|Aronstein, Katherine - Kate|
Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2008
Publication Date: 10/1/2008
Citation: Murray, K.D., Aronstein, K.A. 2008. Transformations of the Gram-positive honey bee pathogen, Paenibacillus larvae, by electroporation. Journal of Microbiological Methods. 75(2):325-328. Interpretive Summary: One major goal in the study of the American Foulbrood (AFB) bacterium of honey bees is to determine what products are produced by the bacterium during infection which allows it to first, evade the honey bee immune system, and second, to kill the infected bee larvae. These so-called virulence factors would be encoded by genes in the DNA of the bacterium. The genes for the virulence factors may be present on the bacterial chromosome, or as is often the case for disease causing bacteria, they made be present on small DNA molecules separate from the chromosome which are called plasmids. Several previous studies have found natural plasmids occurring in AFB bacteria, but it is unknown as of yet whether there are genes for virulence factors on them. One way to get evidence concerning the presence of virulence genes on plasmids is to put a plasmid into a plasmid-free strain of AFB bacteria, and see if the presence of the plasmid makes the strain more virulent in its bee larvae host in a controlled laboratory setting. Such experiments are hampered in the case of the AFB bacterium because as of yet there has been no reliable way to efficiently introduce plasmids into the bacteria. The present study overcomes that obstacle. The study develops and optimizes a method which allows the introduction of plasmids to AFB bacteria by using high voltage electrical pulses to make pores in the bacterial membrane, thereby allowing entry of plasmids. In addition to benefiting the study of the AFB bacterium’s natural plasmids, the ability to introduce plasmids into cells is also a major tool which can be utilized to inactivate presumed chromosomal virulence genes, in an effort to see if reduced virulence will result. Identification of the genes for virulence factors will allow us to design strategies to block the action of those factors.
Technical Abstract: In this study we developed an electrotransformation method for use with the Gram-positive bacterium Paenibacillus larvae—a deadly pathogen of honey bees. The method is substantially different from the only other electroporation method for a Paenibacillus species found in the literature. Using the type strain of P. larvae, B-2605, we have optimized factors pertaining to cell density at culture harvest, content of the washing/electroporation solution, field strength of the electrical pulse, recovery growth medium, and recovery time period. With the optimized method, we achieved an average transformation efficiency of 1.9 × 105 transformants/µg DNA. We demonstrate that the method is likely generally applicable with any plasmid which is able to replicate in P. larvae, and that the prepared competent cells can be stored frozen for at least 6 weeks before use. In addition, this study provides evidence that strain B-2605 may lack a functional restriction-modification system. The electroporation method herein should facilitate the study of the several previously discovered natural plasmids of P. larvae, and is a step toward developing a genetic system for this species.