Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

John T. Singer, Ryan T. Phennicie, Matthew J. Sullivan, Laura A. Porter, Valerie J. Shaffer, and Carol H. Kim

Fluorescent proteins have been used to visualize different biological processes. One such process is the study of immune response to a bacterial infection in vivo. The organism being studied here is the zebrafish, which has a transparent exoskeleton in its early developmental stages, making visualization of fluorescently labeled pathogens possible. The goal of the study was to develop plasmids producing red-fluorescent-protein (RFP) in a non-toxic manner in a variety of gram negative bacteria. The labeled bacteria could then be introduced into embryonic zebrafish, followed by detection of immune response. So while bacteria were labeled with RFP, macrophages and neutrophils, which are the innate immune cells of the zebrafish, were labeled with green-fluorescent-protein (GFP) helping in detection of any interaction between the two. The plasmid they chose was a broad –host –range mobilizable, IncQ, plasmid called pMMB66EH, that has a tac promoter and a lacI repressor. They used four variants of RFP, which had shorter maturation times and higher brightness. These genes were cloned into pMMB66EH at a site downstream of the tac promoter. Since this plasmid is not self transferable, it had to be mobilized by another plasmid pRK2013 into three pathogens i.e., Edwardsiella tarda, Vibrio anguillarum and Pseudomonas aeruginosa. To be sure that their RFP producing plasmids were stable in the three bacteria, they conducted plasmid stability assays and found that most of their plasmids were stable. Of all plasmid and host combinations tested, they found P. aeruginosa PA14 bearing p67T1 to be the most stable and used this for studying immune response in zebrafish. They injected zebrafish embryos with P. aeruginosa PA14 (p67T1). Red fluorescence was visible using a wide-field epifluorescence microscope at X40 magnification. The bacteria were found to colonize the yolk initially and then spread to other regions such as the pericardium and head. Since the zebrafish was a transgenic variety capable of expressing GFP in macrophages and neutrophils, their movement towards the sites of infection could be seen as well. The most interesting result was that they were able to see phagocytosis of the bacteria by the immune cells. Although their initial plasmid constructs were designed to produce RFP under the regulation of the tac promoter, spontaneous mutations occurring in the lacI repressor resulted in constitutive production of RFP. The relevance of constitutive expression is not clear. In discussion they say that the plasmid that constitutively expresses RFP confers no additional burden on the zebrafish. Not having measured burden of any of the other plasmids that regulate the production of RFP, it is hard to say that constitutively expressed plasmids provide any benefit.
To summarize, the authors aimed to create a set of plasmids that would express RFP in a non-toxic manner in a variety of bacterial hosts that could be used in studying immune response in zebrafish. They were successful in creating plasmids that could be transferred to members of gamma-proteobacteria only. To transfer their plasmids to more unrelated bacteria, use of an IncP plasmid as the helper would be better.

Diya Sen
Graduate student
University of Idaho