype belonging to the genuses Tequatrovirus (PsgM02F) and Phapecoctavirus (PsgM04F) of the subfamilies Straboviridae (PsgM02F) and Stephanstirmvirinae (PsgM04F), according to recent bacterial viruses' taxonomy, based on their complete genome sequences. The 165,282 bp (PsgM02F) and 151,205 bp (PsgM04F) genomes do not feature any lysogenic-related (integrase) genes and, hence, can safely be assumed to follow a lytic lifestyle. While phage PsgM02F produced a morphogenesis yield of 124 virions per host cell, phage PsgM04F produced only 12 virions per host cell, indicating that they replicate well in Psg with a 50 min latency period. Genome mechanical analyses established a relationship between genome bendability and virion morphogenesis yield within infected host cells.

Keywords: Pseudomonas syringae pv. garcae; Tequatrovirus and Phapecoctavirus genuses; adsorption features; bacteriophage; coffee bacterial blight; genomic structural features; virion morphogenesis yield.

Figure 1

Morphology of phages PsgM02F (a) and PsgM04F (d) plaques on a lawn of their bacterial host (Psg IBSBF-158) observed under optical microscopy (40× magnification, where bacterial debris around the phage plaques are also clearly noticed), and virion morphotypes (Phage PsgM02F: (b,c); Phage PsgM04F: (e,f) obtained by TEM analysis following negative-staining). The TEM photomicrographs of the two-phage virions allow us to observe the intact head (containing the dsDNA) and uncontracted tail (Phage PsgM02F: (b); Phage PsgM04F: (e), and empty head, contracted sheath and tail tube following translocation of its dsDNA into the host) (Phage PsgM02F: (c); Phage PsgM04F: (f).

Figure 2

Annotated genome maps of phages PsgM02F (a) and PsgM04F (b), displaying GC skew, G + C content and predicted CDS. The colored (except light blue) arrows in the outer ring represent the annotated coding sequences (CDSs) according to the annotation in Supplementary Table S1, whereas the light blue arrows correspond to hypothetical proteins, and black arrows correspond to tRNAs. The arrows represent the direction of transcription (strand + or −).

Figure 3

Proteome-based network analysis, calculated with vConTACT2 and visualized with Cytoskape (version 3.9.1), of phages PsgM02F (a) and PsgM04F (b). The predicted proteomes of phages PsgM02F and PsgM04F were clustered with the proteomes of their closest annotated phages pre-selected from the Millard phage database.

Figure 4

Phylogenetic trees calculated using the soft-core protein clusters from 32 phages connected with phage PsgM02F (highlighted in magenta, (a)) and from 35 phages connected with phage PsgM04F (highlighted in magenta, (b)) (data gathered from GenBank (GCA) and RefSeq (GCF) genome assemblies). Sequences of protein clusters were used for a Maximum Likelihood (ML) phylogenetic reconstruction using 1000 bootstrap replicates.

Figure 5

Statistical characteristics of the four predicted structural features from the PsgM02F and PsgM04F phage genomes.

Figure 6

Heatmap patterns from the results of DNA shape (Propeller Twist, Minor Groove Width, Roll, and Helical Twist) calculations for the assembled genomes of phage PsgM02F (a) and phage PsgM04F (b).

Figure 7

Correlation between the four predicted structural features of the genomes of phage PsgM02F (a) and phage PsgM04F (b).

Figure 8

Dinucleotide correlation frequency patterns in the assembled genomes of phages PsgM02F and PsgM04F. (a) AA dinucleotide, (b) AC dinucleotide, (c) AG dinucleotide, (d) AT dinucleotide, (e) CA dinucleotide, (f) CC dinucleotide, (g) CG dinucleotide, (h) CT dinucleotide, (i) GA dinucleotide, (j) GC dinucleotide, (k) GG dinucleotide, (l) GT dinucleotide, (m) TA dinucleotide, (n) TC dinucleotide, (o) TG dinucleotide, (p) TT dinucleotide. Red line: phage PsgM02F; Blue line: phage PsgM04F.

Figure 9

Dinucleotide frequency in the genomes of phages PsgM02F and PsgM04F.

Figure 10

Heatmap of the differential dinucleotide frequencies between the genomes of phages PsgM02F and PsgM04F.

Figure 11

Predicted intrinsic cyclizability along the phage genome at 7 bp resolution for (a) phage PsgM02F and (b) phage PsgM04F.

Figure 12

Heatmap patterns of the cyclizability values of the genomes of phages PsgM02F (a) and PsgM04F (b).

Figure 13

Distribution of predicted intrinsic cyclizabilities of phage PsgM02F and phage PsgM04F genomes, showing its statistics.

Figure 14

Viral proteomic trees resulting from ViPTree analyses of phages PsgM02F (a1,a2) and PsgM04F (b1,b2) and related phages. (a1,b1) A total of 5633 phage genomes were used as reference sequences to build phylogenetic trees using ViPTree. This figure identifies phages according to their official ICTV classification, with the inner and outer rings indicating their virus family and host group, respectively. (a2,b2) Expanded views of the regions of the trees containing the most closely related phages. The branches containing phage PsgM02F (a2) and phage PsgM04F (b2) are displayed in red. Red star pinpoints the location of phages PsgM02F (a1,a2) and PsgM04F (b1,b2).