Screening of Polyvalent Phage-Resistant Escherichia coli Strains Based on Phage Receptor Analysis

Abstract

Bacteria-based biotechnology processes are constantly under threat from bacteriophage infection, with phage contamination being a non-neglectable problem for microbial fermentation. The essence of this problem is the complex co-evolutionary relationship between phages and bacteria. The development of phage control strategies requires further knowledge about phage-host interactions, while the widespread use of Escherichia coli strain BL21 (DE3) in biotechnological processes makes the study of phage receptors in this strain particularly important. Here, eight phages infecting E. coli BL21 (DE3) via different receptors were isolated and subsequently identified as members of the genera T4virus, Js98virus, Felix01virus, T1virus, and Rtpvirus. Phage receptors were identified by whole-genome sequencing of phage-resistant E. coli strains and sequence comparison with wild-type BL21 (DE3). Results showed that the receptors for the isolated phages, designated vB_EcoS_IME18, vB_EcoS_IME253, vB_EcoM_IME281, vB_EcoM_IME338, vB_EcoM_IME339, vB_EcoM_IME340, vB_EcoM_IME341, and vB_EcoS_IME347 were FhuA, FepA, OmpF, lipopolysaccharide, Tsx, OmpA, FadL, and YncD, respectively. A polyvalent phage-resistant BL21 (DE3)-derived strain, designated PR8, was then identified by screening with a phage cocktail consisting of the eight phages. Strain PR8 is resistant to 23 of 32 tested phages including Myoviridae and Siphoviridae phages. Strains BL21 (DE3) and PR8 showed similar expression levels of enhanced green fluorescent protein. Thus, PR8 may be used as a phage resistant strain for fermentation processes. The findings of this study contribute significantly to our knowledge of phage-host interactions and may help prevent phage contamination in fermentation.

Keywords: Escherichia coli BL21 (DE3); phage contamination; phage receptors; phage resistance; phage-host interaction.

FIGURE 1

Transmission electron micrograph images of Escherichia coli phage vB_EcoM_IME281.

FIGURE 2

Transmission electron micrograph images of Escherichia coli phage vB_EcoM_IME339.

FIGURE 3

Transmission electron micrograph images of Escherichia coli phage vB_EcoM_IME340.

FIGURE 4

Transmission electron micrograph images of Escherichia coli phage vB_EcoM_IME341.

FIGURE 5

Transmission electron micrograph images of Escherichia coli phage vB_EcoM_IME338.

FIGURE 6

Transmission electron micrograph images of Escherichia coli phage vB_EcoS_IME18.

FIGURE 7

Transmission electron micrograph images of Escherichia coli phage vB_EcoS_IME253.

FIGURE 8

Transmission electron micrograph images of Escherichia coli phage vB_EcoS_IME347.

FIGURE 9

One-step growth curves of Escherichia coli phages vB_EcoS_IME18, vB_EcoS_IME253, vB_EcoM_IME281, vB_EcoM_IME338, vB_EcoM_IME339, vB_EcoM_IME340, vB_EcoM_IME341, and vB_EcoS_IME347.

FIGURE 10

Phylogenetic tree based on the terminase large subunit protein amino acid sequences of Escherichia coli phages vB_EcoS_IME18, vB_EcoS_IME253, vB_EcoM_IME281, vB_EcoM_IME338, vB_EcoM_IME339, vB_EcoM_IME340, vB_EcoM_IME341, and vB_EcoS_IME347.

FIGURE 11

Evaluation of recombinant protein expression performance of Escherichia coli PR8. (A) Growth curve of E. coli BL21 (DE3) and PR8. (B) Fluorescence value of EGFP expressed by E. coli BL21 (DE3) and PR8.