Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/29/2000
Publication Date: N/A
Interpretive Summary: Salmonella typhimurium DT104 (DT104) is a Salmonella subtype that can exhibit multiple antibiotic resistance and also is more adept, relative to other Salmonella, at causing disease. In order to study the physical movements and other properties of this microbe, we designed a system in which DT104 makes a fluorescent protein (i.e., green fluorescent protein (GFP)). In order to do so, we attempted to design a plasmid-based system that DT104 could use for making GFP. Plasmids are small circular segments of DNA that bacteria can use for exogenous protein expression. Since exogenous plasmids are not required for DT104 survivability, the GFP plasmid must contain a selection marker such as an antibiotic resistance gene. An antibiotic can then be used to ensure that DT104 maintains the plasmid. Unfortunately, DT104 is endogenously resistant to the antibiotics mostly commonly used for plasmid maintenance. Using a new Zeocin**TM resistance-based plasmid, we were able to create a plasmid that can be used for GFP production in DT104. This system did not appear to perturb physiologic or virulence functions of DT104. For application purposes, this system was used to evaluate the ability of microcin-producing E. coli to kill DT104.
Technical Abstract: Salmonella typhimurium is a Salmonella serovar that can exhibit multiple antibiotic resistance and also has the potential to behave as a "superpathogen." In order to study the physical movements and other properties of this microbe, we wished to express green fluorescent protein (GFP) in a plasmid-based system. However, the antibiotic resistance profile of certain Salmonella typhimurium isolates is extensive (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, erythromycin, kanamycin, neomycin, gentamicin, apramycin, etc.) thus most conventional antibiotic selection-based plasmids are of little value. The Zeocin**TM resistance-based plasmid, pCR-Blunt II-TOPO (Invitrogen), proved to be a useful tool as we were able to clone a GFP-like gene (ECFP, Clontech) into pCR-Blunt II-TOPO and express a fluorescent protein in multiple antibiotic resistant Salmonella typhimurium and in multiresistant Salmonella dublin. This heterologous expression system did not appear to perturb physiologic functions, including cellular invasion which is a major facet of virulence, and was used to evaluate the effects of microcin 24 on multiresistant Salmonella.