title: Generation of a Stable Plasmid forIn VitroandIn VivoStudies of Staphylococcus Species.
abstract: A major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as duringin vivomodels of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid inStaphylococcus aureusUSA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools forStaphylococcusspecies. LAC-p01 was genetically manipulated into anEscherichia coli-S. aureusshuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded (dsoandsso) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use inS. aureusUSA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable inStaphylococcus epidermidisMoreover, pKK22 was maintained for 7 days postinoculation during a murine model ofS. aureussystemic infection and successfully complemented anhlamutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during bothin vitroandin vivoexperiments are powerful new tools for studies ofStaphylococcusIMPORTANCEPlasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation duringin vivostudies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from anS. aureusUSA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generationsin vitroandin vivowithout antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies ofStaphylococcusspecies.
authors.affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Christina N
authors.initials: CN
authors.lastname: Krute
authors.affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Kelsey L
authors.initials: KL
authors.lastname: Krausz
authors.affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Mary A
authors.initials: MA
authors.lastname: Markiewicz
authors.affiliation: Department of Pharmaceutical Sciences, University of New Mexico College of Pharmacy, Albuquerque, New Mexico, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Joyner
authors.affiliation: Department of Pharmaceutical Sciences, University of New Mexico College of Pharmacy, Albuquerque, New Mexico, USA.
authors.forename: Srijana
authors.initials: S
authors.lastname: Pokhrel
authors.affiliation: Department of Pharmaceutical Sciences, University of New Mexico College of Pharmacy, Albuquerque, New Mexico, USA.
authors.forename: Pamela R
authors.initials: PR
authors.lastname: Hall
authors.affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA jbose@kumc.edu.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Bose