High efficiency generalized transduction in Escherichia coli O157:H7

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Enterohemorrhagic E. coli O157:H7 genetic manipulation is limited to recombineering.
• Bacteriophage 933W, a prophage in E. coli O157:H7 EDL933, encodes Shiga toxin genes (stx2AB).
• Shiga toxin causes Hemolytic Uremic Syndrome.

Purpose of the Study:

• To investigate bacteriophage 933W as a tool for genetic manipulation in E. coli O157:H7.
• To assess the efficiency of bacteriophage 933W in transducing genes and markers.

Main Methods:

• Replaced stx2AB genes with a kanamycin cassette in E. coli O157:H7 using recombineering.
• Induced prophage 933W with UV light.
• Collected bacteriophage lysates and used them for transduction experiments in E. coli O157:H7 and E. coli K-12.

Main Results:

• Bacteriophage 933W lysates successfully transduced point mutations and tetracycline resistance cassettes.
• Transduction was efficient in both E. coli O157:H7 and E. coli K-12.
• Co-transduction experiments suggested a maximal transferred DNA size of 61 kB, consistent with the bacteriophage genome.
• Transductants were kanamycin-sensitive, indicating the presence of host DNA in transducing particles.

Conclusions:

• Bacteriophage 933W can be utilized for gene transduction in E. coli O157:H7.
• This provides a novel method for genetic manipulation beyond existing recombineering techniques.
• The findings open new avenues for studying Shiga toxin and related pathogens.