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Title:   Author  item
                                            PLAUT, R - Food And Drug Administration(FDA)item
                                            RIGGINS, D - Food And Drug Administration(FDA)item
                                            Boatwright, Jr, Williamitem
                                            Loving, Crystalitem
                                            Register, Karenitem
                                            Nicholson, Tracyitem
                                            STIBITZ, S - Food And Drug Administration(FDA)
  Submitted to: Molecular Genetics of Bacteria and Phage
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 8/1/2014
                                    Publication  Date: 8/1/2014
                                    Citation: Plaut, R.D., Riggins, D.P., Boatwright Jr, W.D., Loving, C.L., Register, K.B., Nicholson, T.L., Stibitz, S. 2014. Introduction of plasmid DNA into an ST398 livestock-associated methicillin-resistant Staphylococcus aureus strain. 2014 Molecular Genetics of Bacteria and Phages Meeting. Paper No. 47. p. 3.Interpretive Summary:
                                Technical Abstract: MRS926 is a livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) strain of sequence type (ST) 398. In order to facilitate in vitro and in vivo studies of this strain, we sought to tag it with a fluorescent marker. We cloned a codon-optimized gene for TurboGFP into a shuttle vector, flanked by homology to the region of the chromosomal gene glmS. Initial attempts to transfer plasmid DNA to MRS926 were unsuccessful, despite prior passage of DNA through the restriction enzyme-deficient S. aureus strain RN4220. Following examination of the whole genome sequence of another livestock-associated ST398 strain, we hypothesized that RN4220-passaged plasmid DNA was susceptible to restriction by an EcoRII endonuclease present in MRS926. We therefore engineered a strain in which the MRS926 gene encoding the EcoRII methylase was integrated into the chromosome of RN4220. Electrotransformation of plasmid DNA into the EcoRII methylase-proficient engineered strain followed by phage transduction led to the successful transfer of the plasmid to MRS926, as evidenced by the appearance of fluorescent transductants. Shifting to the non-permissive temperature for plasmid replication subsequently led to the isolation of a fluorescent strain in which the plasmid had integrated at the desired chromosomal location. This method for transferring DNA into S. aureus strains carrying the EcoRII endonuclease should facilitate further genetic manipulation of such strains.


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