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Characterization and Partial Purification of an Inhibitory Factor Secreted by Bacillus anthracis and Aeromonas jandaei
Author(s) -
McCartney Julia,
Nesemeier Jacob,
Peterson Nancy
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.620.4
Subject(s) - bacteria , microbiology and biotechnology , agar , biology , agar plate , mold , growth inhibition , bacillus anthracis , food science , chemistry , botany , biochemistry , in vitro , genetics
Over the past 40 years, there has been a dramatic increase in the number of amphibious species that have become extinct or endangered. One cause of this increase is due to parasitic colonization of water mold which has been shown to be a significant cause of frog embryo mortality. Bacteria found on frog egg masses appear to have the ability to inhibit water mold growth. We tested two bacteria species, Bacillus anthracis and Aeromonas jandaei , for their ability to inhibit water mold growth. A narrow ring of bacteria was streaked on LB agar at the periphery of an 8′ petri dish and a 9 mm plug of water mold was placed at the center of the dish. The diameter of water mold growth was measured daily. B. anthracis and A. jandaei were plated 0, 1, and 2 days before adding the water mold plug; B. anthracis inhibited growth 11%, 38% and 93% respectively and A. jandaei inhibited water mold growth 20%, 47% and 91% respectively. We found the inhibition to be nutrient dependent; higher inhibition was observed when assayed on high protein compared to high sugar medium. Growth of bacteria on high protein medium resulted in an increase in the pH over time. However, when we grew water mold on pH‐buffered medium we showed that pH difference could not account for the all inhibition seen in our bioassays. After confirming inhibition, partial purification of the inhibitory factor was investigated. Bacteria were grown for 3–6 days in LB broth. After centrifugation to remove bacteria, the conditioned growth medium was tested for inhibitory factor using a similar bioassay described above. The conditioned media was also tested for heat resistance, showing stability up to 64°C for 30 minutes. The inhibitory factor was then extracted into ethyl acetate and separated using a silica column. Fractions were tested by bioassay and the active fractions were analyzed by NMR. We were able to detect low levels of contaminant plasticizer, Di‐octylphthalate, in both bioactive and inactive fractions. However, we were not able to detect the inhibitory factor itself. We are currently working to scale up the amount of conditioned media for the isolation of the active component. Taken together, these data show that the inhibitory factor works at a distance from the water mold indicating that it is secreted and diffusible from the bacteria. This suggests that populations of bacteria living in egg masses could serve to inhibit water mold colonization and increase survivorship of frog embryos. Further, the concentration of inhibitory molecule needed to affect growth is small, indicating that future practical application would allow small scale use. Support or Funding Information We would like to thank Dr. Ruthig, Dr. Boaz, and Kelsey LaMartina for technical assistance. Funding for this work was kindly provided by the North Central College Summer Grants and the North Central College Student Governing Association.