Characterization of a 3.3-kb plasmid of Escherichia coli O157:H7 and evaluation of stability of genetically engineered derivatives of this plasmid expressing green fluorescence

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 (strain 86-24) harbors a 3.3-kb plasmid (pSP70) that does not encode a selectable phenotype. A 1.1-kb fragment of DNA encoding kanamycin resistance (Kanr) was inserted by in vitro transposon mutagenesis at a random location on pSP70 to construct pSP70-Kanr that conferred Kanr to the host E. coli strain. Oligonucleotides complementary to 5′ and 3′ ends of the fragment encoding Kanr were used for initiating nucleotide sequencing from the plus and minus strands of pSP70, and thereafter primer walking was used to determine nucleotide sequence of pSP70. Analysis of nucleotide sequence revealed that pSP70 contained 3306 base pairs in its genome and that the genome was almost 100% identical to nucleotide sequences of small plasmids identified in EHEC O157:H7 isolates from Germany and Japan. A DNA cassette encoding a green fluorescent protein (GFP), ampicillin resistance (Ampr), and a double transcriptional terminator (DT) was cloned in pSP70 either at the BamHI site (created by deletion of mobA by PCR) or at the NsiI site located downstream of mobA to generate pSP70 ΔmobA-GFP/Ampr/DT (pSM431) and pSP70-GFP/Ampr/DT (pSM433), respectively. Introduction of pSM431 or pSM433 into EHEC O157:H7 yielded ampicillin-resistant colonies that glowed green under UV illumination. Consecutive subcultures of EHEC O157:H7, carrying pSM431 or pSM433 under conditions simulating the environment of bovine intestine (no selective antibiotic, incubation temperature of 39 °C, with or without oxygen), demonstrated that these plasmids were highly stable as greater than 95% of the isolates recovered from these subcultures were positive for green fluorescence. These findings indicate that EHEC O157:H7 carrying pSM431 or pSM433 would be useful for studying persistence and shedding of this important food-borne pathogen in cattle.