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Title: Analysis of pMA67, a predicted rolling-circle replicating, mobilizable, tetracycline-resistance plasmid from the honey bee pathogen, Paenibacillus larvae

item Murray, Keith
item Aronstein, Katherine

Submitted to: Plasmid Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2007
Publication Date: 3/23/2007
Citation: Murray, K.D., Aronstein, K.A., De Leon, J.H. 2007. Analysis of pMA67, a predicted rolling-circle replicating, mobilizable, tetracycline-resistance plasmid from the honey bee pathogen, Paenibacillus larvae. Plasmid. 58(2):89-100.

Interpretive Summary: American foulbrood is the most serious disease of honey bees caused by bacteria. Beekeepers have controlled the disease for decades by supplying their bees with Terramycin, whose active ingredient is the antibiotic oxytetracycline (OTC). Within the past decade, many beekeepers have noticed that Terramycin has become less effective for controlling this disease. The reason for this is that some bacteria have become resistant to the OTC antibiotic. We recently discovered a DNA molecule in the AFB bacteria which carries a gene called “tetL”. This tetL gene was considered a strong candidate for causing the resistance to OTC. This DNA molecule is not a part of the bacterial chromosome, but is a small DNA molecule called a plasmid. This work thoroughly characterizes all the genes and other components of this plasmid. This characterization revealed that the plasmid is a member of a larger family of plasmids which are found in many bacterial species, and revealed several features of the replication mechanisms of the plasmid. The characterization also revealed that the plasmid is likely to be able to be physically transferred from one bacterial cell to another, and therefore can spread antibiotic resistance among different AFB bacterial cells or even different species of bacteria. We confirmed that this plasmid can confer antibiotic resistance to two other bacterial species. We also confirmed in this study that it is in fact the “tetL” gene on the plasmid that is responsible for the antibiotic resistance. We found that the plasmid is retained very well in the AFB bacteria even in the absence of any OTC treatment, and so is unlikely to significantly decline in AFB cells in nature, even if beekeepers’ use of OTC totally ceases. We believe that knowledge of the basic mechanisms of antibiotic resistance as well as plasmid replication mechanisms can aid in planning future strategies to combat honey bee bacterial infections.

Technical Abstract: This work characterizes a recently discovered natural tetracycline-resistance plasmid called pMA67 from Paenibacillus larvae—a Gram-positive bacterial pathogen of honey bees. We provide evidence that pMA67 replicates by the rolling circle mechanism, and sequence comparisons place it in the pMV158 family of rolling circle replicons. The plasmid contains predicted rep, cop and rnaII genes for control of replication initiating at a predicted double-strand origin. The efficiency of replication is sufficient to render the plasmid segregationally stable in P. larvae. The plasmid has an ssoT single-strand origin, which is shown to allow replication of the plasmid in a Gram-positive bacterium from a different genus, and also poorly in a Gram-negative bacterium, supporting the hypothesis that ssoT origins confer a broad host range. The plasmid is expected to be mobilizable due to the presence of a mob gene and an oriT site. The plasmid contains a tetL gene, whose predicted amino acid sequence implies a relatively ancient divergence from all previously known plasmid-encoded tetL genes. We confirm that the tetL gene alone is sufficient for conferring resistance to tetracyclines. Sequence comparisons, mostly with the well-characterized pMV158, allow us to predict promoters, DNA and RNA secondary structures, DNA and protein motifs, and other elements.