title: 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 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.
authors.affiliation: 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., Mexico.
authors.forename: Ruth
authors.initials: R
authors.lastname: Reyes-Cortés
authors.affiliation: 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., Mexico.
authors.forename: Eva
authors.initials: E
authors.lastname: Martínez-Peñafiel
authors.affiliation: Laboratorio de Microbiología y Mutagénesis Ambiental, Escuela de Biología, Universidad Industrial de Santander, Bucaramanga, Colombia.
authors.forename: Francisco
authors.initials: F
authors.lastname: Martínez-Pérez
authors.affiliation: 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., Mexico.
authors.forename: Mireya
authors.initials: M
authors.lastname: de la Garza
authors.affiliation: 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., Mexico.
authors.forename: Luis
authors.initials: L
authors.lastname: Kameyama
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000378:metabolism; D003090:Coliphages / Q000254:growth & development*; D004251:DNA Transposable Elements; D048168:Escherichia coli K12 / Q000235:genetics* / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D009154:Mutation
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008070:Lipopolysaccharides; D016695:Glycosyltransferases; C080934:lipooligosaccharide 1,5-heptosyltransferase; D002238:Carbohydrate Epimerases; C580834:hldD protein, E coli