A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12

Abstract

We have developed a direct and efficient strategy, based on a three-step method, to select bacterial cell-envelope mutants resistant to bacteriophage infection. Escherichia coli K-12 strain W3110 underwent classical transposon mutagenesis followed by replica plating and selection for mutants resistant to infection by coliphage mEp213. To verify that phage resistance was due to mutations in the cell envelope, we transformed host cells with the viral genome using electroporation and selected those in which virions were subsequently detected in the supernatant. Among the nine mutants resistant to coliphage infection that we selected, six were in the fhuA gene, two were mutated in the waaC gene, and one was mutated in the gmhD gene. The latter two gene products are involved in the synthesis of lipopolysaccharide (LPS). The efficiency of plating and adsorption of phage mEp213 was affected in these mutants. We verified that LPS is required for the efficient infection of phage l as well. We propose that this mutation-andselection strategy can be used to find host factors involved in the initial steps of phage infection for any cognate pair of phage and bacteria.

K5 Capsule and Lipopolysaccharide Are Important in Resistance to T4 Phage Attack in Probiotic E. coli Strain Nissle 1917

“…Bacteria-phage interactions and subsequent evolution is a fascinating research focus and in certain cases, these interaction leads to the emergence of new gastrointestinal pathogens which generally exhibit enhanced virulence and antibiotic resistance (Bielaszewska et al, 2011). On the other hand, bacterial strains have evolved to modify their surface structures to counteract these phage infections (Nordstrom and Forsgren, 1974; Reyes-Cortes et al, 2012; Denes et al, 2015). Owing to the clinical significance of EcN (Rozanska et al, 2014; Scaldaferri et al, 2016; Sonnenborn, 2016) and it’s observed anti-shiga toxin effect against EHEC strains (Reissbrodt et al, 2009; Rund et al, 2013), it is of high priority to understand the phage sensitivity of EcN prior to its prescription in the treatment of EHEC infections.…”

Characterization of Pseudomonas aeruginosa Phage C11 and Identification of Host Genes Required for Virion Maturation

“…Totally 57 host genes were identified, the majority of them had not been found associated with phage infections previously8. Phage receptor related genes were also screened in E. coli phage mEp213 infection by employing a novel strategy to select bacterial cell-envelope mutants resistant to phage infection9. More recently, it’s found that the genome injection of E. coli phage HK97 required the glucose transporter PtsG and the periplasmic chaperone FkpA encoded by the host genes10.…”

Random Transposon Mutagenesis for Cell-Envelope Resistant to Phage Infection

“…Complementation assay: To carry out the complementation assay, plasmids pWaaC and pGmhD were transformed into their respective mutants: PRM4, PRM11, and PRM12 [ 3 ]. Then, their susceptibility to phage mEp213 was tested by overlay assay.…”

“…To better illustrate the advantages provided by this strategy, we briefl y describe the results obtained with E. coli W3110 and its phage mEp213 [ 3 ] . Firstly, we generated kanamycin-resistant mutants with miniTn10Km r transposon, and then we selected 12 phage-resistant mutants by replica-plating.…”