A generalized transducing phage (ϕIF3) for the genomically sequenced Serratia marcescens strain Db11: a tool for functional genomics of an opportunistic human pathogen

Abstract

A bacteriophage (ϕIF3) capable of mediating generalized transduction in Serratia marcescens strain Db11 has been isolated and characterized. The genome of this Serratia strain has recently been sequenced and is likely to become the reference strain for S. marcescens researchers. ϕIF3 is most likely a virulent phage, which can transduce markers at frequencies of 10−6 transductants per p.f.u. It has a lipopolysaccharide receptor and was determined to have a latent period of 50 min and a burst size of approximately 100 phages. The phage DNA was resistant to digestion with restriction enzymes. Electron microscopy showed ϕIF3 to be a member of the family Myoviridae. This is the first report of a generalized transducing phage able to infect Db11 and this phage will be a valuable tool for functional genomic analysis of the pathogen host.

The Pseudomonas aeruginosa generalized transducing phage φPA3 is a new member of the φKZ-like group of ‘jumbo’ phages, and infects model laboratory strains and clinical isolates from cystic fibrosis patients

“…Treated sewage effluent was collected from Milton, Cambridge. Phage isolation and sample preparation were carried out as described by Petty et al (2006). Phages obtained were plaque-purified four times.…”

“…Phages obtained were plaque-purified four times. High-titre lysates were obtained as described by Petty et al (2006), and following removal of the lysate, an additional spin (1 min, 20 000 g) was carried out in order to remove any 'glutinous' precipitate, which often carried over after the initial spin.…”

“…DNA was extracted from phage virions using the Lambda DNA extraction protocol from the Phase Lock Gel (PLG) kit by 5 PRIME. Lysates isolated as described in Petty et al (2006) were used for all phage DNA extractions. Phage DNA digestions were performed for 2 h at 37 uC and assayed by electrophoresis on a 1 % agarose gel.…”

“…Phage adsorption experiments were performed as described by Petty et al (2006). Briefly, 10 ml overnight cultures of PAO1 and E. coli DH5a were infected with QPA3 (m.o.i.…”

“…A QPA3 one-step growth curve was conducted as described by Petty et al (2006). Overnight cultures of PAO1 were diluted to OD 600 0.02 in 25 ml LB in a sterile 250 ml conical flask and grown in a shaking waterbath at 37 uC, 300 r.p.m.…”