Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/3/2007
Publication Date: 10/4/2007
Citation: Sharma, V.K., Stanton, T.B. 2007. Characterization of a cryptic plasmid from Escherichia coli O157:H7 and evaluation of the expression of green-fluorescent protein from genetically engineered derivatives of this plasmid [abstract]. 67th North Central Branch, American Society for Microbiology Meeting. p. 5.
Technical Abstract: Escherichia coli O157:H7 strain 86-24 harbors a 3.3 kb cryptic plasmid (pSP). Our objectives were to clone a DNA cassette expressing a green-fluorescent protein (GFP) from a lac promoter and an ampicillin resistance (Amp**r) gene on pSP, and to monitor both the expression of GFP and the stability of pSP-GFP/Amp**r derivatives. In order to identify sites to clone GFP/Amp**r cassette on pSP, a transposon expressing kanamycin resistance (Kan**r) was introduced into pSP in vitro, and derivatives expressing Kan**r (pSP Kan) were identified in an E. coli K-12 background. Primers complementary to the 5’ and 3’ ends of transposon were used to initiate nucleotide sequence analysis of pSP Kan by primer walking. The nucleotide sequence analysis revealed that pSP contained 3305 base pairs in its genome and this genome was 100% homologous (with respect to its nucleotide sequence and number and arrangement of ORFs) to the genome of small plasmids identified in E. coli O157:H7 isolates from Germany (Haarmann et al., 1998) and Japan (Makino et al., 1998). Two sites were selected on pSP for cloning of GFP/Amp**r cassette. In one derivative GFP/Amp**r cassette substituted the ORF mobA (pSP-GFP/Amp**r delta mobA), and in the second derivative GFP/Amp**r cassette was cloned at the Nsi1 site located downstream of mobA (pSP-GFP/Amp**r Nsi1). Introduction of pSP-GFP/Amp**r delta mobA or pSP-GFP/Amp**r Nsi1 into strain 86-24 resulted in the identification of Amp**r colonies that glowed green under UV illumination. Demonstrating both the stable maintenance of plasmid constructs and the expression of GFP under various laboratory conditions are important first considerations in using the GFP marker to track 86-24 in bovine fecal and tissue samples.