Characterization and complete genome sequence of Privateer, a highly prolate Proteus mirabilis podophage

Abstract

The Gram-negative bacterium Proteus mirabilis causes a large proportion of catheter-associated urinary tract infections, which are among the world's most common nosocomial infections. Here, we characterize P. mirabilis bacteriophage Privateer, a prolate podophage of the C3 morphotype isolated from Texas wastewater treatment plant activated sludge. Basic characterization assays demonstrated Privateer has a latent period of ~40 min and average burst size around 140. In the 90.7 kb Privateer genome, 43 functions were assigned for the 144 predicted protein-coding genes. Genes encoding DNA replication proteins, DNA modification proteins, four tRNAs, lysis proteins, and structural proteins were identified. Cesium-gradient purified Privateer particles analyzed via LC-MS/MS verified the presence of several predicted structural proteins, including a longer, minor capsid protein apparently produced by translational frameshift. Comparative analysis demonstrated Privateer shares 83% nucleotide similarity with Cronobacter phage vB_CsaP_009, but low nucleotide similarity with other known phages. Predicted structural proteins in Privateer appear to have evolutionary relationships with other prolate podophages, in particular the Kuraviruses.

Keywords: Bacteriophage; Genomics; Prolate; Proteus; Urinary tract infection.

Figure 1. Privateer isolation and imaging.

(A) Privateer clear plaques with a diameter of ~0.15-mm on P. mirabilis ATCC 35659 lawn. (B) The blue band containing the purified Privateer particles following CsCl step-gradient ultracentrifugation. (C) Privateer transmission electron micrograph displaying virions ~145 × 35 nm, with a stubby ~10-nm tail.

Figure 2. Characterization of Privateer infection.

(A) Privateer adsorption curve, where phages not adsorbed to host cells were detected relative to input. P/Po = free phages at time point/free phages at 0 min. (B) Privateer one-step growth curve, where plaque-forming units were quantified after MOI = 0.01 infection. (C) Lysis curve at MOI = 10, where OD₅₅₀ of host liquid culture was measured over time. Representative instances of three replicates are shown.

Figure 3. Privateer genome plot.

The predicted genes are color-coded corresponding to the functional categories of their protein products. The label for virion-associated proteins detected in purified phage particles by mass spectrometry are bordered with red.

Figure 4. Comparative genomics among similar phiEco32-like phages.

Genome organization and comparison of protein identities for Proteus phage Privateer with selected C3 morphotype podophages. Cronobacter phage vB_CsaP_009 and Cronobacter phage vB_CsaP_GAP52 have the highest nucleotide identity with Privateer, and are all unclassified within the Podoviridae family. Escherichia phage Paul, Escherichia phage vB_EcoP_SU10, and Escherichia virus phiEco32 are phages in the Kuravirus genus. Proteins sharing significant sequence identity with at least 10⁻²⁰ BLASTp expectation value are linked via gray bands (see “Materials and Methods”).

Figure 5. Analysis of Privateer virion proteins.

Proteins of phage Privateer identified by SDS-PAGE and mass spectrometry. (A) CsCl purified phage particles were separated on a 4–20% Tris-glycine SDS-PAGE gel. Molecular masses (in kiloDalton) of the protein ladder are displayed to the left of the gel. The white and black arrowheads indicate the expected location for minor and major capsid bands, respectively. (B) Table of mass spectrometry results for trypsin-digested Privateer proteins from whole phage particles. The total spectrum count is equal to the total number of total peptide spectral matches assigned to the protein, and the unique peptide count is equal to the number of peptide sequences exclusive to the protein. (C) Aligned sequences of the annotated major and minor capsid proteins. The highlighted regions indicate peptides identified via mass spectrometry.

Figure 6. Phylogenetic tree based on phage morphogenesis proteins.

The phylogenetic tree was built using four conserved structural proteins: portal, scaffold, major capsid and tail tubular protein. Branch support values are displayed in red. See “Materials and Methods” for additional details and accessions.