Abstract
We present a detailed analysis of the genome architecture, structural proteome and infection-related properties of three Pseudomonas phages, designated LUZ7, LIT1 and PEV2. These podoviruses encapsulate 72.5 to 74.9kb genomes and lyse their host after 25min aerobic infection. PEV2 can successfully infect under anaerobic conditions, but its latent period is tripled, the lysis proceeds far slower and the burst size decreases significantly. While the overall genome structure of these phages resembles the well-studied coliphage N4, these Pseudomonas phages encode a cluster of tail genes which displays significant similarity to a Pseudomonasaeruginosa (cryptic) prophage region. Using ESI-MS/MS, these tail proteins were shown to be part of the phage particle, as well as ten other proteins including a giant 370kDa virion RNA polymerase. These phages are the first described representatives of a novel kind of obligatory lytic P. aeruginosa-infecting phages, belonging to the widespread “N4-like viruses” genus.
Highlights
Bacteriophage N4, a lytic podovirus, was originally isolated from the sewers of Genoa, Italy, using Escherichia coli K-12 as host organism (Schito et al, 1967)
N4 uses three different RNA polymerases (RNAPs) during its life cycle, including a giant virionencapsulated RNAP of 3500 amino acids that is co-injected with the viral DNA upon phage infection
As seen by mass spectrometry, the LUZ7 and LIT1/PEV2 particles contain a giant virionencapsulated RNAP (vRNAP) (3,398 amino acids) (Table 1) which is devoid of cysteine residues; this could be an important requirement for the enzyme to pass through the tail into the cell and/or through the host periplasm, which contains proteins that catalyze disulfide bond formation (Ritz and Beckwith, 2001)
Summary
Bacteriophage N4, a lytic podovirus, was originally isolated from the sewers of Genoa, Italy, using Escherichia coli K-12 as host organism (Schito et al, 1967). N4 remained a genetic orphan for more than 40 years, being the only known phage that did not require the host RNAP for transcription of its early genes (Choi et al, 2008; Lavigne et al, 2008). One of the middle gene products, the N4 single-strand-DNA binding protein, activates the E. coli σ70-holoenzyme to carry out transcription from the late phage promoters (Beck et al, 2007).
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