Isolation of generalized transducing bacteriophages for uropathogenic strains of Escherichia coli

Abstract

The traditional genetic procedure for random or site-specific mutagenesis in Escherichia coli K-12 involves mutagenesis, isolation of mutants, and transduction of the mutation into a clean genetic background. The transduction step reduces the likelihood of complications due to secondary mutations. Though well established, this protocol is not tenable for many pathogenic E. coli strains, such as uropathogenic strain CFT073, because it is resistant to known K-12 transducing bacteriophages, such as P1. CFT073 mutants generated via a technique such as lambda Red mutagenesis may contain unknown secondary mutations. Here we describe the isolation and characterization of transducing bacteriophages for CFT073. Seventy-seven phage isolates were acquired from effluent water samples collected from a wastewater treatment plant in Madison, WI. The phages were differentiated by a host sensitivity-typing scheme with a panel of E. coli strains from the ECOR collection and clinical uropathogenic isolates. We found 49 unique phage isolates. These were then examined for their ability to transduce antibiotic resistance gene insertions at multiple loci between different mutant strains of CFT073. We identified 4 different phages capable of CFT073 generalized transduction. These phages also plaque on the model uropathogenic E. coli strains 536, UTI89, and NU14. The highest-efficiency transducing phage, ΦEB49, was further characterized by DNA sequence analysis, revealing a double-stranded genome 47,180 bp in length and showing similarity to other sequenced phages. When combined with a technique like lambda Red mutagenesis, the newly characterized transducing phages provide a significant development in the genetic tools available for the study of uropathogenic E. coli.

Fig. 1.

Dendrogram of the 77 phage isolates generated from a hierarchical cluster analysis of the plaque fingerprints of each phage on 23 different E. coli strains. Clusters represent groups of phage with identical host plaque patterns. The starred isolates are capable of generalized transduction in CFT073.

Fig. 2.

Colony PCR with dsdA-specific (A) and lacZ-specific (B) primers on transductants from each phage in duplicate. WAM3686 (ΔdsdA::kan) and WAM2909 (ΔlacZ::cm) were included as controls.

Fig. 3.

Electron micrographs of the 4 confirmed transducing phages. (A) ΦEB49; (B) ΦEB5; (C) ΦEB32; (D) ΦEB47.

Fig. 4.

Annotated map of the 47,180-bp ΦEB49 genome. The computer-based genome annotation software DNAMaster (http://cobamide2.bio.pitt.edu/) predicted the location of 74 ORFs and 2 tRNAs.