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.

Microbiology. 2012 Dec;158(Pt 12):3063-71.

Reyes-Cortés R, Martínez-Peñafiel E, Martínez-Pérez F, de la Garza M, Kameyama L.

 

Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, Av. Instituto Politécnico Nacional No. 2508, C.P. 7360, México D.F., México

Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Av. Instituto Politécnico Nacional No. 2508, C.P. 7360, México D.F., México

Laboratorio de Microbiología y Mutagénesis Ambiental, Escuela de Biología, Universidad Industrial de Santander, Bucaramanga, Colombia

 

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 λ as well. We propose that this mutation-and-selection strategy can be used to find host factors involved in the initial steps of phage infection for any cognate pair of phage and bacteria.

PMID: 23103976

 

Supplements:

It is well known that an incomplete treatment, incorrect use or abuse of antibiotics leads multi-resistant bacteria. An alternative approach which is seriously reconsidered nowadays is bacteriophage therapy.

Briefly, bacteriophage (phage or bacterial virus) infection begins with the external membrane receptor recognition. Phage genome is then ejected into the cytoplasm, and different ORFs are expressed, in which the majority of proteins are required for the development and maturation of the virion particle.

The main structure contributing to cell-survival, the cell-envelope, is also the shield for phage infections. Nevertheless, up to date, reports related to the molecular mechanism of phage infections at the envelope level are scarce.

In this article, we proposed a simple and efficient strategy to select host mutants, resistant to phage infections, at the cell envelope level. This is based on three step method: 1) Random transposon mutagenesis and selection of phage-resistant mutants by replica plating 2) Electroporation of the phage DNA into the mutant-hosts and detection of the viral progeny in the supernatant, and 3) Sequencing the host-chromosomal regions adjacent to the transposon insertion.

This was tested using the host Escherichia coli K-12 strain W3110, and the “lambda-like” phage mEp213. All the selected mutants were related to the cell envelope. We confirmed the involvement of the OM receptor FhuA. In addition, the mutations on waaC (LPS heptosyltransferase I) and gmhD (ADP-L-glycero-D-mannoheptose-6-epimerase) genes, whose products are involved in LPS synthesis, also are required for an efficient phage infection.

A wider knowledge of the initial process of phage infection would eventually contribute to a better phage therapy.

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